CN118234707A

CN118234707A - Cyclic lipids and methods of use thereof

Info

Publication number: CN118234707A
Application number: CN202280075015.7A
Authority: CN
Inventors: M·贾亚拉曼; S·斯库利
Original assignee: Reinagard Medical Management Co
Current assignee: Reinagard Medical Management Co
Priority date: 2021-09-14
Filing date: 2022-09-14
Publication date: 2024-06-21

Abstract

The present disclosure details methods for delivering nucleic acid sequences, polypeptides or peptides for use in various lipids, compositions and/or optimization systems and delivery vehicles for vaccination against infectious agents.

Description

Cyclic lipids and methods of use thereof

Cross Reference to Related Applications

The present application claims the benefits and priorities of U.S. provisional patent application No. 63/244,146, filed on 9 months 14, 2021, U.S. provisional patent application No. 63/293,286, filed on 12 months 23, 2021, and U.S. provisional patent application No. 63/336,008, filed on 4 months 28, 2022, each of which is incorporated herein by reference in its entirety.

Technical Field

The present disclosure relates to optimized systems for delivery of nucleic acid sequences, polypeptides or peptides and methods of using these optimized systems for treating diseases, disorders and/or conditions.

Background

Proteins have been the standard of care, but in the last few years the use of nucleic acids as a therapeutic modality for a variety of diseases and therapeutic indications has become increasingly prominent. Multiple companies have shown that nucleic acids (e.g., siRNA, mRNA, circular RNA, DNA, ASO, etc.) may be more effective when compared to protein-based therapies, but that targeted delivery systems are needed for both nucleic acid and protein therapeutics to ensure that the therapeutic is localized to the targeted cell, tissue, or organ.

Current delivery systems, including lipid-based delivery systems such as lipid nanoparticles, focus on protecting the cargo being delivered (cargo), but not on the lipid being used by the delivery system, and generally not on the cargo or the localized delivery of the delivery system. There is a need in the art for improved lipid-based delivery systems.

Disclosure of Invention

The present disclosure provides novel lipids useful in delivery vehicles for delivery systems and a directional exploration platform for screening and developing targeting systems for targeted delivery of nucleic acid and protein therapeutics, e.g., to immune cells.

In one aspect of the present disclosure, provided herein is a lipid having any one of formulas (CY), (CY-I) to (CY-IX), or a pharmaceutically acceptable salt or solvate thereof, or any lipid in table (I), or a salt or solvate thereof, see below, collectively and each individually referred to as a "lipid of the present disclosure".

In one aspect of the present disclosure, provided herein is a pharmaceutical composition comprising:

a) A polynucleotide encoding at least one protein of interest, and

B) Delivery vehicle comprising at least one lipid

Wherein the composition elicits an immune response in the subject.

In one aspect, the polynucleotide is DNA.

In one aspect, the polynucleotide is RNA.

In one aspect, the RNA is short interfering RNA (siRNA).

In one aspect, the siRNA inhibits or suppresses expression of a target of interest in a cell.

In one aspect, the inhibition or inhibition is about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30％、20-40％、20-50％、20-60％、20-70％、20-80％、20-90％、20-95％、20-100％、30-40％、30-50％、30-60％、30-70％、30-80％、30-90％、30-95％、30-100％、40-50％、40-60％、40-70％、40-80％、40-90％、40-95％、40-100％、50-60％、50-70％、50-80％、50-90％、50-95％、50-100％、60-70％、60-80％、60-90％、60-95％、60-100％、70-80％、70-90％、70-95％、70-100％、80-90％、80-95％、80-100％、90-95％、90-100％ or 95-100%.

In one aspect, the polynucleotide is substantially circular.

In one aspect, the polynucleotide comprises an Internal Ribosome Entry Site (IRES) sequence operably linked to a payload sequence region.

In one aspect, the IRES sequence comprises a sequence derived from picornavirus complement DNA, encephalomyocarditis virus (EMCV) complement DNA, poliovirus complement DNA, or an antennapedia gene from drosophila melanogaster (Drosophila melanogaster).

In one aspect, the polynucleotide comprises a termination element, wherein the termination element comprises at least one termination codon.

In one aspect, the polynucleotide comprises a regulatory element.

In one aspect, the polynucleotide comprises at least one masking agent.

In one aspect, in vitro transcription is used to produce a substantially circular polynucleotide.

In one aspect, the payload sequence region comprises a non-coding nucleic acid sequence.

In one aspect, the payload sequence region comprises a coding nucleic acid sequence.

In one aspect, the coding nucleic acid sequence encodes a protein of interest of campylobacter jejuni (Campylobacter jejuni). In one aspect, the encoding nucleic acid sequence encodes a protein of interest of clostridium difficile (Clostridium difficile). In one aspect, the coding nucleic acid sequence encodes a protein of interest of Entamoeba histolytica (Entamoeba histolytica). In one aspect, the coding nucleic acid sequence encodes a protein of interest of enterotoxin B. In one aspect, the coding nucleic acid sequence encodes a protein of interest of Norwalk virus (Norwalk virus) or Norwalk virus (norovirus). In one aspect, the coding nucleic acid sequence encodes a protein of interest of helicobacter pylori (Helicobacter pylori). In one aspect, the encoding nucleic acid sequence encodes a protein of interest of rotavirus (rotavirus). In one aspect, the coding nucleic acid sequence encodes a protein of interest of candida yeast (CANDIDA YEAST). In one aspect, the coding nucleic acid sequence encodes a protein of interest of a coronavirus (coronavirus). In one aspect, the coding nucleic acid sequence encodes a protein of interest of SARS-CoV. In one aspect, the coding nucleic acid sequence encodes a protein of interest of SARS-CoV-2. In one aspect, the coding nucleic acid sequence encodes a protein of interest of MERS-CoV. In one aspect, the encoding nucleic acid sequence encodes a protein of interest of enterovirus 71. In one aspect, the coding nucleic acid sequence encodes a protein of interest of Epstein-Barr virus (Epstein-Barr virus). In one aspect, the coding nucleic acid sequence encodes a protein of interest of a gram-negative bacterium. In one aspect, the gram negative bacterium is bordetella (Bordetella). in one aspect, the coding nucleic acid sequence encodes a protein of interest of a gram positive bacterium. In one aspect, the gram positive bacterium is clostridium tetani (Clostridium tetani). In one aspect, the gram positive bacterium is Francisella tularensis (FRANCISELLA TULARENSIS). In one aspect, the gram positive bacterium is a Streptococcus (Streptococcus) bacterium. In one aspect, the gram positive bacterium is a Staphylococcus (Staphylococcus) bacterium. In one aspect, the coding nucleic acid sequence encodes a protein of interest for hepatitis. In one aspect, the coding nucleic acid sequence encodes a protein of interest of human cytomegalovirus (Human Cytomegalovirus). In one aspect, the coding nucleic acid sequence encodes a protein of interest of human immunodeficiency virus (Human Immunodeficiency Virus). In one aspect, the coding nucleic acid sequence encodes a protein of interest of human papillomavirus (Human Papilloma Virus). In one aspect, the coding nucleic acid sequence encodes a protein of interest of an influenza virus. In one aspect, the coding nucleic acid sequence encodes a protein of interest of JC virus (John Cunningham Virus). In one aspect, the coding nucleic acid sequence encodes a protein of interest of a Mycobacterium (Mycobacterium). In one aspect, the coding nucleic acid sequence encodes a protein of interest of a poxvirus (Poxviruses). In one aspect, the coding nucleic acid sequence encodes a protein of interest of pseudomonas aeruginosa (Pseudomonas aeruginosa). In one aspect, the coding nucleic acid sequence encodes a protein of interest of respiratory syncytial virus (Respiratory Syncytial Virus). In one aspect, the coding nucleic acid sequence encodes a protein of interest of the rubella virus (Rubella virus). In one aspect, the coding nucleic acid sequence encodes a protein of interest of varicella zoster virus (VARICELLA ZOSTER VIRUS). In one aspect, the encoding nucleic acid sequence encodes a protein of interest of chikungunya virus (Chikungunya virus). In one aspect, the coding nucleic acid sequence encodes a protein of interest of Dengue virus (Dengue virus). In one aspect, the coding nucleic acid sequence encodes a protein of interest of rabies virus (Rabies virus). In one aspect, the encoding nucleic acid sequence encodes a protein of interest of trypanosoma cruzi (Trypanosoma cruzi) and/or chagas disease (CHAGAS DISEASE). In one aspect, the coding nucleic acid sequence encodes a protein of interest of Ebola virus (Ebola virus). In one aspect, the coding nucleic acid sequence encodes a protein of interest of plasmodium falciparum (Plasmodium falciparum). In one aspect, the coding nucleic acid sequence encodes a protein of interest of a Marburg virus (Marburg virus). In one aspect, the coding nucleic acid sequence encodes a protein of interest of japanese encephalitis virus (Japanese encephalitis virus). In one aspect, the coding nucleic acid sequence encodes a protein of interest of the st. In one aspect, the coding nucleic acid sequence encodes a protein of interest of West Nile Virus (West Nile Virus). In one aspect, the coding nucleic acid sequence encodes a protein of interest of yellow fever virus (Yellow Fever virus). In one aspect, the coding nucleic acid sequence encodes a protein of interest of bacillus anthracis (Bacillus anthracis). In one aspect, the coding nucleic acid sequence encodes a protein of interest of a Botulinum toxin (Botulinum toxin). In one aspect, the coding nucleic acid sequence encodes a protein of interest of ricin (Ricin). in one aspect, the coding nucleic acid sequence encodes Shiga toxin (Shiga toxin) and/or a protein of interest of the Shiga toxin.

In one aspect, the polynucleotide comprises at least one modification.

In one aspect, at least 20% of the bases are modified. In one aspect, at least 30% of the bases are modified. In one aspect, at least 40% of the bases are modified. In one aspect, at least 50% of the bases are modified. In one aspect, at least 60% of the bases are modified. In one aspect, at least 70% of the bases are modified. In one aspect, at least 80% of the bases are modified. In one aspect, at least 90% of the bases are modified. In one aspect, at least 100% of the bases are modified. In one aspect, a particular base comprises at least one modification.

In one aspect, the base is adenine. In one aspect, at least 20% of the adenine bases are modified. In one aspect, at least 30% of the adenine bases are modified. In one aspect, at least 40% of the adenine bases are modified. In one aspect, at least 50% of the adenine bases are modified. In one aspect, at least 60% of the adenine bases are modified. In one aspect, at least 70% of the adenine bases are modified. In one aspect, at least 80% of the adenine bases are modified. In one aspect, at least 90% of the adenine bases are modified. In one aspect, at least 100% of the adenine bases are modified.

In one aspect, the base is guanine. In one aspect, at least 20% of the guanine bases are modified. In one aspect, at least 30% of the guanine bases are modified. In one aspect, at least 40% of the guanine bases are modified. In one aspect, at least 50% of the guanine bases are modified. In one aspect, at least 60% of the guanine bases are modified. In one aspect, at least 70% of the guanine bases are modified. In one aspect, at least 80% of the guanine bases are modified. In one aspect, at least 90% of the guanine bases are modified. In one aspect, at least 100% of the guanine bases are modified.

In one aspect, the base is cytosine. In one aspect, at least 20% of the cytosine bases are modified. In one aspect, at least 30% of the cytosine bases are modified. In one aspect, at least 40% of the cytosine bases are modified. In one aspect, at least 50% of the cytosine bases are modified. In one aspect, at least 60% of the cytosine bases are modified. In one aspect, at least 70% of the cytosine bases are modified. In one aspect, at least 80% of the cytosine bases are modified. In one aspect, at least 90% of the cytosine bases are modified. In one aspect, at least 100% of the cytosine bases are modified.

In one aspect, the base is uracil. In one aspect, at least 20% of uracil bases are modified. In one aspect, at least 30% of uracil bases are modified. In one aspect, at least 40% of uracil bases are modified. In one aspect, at least 50% of uracil bases are modified. In one aspect, at least 60% of uracil bases are modified. In one aspect, at least 70% of uracil bases are modified. In one aspect, at least 80% of uracil bases are modified. In one aspect, at least 90% of uracil bases are modified. In one aspect, at least 100% of uracil bases are modified.

In one aspect, the at least one modification is pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl uridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 5-propynyl-uridine, 1-taurinomethyl-pseudouridine, 5-methyl-uridine, 5-taurinomethyl-2-thio-uridine, 1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetyl-cytidine, 5-formyl-cytidine, N4-methyl-cytidine, 5-hydroxymethyl-cytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1-methyladenosine, N6-isopentenyl adenosine, N6- (cis-hydroxyisopentenyl) adenosine, 2-methylthio-N6- (cis-hydroxyisopentenyl) adenosine, N6-glycylcarbamoyl adenosine, N6-threonyl carbamoyl adenosine, 2-methylthio-N6-threonyl carbamoyl adenosine, N6, N6-dimethyl adenine, 7-methyl adenine, 2-methylsulfanyl-adenine, and 2-methoxy-adenine, inosine, 1-methyl-inosine, hupesine (wyosine), hupesine (wybutosine), 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine, N2-dimethyl-guanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thioguanosine, N2-methyl-6-thioguanosine, or N2, N2-dimethyl-6-thioguanosine.

In one aspect, the pharmaceutical composition comprises at least one cationic lipid selected from the group consisting of: any lipid in table (I), any lipid having the structure of formula (CY-II), any lipid having the structure of formula (CY-III), any lipid having the structure of formula (CY-IV), any lipid having the structure of formula (CY-V), any lipid having the structure of formula (CY-VI), and combinations thereof.

In one aspect, the cationic lipid is any lipid having the structure of formula (CY-I).

In one aspect, the cationic lipid is selected from the group consisting of compounds CY1, CY2, CY3, CY9, CY10, CY11, CY12, CY22, CY23, CY24, CY30, CY31, CY32, CY33, CY43, CY44, CY45, CY50, CY51, CY52, and CY 53.

In one aspect, the cationic lipid is any lipid having the structure of formula (CY-II).

In one aspect, the cationic lipid is selected from the group consisting of compounds CY4, CY5, CY16, CY17, CY18, CY25, CY26, CY37, CY38, CY39, CY46, CY56, and CY 57.

In one aspect, the cationic lipid is any lipid having the structure of formula (CY-III).

In one aspect, the cationic lipid is selected from the group consisting of compounds CY6, CY14, CY27, CY35, CY47, and CY 55.

In one aspect, the cationic lipid is any lipid having the structure of formula (CY-IV).

In one aspect, the cationic lipid is selected from the group consisting of compounds CY7, CY8, CY19, CY20, CY21, CY28, CY29, CY40, CY41, CY42, CY48, CY49, CY58, CY59, and CY 60.

In one aspect, the cationic lipid is any lipid having the structure of formula (CY-V).

In one aspect, the cationic lipid is any lipid having the structure of formula (CY-VI).

In one aspect, the pharmaceutical composition comprises an additional cationic lipid.

In one aspect, the pharmaceutical composition comprises a neutral lipid.

In one aspect, the pharmaceutical composition comprises an anionic lipid.

In one aspect, the pharmaceutical composition comprises a helper lipid.

In one aspect, the pharmaceutical composition comprises stealth lipids.

In one aspect, the weight ratio of the lipid to the polynucleotide is from about 100:1 to about 1:1.

In one aspect, the pharmaceutical composition delivers a cargo or payload into immune cells of a subject in need thereof. The immune cells may be T cells, such as cd8+ T cells, cd4+ T cells, or T regulatory cells. The immune cells may also be, for example, macrophages or dendritic cells.

In one aspect, the vaccine formulation comprises a pharmaceutical composition.

In one aspect, the vaccine is prepared with any of formulas (I) - (VI).

In one aspect, provided herein is a method of vaccinating a subject against an infectious agent, the method comprising contacting the subject with a vaccine formulation or formulation and eliciting an immune response.

In one aspect, the infectious agent is campylobacter jejuni, clostridium difficile, endo-amoeba histolyticum, enterotoxin B, norwalk or norovirus, helicobacter pylori, rotavirus, candida yeast, coronavirus (including SARS-CoV, SARS-CoV-2 and MERS-CoV), enterovirus 71, epstein-barr virus, gram negative bacteria (including bordetella), gram positive bacteria (including clostridium tetani, franciscensis, streptococci bacteria and staphylococci bacteria), and hepatitis, human cytomegalovirus, human immunodeficiency virus, human papillomavirus, influenza virus, JC virus, mycobacteria, poxvirus, pseudomonas aeruginosa, respiratory syncytial virus, rubella virus, varicella zoster virus, chikungunya virus, dengue virus, rabies virus, trypanosoma and/or calixas virus, ebola virus, malignancies, japanese encephalitis virus, st-lewis virus, shiorubiquitos, shiga toxin, shiga virus, or shiga-like bacteria.

In one aspect, the contacting is enteral (into the intestines), gastrointestinal tract, epidural (into the dura mater), oral (by way of the oral cavity), transdermal, intracerebral (into the brain), intracerebroventricular (into the ventricle), epicutaneous (coated onto the skin), intradermal (into the skin itself), subcutaneous (under the skin), nasal administration (via the nose), intravenous (into the vein), intravenous bolus, intravenous drip, intra-arterial (into the artery), intramuscular (into the muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraparenchymal (into brain tissue), intraperitoneal (infusion or injection into the peritoneum), infusion or injection, Intravesical infusion, intravitreal (via the eye), intracavitary injection (into the pathological cavity), intracavitary (into the fundus of the penis), intravaginal administration, intrauterine, extraamniotic administration, transdermal (via whole skin diffusion for systemic dispersion), transmucosal (via mucosal diffusion), transvaginal, insufflation (sniffing), sublingual, subcchear, enema, eye drops (onto the conjunctiva), ear drops, aural (in the ear or with the aid of the ear), buccal (directed towards the cheek), conjunctival, skin, teeth (to one or more teeth), electroosmosis, intracervical, dou Daona, intratracheal, extracorporeal, hemodialysis, invasive, interstitial, intraabdominal, Intra-amniotic, intra-articular, intra-biliary, intra-bronchial, intra-cystic, intra-cartilage (in cartilage), intra-caudal (in the cauda equina), intra-cerebral-cisterna (in the greater cisterna cerebellum), intra-corneal (in the cornea), intra-dental coronary, intra-coronary (in the coronary artery), intra-corpora cavernosa (in the inflatable space of the corpora cavernosa), intra-discal (in the intervertebral disc), intra-tubular (in the gland duct), intra-duodenal (in the duodenum), intra-dural (in or under the dura mater), intra-epidermal (to the epidermis), intra-esophageal (to the esophagus), intra-gastric (in the stomach), intra-gingival (in the gingiva), intra-ileum (in the distal portion of the small intestine), intra-ocular (in the distal portion of the small intestine), Intralesional (within or directly introduced to a localized lesion), intraluminal (within a lumen), intralymphatic (within a lymph), intramedullary (within a bone marrow cavity of a bone), meningeal (within a meninge), myocardial (within a myocardium), intraocular (within an eye), ovarian (within an ovary), pericardial (within a pericardium), pleural (within a pleura), prostate (within a prostate gland), pulmonary (within a lung or its bronchi), sinus (within a sinus or orbit Zhou Douna), spinal (within a spinal column), synovial (within a synovial cavity of a joint), tendon (within a tendon), testicular (within a testis), intrathecal (within cerebrospinal fluid at any level of the cerebrospinal shaft), intrathecal (within a cerebrospinal fluid at a spinal axis), Intrathoracic (intrathoracic), intratubular (intratubular of the organ), intratumoral (intratumoral), intrathecal (in middle ear (aurus media)), intravascular (in one or more blood vessels), intraventricular (in the chamber), iontophoretic (by means of an electric current in which ions of a soluble salt migrate into body tissue), lavage (flush or rinse open wounds or body cavities), translaryngeal (directly onto the larynx), nasogastric (via the nose and into the stomach), occlusive dressing techniques (topical route application, which is then covered by dressing of an occlusive region), transocular (out of the eye), transoropharyngeal (directly into the mouth and pharynx), parenteral, percutaneous, periarticular, epidural, peri-nerve, periodontal, rectal, respiratory tract (by oral or nasal inhalation into the respiratory tract for local or systemic effects), retrobulbar (postcerebral or retrobulbar), soft tissue, subarachnoid, subconjunctival, submucosa, topical, transplacental (via or across the placenta), transtracheal (across the tracheal wall), transtympanic (across or through the tympanic cavity), ureteral (to ureter), urethral (to urethra), transvaginal, caudal block (euglenoid block), diagnostic, nerve block, biliary tract perfusion, cardiac perfusion, photopheresis, or transspinal.

Drawings

Fig. 1 is a diagram illustrating one embodiment of a directionality discovery platform of the present disclosure.

Fig. 2 is a diagram showing an initial polynucleotide construct of the present disclosure, which may be linear or circular.

Fig. 3A is a diagram illustrating a series of reference polynucleotide constructs of the present disclosure, which may include at least one barcode region (BC) and/or reverse barcode region (CB) and a payload region (P).

Fig. 3B is a diagram depicting a series of reference polynucleotide constructs of the present disclosure, wherein a barcode region (BC) or a reverse barcode region (CB) may overlap with a payload region (P).

Fig. 3C is a diagram depicting a series of reference polynucleotide constructs of the present disclosure, which may include at least one tag and/or label.

Fig. 4A is a diagram depicting a series of circular reference polynucleotide constructs of the present disclosure, which may include at least one barcode region (BC) and/or reverse barcode region (CB) and a payload region (P).

Fig. 4B is a diagram depicting a series of circular reference polynucleotide constructs of the present disclosure, wherein a barcode region (BC) or a reverse barcode region (CB) may overlap with a payload region (P).

Fig. 4C is a diagram illustrating a series of reference polynucleotide constructs of the present disclosure, which may include at least one tag and/or label.

Fig. 5 is a diagram depicting a series of delivery vehicles of the present disclosure.

Detailed Description

I. Introduction to the delivery System

Compared to traditional therapies, nucleic acid therapies emerge as the dominant approach to the treatment of various diseases and therapeutic indications in view of versatility, lower immune response, and higher efficacy. For example, nucleic acid therapy includes the use of: small interfering (siRNA) for reducing translation of messenger RNA (mRNA); mRNA, as a means of generating a target of interest; circular RNAs (ornas), which may provide a sponge that continuously produces polypeptides or peptides or may compete with other RNA molecules; and viral vectors for providing continuous production of an object of interest. However, some nucleic acids are unstable and readily degrade, so they need to be formulated to prevent degradation and aid in intracellular delivery of the nucleic acid.

Current delivery vehicles, including lipid-based delivery vehicles such as lipid nanoparticles and liposomes, focus on protecting cargo, but not on localizing delivery cargo or delivery vehicle to a specific area in the body.

Provided herein is a directional exploration platform for evaluating a targeting system for localized delivery to a specific target area, cell or tissue. As shown in fig. 1, the tropism exploration platform can be used to evaluate Lipid Nanoparticle (LNP) libraries and/or AAV libraries in order to determine the tropism or signature profile of a targeting system in the library. The library can be administered to a subject (e.g., a non-human primate, rabbit, mouse, rat, or another mammal), and organs and tissues of the subject are scanned and/or collected and analyzed to determine the location of identifiers (e.g., barcodes, tags, signals, and/or labels) contained in or associated with LNP or AAV in the library. This analysis provides a tropism signature or profile for each LNP and AAV in the library.

Initial construct architecture

The targeting system of the directionality exploration platform may include an initial construct that encodes or includes a cargo or payload. An example of an initial polynucleotide construct 100, which may be linear or circular, is provided in fig. 2. The initial polynucleotide construct 100 may include at least one payload region 10 that is or encodes a payload or cargo of interest. The initial polynucleotide construct 100 may contain 1 or 2 flanking regions 20, and the flanking regions 20 may be located 5 'of the payload region 10 or 3' of the payload region 10. In some cases, the initial polynucleotide construct 100 does not contain flanking region 20. Flanking region 20 of initial polynucleotide construct 100 may include at least one regulatory region 30. At least one flanking region 20 of the initial polynucleotide construct 100 may include at least one identifier region 40. The identifier area 40 may be, but is not limited to, a bar code, a label, a signal, and/or a tag. In addition, the identifier region 40 may be located within the payload region 10 or may be located in the payload region 10 and the at least one flanking region 20.

In some embodiments, the initial construct comprises from about 5 to about 10,000 residues. As one non-limiting example, the length of the initial construct can be 5 to 30, 5 to 50, 5 to 100, 5 to 250, 5 to 500, 5 to 1,000, 5 to 1,500, 5 to 3,000, 5 to 5,000, 5 to 7,000, 5 to 10,000, 30 to 50, 30 to 100, 30 to 250, 30 to 500, 30 to 1,000, 30 to 1,500, 30 to 3,000, 30 to 5,000, 30 to 7,000, 30 to 10,000, 100 to 250, 100 to 500, 100 to 1,000, 100 to 1,500, 100 to 3,000, 100 to 5,000, 100 to 7,000, 100 to 10,000, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 3,000, 500 to 7,000, 500 to 10,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 3,000, 5,000, 3,000 to 3,000, 5,000, 5 to 3,000.

In some embodiments, the payload region is greater than about 5 residues in length, such as, but not limited to, at least or greater than about 5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、40、45、50、55、60、70、80、90、100、120、140、160、180、200、250、300、350、400、450、500、600、700、800、900、1,000、1,100、1,200、1,300、1,400、1,500、1,600、1,700、1,800、1,900、2,000、2,500、3,000、4,000、5,000、6,000、7,000、8,000、9,000、10,000 or more than 10,000 residues.

In some embodiments, the flanking regions may independently range from 0 to 10,000 residues in length, such as, but not limited to, at least 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、40、45、50、55、60、70、80、90、100、120、140、160、180、200、250、300、350、400、450、500、600、700、800、900、1,000、1,100、1,200、1,300、1,400、1,500、1,600、1,700、1,800、1,900、2,000、2,500、3,000、4,000、5,000、6,000、7,000、8,000、9,000 and 10,000.

In some embodiments, the length of the regulatory region may independently be in the range of 0 to 3,000 residues, such as, but not limited to, at least 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、40、45、50、55、60、70、80、90、100、120、140、160、180、200、250、300、350、400、450、500、600、700、800、900、1,000、1,100、1,200、1,300、1,400、1,500、1,600、1,700、1,800、1,900、2,000、2,500 and 3,000.

In some embodiments, the initial construct may be circularized or concatenated to produce a molecule that facilitates interaction between the 3 'and 5' ends of the initial construct.

Reference construct architecture

An initial construct that includes at least one identifier (e.g., a bar code, a tag, a signal, and/or a label) is referred to as a reference construct. The reference polynucleotide construct may comprise 1,2,3, 4, 5, 6, 7, 8, 9 or 10 or more identifiers, which may be the same or different throughout the reference polynucleotide construct.

In some embodiments, the length of the identifier region may independently be in the range of 1 to 3,000 residues, such as, but not limited to, at least 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、40、45、50、55、60、70、80、90、100、120、140、160、180、200、250、300、350、400、450、500、600、700、800、900、1,000、1,100、1,200、1,300、1,400、1,500、1,600、1,700、1,800、1,900、2,000、2,500 and 3,000. As one non-limiting example, the length of the identifier region can be 1-5 residues, 2-5 residues, 3-5 residues, 2-7 residues, 3-7 residues, 1-10 residues, 2-10 residues, 3-10 residues, 5-10 residues, 7-10 residues, 1-15 residues, 2-15 residues, 3-15 residues, 5-15 residues, 7-15 residues, 10-15 residues, 12-15 residues, 1-20 residues, 2-20 residues, 3-20 residues, 5-20 residues, 7-20 residues, 10-20 residues, 12-20 residues, 15-20 residues, 17-20 residues, 1-25 residues, 2-25 residues, 3-25 residues, 5-25 residues, 7-25 residues, 10-25 residues, 12-25 residues, 15-25 residues, 17-25 residues, 20-25 residues, 1-30 residues, 2-30 residues, 3-30 residues, 5-30 residues, 7-30 residues, 10-30 residues, 12-30 residues, 15-30 residues, 17-30 residues, 20-30 residues, 25-30 residues, and, 1-35 residues, 2-35 residues, 3-35 residues, 5-35 residues, 7-35 residues, 10-35 residues, 12-35 residues, 15-35 residues, 17-35 residues, 20-35 residues, 25-35 residues, 30-35 residues, 1-40 residues, 2-40 residues, 3-40 residues, 5-40 residues, 7-40 residues, 10-40 residues, 12-40 residues, 15-40 residues, 17-40 residues, 20-40 residues, 25-40 residues, 30-40 residues, 35-40 residues, 1-45 residues, 2-45 residues, 3-45 residues, 5-45 residues, 7-45 residues, 10-45 residues, 12-45 residues, 15-45 residues, 17-45 residues, 20-45 residues, 25-45 residues, and, 30-45 residues, 35-45 residues, 40-45 residues, 1-50 residues, 2-50 residues, 3-50 residues, 5-50 residues, 7-50 residues, 10-50 residues, 12-50 residues, 15-50 residues, 17-50 residues, 20-50 residues, 25-50 residues, 30-50 residues, 35-50 residues, 40-50 residues or 45-50 residues.

Non-limiting examples of reference polynucleotide constructs, which may be linear or circular, having at least one identifier are provided in fig. 3A, 3B, and 3C. Non-limiting examples of circular reference polynucleotide constructs having at least one identifier are provided in fig. 4A, 4B, and 4C. In fig. 3A, 3B, 4A and 4B, the reference polynucleotide construct includes a payload region (referred to as "P" in the figures) and at least one identifier region (referred to as "BC" in the figures) and/or a reverse identifier region (referred to as "CB" in the figures). In fig. 3C and 4C, the reference polynucleotide construct includes a payload region (referred to as "P" in the figures) and at least one identifier portion associated with the reference polynucleotide construct.

In some embodiments, the identifier region in the reference construct overlaps with the payload region. As used herein, "overlapping" means that at least one nucleotide of the identifier region extends into the payload region. In some aspects, the identifier region overlaps the payload region by 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 nucleotides, 49 nucleotides, 50 nucleotides or more than 50 nucleotides. In some aspects, the identifier region overlaps the payload region by 1-5 nucleotides, 2-5 nucleotides, 3-5 nucleotides, 2-7 nucleotides, 3-7 nucleotides, 1-10 nucleotides, 2-10 nucleotides, 3-10 nucleotides, 5-10 nucleotides, 7-10 nucleotides, 1-15 nucleotides, 2-15 nucleotides, 3-15 nucleotides, 5-15 nucleotides, 7-15 nucleotides, 10-15 nucleotides, 12-15 nucleotides, 1-20 nucleotides, 2-20 nucleotides, 3-20 nucleotides, 5-20 nucleotides, 7-20 nucleotides, 10-20 nucleotides, 12-20 nucleotides, 15-20 nucleotides, 17-20 nucleotides, 1-25 nucleotides, 2-25 nucleotides, 3-25 nucleotides, 5-25 nucleotides, 7-25 nucleotides, 10-25 nucleotides, 12-25 nucleotides, 15-25 nucleotides, 17-25 nucleotides, 20-25 nucleotides, 1-30 nucleotides, 2-30 nucleotides, 3-30 nucleotides, 5-30 nucleotides, 7-30 nucleotides, 10-30 nucleotides, 12-30 nucleotides, 15-30 nucleotides, 17-30 nucleotides, 20-30 nucleotides, 25-30 nucleotides, 1-35 nucleotides, 2-35 nucleotides, 3-35 nucleotides, 5-35 nucleotides, 7-35 nucleotides, 10-35 nucleotides, 12-35 nucleotides, 15-35 nucleotides, 17-35 nucleotides, 20-35 nucleotides, 25-35 nucleotides, 30-35 nucleotides, 1-35 nucleotides, 2-35 nucleotides, 3-35 nucleotides, 5-35 nucleotides, 7-35 nucleotides, 10-35 nucleotides, 12-35 nucleotides, 15-35 nucleotides, 17-35 nucleotides, 20-35 nucleotides, 25-35 nucleotides, 30-35 nucleotides, 1-40 nucleotides, 2-40 nucleotides, 3-40 nucleotides, 5-40 nucleotides, 7-40 nucleotides, 10-40 nucleotides, 12-40 nucleotides, 15-40 nucleotides, 17-40 nucleotides, 20-40 nucleotides, 25-40 nucleotides, 30-40 nucleotides, 35-40 nucleotides, 1-45 nucleotides, 2-45 nucleotides, 3-45 nucleotides, 5-45 nucleotides, 7-45 nucleotides, 10-45 nucleotides, 12-45 nucleotides, 15-45 nucleotides, 17-45 nucleotides, 20-45 nucleotides, 25-45 nucleotides, 30-45 nucleotides, 35-45 nucleotides, 40-45 nucleotides, 1-50 nucleotides, 2-50 nucleotides, 3-50 nucleotides, 5-50 nucleotides, 7-50 nucleotides, 10-50 nucleotides, 12-50 nucleotides, 15-50 nucleotides, 17-50 nucleotides, 20-50 nucleotides, 25-50 nucleotides, 30-50 nucleotides, 35-50 nucleotides, 40-50 nucleotides or 45-50 nucleotides.

In some embodiments, the reference polynucleotide construct comprises a payload region and an identifier region. The identifier region may be located 5 'of the payload region, 3' of the payload region, or the identifier region may overlap with the 5 'or 3' end of the payload region.

In some embodiments, the reference polynucleotide construct comprises a payload region and two identifier regions. Each identifier region may be located independently 5 'of the payload region, 3' of the payload region, or the identifier region may overlap with the 5 'or 3' end of the payload region.

As one non-limiting example, the first identifier region is located 5 'of the payload region and the second identifier region is located 3' of the payload region. As one non-limiting example, the first identifier region and the second identifier region are located 5' of the payload region. As one non-limiting example, the first identifier region and the second identifier region are located 3' of the payload region.

As one non-limiting example, the first identifier region is inverted and located 5 'of the payload region, and the second identifier region is located 3' of the payload region. As one non-limiting example, the first identifier region is inverted and located 5 'of the payload region, and the second identifier region is inverted and located 3' of the payload region. As one non-limiting example, the first identifier region is located 5 'of the payload region and the second identifier region is inverted and located 3' of the payload region. As a non-limiting example, the first identifier region and the second identifier region are both inverted and located 5' of the payload region. As one non-limiting example, the first identifier region and the second identifier region are located 5' of the payload region, and the first identifier region is inverted. As one non-limiting example, the first identifier region and the second identifier region are located 5' of the payload region, and the second identifier region is inverted. As one non-limiting example, the first identifier region and the second identifier region are both inverted and located 3' of the payload region. As one non-limiting example, the first identifier region and the second identifier region are located 3' of the payload region, and the first identifier region is inverted. As one non-limiting example, the first identifier region and the second identifier region are located 3' of the payload region, and the second identifier region is inverted.

As one non-limiting example, the first identifier region is located 5 'of and overlaps the payload region, and the second identifier region is located 3' of the payload region. As one non-limiting example, the first identifier region is located 5 'of the payload region and the second identifier region is located 3' of and overlaps with the payload region.

As one non-limiting example, the first identifier region and the second identifier region are located 5' of the payload region, and the second identifier region overlaps the payload region. As one non-limiting example, the first identifier region and the second identifier region are located 3' of the payload region, and the first identifier region overlaps the payload region.

As one non-limiting example, the first identifier region is inverted, located 5 'of and overlapping the payload region, and the second identifier region is located 3' of the payload region. As one non-limiting example, the first identifier region is inverted and located 5 'of the payload region, and the second identifier region is located 3' of and overlaps the payload region. As one non-limiting example, the first identifier region is inverted, located 5 'of the payload region, the second identifier region is located 3' of the payload region, and both the first identifier region and the second identifier region overlap the payload region.

As one non-limiting example, the first identifier region is inverted, located 5 'of and overlapping the payload region, and the second identifier region is inverted, located 3' of the payload region. As one non-limiting example, the first identifier region is inverted and located 5 'of the payload region, and the second identifier region is inverted, located 3' of the payload region and overlapping the payload region. As one non-limiting example, the first identifier region is inverted and located 5 'of the payload region and the second identifier region is inverted and located 3' of the payload region, and both the first identifier region and the second identifier region overlap the payload region.

As one non-limiting example, the first identifier region is located 5 'of and overlaps the payload region, and the second identifier region is inverted and located 3' of the payload region. As one non-limiting example, the first identifier region is located 5 'of the payload region and the second identifier region is inverted, located 3' of the payload region and overlaps the payload region. As one non-limiting example, the first identifier region is located 5 'of the payload region and the second identifier region is inverted and located 3' of the payload region, and both the first identifier region and the second identifier region overlap the payload region.

As one non-limiting example, the first identifier region and the second identifier region are both inverted and located 5' of the payload region, and the second identifier region overlaps the payload region. As one non-limiting example, the first identifier region and the second identifier region are located 5' of the payload region, and the first identifier region is inverted and the second identifier region overlaps the payload region. As one non-limiting example, the first identifier region and the second identifier region are located 5' of the payload region, and the second identifier region is inverted and overlaps the payload region. As one non-limiting example, the first identifier region and the second identifier region are both inverted and located 3' of the payload region, and the first identifier region overlaps the payload region. As one non-limiting example, the first identifier region and the second identifier region are located at the payload region 3', and the first identifier region is inverted and overlaps the payload region. As one non-limiting example, the first identifier region and the second identifier region are located 3' of the payload region, and the second identifier region is inverted and the first payload region overlaps the payload region.

In some embodiments, at least one identifier moiety can be associated with a reference polynucleotide construct. The reference polynucleotide construct may have 1,2, 3, 4,5, 6, 7, 8, 9, or 10 or more identifier moieties associated with the reference polynucleotide construct, which may be the same moiety or different moieties associated with the reference polynucleotide construct. Each identifier portion may be independently located on a flanking region 5 'of the payload region, on a flanking region 3' of the payload region, or the location of the identifier portion may span the 5 'or 3' end of the payload region and the flanking region. in some aspects, the location of the identifier portion can include one or more nucleotides of the payload region, such as, but not limited to, 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 nucleotides, 49 nucleotides, 50 nucleotides or more than 50 nucleotides. In some aspects, the location of the identifier portion may include one or more nucleotides of the payload region, such as, but not limited to, 1-5 nucleotides, 2-5 nucleotides, 3-5 nucleotides, 2-7 nucleotides, 3-7 nucleotides, 1-10 nucleotides, 2-10 nucleotides, 3-10 nucleotides, 5-10 nucleotides, 7-10 nucleotides, 1-15 nucleotides, 2-15 nucleotides, 3-15 nucleotides, 5-15 nucleotides, 7-15 nucleotides, 10-15 nucleotides, 12-15 nucleotides, 1-20 nucleotides, 2-20 nucleotides, 3-20 nucleotides, 5-20 nucleotides, 7-20 nucleotides, 10-20 nucleotides, 12-20 nucleotides, 15-20 nucleotides, 17-20 nucleotides, 1-25 nucleotides, 2-25 nucleotides, 3-25 nucleotides, 5-25 nucleotides, 7-25 nucleotides, 10-25 nucleotides, 12-25 nucleotides, 15-25 nucleotides, 17-25 nucleotides, 20-25 nucleotides, 1-30 nucleotides, 2-30 nucleotides, 3-30 nucleotides, 5-30 nucleotides, 7-30 nucleotides, 10-30 nucleotides, 12-30 nucleotides, 15-30 nucleotides, 17-30 nucleotides, 20-30 nucleotides, 25-30 nucleotides, 1-35 nucleotides, 2-35 nucleotides, 3-35 nucleotides, 5-35 nucleotides, 7-35 nucleotides, 10-35 nucleotides, 12-35 nucleotides, 15-35 nucleotides, 17-35 nucleotides, 20-35 nucleotides, 25-35 nucleotides, 30-35 nucleotides, 1-35 nucleotides, 2-35 nucleotides, 3-35 nucleotides, 5-35 nucleotides, 7-35 nucleotides, 10-35 nucleotides, 12-35 nucleotides, 15-35 nucleotides, 17-35 nucleotides, 20-35 nucleotides, 25-35 nucleotides, 30-35 nucleotides, 1-40 nucleotides, 2-40 nucleotides, 3-40 nucleotides, 5-40 nucleotides, 7-40 nucleotides, 10-40 nucleotides, 12-40 nucleotides, 15-40 nucleotides, 17-40 nucleotides, 20-40 nucleotides, 25-40 nucleotides, 30-40 nucleotides, 35-40 nucleotides, 1-45 nucleotides, 2-45 nucleotides, 3-45 nucleotides, 5-45 nucleotides, 7-45 nucleotides, 10-45 nucleotides, 12-45 nucleotides, 15-45 nucleotides, 17-45 nucleotides, 20-45 nucleotides, 25-45 nucleotides, 30-45 nucleotides, 35-45 nucleotides, 40-45 nucleotides, 1-50 nucleotides, 2-50 nucleotides, 3-50 nucleotides, 5-50 nucleotides, 7-50 nucleotides, 10-50 nucleotides, 12-50 nucleotides, 15-50 nucleotides, 17-50 nucleotides, 20-50 nucleotides, 25-50 nucleotides, 30-50 nucleotides, 35-50 nucleotides, 40-50 nucleotides or 45-50 nucleotides.

In some embodiments, one identifier moiety can be associated with a reference polynucleotide construct. As one non-limiting example, the identifier moiety can be associated with the reference polynucleotide construct on the 5' end of the reference polynucleotide construct. As one non-limiting example, the identifier moiety can be associated with the reference polynucleotide construct on the 5' flanking region. As one non-limiting example, the identifier moiety can be associated with the reference polynucleotide construct on the 3' flanking region. As one non-limiting example, the identifier moiety can be associated with the reference polynucleotide construct on the 3' end of the reference polynucleotide construct. As one non-limiting example, the identifier moiety can be associated with the reference polynucleotide construct over a payload region. As one non-limiting example, the reference polynucleotide construct comprises an identifier portion, and the position of the identifier portion spans the 5 'end and 5' flanking regions of the payload region. As one non-limiting example, the reference polynucleotide construct comprises an identifier portion, and the position of the identifier portion spans the 3 'end and the 3' flanking region of the payload region.

In some embodiments, two identifier moieties are associated with a reference polynucleotide construct. As one non-limiting example, the first identifier portion and the second identifier portion are located on the 5' flanking region. As one non-limiting example, the first identifier portion and the second identifier portion are located on a payload area. As one non-limiting example, the first identifier portion and the second identifier portion are located on 3' flanking regions. As one non-limiting example, the first identifier portion and the second identifier portion are located on the 5' end of the reference polynucleotide construct. As one non-limiting example, the first identifier portion and the second identifier portion are located on the 3' end of the reference polynucleotide construct.

As one non-limiting example, the first identifier portion is located on the 5 'end of the reference polynucleotide construct and the second identifier portion is located on the 5' flanking region. As one non-limiting example, the first identifier portion is located on the 5' end of the reference polynucleotide construct and the second identifier portion is located on the payload region. As one non-limiting example, the first identifier portion is located on the 5 'end of the reference polynucleotide construct and the second identifier portion is located on the 3' flanking region. As one non-limiting example, the first identifier portion is located on the 5 'end of the reference polynucleotide construct and the second identifier portion is located across the 5' flanking region and the payload region. As one non-limiting example, the first identifier portion is located on the 5 'end of the reference polynucleotide construct and the second identifier portion is located across the 3' flanking region and the payload region. As one non-limiting example, the first identifier portion is located on the 5 'end of the reference polynucleotide construct and the second identifier portion is located on the 3' end of the reference polynucleotide construct.

As one non-limiting example, the first identifier portion is located on the 5' flanking region and the second identifier portion is located on the payload region. As one non-limiting example, the first identifier portion is located on the 5 'flanking region and the second identifier portion is located on the 3' flanking region. As one non-limiting example, the first identifier portion is located on the 5 'flanking region and the second identifier portion is located across the 5' flanking region and the payload region. As one non-limiting example, the first identifier portion is located on the 5 'flanking region and the second identifier portion is located across the 3' flanking region and the payload region. As one non-limiting example, the first identifier portion is located on the 5 'flanking region and the second identifier portion is located on the 5' end of the reference polynucleotide construct. As one non-limiting example, the first identifier portion is located on the 5 'flanking region and the second identifier portion is located on the 3' end of the reference polynucleotide construct.

As one non-limiting example, the first identifier portion is located across the 5 'flanking region and the payload region, and the second identifier portion is located on the 5' end of the reference polynucleotide construct. As one non-limiting example, the first identifier portion is located across the 5 'flanking region and the payload region, and the second identifier portion is located on the 5' flanking region. As one non-limiting example, the first identifier portion is located across the 5' flanking region and the payload region, and the second identifier portion is located on the payload region. As one non-limiting example, the location of the first identifier portion spans the 5 'flanking region and the payload region, and the location of the second identifier portion spans the 3' flanking region and the payload region. As one non-limiting example, the first identifier portion is located across the 5 'flanking region and the payload region, and the second identifier portion is located on the 3' flanking region. As one non-limiting example, the first identifier portion is located across the 5 'flanking region and the payload region, and the second identifier portion is located on the 3' end of the reference polynucleotide construct.

As one non-limiting example, the first identifier portion is located on the payload region and the second identifier portion is located on the 5' end of the reference polynucleotide construct. As one non-limiting example, the first identifier portion is located on the payload area and the second identifier portion is located on the 5' flanking area. As one non-limiting example, the first identifier portion is located on the payload area and the second identifier portion is located across the 5' flanking area and the payload area. As one non-limiting example, the first identifier portion is located on the payload area and the second identifier portion is located across the 3' flanking area and the payload area. As one non-limiting example, the first identifier portion is located on the payload area and the second identifier portion is located on the 3' flanking area. As one non-limiting example, the first identifier portion is located on the payload region and the second identifier portion is located on the 3' end of the reference polynucleotide construct.

As one non-limiting example, the first identifier portion is located across the 3 'flanking region and the payload region, and the second identifier portion is located on the 5' end of the reference polynucleotide construct. As one non-limiting example, the first identifier portion is located across the 3 'flanking region and the payload region, and the second identifier portion is located on the 5' flanking region. As one non-limiting example, the location of the first identifier portion spans the 3 'flanking region and the payload region, and the location of the second identifier portion spans the 5' flanking region and the payload region. As one non-limiting example, the first identifier portion is located across the 3' flanking region and the payload region, and the second identifier portion is located on the payload region. As one non-limiting example, the first identifier portion is located across the 3 'flanking region and the payload region, and the second identifier portion is located on the 3' flanking region. As one non-limiting example, the first identifier portion is located across the 3 'flanking region and the payload region, and the second identifier portion is located on the 3' end of the reference polynucleotide construct.

As one non-limiting example, the first identifier portion is located across the 3 'flanking region and the payload region, and the second identifier portion is located on the 5' flanking region. As one non-limiting example, the first identifier portion is located across the 5' flanking region and the payload region, and the second identifier portion is located on the payload region. As one non-limiting example, the location of the first identifier portion spans the 5 'flanking region and the payload region, and the location of the second identifier portion spans the 3' flanking region and the payload region. As one non-limiting example, the first identifier portion is located across the 5 'flanking region and the payload region, and the second identifier portion is located on the 3' flanking region. As one non-limiting example, the first identifier portion is located across the 5 'flanking region and the payload region, and the second identifier portion is located on the 3' end of the reference polynucleotide construct.

As one non-limiting example, the first identifier portion is located on the 3 'flanking region and the second identifier portion is located on the 5' end of the reference polynucleotide construct. As one non-limiting example, the first identifier portion is located on the 3 'flanking region and the second identifier portion is located on the 5' flanking region. As one non-limiting example, the first identifier portion is located on the 3 'flanking region and the second identifier portion is located across the 5' flanking region and the payload region. As one non-limiting example, the first identifier portion is located on the 3' flanking region and the second identifier portion is located on the payload region. As one non-limiting example, the first identifier portion is located on the 3 'flanking region and the second identifier portion is located across the 3' flanking region and the payload region. As one non-limiting example, the first identifier portion is located on the 3 'flanking region and the second identifier portion is located on the 3' end of the reference polynucleotide construct.

As one non-limiting example, the first identifier portion is located on the 3 'end of the reference polynucleotide construct and the second identifier portion is located on the 5' end of the reference polynucleotide construct. As one non-limiting example, the first identifier portion is located on the 3 'end of the reference polynucleotide construct and the second identifier portion is located on the 5' flanking region. As one non-limiting example, the first identifier portion is located on the 5 'end of the reference polynucleotide construct and the second identifier portion is located across the 5' flanking region and the payload region. As one non-limiting example, the first identifier portion is located on the 3' end of the reference polynucleotide construct and the second identifier portion is located on the payload region. As one non-limiting example, the first identifier portion is located on the 5 'end of the reference polynucleotide construct and the second identifier portion is located across the 3' flanking region and the payload region. As one non-limiting example, the first identifier portion is located on the 3 'end of the reference polynucleotide construct and the second identifier portion is located on the 3' flanking region.

In some embodiments, three identifier portions are associated with a reference polynucleotide construct.

In some embodiments, four identifier portions are associated with a reference polynucleotide construct.

In some embodiments, five identifier moieties are associated with a reference polynucleotide construct.

In some embodiments, six identifier moieties are associated with a reference polynucleotide construct.

In some embodiments, seven identifier portions are associated with a reference polynucleotide construct.

In some embodiments, eight identifier portions are associated with a reference polynucleotide construct.

In some embodiments, nine identifier moieties are associated with a reference polynucleotide construct.

In some embodiments, ten identifier moieties are associated with the reference polynucleotide construct.

II. cargo and payload

The initial construct and reference construct of the present disclosure may comprise, encode, or be conjugated to a cargo or payload. As used herein, the term "cargo" or "payload" may refer to one or more molecules or structures encompassed in a delivery vehicle for delivery to or into a cell or tissue. Non-limiting examples of cargo may include nucleic acids, polypeptides, peptides, proteins, liposomes, tags, labels, small chemical molecules, large biological molecules, and any combinations or fragments thereof. In the initial construct and the reference construct, the region of the construct that contains or encodes the cargo or payload is referred to as the "cargo region" or "payload region".

In some embodiments, the cargo or payload is or encodes a biologically active molecule, such as, but not limited to, a therapeutic protein. As used herein, the term "biologically active" refers to the property of any agent that is active in a biological system, and in particular in an organism. For example, an agent that has a biological effect on an organism when administered to the organism is considered to be biologically active. In some embodiments, the cargo or payload is or encodes one or more prophylactically or therapeutically active proteins, polypeptides, or other factors. As one non-limiting example, the cargo or payload may be or encode an agent that enhances tumor killing activity in cancer, such as, but not limited to TRAIL or Tumor Necrosis Factor (TNF). As another non-limiting example, the cargo or payload may be a pharmaceutical encoding a suitable agent for treating a disorder such as: muscular dystrophy (e.g., cargo or payload is or encodes a actin), cardiovascular disease (e.g., cargo or payload is or encodes SERCA2a, GATA4, tbx5, mef2C, hand, myocd, etc.), neurodegenerative disease (e.g., cargo or payload is or encodes NGF, BDNF, GDNF, NT-3, etc.), chronic pain (e.g., cargo or payload is or encodes GlyRal), enkephalin or glutamate decarboxylase (e.g., cargo or payload is or encodes tid 65, GAD67 or another isoform), lung disease (e.g., CFTR for cargo or payload), hemophilia (e.g., factor VIII or factor IX for cargo or payload), neoplasia (e.g., PTEN、ATM、ATR、EGFR、ERBB2、ERBB3、ERBB4、Notchl、Notch2、Notch3、Notch4、AKT、AKT2、AKT3、HIF、HI Fla、HIF3a、Met、HRG、Bcl2、PPARα、PPARγ、WT1( Wilms Tumor (Wilms Tumor)), FGF receptor family members (5 members: 1.2, 3, 4, 5), CDKN2a, APC, RB (retinoblastoma), MEN1, VHL, BRCA1, BRCA2, androgen Receptor (AR), TSG101, IGF receptor, igfl (4 variants), IGF2 (3 variants), igfl receptor, IGF2 receptor, bax, bcl2, family of apoptotic proteases (9 members: 1, 2, 3,4, 6, 7, 8, 9, 12), kras, ape), age-related macular degeneration (e.g., the cargo or payload is or encodes Aber, ccl2, cc2, ceruloplasmin (ceruloplasmin; cp), timp3, cathepsin D, vldlr), schizophrenia (e.g., neuregulin (Nrgl), erb4 (receptor for neuregulin), polyprotein (Complexin) -1 (Cplxl), tphl tryptophan hydroxylase, tph2 tryptophan hydroxylase 2, neurexin 1, GSK3a, GSK3b, 5-HIT (Slc 6a 4), COMT, DRD (Drdla), slc6A3, DAOA, DTNBPI, dao (Daol)), trinucleotide repeat disorders (e.g., HTT (huntington Dx), tph2 tryptophan hydroxylase 2, neurexin 1, GSK3b, 5-HIT 6a 4), SBMA/SMAXI/AR (Kennedy Dx), FXN/X25 (Friedrich's Ataxia), ATX3 (Mandarin-Joseph Dx), ATXNI and ATXN2 (spinocerebellar ataxia), DMPK (myotonic dystrophy), atropin-1 and Atnl (DRPLA Dx), CBP (Creb-BP-global instability), VLDLR (Alzheimer's disease), atxn, atxn10, fragile X syndrome (e.g., cargo or payload is or encodes FMR 2), FXRI, FXR2, mGLUR), secretase-related disorders (e.g., cargo or payload is or encodes APH-1 (α and β), presenilin (Psenl), arrestin (nicastrin; Ncstn), PEN-2), ALS (e.g., the cargo or payload is or encodes SOD1, ALS2, STEX, FUS, TARDBP, VEGF (VEGF-a, VEGF-b, VEGF-c)), autism (e.g., the cargo or payload is or encodes Mecp2 BZRAP, MDGA, sema5A, axonal protein 1), alzheimer's disease (e.g. cargo or payload or code El, CHIP, UCH, UBB, tau, LRP, PICALM, clusterin, PS1, SORL1, CR1, vldlr, ubal, uba, CHIP28 (Aqpl, aquaporin 1), uchll, uchl3, APP), inflammation (e.g. NOD2/CARD15 with cargo or payload or encoding IL-10、IL-1(IL-Ia、IL-Ib)、IL-13、IL-17(IL-17a(CTLA8)、IL-17b、IL-17c、IL-17d、IL-171)、11-23、Cx3crl、ptpn22、TNFa、 for IBD, IL-6, IL-12 (IL-12 a, IL-12 b), CTLA4, cx3 cll), parkinson's Disease (e.g. x-synuclein (Synuclein), and, DJ-1, LRRK2, parkinson's protein (Parkin), PINK 1), blood and coagulation disorders (e.g., anemia, naked lymphocyte syndrome, bleeding disorders, hemophagocytic lymphoproliferative disorders, hemophilia A, hemophilia B, hemorrhagic disorders, leukopenia and disorders, hemophilia B, and other disorders, Sickle cell anemia and thalassemia) (e.g., cargo or payload is or encodes CRAN1、CDA1、RPS19、DBA、PKLR、PK1、NT5C3、UMPH1、PSNI、RHAG、RH50A、NRAMP2、SPTB、ALAS2、ANH1、ASB、ABCB7、ABC7、ASAT、TAPBP、TPSN、TAP2、ABCB3、PSF2、RING11、MHC2TA、C2TA、RFX5、RFXAP、RFX5、TBXA2R、P2RX1、P2X1、HF1、CFH、HUS、MCFD2、FANCA、FAC A、FA1、FA、FA A、FAAP95、FAAP90、FLJ34064、FANCB、FANCC、FACC、BRCA2、FANCDI、FANCD2、FANCD、FACD、FAD、FANCE、FACE、FANCF、XRCC9、FANCG、BR1PI、BACH1、FANCJ、PHF9、FANCL、FANCM、KIAA1596、PRF1、HPLH2、UNC13D、MUNC13-4、HPLH3、HLH3、FHL3、F8、FSC、PI、ATT、F5、ITGB2、CD18、LCAMB、LAD、EIF2B1、EIF2BA、EIF2B2、EIF2B3、EIF2B5、LVWM、CACH、CLE、EIF2B4、HBB、HBA2、HBB、HBD、LCRB、HBA1)、B cell non-hodgkin lymphoma or leukemia (e.g., cargo or payload is or encodes BCL7A、BCL7、ALI、TCL5、SCL、TAL2、FLT3、NBS1、NBS、ZNFN1AI、1KI、LYF1、HOXD4、HOX4B、BCR、CML、PHL、ALL、ARNT、KRAS2、RASK2、GMPS、AFIO、ARHGEF12、LARG、KIAA0382、CALM、CLTH、CEBPA、CEBP、CHIC2、BTL、FLT3、KIT、PBT、LPP、NPMI、NUP214、D9S46E、CAN、CAIN、RUNXI、CBFA2、AML1、WHSC1LI、NSD3、FLT3、AF1Q、NPMI、NUMA1、ZNF145、PLZF、PML、MYL、STAT5B、AF1Q、CALM、CLTH、ARL11、ARLTS1、P2RX7、P2X7、BCR、CML、PHL、ALL、GRAF、NF1、VRNF、WSS、NFNS、PTPNII、PTP2C、SHP2、NS1、BCL2、CCND1、PRAD1、BCL1、TCRA、GATA1、GF1、ERYF1、NFE1、ABLI、NQO1、DIA4、NMOR1、NUP214、D9S46E、CAN、CAIN)、 inflammation and immune-related diseases and disorders (e.g., cargo or payload is or encodes KIR3DL1、NKAT3、NKB1、AMB11、K1R3DS1、IFNG、CXCL12、TNFRSF6、APT1、FAS、CD95、ALPS1A、IL2RG、SCIDX1、SCIDX、IMD4、CCL5、SCYA5、D17S136E、TCP228、IL10、CSIF、CMKBR2、CCR2、CMKBR5、CCCKR5(CCR5)、CD3E、CD3G、AICDA、AID、HIGM2、TNFRSF5、CD40、UNG、DGU、HIGM4、TNFSFS、CD40LG、HIGM1、IGM、FOXP3、IPEX、AIID、XPID、PIDX、TNFRSF14B、TACI)、 inflammation (e.g., cargo or payload is or encodes IL-10、IL-1(IL-IA、IL-IB)、IL-13、IL-17(IL-17a(CTLA8)、IL-17b、IL-17c、IL-17d、IL-171)、11-23、Cx3crl、ptpn22、TNFa、 for NOD2/CARD15、IL-6、IL-12(IL-12a、IL-12b)、CTLA4、Cx3cII)、JAK3、JAKL、DCLREIC、ARTEMIS、SCIDA、RAG1、RAG2、ADA、PTPRC、CD45、LCA、IL7R、CD3D、T3D、IL2RG、SCIDXI、SCIDX、IMD4)、 metabolism of IBD), Liver, a part of the liver, Kidney and protein diseases and conditions (e.g., cargo or payload is or encodes TTR、PALB、APOA1、APP、AAA、CVAP、ADI、GSN、FGA、LYZ、TTR、PALB、KRT18、KRT8、CIRH1A、NAIC、TEX292、KIAA1988、CFTR、ABCC7、CF、MRP7、SLC2A2、GLUT2、G6PC、G6PT、G6PT1、GAA、LAMP2、LAMPB、AGL、GDE、GBE1、GYS2、PYGL、PFKM、TCF1、HNF1A、MODY3、SCOD1、SCO1、CTNNB1、PDGFRL、PDGRL、PRLTS、AX1NI、AXIN、CTNNB1、TP53、P53、LFS1、IGF2R、MPRI、MET、CASP8、MCH5、UMOD、HNFJ、FJHN、MCKD2、ADMCKD2、PAH、PKU1、QDPR、DHPR、PTS、FCYT、PKHD1、ARPKD、PKD1、PKD2、PKD4、PKDTS、PRKCSH、G19P1、PCLD、SEC63)、 muscle/bone diseases and conditions (e.g., cargo or payload is or encodes DMD、BMD、MYF6、LMNA、LMN1、EMD2、FPLD、CMDIA、HGPS、LGMDIB、LMNA、LMNI、EMD2、FPLD、CMDIA、FSHMD1A、FSHD1A、FKRP、MDC1C、LGMD2I、LAMA2、LAMM、LARGE、KIAA0609、MDC1D、FCMD、TTID、MYOT、CAPN3、CANP3、DYSF、LGMD2B、SGCG、LGMD2C、DMDA1、SCG3、SGCA、ADL、DAG2、LGMD2D、DMDA2、SGCB、LGMD2E、SGCD、SGD、LGMD2F、CMD1L、TCAP、LGMD2G、CMD1N、TRIM32、HT2A、LGMD2H、FKRP、MDCIC、LGMD21、TTN、CMD1G、TMD、LGMD2J、POMT1、CAV3、LGMD1C、SEPN1、SELN、RSMD1、PLEC1、PLTN、EBS1、LRP5、BMND1、LRP7、LR3、OPPG、VBCH2、CLCN7、CLC7、OPTA2、OSTMI、GL、TCIRG1、TIRC7、OC116、OPTB1、VAPB、VAPC、ALS8、SMN1、SMA1、SMA2、SMA3、SMA4、BSCL2、SPG17、GARS、SMAD1、CMT2D、HEXB、IGHMBP2、SMUBP2、CATF1、SMARD1)、 nerve and neuronal diseases and conditions (e.g., cargo or payload is or encodes SOD1、ALS2、STEX、FUS、TARDBP、VEGF(VEGF-a、VEGF-b、VEGF-c)、APP、AAA、CVAP、ADI、APOE、AD2、PSEN2、AD4、STM2、APBB2、FE65LI、NOS3、PLAU、URK、ACE、DCPI、ACEI、MPO、PAC1PI、PAXIPIL、PTIP、A2M、BLMH、BMH、PSEN1、AD3、Mecp2、BZRAP1、MDGA2、Sema5A、 axon protein 1、GLO1、MECP2、RTT、PPMX、MRX16、MRX79、NLGN3、NLGN4、KIAA1260、AUTSX2、FMR2、FXR1、FXR2、mGLUR5、HD、IT15、PRNP、PRIP、JPH3、JP3、HDL2、TBP、SCA17、NR4A2、NURR1、NOT、TINUR、SNCAIP、TBP、SCA17、SNCA、NACP、PARK1、PARK4、DJI、PARK7、LRRK2、PARK8、PINK1、PARK6、UCHL1、PARK5、SNCA、NACP、PARK1、PARK4、PRKN、PARK2、PDJ、DBH、NDUFV2、MECP2、RTT、PPMX、MRX16、MRX79、CDKL5、STK9、MECP2、RTT、PPMX、MRX16、MRX79、x- synuclein, DJ-1, neuregulin-l (Nrgl), erb4 (neuregulin receptor), polyprotein-l (Cplxl), tphl tryptophan hydroxylase, tpp 2, tryptophan hydroxylase 2, axon protein 1, GSK3 GSK3a, GSK3b, 5-HTT (Slc 6a 4), CONT, DRD (Drdla), SLC6A, DAOA, DTNBP1, dao (Daol), APH-l (alpha and beta), presenilin (Psenl), altin, (Ncstn), PEN-2, nosl, parpl, Natl, nat2, HTT, SBMA/SMAX1/AR, FXN/X25, ATX3, TXN, ATXN2, DMPK, atrophin-1, atnl, CBP, VLDLR, atxn, and AtxnlO) and ocular diseases and disorders (e.g., aber, ccl2, cc2, ceruloplasmin (cp), timp3, cathepsin -D、Vldlr、Ccr2、CRYAA、CRYA1、CRYBB2、CRYB2、PITX3、BFSP2、CP49、CP47、CRYAA、CRYAI、PAX6、AN2、MGDA、CRYBA1、CRYB1、CRYGC、CRYG3、CCL、LIM2、MP19、CRYGD、CRYG4、BFSP2、CP49、CP47、HSF4、CTM、HSF4、CTM、MIP、AQPO、CRYAB、CRYA2、CTPP2、CRYBB1、CRYGD、CRYG4、CRYBB2、CRYB2、CRYGC、CRYG3、CCL、CRYAA、CRYAI、GJA8、CX50、CAE1、GJA3、CX46、CZP3、CAE3、CCM1、CAM、KRIT1、APOA1、TGFBI、CSD2、CDGG1、CSD、BIGH3、CDG2、TACSTD2、TROP2、M1SI、VSX1、RINX、PPCD、PPD、KTCN、COL8A2、FECD、PPCD2、PIP5K3、CFD、KERA、CNA2、MYOC、TIGR、GLCIA、JO AG、GPOA、OPTN、GLC1E、FIP2、HYPL、NRP、CYP1BI、GLC3A、OPA1、NTG、NPG、CYP1BI、GLC3A、CRB1、RP12、CRX、CORD2、CRD、RPGRIPI、LCA6、CORD9、RPE65、RP20、AIPL1、LCA4、GUCY2D、GUC2D、LCA1、CORD6、RDH12、LCA3、ELOVL4、ADMD、STGD2、STGD3、RDS、RP7、PRPH2、PRPH、AVMD、AOFMD, and VMD 2).

In some embodiments, the cargo or payload is or encodes a factor that can affect cell differentiation. As one non-limiting example, expression of one or more of Oct4, klf4, sox2, c-Myc, L-Myc, dominant negative p53, nanog, glisl, lin, TFIID, mir-302/367, or other miRNAs may render the cell into an induced pluripotent stem (induced pluripotent stem; iPS) cell.

In some embodiments, the cargo or payload is or encodes a factor for transdifferentiating (TRANSDIFFERENTIATING) cells. Non-limiting examples of factors include: for cardiomyocytes, one or more of GATA4, tbx5, mef2C, myocd, hand2, SRF, mespl, SMARCD 3; for neural cells Ascii, nurrl, lmxlA, bm, mytll, neuroDl, foxA2; and for hepatocytes, hnf4a, foxal, foxa2 or Foxa3.

Polypeptides, proteins and peptides

The initial and reference constructs of the present disclosure may comprise, be encoded as, be conjugated to, or be a cargo or payload of a polypeptide, protein, or peptide. As used herein, the term "polypeptide" generally refers to a polymer of amino acids linked by peptide bonds and encompasses "proteins" and "peptides". Polypeptides for use in the present disclosure include all polypeptides, proteins and/or peptides known in the art. Non-limiting classes of polypeptides include antigens, antibodies, antibody fragments, cytokines, peptides, hormones, enzymes, oxidants, antioxidants, synthetic polypeptides and chimeric polypeptides.

As used herein, the term "peptide" generally refers to a shorter polypeptide of about 50 amino acids or less. Peptides having only two amino acids may be referred to as "dipeptides". Peptides having only three amino acids may be referred to as "tripeptides". A polypeptide generally refers to a polypeptide having from about 4 to about 50 amino acids. The peptides may be obtained via any method known to those skilled in the art. In some embodiments, the peptide may be expressed in culture. In some embodiments, the peptide may be obtained via chemical synthesis (e.g., solid phase peptide synthesis).

In some embodiments, the initial and baseline constructs of the present disclosure may comprise, encode, or be conjugated to a cargo or payload that is a simple protein that produces amino acids and occasionally small carbohydrates upon hydrolysis. Non-limiting examples of simple proteins include albumin, peptoids, globulins, gluten, histones, and protamine.

In some embodiments, the initial and baseline constructs of the present disclosure may comprise, encode, or be conjugated to a cargo or payload, which is a conjugated protein, which may be a simple protein associated with a non-protein. Non-limiting examples of conjugated proteins include glycoproteins, haemoglobin, lecithin proteins, nucleoprotein and phosphoproteins.

In some embodiments, the initial and baseline constructs of the present disclosure may comprise, encode, or be conjugated to a cargo or payload that is a derivative protein that is a protein derived from a simple or conjugated protein by chemical or physical means. Non-limiting examples of derivatized proteins include denatured proteins and peptides.

In some embodiments, the polypeptide, protein, or peptide may be unmodified.

In some embodiments, the polypeptide, protein, or peptide may be modified. Types of modifications include, but are not limited to, phosphorylation, glycosylation, acetylation, ubiquitination/threonization, methylation, palmitoylation, quinone, amidation, myristoylation, pyrrolidone carboxylic acid, hydroxylation, phosphopantetheine (Phosphopantetheine), prenylation, GPI anchoring, oxidation, ADP ribosylation, sulfation, S-nitrosylation, citrullination, nitration, gamma-carboxyglutamic acid, formylation, hypusine, tropaquine (Topaquinone; TPQ), bromination, lysine Tropaquine (LTQ), tryptophan quinone (Tryptophan tryptophylquinone; TTQ), iodination and Cysteine Tryptophan Quinone (CTQ). In some aspects, the polypeptide, protein, or peptide may be modified by post-transcriptional modification that may affect its structure, subcellular localization, and/or function.

In some embodiments, the polypeptide, protein, or peptide may be modified using phosphorylation. Phosphorylating or adding phosphate groups to serine, threonine or tyrosine residues is one of the most common modified forms of proteins. Protein phosphorylation plays an important role in fine-tuning the signal in the intracellular signaling cascade.

In some embodiments, the polypeptide, protein, or peptide may be modified using ubiquitination, which is a covalent linkage of ubiquitin to a protein of interest. Ubiquitination-mediated protein turnover has been shown to play a role in driving the cell cycle as well as in protein-degradation-independent intracellular signaling pathways.

In some embodiments, the polypeptide, protein, or peptide may be modified using acetylation and methylation that may play a role in regulating gene expression. As one non-limiting example, acetylation and methylation may mediate the formation of chromatin domains (e.g., true chromatin and heterochromatin), which may have an effect on mediating gene silencing.

In some embodiments, the polypeptide, protein, or peptide may be modified using glycosylation. Glycosylation is one of the large number of glycan groups attached and is a modification that occurs in about half of all proteins and that plays a role in biological processes including, but not limited to, embryonic development, cell division, and protein structure regulation. Two major types of protein glycosylation are N-glycosylation and O-glycosylation. For N-glycosylation, glycans are attached to asparagine; and for O-glycosylation, glycans are linked to serine or threonine.

In some embodiments, the polypeptide, protein, or peptide may be modified using threonization. Sumoylation adds SUMO (small ubiquitin-like modifier) to proteins and is a post-translational modification similar to ubiquitination.

Antibodies to

As used herein, the term "antibody" refers in its broadest sense and specifically covers various embodiments, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies formed from at least two intact antibodies), and antibody fragments (e.g., bifunctional antibodies), so long as they exhibit the desired biological activity (e.g., "functionality"). Antibodies are predominantly amino acid-based molecules, which are monomeric or polymeric polypeptides comprising at least one amino acid region derived from a known or parent antibody sequence and at least one amino acid region derived from a non-antibody sequence. Antibodies may comprise one or more modifications (including, but not limited to, addition of sugar moieties, fluorescent moieties, chemical tags, etc.). For purposes herein, an "antibody" may comprise heavy and light chain variable domains and an Fc region.

The cargo or payload may comprise or may encode a polypeptide that forms one or more functional antibodies.

In some embodiments, the cargo or payload may comprise or may encode a polypeptide formed or used as any antibody, including but not limited to antibodies known in the art and/or commercially available antibodies that may be therapeutic, diagnostic, or for research purposes. In addition, the cargo or payload may comprise or encode fragments of such antibodies or antibodies, such as, but not limited to, variable domains or Complementarity Determining Regions (CDRs).

As used herein, the term "primary antibody" refers to a typical iso-tetrasaccharide protein of about 150,000 daltons, which is composed of two identical light (L) chains and two identical heavy (H) chains. Genes encoding antibody heavy and light chains are known and their respective constituent segments have been well characterized and described (Matsuda, F. Et al 1998.The Journal of Experimental Medicine.188 (11); 2151-62 and Li, A. Et al 2004.Blood.103 (12): 4602-9, the contents of each of which are incorporated herein by reference in their entirety). Each light chain is linked to the heavy chain by one covalent disulfide bond, whereas the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (V _H) at one end followed by multiple constant domains. Each light chain has a variable domain at one end (V _L) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain. As used herein, the term "light chain" refers to the component of an antibody from any vertebrate species that is classified into one of two distinct types (based on the amino acid sequence of the constant domain, referred to as kappa and lambda). Antibodies can be classified into different classes depending on the amino acid sequence of the constant domain of their heavy chain. There are five main classes of intact antibodies: igA, igD, igE, igG and IgM, and several of these antibodies can be further divided into subclasses (isotypes), such as IgG1, igG2, igG3, igG4, igA, and IgA2.

As used herein, the term "variable domain" refers to a particular antibody domain found on both the heavy and light chains of an antibody, which varies widely in sequence among antibodies and is used for the binding and specificity of each particular antibody for its particular antigen. The variable domain comprises a hypervariable region. As used herein, the term "hypervariable region" refers to a region within the variable domain that comprises amino acid residues responsible for antigen binding. The amino acids present within the hypervariable region determine the structure of the Complementarity Determining Regions (CDRs) that become part of the antigen binding site of the antibody. As used herein, the term "CDR" refers to a region in an antibody that comprises a structure complementary to its antigen or epitope of interest. Other portions of the variable domain that do not interact with the antigen are referred to as Framework (FW) regions. An antigen binding site (also referred to as an antigen combining site or paratope) comprises amino acid residues necessary for interaction with a particular antigen. The exact residues that make up the antigen binding site are typically clarified by co-crystallization with the bound antigen, however, computational evaluation may also be used based on comparison with other antibodies (Strohl, w.r.therapeutic Antibody engineering, wood head Publishing, philiadelphia pa.2012, chapter 3, pages 47-54, the contents of which are incorporated herein by reference in their entirety). Determining the residues that make up a CDR may include using numbering schemes including, but not limited to, the numbering schemes taught by: kabat [ Wu, T.T. et Al, 1970, JEM,132 (2): 211-50 and Johnson, G.et Al, 2000,Nucleic Acids Res.28 (1): 214-8, the contents of each of which are incorporated herein by reference in their entirety ], chothia [ Chothia and Lesk, J.mol. Biol.196,901 (1987), chothia et Al, nature 342,877 (1989) and Al-Lazikani, B.et Al, 1997, J.mol. Biol.273 (4): 927-48, the contents of each of which are incorporated herein by reference in their entirety ], lefranc (Lefranc, M.P. et Al, 2005,Immunome Res.1:3) and Honygger (Honygger, A. And Pluckthun, A.2001.J.mol. Biol.309 (3): 657-70, the contents of which are incorporated herein by reference in their entirety.

The V _H and V _L domains each have three CDRs. The V _L CDRs are referred to herein as CDR-L1, CDR-L2 and CDR-L3 in the order of appearance as they move along the variable domain polypeptide from N-terminus to C-terminus. The V _H CDRs are referred to herein as CDR-H1, CDR-H2 and CDR-H3 in the order of appearance as they move along the variable domain polypeptide from N-terminus to C-terminus. Each of the CDRs has an advantageous typical structure, which comprises, in addition to CDR-H3, an amino acid sequence that is highly variable in sequence and length between antibodies, thereby generating a variety of three-dimensional structures in the antigen binding domain. In some cases, CDR-H3 can be analyzed in a panel of related antibodies to assess antibody diversity.

Various methods of determining CDR sequences are known in the art and can be applied to known antibody sequences. The system described by Kabat, also referred to as "according to Kabat numbering", "Kabat definition" and "Kabat labelling", provides a well-defined residue numbering system for any variable domain of an antibody, and provides the exact residue boundaries defining the three CDRs of each chain. (Kabat et al Sequences of Proteins of Immunological Interest, national Institutes of Health, bethesda, md. (1987) and (1991), the contents of which are incorporated by reference in their entirety). Kabat CDRs comprise about residues 24-34 (CDR 1), 50-56 (CDR 2), and 89-97 (CDR 3) in the light chain variable domain, and residues 31-35 (CDR 1), 50-65 (CDR 2), and 95-102 (CDR 3) in the heavy chain variable domain. Chothia and colleagues found that some of the sub-portions within the Kabat CDRs adopt nearly identical peptide backbone conformations, despite the tremendous diversity at the amino acid sequence level. (Chothia et al (1987) J.mol. Biol.196:901-917; And Chothia et al (1989) Nature 342:877-883, the contents of each of which are incorporated herein by reference in their entirety). These CDRs may be referred to as "Chothia CDRs", "Chothia numbering" or "according to Chothia numbering" and comprise about residues 24-34 (CDR 1), 50-56 (CDR 2) and 89-97 (CDR 3) in the light chain variable domain and residues 26-32 (CDR 1), 52-56 (CDR 2) and 95-102 (CDR 3) in the heavy chain variable domain. Mol. Biol.196:901-917 (1987). The system described by MacCallum, also referred to as "numbering according to MacCallum" or "MacCallum", comprises about residues 30-36 (CDR 1), 46-55 (CDR 2) and 89-96 (CDR 3) in the light chain variable domain and residues 30-35 (CDR 1), 47-58 (CDR 2) and 93-101 (CDR 3) in the heavy chain variable domain. (MacCallum et al ((1996) J.mol. Biol.262 (5): 732-745), the contents of which are incorporated herein by reference in their entirety). The system described by AbM, also referred to as "according to AbM numbering" or "AbM numbering", comprises about residues 24-34 (CDR 1), 50-56 (CDR 2) and 89-97 (CDR 3) in the light chain variable domain and residues 26-35 (CDR 1), 50-58 (CDR 2) and 95-102 (CDR 3) in the heavy chain variable domain. IMGT (international immunogenetics information system (INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM)) numbering of variable regions, which is numbering (Lefranc,M.-P.,"The IMGT unique numbering for immunoglobulins,T cell Receptors and Ig-like domains",The Immunologist,7,132-136(1999), for residues in immunoglobulin variable heavy or light chains according to the method of IIMGT and is incorporated herein by reference in its entirety, may also be used. As used herein, "IMGT sequence number" or "numbering according to IMTG" refers to the sequence number of the coding variable region according to IMGT. For the heavy chain variable domain, when numbered according to IMGT, the hypervariable region is in the range of amino acid positions 27 to 38 for CDR1, amino acid positions 56 to 65 for CDR2, and amino acid positions 105 to 117 for CDR 3. For the light chain variable domain, when numbered according to IMGT, the hypervariable region is in the range of amino acid positions 27 to 38 for CDR1, amino acid positions 56 to 65 for CDR2, and amino acid positions 105 to 117 for CDR 3.

In some embodiments, the cargo or payload may comprise or may encode antibodies that have been produced using methods known in the art, such as, but not limited to, immunization and display techniques (e.g., phage display, yeast display, and ribosome display), hybridoma techniques, heavy and light chain variable region cDNA sequences selected from hybridomas or other sources.

In some embodiments, the cargo or payload may comprise or may encode antibodies developed using any naturally occurring or synthetic antigen. As used herein, an "antigen" is an entity that induces or induces an immune response in an organism. An immune response is characterized by the response of cells, tissues and/or organs of an organism to the presence of a foreign entity. Such immune responses typically cause an organism to produce one or more antibodies to a foreign entity (e.g., an antigen or a portion of an antigen). As used herein, "antigen" also refers to a binding partner for a specific antibody or a binding agent in a display library.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous cells (or clones), i.e., the individual antibodies comprising the population are identical and/or bind to the same epitope, except for possible variants (such variants typically being present in minor amounts) that may be produced during the production of the monoclonal antibody. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies herein include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies.

As used herein, the term "humanized antibody" refers to a chimeric antibody that comprises a minimal portion from one or more non-human (e.g., murine) antibody sources and the remainder derived from one or more human immunoglobulin sources. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of an antibody from the recipient are replaced by residues from a hypervariable region of an antibody (donor antibody) of the desired specificity, affinity and/or capacity from a non-human species such as mouse, rat, rabbit or non-human primate.

In some embodiments, the cargo or payload may comprise or encode an antibody mimetic. As used herein, the term "antibody mimetic" refers to any molecule that mimics the function or action of an antibody and that binds specifically and with high affinity to its molecular target. In some embodiments, the antibody mimetic may be a monofunctional antibody designed to incorporate the fibronectin type III domain (Fn 3) as a protein backbone. In some embodiments, antibody mimics may be those known in the art, including but not limited to affinity antibody molecules, affilin, affitin, anti-carrier (anticalin), high affinity multimers, xin Tien (Centyrin), DARPINS ^TM, fenomo (fynomer), kunitz domain (Kunitz domain), and domain peptides. In other embodiments, the antibody mimetic may include one or more non-peptide regions.

Antibody fragments and variants

In some embodiments, the cargo or payload may comprise or encode an antibody fragment comprising an antigen binding region from a full length antibody. Non-limiting examples of antibody fragments include Fab, fab ', F (ab') ₂, and Fv fragments, bifunctional antibodies, linear antibodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen binding fragments, known as "Fab" fragments, each having a single antigen binding site. A residual "Fc" fragment was also generated, the name of which reflects its ability to crystallize readily. Pepsin treatment resulted in a F (ab') ₂ fragment that had two antigen binding sites and was still able to crosslink the antigen. The compounds and/or compositions of the present disclosure may comprise one or more of these fragments.

In some embodiments, the Fc region may be a modified Fc region, wherein the Fc region may have single amino acid substitutions compared to the corresponding sequence of the wild-type Fc region, wherein the single amino acid substitutions result in an Fc region having the preferred characteristics of those of the wild-type Fc region. Non-limiting examples of Fc properties that can be altered by single amino acid substitutions include binding properties or reaction to pH conditions.

As used herein, the term "Fv" refers to an antibody fragment comprising the smallest fragment on an antibody required to form an intact antigen binding site. These regions consist of dimers of one heavy and one light chain variable domain that are closely, non-covalently associated. Fv fragments may be produced by proteolytic cleavage but are largely unstable. Recombinant methods for generating stable Fv fragments are known in the art, typically via insertion of a flexible linker between the light and heavy chain variable domains to form a single chain Fv (scFv), or via the introduction of a disulfide bridge between the heavy and light chain variable domains.

As used herein, the term "single chain Fv" or "scFv" refers to a fusion protein of V _H and V _L antibody domains, wherein the domains are joined together into a single polypeptide chain by a flexible peptide linker. In some embodiments, fv polypeptide linkers enable the scFv to form a desired structure for antigen binding. In some embodiments, scfvs are used in conjunction with phage display, yeast display, or other display methods, where they can be expressed in conjunction with a surface member (e.g., phage coat protein) and used to identify high affinity peptides of a given antigen.

As used herein, the term "antibody variant" refers to a modified antibody (relative to a native or starting antibody) or a biological molecule (e.g., an antibody mimetic) that is similar in structure and/or function to the native or starting antibody. Antibody variants may differ in their amino acid sequence, composition or structure as compared to the original antibody. Antibody variants may include, but are not limited to, antibodies with altered isotypes (e.g., igA, igD, igE, igG ₁、IgG₂、IgG₃、IgG₄ or IgM), humanized variants, optimized variants, multispecific antibody variants (e.g., bispecific variants), and antibody fragments.

Multispecific antibodies

In some embodiments, the cargo or payload may be or encode an antibody that binds to more than one epitope. As used herein, the term "multifunctional antibody" or "multispecific antibody" refers to an antibody in which two or more variable regions bind to different epitopes. Epitopes may be on the same or different targets. In certain embodiments, the multispecific antibody is a "bispecific antibody" that recognizes two different epitopes on the same or different antigens.

In some embodiments, the method can be performed byMultispecific antibodies are prepared using methods described in International patent publication WO201109726, the disclosure of which is incorporated herein by reference in its entirety. First, a library of homologous, naturally occurring antibodies (i.e., mammalian cell surface display) is generated by any method known in the art, and then screened for multispecific antibodies that specifically bind to two or more antigens of interest by FACSAria or another screening method. In some embodiments, the identified multispecific antibodies are further evolved by any method known in the art to produce a set of modified multispecific antibodies. These modified multispecific antibodies are screened for binding to the antigen of interest. In some embodiments, the multispecific antibodies can be further optimized by screening the evolved modified multispecific antibodies for optimized or desired features.

In some embodiments, the method can be performed byMultispecific antibodies are prepared using methods described in U.S. publication No. US20150252119, the contents of which are incorporated herein by reference in their entirety. In one approach, the variable domains of two parent antibodies (where the parent antibodies are monoclonal antibodies) are evolved in a manner such that a single light chain is functionally complementary to the heavy chains of two different parent antibodies, using any method known in the art. Another approach entails evolving the heavy chain of a single parent antibody to recognize a second antigen of interest. The third method involves evolving the light chain of the parent antibody to recognize the second antigen of interest. Methods for polypeptide evolution are described in international publication WO2012009026, the contents of which are incorporated herein by reference in their entirety, and include, as one non-limiting example, integrated site evolution (CPE), combined Protein Synthesis (CPS), integrated site insertion (CPI), integrated site deletion (CPD), or any combination thereof. The Fc region of the multispecific antibody described in U.S. publication No. US20150252119 can be produced using the knob-in-hole approach (knob-in-hole approach) or any other approach that enables the Fc domain to form a heterodimer. The resulting multispecific antibodies can be further evolved to obtain improved features or characteristics, such as binding affinity for an antigen of interest.

Bispecific antibodies

In some embodiments, the cargo or payload may be or encode a bispecific antibody. As used herein, the term "bispecific antibody" refers to an antibody capable of binding two different antigens. Such antibodies typically comprise regions from at least two different antibodies. Such antibodies typically comprise antigen binding regions from at least two different antibodies. For example, bispecific monoclonal antibodies (BsMAb, bsAb) are artificial proteins composed of fragments of two different monoclonal antibodies, thus enabling BsAb to bind to two different types of antigens.

In some cases, the cargo or payload may be or encode a bispecific antibody comprising antigen binding regions from two different anti-tau antibodies. For example, such bispecific antibodies may comprise binding regions from two different antibodies.

Bispecific antibody frameworks may include any of those described in: rietvmuler, g.,2012.Cancer Immunity.12:12-18; marvin, J.S. et al 2005.Acta Pharmacologica Sinica.26 (6): 649-58; and Schaefer, W.et al, 2011.PNAS.108 (27): 11187-92, the respective contents of which are incorporated herein by reference in their entirety.

A new BsMAb generation, called a "three-function bispecific" antibody, has been developed. These antibodies consist of two heavy chains and two light chains, each from two different antibodies, with two Fab regions (arms) directed against two antigens and the Fc region (foot) comprising two heavy chains and forming a third binding site.

Of the two paratopes on top of the variable domain forming the bispecific antibody, one can be directed against the antigen of interest and the other against a T lymphocyte antigen, such as CD3. In the case of a trifunctional antibody, the Fc region may additionally bind to cells expressing Fc receptors, such as macrophages, natural Killer (NK) cells or dendritic cells. In summary, the targeted cells are linked to one or two cells in the immune system, which then destroy the targeted cells.

Other types of bispecific antibodies have been designed to overcome certain problems, such as short half-life, immunogenicity, and side effects caused by cytokine release. It includes chemically linked Fab (consisting of only Fab regions), and various types of bivalent and trivalent single chain variable fragments (scFv), fusion proteins that mimic the variable domains of both antibodies. These newer forms, which have been developed recently, are bispecific T cell binding molecules (BiTE) and mAb2, engineered to contain antibodies that replace the Fcab antigen binding fragment of the Fc constant region.

Using molecular genetics, two scFv can be engineered in tandem into a single polypeptide, separated by a linker domain, known as a "tandem scFv" (tascFv). TascFv has been found to be poorly soluble and require refolding when produced in bacteria, or it can be manufactured in mammalian cell culture systems, avoiding refolding requirements, but possibly causing poor yields. Construction of tascFv of genes with two different scfvs resulted in a "bispecific single chain variable fragment" (double scFv). Commercial companies have only developed two types tascFv clinically; both are bispecific agents against tumor indications that were actively developed by Micromet at an early stage, and are described as "bispecific T cell engagement molecules (bites)". Bonauzumab (Blinatumomab) is anti-CD 19/anti-CD 3 bispecific tascFv, which potentiates T-cell responses to phase 2B cell non-Hodgkin lymphoma. MT110 is an anti-EP-CAM/anti-CD 3 bispecific tascFv which enhances the T cell response to phase 1 solid tumors. Affimed bispecific tetravalent "tandabs" are also being studied.

In some embodiments, the cargo or payload may be or encode an antibody comprising a single antigen binding domain. These molecules are extremely small, with a molecular weight approximately one tenth of that observed for a full-size mAb. Other antibodies may include "nanobodies" derived from the antigen binding variable heavy chain region (V _HH) of heavy chain antibodies found in camels and Luo Mazhong, which do not have a light chain.

PCT publication WO2014144573 to Memorial Sloan-KETTERING CANCER CENTER, the contents of which are incorporated herein by reference in their entirety, discloses and claims a multimerization technique for preparing dimeric multispecific binders (e.g., fusion proteins comprising antibody components) with improved properties relative to multispecific binders that do not have dimerization capabilities.

In some cases, the cargo or payload may be or encode a tetravalent bispecific antibody (TetBiAb as disclosed and claimed in PCT publication WO2014144357, the contents of which are incorporated herein in their entirety). The TetBiAb is characterized by a second pair of Fab fragments with a second antigen specificity linked to the C-terminus of the antibody, thus providing a molecule that is bivalent for each of the two antigen specificities. Tetravalent antibodies were generated by genetic engineering methods by covalently linking an antibody heavy chain to a Fab light chain (which associates with its cognate, co-expressed Fab heavy chain).

In some aspects, the cargo or payload may be or encode a biosynthetic antibody as described in U.S. patent No. 5,091,513 (the contents of which are incorporated herein by reference in their entirety). Such antibodies may include one or more sequences of amino acids that constitute a region that serves as a binding site for a Biosynthetic Antibody (BABS). Sites comprise 1) non-covalently associated or disulfide bonded synthetic V _H and V _L dimers, 2) V _H-V_L or V _L-V_H single chains, wherein V _H and V _L are linked by a polypeptide linker, or 3) individual V _H or V _L domains. The binding domain comprises linked CDR and FR regions, which may be derived from individual immunoglobulins. Biosynthetic antibodies may also include other polypeptide sequences that serve as, for example, enzymes, toxins, binding sites, or attachment sites to immobilized media or radioactive atoms. Methods for preparing biosynthetic antibodies, for designing BABS with any specificity that can be elicited by in vivo production of antibodies, and for preparing analogs thereof are disclosed.

In some embodiments, the cargo or payload may be or encode an antibody having the antibody acceptor framework taught in U.S. patent No. 8,399,625. Such antibody acceptor frameworks may be particularly suitable for accepting CDRs from an antibody of interest. In some cases, CDRs from anti-tau antibodies known in the art or developed according to the methods presented herein may be used.

Miniaturized antibodies

In some embodiments, the cargo or payload may be or encode a "mini" antibody. A preferred example of mAb miniaturization includes Small Modular Immunopharmaceuticals (SMIPs) from Trubion Pharmaceuticals. These molecules (which may be monovalent or bivalent) are recombinant single-chain molecules containing one V _L, one V _H antigen-binding domain, and one or two constant "effector" domains (all linked by a linker domain). It is possible that such molecules may provide increased tissue or tumor penetration as claimed by the fragments while retaining the advantage of immune effector function conferred by the constant domains. At least three "miniaturized" SMIPs have entered clinical development. TRU-015, an anti-CD 20 SMIP developed in concert with Wyeth, is the most advanced project, and has progressed to stage 2 for Rheumatoid Arthritis (RA). Earlier attempts in Systemic Lupus Erythematosus (SLE) and B-cell lymphomas eventually discontinued. Trubion and Facet Biotechnology cooperate to develop TRU-016, an anti-CD 37 SMIP, a project that has reached stage 2, for the treatment of CLL and other lymphoneoplastic tumors. Wyeth has approved anti-CD 20 SMIP SBI-087 for use in the treatment of autoimmune diseases, including RA, SLE and possibly multiple sclerosis, but these programs are still in the earliest stages of clinical testing.

Bifunctional antibodies

In some embodiments, the cargo or payload may be or encode a bifunctional antibody. As used herein, the term "bifunctional antibody" refers to a small antibody fragment having two antigen binding sites. The bifunctional antibody comprises a heavy chain variable domain V _H, which is linked to a light chain variable domain V _L in the same polypeptide chain. By using a linker that is too short to allow pairing between two domains on the same strand, the domains are forced to pair with the complementary domain of the other strand and two antigen binding sites are created.

The bifunctional antibody is a functional bispecific single chain antibody (bscAb). These bivalent antigen binding molecules are composed of non-covalent dimers of scFv and can be produced in mammalian cells using recombinant methods. (see, e.g., mack et al, proc. Natl. Acad. Sci.,92:7021-7025,1995). Few bifunctional antibodies have entered clinical development. In a Beckman Research Institute of the City of Hope sponsored study (clinical.gov NCT 00647153), an iodine 123 labeled bifunctional antibody version of the anti-CEA chimeric antibody cT84.66 has been evaluated for preoperative immunoscintigraphy detection of colorectal cancer.

Monoclonal antibodies

In some embodiments, the cargo or payload may be or encode a "monoclonal antibody (unibody)", wherein the hinge region has been removed from the IgG4 molecule. Although IgG4 molecules are unstable and can exchange light-heavy chain heterodimers with each other, the deletion of the hinge region can completely prevent heavy-heavy chain pairing, leaving a highly specific monovalent light/heavy chain heterodimer while preserving the Fc region to ensure stability and in vivo half-life. This configuration minimizes the risk of immune activation or oncogenic growth, as IgG4 interacts poorly with FcR and monovalent monoclonal antibodies fail to promote intracellular signaling complex formation. However, these views are supported primarily by laboratory rather than clinical evidence. Other antibodies may be "miniaturized" antibodies, which are compacted 100kDa antibodies.

Intracellular antibodies

In some embodiments, the cargo or payload may be or encode an intracellular antibody. The term "intracellular antibody (intrabody)" refers to a form of antibody that is not secreted by the cell from which it is produced, but instead targets one or more intracellular proteins. Intracellular antibodies can be used to affect a variety of cellular processes including, but not limited to, intracellular migration, transcription, translation, metabolic processes, proliferation signaling, and cell division. In some embodiments, the methods of the present disclosure may include intracellular antibody-based therapies. In some such embodiments, the variable domain sequences and/or CDR sequences disclosed herein can be incorporated into one or more constructs for use in intracellular antibody-based therapies. For example, an intracellular antibody may target one or more glycosylated intracellular proteins or may modulate the interaction between one or more glycosylated intracellular proteins and an alternative protein.

More than twenty years ago, intracellular antibodies against intracellular targets were first described (Biocca, neuberger and Cattaneo EMBO j.9:101-108,1990, the contents of which are incorporated herein by reference in their entirety). Intracellular expression of intracellular antibodies in different compartments of mammalian cells allows blocking or modulating the function of endogenous molecules (Biocca et al, EMBO J.9:101-108,1990; colby et al, proc. Natl. Acad. Sci. U.S. A.101:17616-21,2004, the contents of which are incorporated herein by reference in their entirety). Intracellular antibodies can alter protein folding, protein-protein, protein-DNA, protein-RNA interactions, and protein modifications. It can induce phenotypic gene knockdown and act as a neutralizing agent by binding directly to the antigen of interest, by diverting its intracellular migration or by inhibiting its association with a binding partner. It is mainly used as a research tool and emerges as a therapeutic molecule for the treatment of human diseases (e.g. viral lesions, cancer and misfolded diseases). Regarding their use in therapy, the rapidly growing bio-market for recombinant antibodies provides intracellular antibodies with enhanced binding specificity, stability and solubility, as well as lower immunogenicity.

In some embodiments, the intracellular antibodies have advantages over interfering RNAs (irnas); for example, iRNA has been shown to exert a variety of nonspecific effects, while intracellular antibodies have been shown to have high specificity and affinity for the antigen of interest. Furthermore, as a protein, intracellular antibodies have a much longer active half-life than iRNA. Thus, when the active half-life of an intracellular target molecule is long, the process of gene silencing via iRNA can be slow, while the effect of intracellular antibody expression can be nearly instantaneous. Finally, it is possible to design intracellular antibodies to block certain binding interactions of specific target molecules while retaining other effects.

Intracellular antibodies are typically single chain variable fragments (scFv) expressed by recombinant nucleic acid molecules and engineered to survive inside the cell (e.g., in the cytoplasm, endoplasmic reticulum, or extracellular matrix). For example, an intracellular antibody can be used to eliminate the function of a protein to which the intracellular antibody binds. Expression of the intracellular antibody may also be regulated via the use of an inducible promoter in a nucleic acid expression vector comprising the intracellular antibody. Intracellular antibodies for the viral genomes of the present disclosure can be generated using methods known in the art, such as the methods disclosed and reviewed in: marasco et al, 1993Proc.Natl. Acad.Sci.USA,90:7889-7893; Chen et al 1994,Hum.Gene Ther.5:595-601; chen et al, 1994, proc.Natl. Acad. Sci. USA,91:5932-5936; maciejewski et al, 1995, nature Med.,1:667-673; marasco,1995, immunotech,1:1-19; MHASHILKAR et al, 1995,EMBO J.14:1542-51; chen et al 1996,Hum.Gene Therap, 7:1515-1525; Marasco, gene Ther.4:11-15,1997; rondon and Marasco,1997, annu. Rev. Microbiol.51:257-283; cohen et al 1998,Oncogene 17:2445-56; proba et al, 1998, J.mol. Biol.275:245-253; cohen et al 1998, oncogene17:2445-2456; hassanzadeh et al, 1998,FEBS Lett.437:81-6; Richardson et al 1998,Gene Ther.5:635-44; ohage and Steipe,1999, J.mol. Biol.291:1119-1128; ohage et al, 1999, J.mol. Biol.291:1129-1134; wirtz and stepe, 1999,Protein Sci.8:2245-2250; zhu et al 1999,J.Immunol.Methods 231:207-222; Arafat et al, 2000,Cancer Gene Ther.7:1250-6; der Maur et al, 2002, J.biol. Chem.277:45075-85; MHASHILKAR et al, 2002,Gene Ther.9:307-19; and Wheeler et al 2003,FASEB J.17:1733-5; and references cited therein). In particular, CCR5 intracellular antibodies have been produced by Steinberger et al, 2000,Proc.Natl.Acad.Sci.USA 97:805-810. See generally Marasco,WA,1998,"Intrabodies:Basic Research and Clinical Gene Therapy Applications"Springer:New York; and reviews of scFv, see Pluckaphun, "The Pharmacology of Monoclonal Antibodies",1994, volume 113, rosenburg and Moore, springer-Verlag, new York, pages 269-315; the contents of each of which are each incorporated by reference in their entirety.

Sequences from donor antibodies can be used to generate intracellular antibodies. Intracellular antibodies are typically expressed recombinantly in cells as single domain fragments (e.g., isolated V _H and V _L domains) or single chain variable fragment (scFv) antibodies. For example, intracellular antibodies are typically expressed as a single polypeptide to form single chain antibodies comprising variable domains of heavy and light chains joined by a flexible linker polypeptide. Intracellular antibodies typically lack disulfide bonds and are capable of modulating the expression or activity of a gene of interest via their specific binding activity. Single chain antibodies may also be expressed as single chain variable region fragments joined to the constant region of the light chain.

As known in the art, intracellular antibodies can be engineered into recombinant polynucleotide vectors to encode a codon cell migration signal at their N or C terminus to allow expression at high concentrations in the subcellular compartment in which the protein of interest is located. For example, an intracellular antibody targeting the Endoplasmic Reticulum (ER) is engineered to incorporate a leader peptide and optionally a C-terminal ER retention signal. Intracellular antibodies intended to exert activity in the nucleus are engineered to include nuclear localization signals. The lipid moiety binds to the intracellular antibody to tether the intracellular antibody to the cytoplasmic side of the plasma membrane. Intracellular antibodies may also be targeted to function in the cytosol. For example, cytoplasmic intracellular antibodies are used to sequester factors within the cytosol, thereby preventing their transport to their natural cellular destination.

There are certain technical challenges with respect to intracellular antibody expression. In particular, the conformational folding and structural stability of the protein of a newly synthesized intracellular antibody within a cell is affected by the reducing conditions of the intracellular environment.

The intracellular antibodies of the present disclosure are promising therapeutics for the treatment of misfolded diseases, including tauopathies, prion diseases, alzheimer's, parkinson's and Huntington's, due to their virtually unlimited ability to specifically recognize different conformations of proteins, including pathological isoforms, and because they can target potential aggregation sites, both intracellular and extracellular. These molecules can act as neutralizing agents for amyloidogenic proteins by preventing aggregation of the amyloidogenic proteins, and/or as molecular schedulers for intracellular traffic by rerouting proteins from their potential aggregation sites.

Maximum antibody

In some embodiments, the cargo or payload may be or encode a bivalent scFV fused to the amino terminus of an Fc (CH 2-CH3 domain) of an IgG, for a maximum antibody (maxibody).

Chimeric Antigen Receptor (CAR)

In some embodiments, the cargo or payload can be or encode a Chimeric Antigen Receptor (CAR) that, when transduced into immune cells (e.g., T cells and NK cells), can redirect the immune cells toward targets (e.g., tumor cells) that express molecules recognized by the extracellular target portion of the CAR.

As used herein, the term "Chimeric Antigen Receptor (CAR)" refers to a synthetic receptor that mimics a TCR on the surface of a T cell. In general, CARs consist of an extracellular targeting domain, a transmembrane domain/region, and an intracellular signaling/activation domain. In standard CAR receptors, the components: extracellular targeting domain, transmembrane domain and intracellular signaling/activation domain are linearly constructed as a single fusion protein. The extracellular region comprises a targeting domain/moiety (e.g., scFv) that recognizes a specific tumor antigen or other tumor cell surface molecule. The intracellular region may contain a signaling domain of the TCR complex (e.g., a signaling region of cd3ζ) and/or one or more costimulatory signaling domains, such as those from CD28, 4-1BB (CD 137) and OX-40 (CD 134). For example, a "first generation CAR" has only a CD3 zeta signaling domain, while to increase T cell persistence and proliferation, a costimulatory intracellular domain is added, resulting in a second generation CAR having a CD3 zeta signaling domain plus one costimulatory signaling domain, and a third generation CAR having a CD3 zeta signaling domain plus two or more costimulatory signaling domains. When expressed by T cells, the CAR confers antigen specificity to the T cells determined by the extracellular targeting portion of the CAR. In some aspects, one or more elements, such as homing (home) and suicide genes, may be added to produce a more competent and safer CAR architecture (so-called fourth generation CAR).

In some embodiments, the extracellular targeting domain is joined to the intracellular signaling domain via a hinge (also referred to as a spacer domain or spacer) and a transmembrane region. The hinge connects the extracellular targeting domain with a transmembrane domain that passes through the cell membrane and is linked to an intracellular signaling domain. Due to the size of the target protein to which the targeting moiety binds, as well as the size and affinity of the targeting domain itself, it may be desirable to alter the hinge to optimize the potency of the CAR transformed cells to cancer cells. Upon recognition and binding of the targeting moiety to the target cell, the intracellular signaling domain generates an activation signal to the CAR T cell that is further amplified by the "second signal" of the one or more intracellular co-stimulatory domains. Once activated, CAR T cells can destroy target cells.

In some embodiments, the CAR can be split into two parts, each part connecting the dimerization domain such that the input triggering dimerization facilitates assembly of the complete functional receptor. Wu and Lim report resolution CAR (split CAR) in which the extracellular CD19 binding domain and intracellular signaling element are separated and linked to FKBP domain and FRB x (FKBP-rapamycin bound T2089L mutant) domain heterodimerized in the presence of rapamycin analog AP 21967. Split receptors (split receptors) are assembled in the presence of AP21967 and activate T cells along with specific antigen binding (Wu et al, science,2015,625 (6258): aab4077, the contents of which are incorporated herein by reference in their entirety).

In some embodiments, the CAR may be designed as an inducible CAR that has incorporated a Tet-On inducible system into the CD19CAR construct. CD19CAR is activated only in the presence of doxycycline (Dox). Sakemura reports that Tet-CD19CAR T cells in the presence of Dox are equivalently cytotoxic to cd19+ cell lines compared to conventional CD19CAR T cells, and have equivalent cytokine production and proliferation following CD19 stimulation ((Sakemura et al, cancer immune. Res., 21, electronic version, 2016; the contents of which are incorporated herein by reference in their entirety). A dual system provides more flexibility for turning CAR expression on and off in transduced T cells.

In some embodiments, the cargo or payload may be or encode a first generation CAR, or a second generation CAR, or a third generation CAR, or a fourth generation CAR. In some embodiments, the cargo or payload can be or encode a complete CAR construct consisting of an extracellular domain, a hinge, and a transmembrane domain, and an intracellular signaling region. In other embodiments, the cargo or payload may be or encode components of the complete CAR construct, including extracellular targeting moieties, hinge regions, transmembrane domains, intracellular signaling domains, one or more co-stimulatory domains, and other additional elements that improve CAR architecture and functionality, including but not limited to leader sequences, homing elements, and safety switches, or combinations of such components.

In some embodiments, the cargo or payload may be or encode a tunable CAR. The reversible on-off switching mechanism allows management of acute toxicity caused by excessive CAR-T cell expansion. Ligand-conferred CAR modulation may be effective in counteracting tumor escape induced by antigen loss, thereby avoiding functional exhaustion due to tonic signaling due to long-term antigen exposure and improving persistence of CAR-expressing cells in vivo. Tunable CARs can be used to down-regulate CAR expression to limit the goal of tissue toxicity caused by oncolytic syndrome. Down-regulating CAR expression after anti-tumor efficacy can prevent (1) tumor toxicity off-target caused by antigen expression in normal tissues; (2) in vivo antigen independent activation.

Extracellular targeting domains/moieties

In some embodiments, the extracellular target portion of the CAR can be any agent that recognizes and binds to a given target molecule (e.g., a neoantigen on a tumor cell) with high specificity and affinity. The target portion may be: antibodies and variants thereof that specifically bind to a target molecule on a tumor cell; or an aptamer selected from a pool of random sequences based on its ability to bind to a target molecule on a tumor cell; or a variant or fragment thereof that binds to a target molecule on a tumor cell; or an antigen recognition domain from a primary T Cell Receptor (TCR) (e.g., a CD4 extracellular domain that recognizes HIV-infected cells); or an exogenously recognizing component, such as a cytokine that causes recognition of the attachment of a target cell carrying a cytokine receptor or a natural ligand for the receptor.

In some embodiments, the targeting domain of the CAR can be an Ig NAR, fab fragment, fab ' fragment, F (ab) '2 fragment, F (ab) '3 fragment, fv, single chain variable fragment (scFv), diav, (scFv) 2, minibody, diabody, triabody, tetrafunctional antibody, disulfide stabilized Fv protein (dsFv), a monoclonal antibody, a nanobody, or an antigen-binding region derived from an antibody that specifically recognizes a target molecule (e.g., a tumor-specific antigen (TSA)). In one embodiment, the targeting moiety is an scFv antibody. The scFv domain is capable of maintaining CAR T cells in proximity to cancer cells and triggering T cell activation when expressed on the surface of the CAR T cells and subsequently binds to a target protein on the cancer cells. scFv can be produced using conventional recombinant DNA technology and are discussed in this disclosure.

In some embodiments, the targeting moiety of the CAR construct can be an aptamer, such as a peptide aptamer, that specifically binds to a target molecule of interest. Peptide aptamers may be selected from a pool of random sequences based on their ability to bind to target molecules of interest.

In some embodiments, the targeting moiety of the CAR construct can be a natural ligand of the target molecule or a variant and/or fragment thereof capable of binding to the target molecule. In some aspects, the targeting moiety of the CAR can be a receptor for a target molecule, e.g., full length human CD27, as a CD70 receptor, can be fused in-frame with the signaling domain of CD3 zeta, thereby forming a CD27 chimeric receptor as an immunotherapeutic agent for CD 70-positive malignancies.

In some embodiments, the targeting portion of the CAR can recognize a Tumor Specific Antigen (TSA), such as a cancer neoantigen that is expressed on tumor cells to a limited extent.

As one non-limiting example, a CAR of the present disclosure may comprise an extracellular targeting domain capable of binding to a tumor-specific antigen selected from the group consisting of: 5T4, 707-AP, A33, AFP (alpha fetoprotein), AKAP-4 (A kinase anchoring protein 4), ALK, alpha 5 beta 1-integrin, androgen receptor, annexin II, alphA-Actin-4, ART-4, B1, B7H3, B7H4, BAGE (B melanoma antigen), BCMA, BCR-ABL fusion protein, beta-catenin, BKT-antigen, BTA, CA-I (carbonic anhydrase I), CA50 (cancer antigen 50), CA125, CA15-3, CA195, CA242, calretinin, CAIX (carbonic anhydrase), cytotoxic T lymphocyte recognized antigen on melanoma (cytoxic T-lymphocyte recognized antigen on melanoma; CAMEL), CAM43, CAP-1, apoptosis protease -8/m、CD4、CD5、CD7、CD19、CD20、CD22、CD23、CD25、CD27/m、CD28、CD30、CD33、CD34、CD36、CD38、CD40/CD154、CD41、CD44v6、CD44v7/8、CD45,CD49f、CD56、CD68\KP1、CD74、CD79a/CD79b、CD103、CD123、CD133、CD138、CD171、cdc27/m、CDK4( cyclin-dependent kinase 4), CDKN2A, CDS, carcinoembryonic antigen (carcinoembryonic antigen; CEA), CEACAM5, CEACAM6, stained particulate, c-Met, c-Myc, coa-1, cscp, CT7, CT10, cyclophilin B, cyclin B1, cytoplasmic tyrosine kinase, cytokeratin, DAM-10, DAM-6, dek-can fusion protein, myotonin (desmin), DEP domain containing 1 (DEP domain containing 1; DEPDC 1), E2A-PRL, EBNA, epidermal growth factor receptor (EPIDERMAL GROWTH FACTOR RECEPTOR; EGF-R), epithelial glycoprotein-1 (EPITHELIAL GLYCOPROTEIN-1; EGP-1) (TROP-2), EGP-2, EGP-40, epidermal growth factor receptor (EPIDERMAL GROWTH FACTOR RECEPTOR; EGFR), egfrvlll, EF-2, ELF2M, EMMPRIN, epithelial cell adhesion molecule (EPITHELIAL CELL adhesion molecule; EpCAM), ephA2, epstein-barr virus antigen, erb (ErbB 1; erbB3; erbB 4), epithelial tumor antigen (EPITHELIAL TUMOR ANTIGEN; ETA), ETV6-AML1 fusion protein, fibroblast activation protein (fibroblast activation protein; FAP), folate-binding protein (folate-binding protein; FBP), FGF-5, folate receptor, FOS-associated antigen 1, fucosyl GM1, G250, gap (gap-1; GAGE-2), galectin, GD2 (ganglioside), GD3, glial fibrillary acidic protein (glial fibrillary acidic protein; GFAP), GM2 (carcinoembryonic antigen-immunogenicity-1; OFA-I-1), gnT-V, gp, H4-RET, HAGE (helicase antigen), HER-2/neu, hypoxia inducible factor (hypoxia inducible factor;HIF)、HIF-1、HIF-2、HLa-a2、HLa-a*0201-R170I、HLa-al l、HMWMAA、Hom/Mel-40、HSP70-2M( heat shock protein 70), HST-2, HTgp-175, hTERT (or hTRT), human papillomavirus-E6/human papillomavirus-E7 and E6, iCE (immunocapture EIA), IGF-1R, IGH-IGK, IL-2R, IL-5, Integrin-linked kinase (integrin-LINKED KINASE; ILK), IMP3 (insulin-like growth factor II mRNA-binding protein 3), interferon regulatory factor 4 (interferon regulatory factor 4; IRF 4), KDR (kinase insert domain receptor), KIAA0205, KRAB-zinc finger protein (KID) -3; KID31, KSA (17-1A), K-ras, LAGE, LCK, LDLR/FUT (LDLR-fucosyltransferase AS fusion protein), leY (Lewis Y), MAD-CT-1, MAGE (tyrosinase, melanomA-Associated antigen) (MAGE-1; MAGE-3), melanin-a tumor antigen (MART), MART-2/Ski, MC1R (melanocortin 1 receptor), MDM2, mesothelin, MPHOSPH1, muscle-specific actin (muscle-SPECIFIC ACTIN; MSA), mammalian target of rapamycin (MAMMALIAN TARGETS of rapamycin; mTOR), MUC-1, MUC-2, MUM-1 (melanomA-Associated antigen (mutant) 1), MUM-2, MUM-3, myosin/m, MYL-RAR, NA88-A, N-acetamido glucose transferase, neo-PAP, NF-KB (nuclear factor- κb), neurofilament, neuron-specific enolase (neuron-specific enolase; NSE), notch receptors, nuMa, N-Ras, NY-BR-1, NY-CO-1, NY-ESO-1, oncostatin M, OS-9, OY-TES1, p53 mutants, p190 mini bcr-abl, pl5 (58), pl85erbB2, pl80erbB-3, PAGE (prostate related gene), prostatophosphoric acid phosphatase (prostatic acid phosphatase; PAP), PAX3, PAX5, platelet-derived growth factor receptor (PLATELET DERIVED growth factor receptor; PDGFR), cytochrome P450 involved in piperidine and pyrrolidine use (PIPA), pml-rarα fusion protein, PR-3 (protease 3), prostate specific antigen (prostate SPECIFIC ANTIGEN; PSA), PSM, prostate stem cell antigen (Prostate STEM CELL ANTIGEN; PSMA), PRAME (antigen of melanoma preferentially expressed), PTPRK, RAGE (renal tumor antigen), raf (A-Raf, B-Raf, and C-Raf), ras, receptor tyrosine kinase, RCAS1, RGSS, ROR1 (receptor tyrosine kinase-like orphan receptor 1), RU1, RU2, SAGE, SART-1, SART-3, SCP-1, SDCCAG, SP-17 (sperm protein 17), src family, SSX (synovial sarcoma X breakpoint) -1, SSX-2 (HOM-MEL-40), and, SSX-3, SSX-4, SSX-5, STAT-3, STAT-5, STAT-6, STEAD, STn, survivin, syk-ZAP70, TA-90 (Mac-2 binding protein\cyclophilin C-associated protein), TAAL, TACSTD1 (tumor-associated calcium signal transducer 1), TACSTD2, TAG-72-4, TAGE, TARP (T cell receptor gamma-substituted reading frame protein), TEL/AML1 fusion protein, TEM1, TEM8 (endosialin or CD 248), TGF beta, TIE2, TLP, TMPRSS2 ETS fusion genes, TNF-receptors (TNF-alpha receptor, TNF-beta receptor; or TNF-gamma receptor), transferrin receptor, TPS, tyrosine-related protein 1 (tyrosine related protein 1; TRP-1), TRP-2/INT2, TSP-180, VEGF receptor, WNT, WT-1 (Wilm's tumor) antigen, and XAGE.

In some embodiments, the cargo or payload may be or encode a CAR comprising a universal immunoreceptor having a targeting moiety capable of binding to a labeled antigen.

In some embodiments, the cargo or payload may be or encode a CAR comprising a targeting moiety capable of binding to a pathogen antigen.

In some embodiments, the cargo or payload can be or encode a CAR comprising a targeting moiety capable of binding to a non-protein molecule (e.g., a tumor-associated glycolipid and carbohydrate).

In some embodiments, the cargo or payload can be or encode a CAR comprising a targeting moiety capable of binding to a component within a tumor microenvironment, including proteins expressed in various tumor stromal cells, including tumor-associated macrophages (TAMs), immature monocytes, immature dendritic cells, immunosuppressive cd4+cd25+ regulatory T cells (tregs), and MDSCs.

In some embodiments, the cargo or payload may be or encode a CAR comprising a targeting moiety capable of binding to: cell surface adhesion molecules, surface molecules of inflammatory cells that occur in autoimmune diseases, or TCRs that elicit autoimmunity. As one non-limiting example, the targeting moiety of the present disclosure may be an scFv antibody that recognizes a Tumor Specific Antigen (TSA), such as an scFv that specifically recognizes and binds human mesothelin, an scFv of SS1 and HN1, an scFv of GD2, a CD19 antigen binding domain, a NKG2D ligand binding domain, a human anti-mesothelin scFv, an anti-CS 1 binding agent, an anti-BCMA binding domain, an anti-CD 19 scFv antibody, a gfrα4 antigen binding fragment, an anti-CLL-1 (C-lectin-like molecule 1) binding domain, a CD33 binding domain, a GPC3 (glypican-3) binding domain, a gfrα4 (glycosyl-phosphatidic acid inositol (GPI) -linked GDNF family α -receptor 4 cell surface receptor) binding domain, a CD123 binding domain, an anti-ROR 1 antibody or fragment thereof, a scFv specific for GPC-3, a scFv for CSPG4, and a scFv for folate receptor α.

Intracellular signaling domains

Upon binding to its target molecule, the intracellular domain of the CAR fusion polypeptide transmits a signal to the immune effector cell, thereby activating at least one of the normal effector functions of the immune effector cell, including lytic activity (e.g., cytokine secretion) or helper activity. Thus, the intracellular domain comprises the "intracellular signaling domain" of the T Cell Receptor (TCR).

In some aspects, an intact intracellular signaling domain may be employed. In other aspects, a truncated portion of the intracellular signaling domain may be used instead of the complete strand, so long as it transduces the effector function signal.

In some embodiments, the intracellular signaling domain may contain a signaling motif known as an immune receptor tyrosine based activation motif (ITAM). Examples of ITAM-containing cytoplasmic signaling sequences include those derived from TCR cd3ζ, fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, CD5, CD22, CD79a, CD79b, and CD66 d. In one example, the intracellular signaling domain is a CD3 zeta (CD 3 zeta) signaling domain.

In some embodiments, the intracellular region further comprises one or more costimulatory signaling domains that provide additional signals to the immune effector cell. These co-stimulatory signaling domains bind to the signaling domains, which can further improve the expansion, activation, memory, persistence, and tumor eradication efficiency of CAR-engineered immune cells (e.g., CAR T cells). In some cases, the costimulatory signaling region comprises 1, 2,3, or 4 cytoplasmic domains with one or more intracellular signaling and/or costimulatory molecules. The costimulatory signaling domain may be the intracellular/cytoplasmic domain of a costimulatory molecule, including but not limited to CD2, CD7, CD27, CD28, 4-1BB (CD 137), OX40 (CD 134), CD30, CD40, ICOS (CD 278), GITR (glucocorticoid-induced tumor necrosis factor receptor), LFA-1 (lymphocyte function-associated antigen-1), LIGHT, NKG2C, B7-H3. In one example, the costimulatory signaling domain is derived from the cytoplasmic domain of CD 28. In another example, the costimulatory signaling domain is derived from the cytoplasmic domain of 4-1BB (CD 137). In another example, the costimulatory signaling domain can be the intracellular domain of GITR as taught in U.S. patent No. 9,175,308; the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the intracellular region may comprise a functional signaling domain from a protein selected from the group consisting of: class I MHC molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation proteins (SLAM), such as CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, CD F-10, SLAMF6, SLAMF7, activated NK cell receptor, BTLA, toll ligand receptor 、OX40、CD2、CD7、CD27、CD28、CD30、CD40、CDS、ICAM-1、LFA-1(CD11a/CD18)、4-1BB(CD137)、B7-H3、CDS、ICAM-1、ICOS(CD278)、GITR、BAFFR、LIGHT、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、NKp44、NKp30、NKp46、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、IL-15Ra、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、NKG2D、NKG2C、NKD2C SLP76、TNFR2、TRANCE/RANKL、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、CD69、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、CD270(HVEM)、GADS、SLP-76、PAG/Cbp、CD19a、, ligands that specifically bind to CD83, DAP 10, TRIM, ZAP70, killer immunoglobulin receptor (Killer immunoglobulin receptor; KIR) such as KIR2DL1、KIR2DL2/L3、KIR2DL4、KIR2DL5A、KIR2DL5B、KIR2DS1、KIR2DS2、KIR2DS3、KIR2DS4、KIR2DS5、KIR3DL1/S1、KIR3DL2、KIR3DL3 and KIR2DP1; lectin-associated NK cell receptors such as Ly49, ly49A and Ly49C.

In some embodiments, the intracellular signaling domains of the present disclosure may contain signaling domains derived from JAK-STAT. In other embodiments, the intracellular signaling domains of the present disclosure can contain signaling domains derived from DAP-12 (death-related protein 12) (Topfer et al, immunol.,2015,194:3201-3212; and Wang et al, cancer immunol.,2015, 3:815-826). DAP-12 is a key signaling receptor in NK cells. The activation signal mediated by DAP-12 plays an important role in triggering NK cell cytotoxicity responses against certain tumor cells and virus-infected cells. The cytoplasmic domain of DAP12 contains an immunoreceptor tyrosine-based activation motif (ITAM). Thus, CARs containing DAP 12-derived signaling domains can be used to adoptively transfer NK cells.

Transmembrane domain

In some embodiments, the CAR can comprise a transmembrane domain. As used herein, the term "transmembrane domain (TM)" broadly refers to an amino acid sequence of about 15 residues in length across the plasma membrane. The transmembrane domain may comprise at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid residues and spans the plasma membrane. In some embodiments, the transmembrane domain may be derived from a natural or synthetic source. The transmembrane domain of the CAR may be derived from any natural membrane-bound protein or transmembrane protein. For example, the transmembrane region may be derived from (i.e., at least comprise the transmembrane region of) the following α, β or ζ chain: t cell receptor, CD3 epsilon, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD33, CD28, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, or CD154.

Or the transmembrane domains of the present disclosure may be synthetic. In some aspects, the synthetic sequences may comprise predominantly hydrophobic residues, such as leucine and valine.

In some embodiments, the transmembrane domain may be selected from the group consisting of: a CD8 a transmembrane domain, a CD4 transmembrane domain, a CD28 transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, and a human IgG4 Fc region.

In some embodiments, the CAR can include an optional hinge region (also referred to as a spacer). The hinge sequence is a flexible short amino acid sequence that facilitates movement of the target binding domain away from the effector cell surface to enable appropriate cell/cell contact, target binding and effector cell activation of the extracellular targeting domain. The hinge sequence may be located between the targeting moiety and the transmembrane domain. The hinge sequence may be any suitable sequence derived or obtained from any suitable molecule. The hinge sequence may be derived from all or a portion of the immunoglobulin (e.g., igG1, igG2, igG3, igG 4) hinge region, i.e., the extracellular region of a type 1 membrane protein (e.g., CD 8a CD4, CD28, and CD 7) that falls between the CH1 and CH2 domains of an immunoglobulin, e.g., an IgG4 Fc hinge, which may be a wild-type sequence or a derivative sequence. Some hinge regions include an immunoglobulin CH3 domain or both a CH3 domain and a CH2 domain. In certain embodiments, the hinge region may be modified relative to IgG1, igG2, igG3, or IgG4, which includes one or more amino acid residues, e.g., 1,2, 3, 4, or 5 residues, substituted with an amino acid residue different from the amino acid residue present in the unmodified hinge.

In some embodiments, the CAR may comprise one or more linkers between any of the domains of the CAR. The length of the linker may be 1-30 amino acids. In this regard, the length of the linker may be 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In other embodiments, the joint may be flexible.

In some embodiments, components including targeting moieties, transmembrane domains, and intracellular signaling domains may be constructed in a single fusion polypeptide. The fusion polypeptide may be the payload of an effector module of the present disclosure.

In some embodiments, the cargo or payload may be or encode a CD 19-specific CAR that targets a different B cell malignancy as well as a Her 2-specific CAR that targets sarcomas, neuroglioblastomas, and advanced Her 2-positive lung malignancy. Series CAR (TanCAR)

In some embodiments, the CAR can be a tandem chimeric antigen receptor (TanCAR) capable of targeting two, three, four, or more tumor-specific antigens. In some aspects, the CAR is bispecific TanCAR comprising two targeting domains that recognize two different TSAs on a tumor cell. Bispecific TanCAR can be further defined as comprising an extracellular region comprising a targeting domain (e.g., an antigen recognition domain) that is specific for a first tumor antigen and a targeting domain (e.g., an antigen recognition domain) that is specific for a second tumor antigen. In other aspects, the CAR is multispecific TanCAR comprising three or more targeting domains configured in a tandem arrangement. The space between the targeting domains in TanCAR can be about 5 to about 30 amino acids in length, for example 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 amino acids.

Splitting CARs

In some embodiments, the CAR component comprising the targeting moiety, transmembrane domain, and intracellular signaling domain can be split into two or more moieties such that it relies on multiple inputs that facilitate assembly of the complete functional receptor. As one non-limiting example, a split CAR consists of two parts assembled in a small molecule dependent manner; one portion of the receptor is characterized by an extracellular antigen binding domain (e.g., scFv), and the other portion has an intracellular signaling domain, such as the cd3ζ intracellular domain.

In other aspects, the split portion of the CAR system can be further modified to increase the signal. As one non-limiting example, the second portion of the cytoplasmic fragment can be anchored to the plasma membrane by incorporating a transmembrane domain (e.g., a CD8 a transmembrane domain) into the construct. Additional ectodomains may also be added to the second portion of the CAR system, e.g., ectodomains that mediate homodimerization. These modifications may increase receptor export activity, i.e., T cell activation.

In some embodiments, the two portions of the split CAR system contain heterodimerization domains that conditionally interact upon binding of the heterodimerization small molecule. Thus, the receptor components assemble in the presence of small molecules, forming an intact system that can then be activated by antigen binding. Any known heterodimerization component can be incorporated into the split CAR system. Other small molecule dependent heterodimerization domains may also be used, including, but not limited to gibberellin-induced dimerization systems (GID 1-GAI), qu Meipu forest (trimethoprim) -SLF-induced ecDHFR and FKBP dimerization, and ABA (abscisic acid) -induced dimerization of PP2C and PYL domains. Dual modulation of inducible assembly (e.g., ligand-dependent dimerization) and degradation (e.g., destabilizing a domain-induced CAR degradation) using split CAR systems can provide more flexibility to control the activity of CAR-modified T cells.

Switchable CAR

In some implementations, the CAR can be a switchable CAR that is temporarily turned on as a controllable CAR in response to a stimulus (e.g., a small molecule). In this CAR design, the system is directly integrated into the hinge domain that separates the scFv domain from the cell membrane domain in the CAR. Such systems make it possible to split or combine different key functions of the CAR, such as activation and co-stimulation within different chains of the receptor complex, thereby mimicking the complexity of the TCR native architecture. This integrated system can switch scFv and antigen interactions between on/off states controlled by the absence/presence of stimulus.

Reversible CAR

In some embodiments, the CAR may be a reversible CAR system. In this CAR architecture, the LID (ligand-induced degradation) domain is incorporated into the CAR system. CARs can be temporarily down-regulated by the addition of ligands for the LID domain.

Inhibitory CAR (iCAR)

In some embodiments, the CAR may be an inhibitory CAR. Inhibitory CARs (icars) refer to bispecific CAR designs in which negative signals are used to enhance tumor specificity and limit normal tissue toxicity. This design incorporates a second CAR with a surface antigen recognition domain and an inhibitory signal domain to limit T cell reactivity even though the activating receptor is engaged at the same time. This antigen recognition domain is directed against normal tissue-specific antigens such that T cells can be activated in the presence of a first protein of interest, but inhibit T cell activation if a second protein that binds to iCAR is present.

As one non-limiting example, CTLA4 and PD1 inhibitory domain-based icars against prostate specific membrane antigen (PMSA) exhibit the ability to selectively limit cytokine secretion, cytotoxicity, and proliferation induced by T cell activation.

Chimeric switch receptors

In some embodiments, the cargo or payload may be or encode a chimeric switch receptor that can switch the negative signal to a positive signal. As used herein, the term "chimeric switch receptor" refers to a fusion protein comprising a first extracellular domain and a second transmembrane and intracellular domain, wherein the first domain comprises a negative signal region and the second domain comprises a positive intracellular signal transduction region. In some aspects, the fusion protein is a chimeric switch receptor comprising the extracellular domain of an inhibitory receptor on a T cell fused to the transmembrane and cytoplasmic domains of a co-stimulatory receptor. The chimeric switch receptor can convert a T cell inhibition signal into a T cell stimulation signal.

As one non-limiting example, a chimeric switch receptor may comprise the extracellular domain of PD-1 fused to the transmembrane and cytoplasmic domain of CD 28. In some aspects, other extracellular domains of inhibitory receptors, such as CTLA-4, LAG-3, TIM-3, KIR, and BTLA, may also be fused to transmembrane and cytoplasmic domains derived from co-stimulatory receptors (e.g., CD28, 4-1BB, CD27, OX40, CD40, GTIR, and ICOS).

In some embodiments, the chimeric switch receptor can comprise a recombinant receptor comprising: an extracellular cytokine binding domain of an inhibitory cytokine receptor (e.g., IL-13 receptor alpha (IL-13 Ralpha 1), IL-10R, and IL-4 Ralpha) fused to an intracellular signaling domain of a stimulatory cytokine receptor (e.g., IL-2R (IL-2 Ralpha, IL-2 Rbeta, and IL-7 Rgamma). One example of such a chimeric cytokine receptor is a recombinant receptor that contains a cytokine binding extracellular domain of IL-4Rα linked to an intracellular signaling domain of IL-7Rα.

In some embodiments, the chimeric switch receptor may be a chimeric tgfβ receptor. Chimeric tgfβ receptors may comprise: an extracellular domain derived from a tgfβ receptor, such as tgfβ receptor 1, tgfβ receptor 2, tgfβ receptor 3, or any other tgfβ receptor or variant thereof; and a non-tgfβ receptor intracellular domain. The non-tgfβ receptor intracellular domain may be an intracellular domain derived from: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CD28, 4-1BB (CD 137), OX40 (CD 134), CD3zeta, CD40, CD27 or a combination thereof.

Activating conditional CAR

In some embodiments, the cargo or payload may be or encode an activating conditional chimeric antigen receptor that is expressed only in activated immune cells. Expression of a CAR may be coupled to an activation-conditional control region that refers to one or more nucleic acid sequences that induce transcription and/or expression of the sequence (e.g., CAR) under its control. Such activation-conditional control regions may be promoters of genes that are upregulated during immune effector cell activation, such as the IL2 promoter or NFAT binding site.

CARs targeting tumor cells with specific proteoglycan markers

In some embodiments, the cargo or payload can be or encode a CAR that targets a particular type of cancer cell. Human cancer cells and cancer metastasis can express unique and otherwise abnormal proteoglycans such as polysaccharide chains (e.g., chondroitin sulfate (chondroitin sulfate; CS), dermatan sulfate (dermatan sulfate; DS or CSB), heparan sulfate (heparan sulfate; HS), and heparin). Thus, the CAR may be fused to a binding moiety that recognizes a cancer-associated proteoglycan. In one example, the CAR can be fused to a VAR2CSA polypeptide (VAR 2-CAR) that binds with high affinity to a particular type of Chondroitin Sulfate A (CSA) linked to proteoglycans. The extracellular ScFv portion of the CAR can be substituted with a VAR2CSA variant comprising at least a minimal CSA binding domain, thereby producing a CAR specific for Chondroitin Sulfate A (CSA) modification. Alternatively, the CAR may be fused to a split protein binding system to produce a Spy-CAR, wherein the scFv portion of the CAR is substituted with one portion of the split protein binding system (e.g., spyTag and Spy capturer) and the cancer recognition molecule (e.g., scFv and or VAR 2-CSA) is linked to the CAR via the split protein binding system.

Nucleic acid

The initial and reference constructs of the present disclosure may comprise a payload region (which may also be referred to as a cargo region), which is a nucleic acid. The term "nucleic acid" in its broadest sense includes any compound and/or substance comprising a polymer of nucleotides that may be referred to as a polynucleotide. Exemplary nucleic acids or polynucleotides include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose Nucleic Acid (TNA), glycol Nucleic Acid (GNA), peptide Nucleic Acid (PNA), locked Nucleic Acid (LNA), or hybrids thereof.

In some embodiments, the payload region comprises a nucleic acid sequence encoding more than one cargo or payload.

In some embodiments, the payload region may be or encode a coding nucleic acid sequence.

In some embodiments, the payload region may be or encode a non-coding nucleic acid sequence.

In some embodiments, the payload region may be or encode both coding and non-coding nucleic acid sequences.

DNA

Deoxyribonucleic acid (DNA) is a molecule that carries genetic information for all living things and consists of two strands that are wrapped around each other to form a shape called a duplex. Each chain has a backbone composed of alternating sugar (deoxyribose) and phosphate groups. Each sugar is linked to one of four bases: adenine (A), cytosine (C), guanine (G) and thymine (T). The two chains are bound together by a bond between adenine and thymine or between cytosine and guanine. The sequence of bases along the backbone serves as instructions for assembling the protein and RNA molecules.

In some embodiments, the payload region may be or encode encoding DNA.

In some embodiments, the payload region may be or encode non-coding DNA.

In some embodiments, the payload region may be or encode both coding and non-coding DNA.

In some embodiments, the DNA may be modified. Types of modifications include, but are not limited to, methylation, acetylation, phosphorylation, ubiquitination, and threification.

Carrier body

In some embodiments, the initial construct and/or reference construct described herein may be, or be encoded by, a vector (e.g., a plasmid or viral vector). In some embodiments, the initial construct and/or the reference construct is or is encoded by a viral vector. The viral vector may be, but is not limited to, a herpes virus (HSV) vector, a retroviral vector, an adenoviral vector, an adeno-associated viral (AAV) vector, a lentiviral vector, and the like. In some embodiments, the viral vector is an AAV vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is an adenovirus vector.

Adeno-associated virus (AAV) vectors

The virus of the parvoviridae (Parvoviridae family) is a non-enveloped icosahedral capsid virus characterized by a single-stranded DNA genome. Parvoviridae consist of two subfamilies: a subfamily parvovirus (Parvovirinae) that infects vertebrates, and a subfamily megaviridae (Densovirinae) that infects invertebrates. The virus family is suitable for use as a biological tool due to its relatively simple structure and ease of manipulation using standard molecular biology techniques. The genome of the virus may be modified to contain minimal components for assembling the functional recombinant virus or viral particle, which are loaded with the desired payload or engineered to express or deliver the desired payload, which may be delivered to the target cell, tissue, organ or organism.

Parvoviridae comprise a genus of dependoviruses (Dependovirus genus) including adeno-associated viruses (AAV) capable of replication in vertebrate hosts including, but not limited to, human, primate, bovine, canine, equine and ovine species.

AAV vector genomes are linear, single stranded DNA (ssDNA) molecules of about 5,000 nucleotides (nt) in length. The AAV vector genome may comprise a payload region and at least one Inverted Terminal Repeat (ITR) or ITR region. ITRs are traditionally flanked by nucleotide sequences encoding non-structural proteins (encoded by the Rep gene) and structural proteins (encoded by the capsid gene or Cap gene). While not wanting to be bound by theory, AAV vector genomes typically comprise two ITR sequences. The AAV vector genome comprises a characteristic T-shaped hairpin structure defined by the self-complementary ends 145 nucleotides of the 5 'and 3' ends of ssDNA, which forms an energetically stable double stranded region. The double-stranded hairpin structure serves a variety of functions including, but not limited to, serving as an origin of DNA replication by serving as a primer for the endogenous DNA polymerase complex of the host virus replicating cell.

In addition to the encoded heterologous payload, the AAV vector genome may comprise all or part of any naturally occurring and/or recombinant AAV serotype nucleotide sequence or variant. AAV variants can have significant homologous sequences at the nucleic acid (genome or capsid) and amino acid level (capsid) to produce constructs that are substantially equivalent in entity and function, which are replicated by and assembled by similar mechanisms. Chiorini et al, J.Vir.71:6823-33 (1997); srivastava et al, J.Vir.45:555-64 (1983); chiorini et al, J.Vir.73:1309-1319 (1999); rutledge et al, J.Vir.72:309-319 (1998); and Wu et al, J.Vir.74:8635-47 (2000), the respective contents of which are incorporated herein by reference in their entirety.

In some embodiments, the AAV vector genome comprises at least one control element that provides replication, transcription, and translation of the coding sequences encoded therein. Not all control elements need be present at all times, so long as the coding sequence is capable of replication, transcription and/or translation in an appropriate host cell. Non-limiting examples of expression control elements include sequences for transcription initiation and/or termination, promoter and/or enhancer sequences, effective RNA processing signals (e.g., splicing and polyadenylation signals), sequences that stabilize cytoplasmic mRNA, sequences that enhance translational efficiency (e.g., kozak consensus sequences), sequences that enhance protein stability, and/or sequences that enhance protein processing and/or secretion.

The AAV vector genomes of the present disclosure may be recombinantly produced and may be based on adeno-associated virus (AAV) parent or reference sequences. As used herein, a "vector genome" is any molecule or portion of a vector that transmits, transduces, or otherwise serves as a heterologous molecule for a nucleic acid, such as described herein.

In addition to single stranded AAV vector genomes (e.g., ssAAV), the present disclosure also provides self-complementary AAV (scAAV) vector genomes. The scAAV vector genome contains DNA strands that anneal together to form double stranded DNA. By skipping the second strand synthesis, scAAV achieves rapid expression in the cell.

In some embodiments, the AAV vector genome is scAAV.

In some embodiments, the AAV vector genome is ssAAV.

In some embodiments, the AAV vector genome can be part of an AAV particle, wherein the serotype of the capsid can be, but is not limited to, AAV1、AAV2、AAV2G9、AAV3、AAV3a、AAV3b、AAV3-3、AAV4、AAV4-4、AAV5、AAV6、AAV6.1、AAV6.2、AAV6.1.2、AAV7、AAV7.2、AAV8、AAV9、AAV9.11、AAV9.13、AAV9.16、AAV9.24、AAV9.45、AAV9.47、AAV9.61、AAV9.68、AAV9.84、AAV9.9、AAV10、AAV11、AAV12、AAV16.3、AAV24.1、AAV27.3、AAV42.12、AAV42-1b、AAV42-2、AAV42-3a、AAV42-3b、AAV42-4、AAV42-5a、AAV42-5b、AAV42-6b、AAV42-8、AAV42-10、AAV42-11、AAV42-12、AAV42-13、AAV42-15、AAV42-aa、AAV43-1、AAV43-12、AAV43-20、AAV43-21、AAV43-23、AAV43-25、AAV43-5、AAV44.1、AAV44.2、AAV44.5、AAV223.1、AAV223.2、AAV223.4、AAV223.5、AAV223.6、AAV223.7、AAV1-7/rh.48、AAV1-8/rh.49、AAV2-15/rh.62、AAV2-3/rh.61、AAV2-4/rh.50、AAV2-5/rh.51、AAV3.1/hu.6、AAV3.1/hu.9、AAV3-9/rh.52、AAV3-11/rh.53、AAV4-8/r11.64、AAV4-9/rh.54、AAV4-19/rh.55、AAV5-3/rh.57、AAV5-22/rh.58、AAV7.3/hu.7、AAV16.8/hu.10、AAV16.12/hu.11、AAV29.3/bb.1、AAV29.5/bb.2、AAV106.1/hu.37、AAV114.3/hu.40、AAV127.2/hu.41、AAV127.5/hu.42、AAV128.3/hu.44、AAV130.4/hu.48、AAV145.1/hu.53、AAV145.5/hu.54、AAV145.6/hu.55、AAV161.10/hu.60、AAV161.6/hu.61、AAV33.12/hu.17、AAV33.4/hu.15、AAV33.8/hu.16、AAV52/hu.19、AAV52.1/hu.20、AAV58.2/hu.25、AAVA3.3、AAVA3.4、AAVA3.5、AAVA3.7、AAVC1、AAVC2、AAVC5、AAV-DJ、AAV-DJ8、AAVF3、AAVF5、AAVH2、AAVrh.72、AAVhu.8、AAVrh.68、AAVrh.70、AAVpi.1、AAVpi.3、AAVpi.2、AAVrh.60、AAVrh.44、AAVrh.65、AAVrh.55、AAVrh.47、AAVrh.69、AAVrh.45、AAVrh.59、AAVhu.12、AAVH6、AAVLK03、AAVH-1/hu.1、AAVH-5/hu.3、AAVLG-10/rh.40、AAVLG-4/rh.38、AAVLG-9/hu.39、AAVN721-8/rh.43、AAVCh.5、AAVCh.5R1、AAVcy.2、AAVcy.3、AAVcy.4、AAVcy.5、AAVCy.5R1、AAVCy.5R2、AAVCy.5R3、AAVCy.5R4、AAVcy.6、AAVhu.1、AAVhu.2、AAVhu.3、AAVhu.4、AAVhu.5、AAVhu.6、AAVhu.7、AAVhu.9、AAVhu.10、AAVhu.11、AAVhu.13、AAVhu.15、AAVhu.16、AAVhu.17、AAVhu.18、AAVhu.20、AAVhu.21、AAVhu.22、AAVhu.23.2、AAVhu.24、AAVhu.25、AAVhu.27、AAVhu.28、AAVhu.29、AAVhu.29R、AAVhu.31、AAVhu.32、AAVhu.34、AAVhu.35、AAVhu.37、AAVhu.39、AAVhu.40、AAVhu.41、AAVhu.42、AAVhu.43、AAVhu.44、AAVhu.44R1、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.46、AAVhu.47、AAVhu.48、AAVhu.48R1、AAVhu.48R2、AAVhu.48R3、AAVhu.49、AAVhu.51、AAVhu.52、AAVhu.54、AAVhu.55、AAVhu.56、AAVhu.57、AAVhu.58、AAVhu.60、AAVhu.61、AAVhu.63、AAVhu.64、AAVhu.66、AAVhu.67、AAVhu.14/9、AAVhu.t 19、AAVrh.2、AAVrh.2R、AAVrh.8、AAVrh.8R、AAVrh.10、AAVrh.12、AAVrh.13、AAVrh.13R、AAVrh.14、AAVrh.17、AAVrh.18、AAVrh.19、AAVrh.20、AAVrh.21、AAVrh.22、AAVrh.23、AAVrh.24、AAVrh.25、AAVrh.31、AAVrh.32、AAVrh.33、AAVrh.34、AAVrh.35、AAVrh.36、AAVrh.37、AAVrh.37R2、AAVrh.38、AAVrh.39、AAVrh.40、AAVrh.46、AAVrh.48、AAVrh.48.1、AAVrh.48.1.2、AAVrh.48.2、AAVrh.49、AAVrh.51、AAVrh.52、AAVrh.53、AAVrh.54、AAVrh.56、AAVrh.57、AAVrh.58、AAVrh.61、AAVrh.64、AAVrh.64R1、AAVrh.64R2、AAVrh.67、AAVrh.73、AAVrh.74、AAVrh8R、AAVrh8RA586R mutant, AAVrh8R R A mutant, AAAV, BAAV, goat AAV, bovine AAV、AAVhE1.1、AAVhEr1.5、AAVhER1.14、AAVhEr1.8、AAVhEr1.16、AAVhEr1.18、AAVhEr1.35、AAVhEr1.7、AAVhEr1.36、AAVhEr2.29、AAVhEr2.4、AAVhEr2.16、AAVhEr2.30、AAVhEr2.31、AAVhEr2.36、AAVhER1.23、AAVhEr3.1、AAV2.5T、AAV-PAEC、AAV-LK01、AAV-LK02、AAV-LK03、AAV-LK04、AAV-LK05、AAV-LK06、AAV-LK07、AAV-LK08、AAV-LK09、AAV-LK10、AAV-LK11、AAV-LK12、AAV-LK13、AAV-LK14、AAV-LK15、AAV-LK16、AAV-LK17、AAV-LK18、AAV-LK19、AAV-PAEC2、AAV-PAEC4、AAV-PAEC6、AAV-PAEC7、AAV-PAEC8、AAV-PAEC11、AAV-PAEC12、AAV-2-pre-miRNA-101、AAV-8h、AAV-8b、AAV-h、AAV-b、AAV SM 10-2、AAV shuffling (Shuffle) 100-1, AAV shuffling 100-3, AAV shuffling 100-7, AAV shuffling 10-2, AAV shuffling 10-6, AAV shuffling 10-8, AAV shuffling 100-2、AAV SM 10-1、AAV SM 10-8、AAV SM 100-3、AAV SM 100-10、BNP61 AAV、BNP62 AAV、BNP63AAV、AAVrh.50、AAVrh.43、AAVrh.62、AAVrh.48、AAVhu.19、AAVhu.11、AAVhu.53、AAV4-8/rh.64、AAVLG-9/hu.39、AAV54.5/hu.23、AAV54.2/hu.22、AAV54.7/hu.24、AAV54.1/hu.21、AAV54.4R/hu.27、AAV46.2/hu.28、AAV46.6/hu.29、AAV128.1/hu.43、 true AAV (ttaV), UPENN AAV 10, japanese AAV 10 serotype 、AAV CBr-7.1、AAV CBr-7.10、AAV CBr-7.2、AAV CBr-7.3、AAV CBr-7.4、AAV CBr-7.5、AAV CBr-7.7、AAV CBr-7.8、AAV CBr-B7.3、AAV CBr-B7.4、AAV CBr-E1、AAV CBr-E2、AAV CBr-E3、AAV CBr-E4、AAV CBr-E5、AAV CBr-e5、AAV CBr-E6、AAV CBr-E7、AAV CBr-E8、AAV CHt-1、AAV CHt-2、AAV CHt-3、AAV CHt-6.1、AAV CHt-6.10、AAV CHt-6.5、AAV CHt-6.6、AAV CHt-6.7、AAV CHt-6.8、AAV CHt-P1、AAV CHt-P2、AAV CHt-P5、AAV CHt-P6、AAV CHt-P8、AAV CHt-P9、AAV CKd-1、AAV CKd-10、AAV CKd-2、AAV CKd-3、AAV CKd-4、AAV CKd-6、AAV CKd-7、AAV CKd-8、AAV CKd-B1、AAV CKd-B2、AAV CKd-B3、AAV CKd-B4、AAV CKd-B5、AAV CKd-B6、AAV CKd-B7、AAV CKd-B8、AAV CKd-H1、AAV CKd-H2、AAV CKd-H3、AAV CKd-H4、AAV CKd-H5、AAV CKd-H6、AAV CKd-N3、AAV CKd-N4、AAV CKd-N9、AAV CLg-F1、AAV CLg-F2、AAV CLg-F3、AAV CLg-F4、AAV CLg-F5、AAV CLg-F6、AAV CLg-F7、AAV CLg-F8、AAV CLv-1、AAV CLv1-1、AAV Clv1-10、AAV CLv1-2、AAV CLv-12、AAV CLv1-3、AAV CLv-13、AAV CLv1-4、AAV Clv1-7、AAV Clv1-8、AAV Clv1-9、AAV CLv-2、AAV CLv-3、AAV CLv-4、AAV CLv-6、AAV CLv-8、AAV CLv-D1、AAV CLv-D2、AAV CLv-D3、AAV CLv-D4、AAV CLv-D5、AAV CLv-D6、AAV CLv-D7、AAV CLv-D8、AAV CLv-E1、AAV CLv-K1、AAV CLv-K3、AAV CLv-K6、AAV CLv-L4、AAV CLv-L5、AAV CLv-L6、AAV CLv-M1、AAV CLv-M11、AAV CLv-M2、AAV CLv-M5、AAV CLv-M6、AAV CLv-M7、AAV CLv-M8、AAV CLv-M9、AAV CLv-R1、AAV CLv-R2、AAV CLv-R3、AAV CLv-R4、AAV CLv-R5、AAV CLv-R6、AAV CLv-R7、AAV CLv-R8、AAV CLv-R9、AAV CSp-1、AAV CSp-10、AAV CSp-11、AAV CSp-2、AAV CSp-3、AAV CSp-4、AAV CSp-6、AAV CSp-7、AAV CSp-8、AAV CSp-8.10、AAV CSp-8.2、AAV CSp-8.4、AAV CSp-8.5、AAV CSp-8.6、AAV CSp-8.7、AAV CSp-8.8、AAV CSp-8.9、AAV CSp-9、AAV.hu.48R3、AAV.VR-355、AAV3B、AAV4、AAV5、AAVF1/HSC1、AAVF11/HSC11、AAVF12/HSC12、AAVF13/HSC13、AAVF14/HSC14、AAVF15/HSC15、AAVF16/HSC16、AAVF17/HSC17、AAVF2/HSC2、AAVF3/HSC3、AAVF4/HSC4、AAVF5/HSC5、AAVF6/HSC6、AAVF7/HSC7、AAVF8/HSC8、AAVF9/HSC9、PHP.B、PHP.A、G2B-26、G2B-13、TH1.1-32, and/or TH1.1-35, and variants thereof.

Reverse terminal repeat (ITR)

In some embodiments, the AAV vector genome may comprise at least one ITR region and a payload region. In some embodiments, the vector genome has two ITRs. The two ITRs flank the payload region at the 5 'and 3' ends. The ITR serves as an origin of replication, comprising a recognition site for replication. The ITRs contain sequence regions that can be complementarily and symmetrically arranged. ITRs incorporated into the vector genome of the present disclosure may be composed of naturally occurring polynucleotide sequences or recombinantly derived polynucleotide sequences.

The ITR can be derived from the same serotype as the capsid or a derivative thereof. The ITRs can have a different serotype than the capsid. In some embodiments, the AAV particle has more than one ITR. In one non-limiting example, an AAV particle has a vector genome comprising two ITRs. In some embodiments, the ITRs have the same serotypes as each other. In another embodiment, the ITRs have different serotypes. Non-limiting examples include zero, one, or two ITRs with the same serotype as the capsid. In some embodiments, both ITRs of the vector genome of the AAV particle are AAV2 ITRs.

Independently, each ITR can be about 100 to about 150 nucleotides in length. ITRs may be about 100-105 nucleotides in length, 106-110 nucleotides in length, 111-115 nucleotides in length, 116-120 nucleotides in length, 121-125 nucleotides in length, 126-130 nucleotides in length, 131-135 nucleotides in length, 136-140 nucleotides in length, 141-145 nucleotides in length, or 146-150 nucleotides in length. In some embodiments, the ITRs are 140-142 nucleotides in length. Non-limiting examples of ITR lengths are those of 102, 140, 141, 142, 145 nucleotides in length, and at least 95% identity thereto.

Promoters

In some embodiments, the payload region of the vector genome comprises at least one element that enhances the specificity and expression of the transgene target (see, e.g., powell et al ,Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy,2015;, the contents of which are incorporated herein by reference in their entirety). Non-limiting examples of elements that enhance transgene target specificity and expression include promoters, endogenous mirnas, post transcriptional regulatory elements (PRE), polyadenylation (PolyA) signal sequences and upstream enhancers (USE), CMV enhancers, and introns.

In some embodiments, a promoter is considered effective when it drives expression of a polypeptide encoded in the payload region of the vector genome of an AAV particle.

In some embodiments, a promoter is considered effective when it drives expression in a targeted cell.

In some embodiments, the promoter drives expression of the payload in the targeted tissue for a period of time. Expression driven by the promoter may last 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, a period of 20 days, 3 weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more than 10 years. Expression may last for 1-5 hours, 1-12 hours, 1-2 days, 1-5 days, 1-2 weeks, 1-3 weeks, 1-4 weeks, 1-2 months, 1-4 months, 1-6 months, 2-6 months, 3-9 months, 4-8 months, 6-12 months, 1-2 years, 1-5 years, 2-5 years, 3-6 years, 3-8 years, 4-8 years, or 5-10 years.

In some embodiments, the expression of the promoter-driven payload is for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 21 years, 22 years, 23 years, 24 years, 25 years, 26 years, 27 years, 28 years, 29 years, 30 years, 31 years, 32 years, 33 years, 34 years, 35 years, 36 years, 37 years, 38 years, 39 years, 40 years, 41 years, 42 years, 43 years, 44 years, 45 years, 46 years, 47 years, 48 years, 49 years, 50 years, 55 years, 60 years, 65 years, or more than 65 years.

Promoters may be naturally occurring or non-naturally occurring. Non-limiting examples of promoters include viral promoters, plant promoters, and mammalian promoters. In some embodiments, the promoter may be a human promoter. In some embodiments, the promoter may be truncated.

Promoters that drive or promote expression in most tissues include, but are not limited to, human elongation factor 1 alpha-subunit (EF 1 alpha), cytomegalovirus (CMV) immediate early enhancer and/or promoter, chicken beta-actin (CBA) and its derivatives CAG, beta Glucuronidase (GUSB) or ubiquitin C (UBC). Tissue-specific expression elements may be used to limit expression of certain cell types, such as, but not limited to, muscle-specific promoters, B-cell promoters, monocyte promoters, leukocyte promoters, macrophage promoters, pancreatic acinar cell promoters, endothelial cell promoters, lung tissue promoters, astrocyte promoters, or nervous system promoters that may be used to limit expression of neurons, astrocytes, or oligodendrocytes.

Non-limiting examples of muscle-specific promoters include the mammalian Muscle Creatine Kinase (MCK) promoter, the mammalian myotonin (DES) promoter, the mammalian troponin I (TNNI 2) promoter, and the mammalian skeletal α -actin (ASKA) promoter (see, e.g., U.S. patent publication US20110212529, the contents of which are incorporated herein by reference in their entirety).

Non-limiting examples of tissue-specific expression elements for neurons include Neuronal Specific Enolase (NSE), platelet Derived Growth Factor (PDGF), platelet derived growth factor B chain (PDGF- β), synaptotagin (Syn), methyl-CpG binding protein 2 (MeCP 2), ca ²⁺/calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR 2), neurofilament light chain (NFL) or heavy chain (NFH), β -globin minigene nβ2, pro-enkephalin (PPE), enkephalin (Enk), and excitatory amino acid transporter 2 (EAAT 2) promoters. Non-limiting examples of tissue-specific expression elements of astrocytes include glial fibrillary acidic protein (glial fibrillary acidic protein; GFAP) and the EAAT2 promoter. Non-limiting examples of tissue-specific expression elements for oligodendrocytes include the Myelin Basic Protein (MBP) promoter.

In some embodiments, the promoter may be less than 1kb. The length of the promoter may be 200、210、220、230、240、250、260、270、280、290、300、310、320、330、340、350、360、370、380、390、400、410、420、430、440、450、460、470、480、490、500、510、520、530、540、550、560、570、580、590、600、610、620、630、640、650、660、670、680、690、700、710、720、730、740、750、760、770、780、790、800 or more than 800 nucleotides. The length of the promoter may be between 200-300、200-400、200-500、200-600、200-700、200-800、300-400、300-500、300-600、300-700、300-800、400-500、400-600、400-700、400-800、500-600、500-700、500-800、600-700、600-800 or 700-800.

In some embodiments, the promoters may be a combination of two or more components of the same or different starting or parent promoters (e.g., without limitation, CMV and CBA). The length of each component may be 200、210、220、230、240、250、260、270、280、290、300、310、320、330、340、350、360、370、380、381、382、383、384、385、386、387、388、389、390、400、410、420、430、440、450、460、470、480、490、500、510、520、530、540、550、560、570、580、590、600、610、620、630、640、650、660、670、680、690、700、710、720、730、740、750、760、770、780、790、800 or more than 800. The length of each component may be between 200-300、200-400、200-500、200-600、200-700、200-800、300-400、300-500、300-600、300-700、300-800、400-500、400-600、400-700、400-800、500-600、500-700、500-800、600-700、600-800 or 700-800. In some embodiments, the promoter is a combination of 382 nucleotide CMV-enhancer sequences and 260 nucleotide CBA-promoter sequences.

In some embodiments, the vector genome comprises a ubiquitous promoter. Non-limiting examples of ubiquitous promoters include CMV, CBA (including derivatives CAG, CBh, etc.), EF-1. Alpha., PGK, UBC, GUSB (hGBp), and UCOE (promoters of HNRPA2B1-CBX 3).

In some embodiments, the promoter is not cell specific.

In some embodiments, the vector genome comprises an engineered promoter.

In some embodiments, the vector genome comprises a promoter from a naturally expressed protein.

Untranslated region (UTR)

By definition, the wild-type untranslated region (UTR) of a gene is transcribed but untranslated. Typically, the 5'utr starts at the transcription start site and ends at the start codon, and the 3' utr starts immediately after the stop codon and continues until the transcription termination signal.

Features that are normally present in fully expressed genes of a particular target organ can be engineered into the UTR to enhance stability and protein production. As one non-limiting example, 5' utrs from mRNA (e.g., albumin, serum amyloid a, apolipoprotein a/B/E, transferrin, alpha fetoprotein, erythropoietin, or factor VIII) that are normally expressed in the liver may be used in the vector genome of AAV particles of the present disclosure to enhance expression in a hepatocyte line or liver.

While not wanting to be bound by theory, the wild-type 5' untranslated region (UTR) includes features that play a role in translation initiation. Kozak sequences are generally known to be involved in the process of ribosomes for initiating translation of multiple genes and are generally included in the 5' utr. The Kozak sequence has a common CCR (a/G) CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (ATG), followed by another 'G'.

In some embodiments, the 5' utr in the vector genome comprises a Kozak sequence.

In some embodiments, the 5' utr in the vector genome does not include a Kozak sequence.

While not wanting to be bound by theory, it is known that wild-type 3' utrs have embedded therein an extension of adenosine and uridine. These AU-rich markers are particularly prevalent in genes with high turnover rates. Based on their sequence and functional characteristics, AU-rich elements (ARE) can be divided into three classes (Chen et al, 1995, the contents of which ARE incorporated herein by reference in their entirety): class I ARE, such as, but not limited to, c-Myc and MyoD, contain several discrete copies of the AUUUA motif within the U-rich region. Class II AREs, such as but not limited to GM-CSF and TNF-a, have two or more overlapping UUAUUUA (U/A) (U/A) nonamers. Class III AREs, such as but not limited to c-Jun and myogenin, ARE less well defined. These U-rich regions do not contain the AUUUA motif. Most proteins bound to ARE known to destabilize the messenger, whereas ELAV family members (most notably HuR) have been reported to increase mRNA stability. HuR binds to ARE of all three classes. Engineering a HuR specific binding site into the 3' utr of a nucleic acid molecule will cause HuR binding and thus in vivo information stabilization.

The introduction, removal or modification of 3' UTR AU-rich elements (AREs) can be used to modulate the stability of polynucleotides. When engineering a particular polynucleotide (e.g., the payload region of a vector genome), one or more ARE copies may be introduced to reduce polynucleotide stability and thereby reduce translation and yield of the resulting protein. Also, ARE can be identified and removed or mutated to increase intracellular stability and thus increase translation and yield of the resulting protein.

In some embodiments, the 3' utr of the vector genome may include an oligo (dT) sequence for templated addition of the poly a tail.

In some embodiments, the vector genome may comprise at least one miRNA seed, binding site, or complete sequence. Micrornas (or mirnas or mirs) are 19-25 nucleotide non-coding RNAs that bind to sites of nucleic acid targets and down-regulate gene expression by reducing nucleic acid molecule stability or by inhibiting translation. The microRNA sequence comprises a "seed" region, i.e., a sequence in the region of positions 2-8 of the mature microRNA that has perfect Watson-Crick complementarity (Watson-Crick complementarity) with the miRNA target sequence of the nucleic acid.

In some embodiments, the vector genome may be engineered to include, alter or remove at least one miRNA binding site, sequence or seed region.

Any UTR from any gene known in the art may be incorporated into the vector genome of an AAV particle. These UTRs, or portions thereof, may be placed in the same orientation as in the gene from which they are selected, or whose orientation or position may be varied. In some embodiments, the UTRs in the vector genome for the AAV particles may be inverted, shortened, elongated, made with one or more other 5 'UTRs or 3' UTRs known in the art. As used herein, the term "change (altered)" when related to a UTR means that the UTR has been changed in some way relative to a reference sequence. For example, the 3 'or 5' UTR may be altered relative to a wild-type or native UTR by a change in orientation or position as taught above, or may be altered by including additional nucleotides, nucleotide deletions, nucleotide exchanges, or translocation.

In some embodiments, the vector genome of the AAV particle comprises at least one artificial UTR that is not a variant of the wild-type UTR.

In some embodiments, the vector genome of the AAV particle comprises a UTR selected from a family of transcripts in which the proteins share common functions, structures, features, or characteristics.

Polyadenylation sequences

In some embodiments, the vector genome comprises at least one polyadenylation sequence between the 3' end of the payload coding sequence and the 5' end of the 3' itr.

In some embodiments, the length of the polyadenylation (poly a) sequence may be in the range of from no to about 500 nucleotides. The polyadenylation sequence may be, but is not limited to, 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499 and 500 nucleotides in length.

In some embodiments, the polyadenylation sequence is 50-100 nucleotides in length. In some embodiments, the polyadenylation sequence is 50-150 nucleotides in length. In some embodiments, the polyadenylation sequence is 50-160 nucleotides in length. In some embodiments, the polyadenylation sequence is 50-200 nucleotides in length. In some embodiments, the polyadenylation sequence is 60-100 nucleotides in length. In some embodiments, the polyadenylation sequence is 60-150 nucleotides in length. In some embodiments, the polyadenylation sequence is 60-160 nucleotides in length. In some embodiments, the polyadenylation sequence is 60-200 nucleotides in length. In some embodiments, the polyadenylation sequence is 70-100 nucleotides in length. In some embodiments, the polyadenylation sequence is 70-150 nucleotides in length. In some embodiments, the polyadenylation sequence is 70-160 nucleotides in length. In some embodiments, the polyadenylation sequence is 70-200 nucleotides in length. In some embodiments, the polyadenylation sequence is 80 to 100 nucleotides in length. In some embodiments, the polyadenylation sequence is 80 to 150 nucleotides in length. In some embodiments, the polyadenylation sequence is 80 to 160 nucleotides in length. In some embodiments, the polyadenylation sequence is 80-200 nucleotides in length. In some embodiments, the polyadenylation sequence is 90-100 nucleotides in length. In some embodiments, the polyadenylation sequence is 90-150 nucleotides in length. In some embodiments, the polyadenylation sequence is 90-160 nucleotides in length. In some embodiments, the polyadenylation sequence is 90-200 nucleotides in length.

Joint

The vector genome may be engineered with one or more spacers or linker regions to separate coding or non-coding regions.

In some embodiments, the payload region of the vector genome may optionally encode one or more linker sequences. In some cases, the linker may be a peptide linker that can be used to attach polypeptides encoded by the payload region (i.e., the antibody light and heavy chains during expression). Some peptide linkers can be cleaved into separate heavy and light chain domains after expression, thereby allowing for assembly of the mature antibody or antibody fragment. Linker cleavage may be enzymatic. In some cases, the linker comprises an enzymatic cleavage site to facilitate intracellular or extracellular cleavage. Some payload regions encode linkers that interrupt polypeptide synthesis during translation of the linker sequence from the mRNA transcript. Such linkers can facilitate translation of individual protein domains from a single transcript. In some cases, two or more linkers are encoded by a payload region of the vector genome.

An Internal Ribosome Entry Site (IRES) is a nucleotide sequence (> 500 nucleotides) that allows translation to be initiated in the middle of an mRNA sequence (Kim, J.H. et al, 2011.PLoS One 6 (4): e18556; the contents of which are incorporated herein by reference in their entirety). The use of an IRES sequence ensures co-expression of genes before and after the IRES, although sequences after the IRES can be transcribed and translated at lower levels than sequences before the IRES sequence.

The 2A peptide is a small "self-cleaving" peptide (18-22 amino acids) derived from viruses such as foot and mouth disease virus (F2A), porcine teschovirus (porcine teschovirus) -1 (P2A), leptospira armyworm (Thoseaasigna) virus (T2A), or equine type A rhinitis virus (E2A). 2A denotes in particular the region of picornavirus polymeric protein that causes ribosome jump at the glycyl-prolyl bond in the C-terminus of the 2A peptide (Kim, J.H. et al, 2011.PLoS One 6 (4): e18556; the contents of which are incorporated herein by reference in their entirety). This jump causes cleavage between the 2A peptide and its immediate downstream peptide. In contrast to IRES linkers, the 2A peptide produces stoichiometric expression of proteins flanking the 2A peptide, and its shorter length may be advantageous in producing viral expression vectors.

Some payload regions encode linkers that comprise furin (furin) cleavage sites. Furin is a calcium-dependent serine endoprotease that cleaves proteins located just downstream of the basic amino acid target sequence (Arg-X- (Arg/Lys) -Arg) (Thomas, G.,2002.Nature Reviews Molecular Cell Biology 3 (10): 753-66; the contents of which are incorporated herein by reference in their entirety). Furin is enriched in the reverse golgi network (trans-golgi network), where it is involved in processing cell precursor proteins. Furin also plays a role in activating a variety of pathogens. This activity can be used to express the polypeptides of the present disclosure.

In some embodiments, the payload region may encode one or more linkers comprising a cathepsin, matrix metalloproteinase, or legumain cleavage site. Such linkers are described, for example, by Cizeau and Macdonald in International publication No. WO2008052322, the contents of which are incorporated herein by reference in their entirety. Cathepsins are a family of proteases with unique mechanisms for cleaving specific proteins. Cathepsin B is a cysteine protease and cathepsin D is an aspartyl protease. Matrix metalloproteinases are a family of calcium-dependent and zinc-containing endopeptidases. Legumain is an enzyme that catalyzes the hydrolysis of (-Asn-Xaa-) bonds of proteins and small molecule substrates.

In some embodiments, the payload region may encode an uncleaved linker. Such linkers may include simple amino acid sequences, such as glycine-rich sequences. In some cases, the linker may comprise a flexible peptide linker comprising glycine and serine residues. The linker may comprise flexible peptide linkers of different lengths, e.g. nxG S4S, where n=1-10, and the length of the encoded linker varies between 5 and 50 amino acids. In one non-limiting example, the linker may be 5xG4S. These flexible linkers are small and do not have side chains, and thus tend not to affect the secondary protein structure, while providing flexible linkers between antibody segments (George, r.a. Et al 2002.Protein Engineering 15 (11): 871-9; hunton, j.s. Et al 1988.PNAS 85:5879-83; and shin, d. Et al 1999.Journal of Immunology.162 (11): 6589-95; each of which is incorporated herein by reference in its entirety). Furthermore, the polarity of serine residues improves solubility and prevents aggregation problems.

In some embodiments, the payload region of the present disclosure may encode small and unbranched serine-rich peptide linkers, such as described by Huston et al in U.S. Pat. No. US5525491, the contents of which are incorporated herein by reference in their entirety. The polypeptides linked by serine-rich linkers have increased solubility encoded by the payload regions of the disclosure.

In some embodiments, the payload regions of the present disclosure may encode artificial linkers, such as those described by Whitlow and Filpula in U.S. patent No. US5856456 and by Ladner et al in U.S. patent No. US 4946778, the respective contents of which are incorporated herein by reference in their entirety.

Introns

In some embodiments, the payload region comprises at least one element for enhancing expression, such as one or more introns or portions thereof. Non-limiting examples of introns include MVM (67-97 bp), F.IX truncated intron 1 (300 bp), beta-globin SD/immunoglobulin heavy chain splice acceptor (250 bp), adenovirus splice donor/immunoglobulin splice acceptor (500 bp), SV40 late splice donor/splice acceptor (19S/16S) (180 bp) and hybrid adenovirus splice donor/IgG splice acceptor (230 bp).

In some embodiments, the intron or intron portion may be 100-500 nucleotides in length. Introns may be 80、90、100、110、120、130、140、150、160、170、171、172、173、174、175、176、177、178、179、180、190、200、210、220、230、240、250、260、270、280、290、300、310、320、330、340、350、360、370、380、390、400、410、420、430、440、450、460、470、480、490 or 500 in length. Introns may be between 80-100、80-120、80-140、80-160、80-180、80-200、80-250、80-300、80-350、80-400、80-450、80-500、200-300、200-400、200-500、300-400、300-500 or 400-500 a long.

Lentiviral vector

Lentiviral vectors are a type of retrovirus that can infect both dividing and non-dividing cells, as their viral capsids can cross the intact membrane of the nucleus of the target cell. Lentiviral vectors have the ability to deliver transgenes into tissues that have long appeared and cannot be treated with stable gene manipulation. Lentiviral vectors have also opened up a fresh view for the wide range of genetic as well as acquired disorders of gene therapy, and the real proposal for their clinical use seems to be imminent.

RNA

Ribonucleic acid (RNA) is a molecule made up of nucleotides that are ribose linked to nitrogen containing bases and phosphate groups. Nitrogenous bases include adenine (A), guanine (G), uracil (U) and cytosine (C). Typically, RNA exists predominantly in single stranded form, but in some cases may also exist in double stranded form. The length, form and structure of RNA vary depending on the purpose of the RNA. For example, the length of RNA can vary from a short sequence (e.g., siRNA) to a long sequence (e.g., lncRNA), can be linear (e.g., mRNA) or circular (e.g., oRNA), and can be coding (e.g., mRNA) or non-coding (e.g., lncRNA) sequences.

In some embodiments, the payload region may be or encode a coding RNA.

In some embodiments, the payload region may be or encode a non-coding RNA.

In some embodiments, the payload region may be or encode both coding and non-coding RNAs.

In some embodiments, the payload region comprises a nucleic acid sequence that enhances expression of the gene. As one non-limiting example, the nucleic acid sequence is messenger RNA (mRNA). As another non-limiting example, the nucleic acid sequence is a circular RNA (oRNA).

In some embodiments, the payload region comprises a nucleic acid sequence that reduces or inhibits expression of a gene. As one non-limiting example, the nucleic acid sequence is a small interfering RNA (siRNA) or a microrna (miRNA).

Messenger RNA (mRNA)

In some embodiments, the initial construct and/or the reference construct may be mRNA. As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide encoding a target of interest and capable of translation to produce the encoded target of interest in vitro, in vivo, in situ, or ex vivo.

Typically, an mRNA molecule comprises at least a coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap, and a poly a tail. In some aspects, one or more structural and/or chemical modifications or alterations may be included in the RNA that may reduce the innate immune response of the cells into which the mRNA is introduced. As used herein, a "structural" feature or modification is a feature or modification in which two or more linked nucleotides are inserted, deleted, repeated, inverted, or randomized in a nucleic acid without significant chemical modification to the nucleotide itself. The structural modification is of a chemical nature and is therefore a chemical modification, as the chemical bond will necessarily break and reform to effect the structural modification. However, structural modifications will result in different nucleotide sequences. For example, the polynucleotide "ATCG" may be chemically modified to "AT-5meC-G".

In general, the shortest length of the region of the initial construct and/or the reference construct may be a length sufficient to encode a nucleic acid sequence of a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, or decapeptide. In another embodiment, the length may be sufficient to encode a peptide of 2-30 amino acids, such as 5-30, 10-30, 2-25, 5-25, 10-25, or 10-20 amino acids. The length may be sufficient to encode a peptide of at least 11, 12, 13, 14, 15, 17, 20, 25, or 30 amino acids, or a peptide of no more than 40 amino acids, such as no more than 35, 30, 25, 20, 17, 15, 14, 13, 12, 11, or 10 amino acids.

Typically, the region of the mRNA encoding the target of interest is greater than about 30 nucleotides in length (e.g., at least or greater than about 35、40、45、50、55、60、70、80、90、100、120、140、160、180、200、250、300、350、400、450、500、600、700、800、900、1,000、1,100、1,200、1,300、1,400、1,500、1,600、1,700、1,800、1,900、2,000、2,500、 and 3,000、4,000、5,000、6,000、7,000、8,000、9,000、10,000、20,000、30,000、40,000、50,000、60,000、70,000、80,000、90,000 or up to and including 100,000 nucleotides in length).

In some embodiments of the present invention, in some embodiments, the mRNA comprises about 30 to about 100,000 nucleotides (e.g., 30 to 50, 30 to 100, 30 to 250, 30 to 500, 30 to 1,000, 30 to 1,500, 30 to 3,000, 30 to 5,000, 30 to 7,000, 30 to 10,000, 30 to 25,000, 30 to 50,000, 30 to 70,000, 100 to 250, 100 to 500, 100 to 1,000, 100 to 1,500, 100 to 3,000, 100 to 5,000, 100 to 7,000, 100 to 10,000, 100 to 25,000, 100 to 50,000, 100 to 70,000, 100 to 100,000, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 3,000, 500 to 5,000, 500 to 7,000, 500 to 10,000, 100 to 10,000 500 to 25,000, 500 to 50,000, 500 to 70,000, 500 to 100,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 3,000, 1,000 to 5,000, 1,000 to 7,000, 1,000 to 10,000, 1,000 to 25,000, 1,000 to 50,000, 1,000 to 70,000, 1,000 to 100,000, 1,500 to 3,000, 1,500 to 5,000, 1,500 to 7,000, 1,500 to 10,000, 1,500 to 25,000, 1,500 to 50,000, 1,500 to 70,000, 1,500 to 100,000, 2,000 to 3,000, 2,000 to 5,000, 2,000 to 7,000, 2,000 to 10,000, 2,000 to 25,000, 2,000 to 50,000, 2,000 to 70,000 and 2,000 to 100,000.

In some embodiments, one or more regions flanking the region encoding the target of interest may independently range in length from 15 to 1,000 nucleotides (e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900 nucleotides or at least 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000 nucleotides).

In some embodiments, the mRNA comprises a tailing sequence that may be in the range of no to 500 nucleotides in length (e.g., at least 60, 70, 80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides). Where the tailed region is a poly-A tail, the length may be determined as a unit of or as a function of poly-A binding protein binding. In this embodiment, the poly-a tail is long enough to bind at least 4 monomers of the poly-a binding protein. The poly a binding protein monomer binds to an extension of approximately 38 nucleotides. Thus, it has been observed that poly-a tails of about 80 nucleotides and 160 nucleotides are functional.

In some embodiments, the mRNA comprises a capping sequence comprising a single cap or a series of nucleotides forming a cap. The capping sequence may be 1 to 10, for example 2-9, 3-8, 4-7, 1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length. In some embodiments, the capping sequence is absent.

In some embodiments, the mRNA comprises a region comprising an initiation codon. The length of the region comprising the start codon may be in the range of 3 to 40, e.g., 5-30, 10-20, 15, or at least 4 or 30 or less nucleotides.

In some embodiments, the mRNA comprises a region comprising a stop codon. The length of the region comprising the stop codon may be in the range of 3 to 40, e.g., 5-30, 10-20, 15, or at least 4 or 30 or less nucleotides.

In some embodiments, the mRNA comprises a region comprising a restriction sequence. The length of the region comprising the restriction sequence may be in the range of 3 to 40, for example 5-30, 10-20, 15, or at least 4 or 30 or less nucleotides.

Untranslated region (UTR)

In some embodiments, the mRNA comprises at least one untranslated region (UTR) flanking a region encoding a target of interest. UTR is transcribed but not translated.

The 5' utr starts at the transcription start site and continues to the start codon, but does not include the start codon; whereas the 3' UTR immediately starts at the stop codon and continues until the transcription termination signal. While not wishing to be bound by theory, UTRs may have regulatory effects on translation and stability of nucleic acids.

The natural 5' utr generally includes features that play a role in translation initiation, as it tends to include Kozak sequences that are generally known to be involved in the process by which ribosomes initiate translation of many genes. The Kozak sequence has a common CCR (a/G) CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), followed by another 'G'. It is also known that the 5' UTR forms a secondary structure involved in elongation factor binding.

It is known that 3' UTRs have an extension of adenosine and uridine embedded therein. These AU-rich markers are particularly prevalent in genes with high turnover rates. Based on their sequence features and functional properties, AU-rich elements (ARE) can be divided into three classes (Chen et al, 1995): class I ARE contain several copies of the auxua motif scattered in the U-rich region. C-Myc and MyoD contain class I AREs. Class II AREs have two or more overlapping UUAUUUA (U/A) (U/A) nonamers. Molecules containing this type of ARE include GM-CSF and TNF-a. Class III ARE less well defined. These U-rich regions do not contain the AUUUA motif. c-Jun and myogenin are examples of such two other well-studied examples. Most proteins bound to ARE known to destabilize the messenger, whereas ELAV family members (most notably HuR) have been reported to increase mRNA stability. HuR binds to ARE of all three classes. Engineering a HuR specific binding site into the 3' utr of a nucleic acid molecule will cause HuR binding and thus in vivo information stabilization. The introduction, removal or modification of 3' UTR AU-rich elements (AREs) can be used to modulate the stability of mRNA. For example, one or more ARE copies may be introduced to make the mRNA less stable and thus reduce translation and reduce the yield of the resulting protein. Alternatively, ARE can be identified and removed or mutated to increase intracellular stability and thus increase translation and yield of the resulting protein.

In some embodiments, the introduction of features that are normally expressed in genes of the organ of interest may enhance the stability of mRNA and protein production in a particular organ and/or tissue. As one non-limiting example, the feature may be UTR. As another example, the feature may be an intron or a portion of an intron sequence.

5' Capping

The 5' cap structure of mRNA is involved in nuclear export, increases mRNA stability and binds to mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability and translation capacity in cells via association of CBP with poly (a) binding protein to form mature circular mRNA species. The cap further aids in removal of the 5' proximal intron during mRNA splicing.

Endogenous mRNA molecules can be capped at the 5 'end, resulting in a 5' -ppp-5 '-triphosphate linkage between the terminal guanosine cap residues of the mRNA molecules and the 5' transcribed sense nucleotides. This 5' -guanylate cap may then be methylated to produce an N7-methyl-guanylate residue. The ribose of the 5 'end of the mRNA and/or the nucleotide transcribed before the end (ANTETERMINAL) may also optionally be 2' -0-methylated. 5' -uncapping via hydrolysis and cleavage of guanylate cap structures can target nucleic acid molecules, such as mRNA molecules, for degradation.

Modification of the mRNA can create a non-hydrolyzable cap structure, thereby preventing uncapping and thus increasing mRNA half-life. Since hydrolysis of the cap structure requires cleavage of the 5'-ppp-5' phosphodiester linkage, modified nucleotides can be used during the capping reaction. For example, vaccinia capping enzymes from NEW ENGLAND Biolabs (Ipswich, MA) and a-thio-guanosine nucleotides can be used according to manufacturer's instructions to create phosphorothioate linkages in the 5' -ppp-5' cap.

Additional modified guanosine nucleotides, such as a-methyl-phosphonate and seleno-phosphate nucleotides, may be used.

Additional modifications include, but are not limited to, 2 '-0-methylation of the 5' end of the mRNA (as mentioned above) and/or ribose of the 5 'end pre-nucleotide at the 2' -hydroxy group of the sugar ring. A variety of different 5 'cap structures can be used to create a 5' cap for a nucleic acid molecule (e.g., an mRNA molecule).

Cap analogs are also referred to herein as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs that differ in their chemical structure from the native (i.e., endogenous, wild-type, or physiological) 5' caps, while retaining cap functionality. The cap analogue may be chemically (i.e. non-enzymatically) or enzymatically synthesized and/or linked to a nucleic acid molecule.

For example, the inverted cap analogue (ARCA) cap contains two guanines linked by a 5' -5' -triphosphate group, wherein one guanine contains an N7 methyl group and a 3' -0-methyl group (i.e., N7,3' -0-dimethyl-guanosine-5 ' -triphosphate-5 ' -guanosine (m ⁷ G-3' mppp-G; which may equivalently be referred to as 3' O-Me-m7G (5 ') ppp (5 ') G). The 3' -0 atom of the other unmodified guanine becomes linked to the 5' -terminal nucleotide of a capping nucleic acid molecule (e.g., mRNA). N7-and 3' -0-methylated guanines provide the terminal portion of the capping nucleic acid molecule (e.g., mRNA).

Another exemplary cap is a mCAP, which is similar to ARCA but has a2 '-0-methyl group on guanosine (i.e., N7,2' -0-dimethyl-guanosine-5 '-triphosphate-5' -guanosine, m ⁷ Gm-ppp-G).

Although cap analogs allow concomitant capping of nucleic acid molecules in an in vitro transcription reaction, up to 20% of transcripts may remain uncapped. This, as well as the structural differences in the cap analogs from the endogenous 5' cap structure of the nucleic acid produced by endogenous cellular transcription mechanisms, can lead to reduced translational capacity and reduced cellular stability.

MRNA can also be capped post-transcriptionally using enzymes to produce a more realistic 5' cap structure. As used herein, the phrase "more realistic" refers to features that closely reflect or mimic endogenous or wild-type features, either structurally or functionally. That is, a "more realistic" feature better represents endogenous, wild-type, natural or physiological cellular function and/or structure than a synthetic feature or analog of the prior art, etc., or outperforms the corresponding endogenous, wild-type, natural or physiological feature in one or more respects. Non-limiting examples of more realistic 5' cap structures are especially those as follows: which has enhanced binding of cap binding proteins, increased half-life, reduced sensitivity to 5 'endonucleases and/or reduced 5' uncapping compared to synthetic 5 'cap structures known in the art (or compared to wild-type, natural or physiological 5' cap structures). For example, recombinant vaccinia virus capping enzymes and recombinant 2 '-0-methyltransferases can create a typical 5' -5 '-triphosphate linkage between the 5' terminal nucleotide of the mRNA and the guanine cap nucleotide, where the cap guanine contains N7 methylation and the 5 'terminal nucleotide of the mRNA contains a 2' -0-methyl group. Such a structure is referred to as Capl structure. This cap results in higher translational capacity and cell stability and reduced activation of the cell pro-inflammatory cytokines compared to, for example, other 5' cap analogue structures known in the art. Cap structures include, but are not limited to, 7mG (5 ^*)ppp(5^*) N, pN2p (cap 0), 7mG (5 ^*)ppp(5^*) NlmpNp (cap 1), and 7mG (5 ^*) -ppp (5') NlmpN mp (cap 2).

In some embodiments, the 5' end cap may comprise an endogenous cap or cap analogue.

In some embodiments, the 5' end cap can comprise a guanine analog. Suitable guanine analogs include, but are not limited to, inosine, nl-methyl-guanosine, 2' fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

IRES sequence

In some embodiments, the mRNA may contain an Internal Ribosome Entry Site (IRES). IRES was first identified as a characteristic picornaviral RNA that plays an important role in initiating protein synthesis in the absence of a 5' cap structure. IRES may serve as the sole ribosome binding site, or may serve as one of the multiple ribosome binding sites of mRNA. An mRNA containing more than one functional ribosome binding site can encode several peptides or polypeptides that are independently translated by the ribosome. Non-limiting examples of IRES sequences that may be used include, but are not limited to, those from the following: picornaviruses (e.g., FMDV), pest viruses (CFFV), polioviruses (PV), encephalomyocarditis viruses (ECMV), foot and Mouth Disease Viruses (FMDV), hepatitis C Viruses (HCV), classical Swine Fever Viruses (CSFV), murine Leukemia Viruses (MLV), simian Immunodeficiency Viruses (SIV), or cricket paralysis viruses (CrPV).

Poly A tail

Long-chain adenine nucleotides (poly a tails) may be added to polynucleotides, e.g., mRNA molecules, during RNA processing to increase stability. Immediately after transcription, the 3 'end of the transcript may be cleaved to release the 3' hydroxyl group. Then, the poly-a polymerase adds a series of adenine nucleotides to the RNA. This process is called polyadenylation and the addition of a length of poly-A tail.

In some embodiments, the poly a tail is greater than 30 nucleotides in length. In another embodiment, the poly-a tail is greater than 35 nucleotides in length (e.g., at least or greater than about 35、40、45、50、55、60、70、80、90、100、120、140、160、180、200、250、300、350、400、450、500、600、700、800、900、1,000、1,100、1,200、1,300、1,400、1,500、1,600、1,700、1,800、1,900、2,000、2,500 and 3,000 nucleotides). In some embodiments, the mRNA comprises about 30 to about 3,000 nucleotides (e.g., 30 to 50, 30 to 100, 30 to 250, 30 to 500, 30 to 750, 30 to 1,000, 30 to 1,500, 30 to 2,000, 30 to 2,500, 50 to 100, 50 to 250, 50 to 500, 50 to 750, 50 to 1,000, 50 to 1,500, 50 to 2,000, 50 to 3,000, 100 to 500, 100 to 750, 100 to 1,000, 100 to 1,500, 100 to 2,000, 100 to 2,500, 100 to 3,000, 500 to 750, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 2,500, 500 to 3,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 3,000, 1,500 to 2,000, 1,500, 1,000 to 2,000, 500 to 3,000, 500 and 3,000.

In some embodiments, the poly a tail is designed relative to the length of the entire mRNA. This design may be based on the length of the region encoding the target of interest, the length of a particular feature or region (e.g., flanking region), or the length of the final product expressed from the mRNA.

In this context, the length of the poly a tail may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% longer than the mRNA or a feature thereof. The poly-A tail can also be designed as part of the mRNA to which it belongs. In this context, the poly-a tail may be the full length of the construct or the full length of the construct minus 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more of the poly-a tail. In addition, engineered binding sites of poly a binding proteins and mRNA conjugation can enhance expression.

In addition, a number of different mRNAs can be linked together with PABP (poly A binding protein) via the 3 'end at the 3' end of the poly A tail using modified nucleotides. Transfection experiments can be performed in relevant cell lines and protein production can be determined by ELISA at 12 hours, 24 hours, 48 hours, 72 hours and day 7 post-transfection.

In some embodiments, the mRNA is designed to include a poly A-G quadruplex (Quartet). The G quadruplet is a cyclic hydrogen bonding array of four guanine nucleotides, which can be formed from G-rich sequences in DNA and RNA. In this embodiment, the G quadruplet is incorporated at the end of the poly a tail.

Stop codon

In some embodiments, the mRNA may include a stop codon. In some embodiments, the mRNA may include two stop codons. In some embodiments, the mRNA may include three stop codons. In some embodiments, the mRNA may include at least one stop codon. In some embodiments, the mRNA may include at least two stop codons. In some embodiments, the mRNA may include at least three stop codons. As a non-limiting example, the stop codon can be selected from TGA, TAA and TAG.

In some embodiments, the mRNA includes a stop codon TGA and one additional stop codon. In another embodiment, the addition stop codon may be a TAA.

Circular RNA (oRNA)

In some embodiments, the initial construct and/or the reference construct is a circular RNA (oRNA). As used herein, the term "oRNA" or "circular RNA" is used interchangeably and may refer to RNA that forms a circular structure via covalent or non-covalent bonds.

In some embodiments, oRNA can be non-immunogenic in mammals (e.g., humans, non-human primates, rabbits, rats, and mice).

In some embodiments oRNA is capable of replication or replicable in the following: cells from aquatic animals (e.g., fish, crabs, shrimps, oysters, etc.), mammalian cells, cells from pets or zoo animals (e.g., cats, dogs, lizards, birds, lions, tigers, and bears, etc.), cells from farm or work animals (e.g., horses, cattle, pigs, chickens, etc.), human cells, cultured cells, primary cells or cell lines, stem cells, progenitor cells, differentiated cells, germ cells, cancer cells (e.g., tumorigenic, metastatic), non-tumorigenic cells (e.g., normal cells), fetal cells, embryonic cells, adult cells, mitotic cells, non-mitotic cells, or any combination thereof.

In some embodiments oRNA has a half-life that is at least that of the linear counterpart. In some embodiments oRNA has an increased half-life relative to the half-life of the linear counterpart. In some embodiments, the half-life is increased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more. In some embodiments, the half-life or persistence of oRNA in the cell is at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours (1 day), 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or more, or any time therebetween. In some embodiments, the half-life or persistence of oRNA in the cell is no more than about 10min to about 7 days, or no more than about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 24 hours (1 day), 36 hours (1.5 days), 48 hours (2 days), 60 hours (2.5 days), 72 hours (3 days), 4 days, 5 days, 6 days, or 7 days.

In some embodiments oRNA has a half-life or persistence in the cell while the cell is dividing. In some embodiments oRNA has a half-life or persistence in the cells after division. In certain embodiments, oRNA has a half-life or persistence in dividing cells of greater than about 10 minutes to about 30 days, or at least about 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 24 hours (1 day), 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or more, or any time therebetween.

In some embodiments oRNA modulates cellular function, e.g., temporarily or chronically. In certain embodiments, the cell function is stably altered, e.g., maintained for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours (1 day), 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or longer. In certain embodiments, the cellular function is temporarily altered, e.g., maintained in a regulatory state for no more than about 30 minutes to about 7 days, or for no more than about 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours (1 day), 36 hours (1.5 day), 48 hours (2 days), 60 hours (2.5 days), 72 hours (3 days), 4 days, 5 days, 6 days, or 7 days.

In some embodiments, oRNA is at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 6,000 nucleotides, at least about 7,000 nucleotides, at least about 8,000 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nucleotides, at least about 12,000 nucleotides, at least about 14,000 nucleotides, at least about 15,000 nucleotides, at least about 16,000 nucleotides, at least about 17,000 nucleotides, at least about 18,000 nucleotides, at least about 19,000 nucleotides, or at least about 20,000 nucleotides. In some embodiments oRNA may be of sufficient size to accommodate the binding site of the ribosome.

In some embodiments, the maximum size of oRNA may be limited by the ability to package and deliver RNA to a target. In some embodiments, oRNA is of a size sufficient to encode the length of the polypeptide, and thus a length of at least 20,000 nucleotides, at least 15,000 nucleotides, at least 10,000 nucleotides, at least 7,500 nucleotides, or at least 5,000 nucleotides, at least 4,000 nucleotides, at least 3,000 nucleotides, at least 2,000 nucleotides, at least 1,000 nucleotides, at least 500 nucleotides, at least 400 nucleotides, at least 300 nucleotides, at least 200 nucleotides, at least 100 nucleotides may be suitable.

In some embodiments, oRNA comprises one or more elements described elsewhere herein. In some embodiments, the elements may be separated from each other by a spacer sequence or linker. In some embodiments, the element can be separated from each other by 1 nucleotide, 2 nucleotides, about 5 nucleotides, about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 80 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, about 800 nucleotides, about 900 nucleotides, about 1000 nucleotides, up to about 1kb, at least about 1000 nucleotides.

In some embodiments, one or more elements are contiguous with each other, e.g., lacking spacer sub-elements.

In some embodiments, one or more elements are conformally flexible. In some embodiments, conformational flexibility is attributed to the sequence being substantially free of secondary structures.

In some embodiments oRNA comprise a secondary or tertiary structure that accommodates the binding site for ribosomes, translations, or rolling circle translations.

In some embodiments oRNA comprises a specific sequence feature. For example, oRNA may comprise a specific nucleotide composition. In some such embodiments, oRNA can include one or more purine-rich regions (adenine or guanosine). In some embodiments oRNA may include one or more AU-rich regions or elements (ARE). In some embodiments oRNA may include one or more adenine-rich regions.

In some embodiments, oRNA comprises one or more modifications described elsewhere herein.

In some embodiments, oRNA comprises one or more expression sequences and is configured for sustained expression in vivo in a cell of a subject. In some embodiments, oRNA is configured such that the expression of one or more expression sequences in the cell at a later point in time is equal to or higher than the earlier point in time. In such embodiments, expression of one or more expression sequences may be maintained at a relatively stable level or may increase over time. Expression of the expressed sequence may be relatively stable for an extended period of time. For example, in some cases, expression of one or more expressed sequences in a cell is not reduced by 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% over a period of at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23, or more days. In some cases, expression of one or more expressed sequences in a cell is maintained at a level that does not vary by more than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% for at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 days or more.

Adjusting element

In some embodiments oRNA comprises a regulatory element. As used herein, a "regulatory element" is a sequence that regulates expression of an expression sequence. The conditioning element may comprise a sequence located near the payload or cargo area. The conditioning element may be operatively connected to the payload or cargo area.

In some embodiments, the regulatory element may increase the amount of payload or cargo expressed as compared to the amount expressed when no regulatory element is present. As one non-limiting example, one adjustment element may increase the amount of payload or cargo expressed by a plurality of payload or cargo sequences connected in series.

In some embodiments, the regulatory element may comprise a sequence that selectively initiates or activates translation of the payload or cargo.

In some embodiments, the regulatory element may comprise a sequence that initiates oRNA or degradation of the payload or cargo. Non-limiting examples of sequences that initiate degradation include, but are not limited to, riboswitch aptamer enzymes (aptazyme) and miRNA binding sites.

In some embodiments, the adjustment element may adjust oRNA the translation of the payload or cargo. The modulation may cause the payload or cargo to increase (enhancer) or decrease (inhibitor). The adjustment element may be located near (e.g., on one or both sides of) the payload or cargo.

In some embodiments, the translation initiation sequence is used as a regulatory element. In some embodiments, the translation initiation sequence comprises an AUG/ATG codon. In some embodiments, the translation initiation sequence comprises any eukaryotic initiation codon, such as, but not limited to, AUG/ATG, CUG/CTG, GUG/GTG, UUG/TTG, ACG, AUC/ATC, AUU, AAG, AUA/ATA, or AGG. In some embodiments, the translation initiation sequence comprises a Kozak sequence. In some embodiments, translation begins under selective conditions (e.g., stress-inducing conditions) with an alternative translation initiation sequence, such as a translation initiation sequence other than AUG/ATG codons. As one non-limiting example, translation of a circular polyribonucleotide may begin with an alternative translation initiation sequence, such as ACG. As another non-limiting example, circular polyribonucleotide translation may begin with an alternative translation initiation sequence CUG/CTG. As another non-limiting example, translation may begin with an alternative translation initiation sequence GUG/GTG. As yet another non-limiting embodiment, translation may begin with a repeat-related non-AUG (RAN) sequence, such as an alternative translation initiation sequence that includes a short extension (e.g., CGG, GGGGCC, CAG, CTG) of the repeat RNA.

Masking agent

Any nucleotide that masks codons flanking the initiation translation may be used to alter the translation initiation position, translation efficiency, length and/or structure of oRNA. In some embodiments, a masking agent may be used near the start codon or alternative start codon to mask or hide the codon to reduce the probability of translation initiation at the masked start codon or alternative start codon. Non-limiting examples of masking agents include antisense Locked Nucleic Acid (LNA) oligonucleotides and exon junction complexes (exon junction complex; EJC). In some embodiments, a masking agent may be used to mask the start codon of oRNA in order to increase the likelihood that translation will be initiated at an alternative start codon.

Translation initiation sequences

In some embodiments oRNA encodes a polypeptide or peptide and may comprise a translation initiation sequence. The translation initiation sequence may include, but is not limited to, an initiation codon, a non-coding initiation codon, a Kozak sequence, or a Shine-Dalgarno sequence. The translation initiation sequence may be located near (e.g., on one or both sides of) the payload or cargo.

In some embodiments, the translation initiation sequence provides conformational flexibility to oRNA. In some embodiments, the translation initiation sequence is within the substantially single-stranded region of oRNA.

ORNA can include more than 1 start codon, such as, but not limited to, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or more than 15 start codons. Translation may be initiated at the first initiation codon or may be initiated downstream of the first initiation codon.

In some embodiments oRNA may start at a codon that is not the first start codon (e.g., AUG). Translation of the circular polyribonucleotide may be initiated at alternative translation initiation sequences such as, but not limited to ACG, AGG, AAG, CUG/CTG, GUG/GTG, AUA/ATA, AUU/ATT, UUFG/TTG. In some embodiments, translation begins under selective conditions, such as stress-induced conditions, with an alternative translation initiation sequence. As one non-limiting example, translation of oRNA may begin with an alternative translation initiation sequence, such as ACG. As another non-limiting example, oRNA translations may begin with the alternative translation initiation sequence CUG/CTG. As yet another non-limiting embodiment, oRNA translations may begin with the alternative translation initiation sequence GTG/GUG. As yet another non-limiting embodiment, oRNA may begin translation at a repeat-related non-AUG (RAN) sequence, such as an alternative translation initiation sequence that includes a short extension of the repetitive RNA (e.g., CGG, GGGGCC, CAG, CTG).

IRES sequence

In some embodiments, oRNA described herein comprises an Internal Ribosome Entry Site (IRES) element capable of engaging a eukaryotic ribosome. In some embodiments, the IRES element is at least about 5 nucleotides, at least about 8 nucleotides, at least about 9 nucleotides, at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 250 nucleotides, at least about 350 nucleotides, or at least about 500 nucleotides. In one embodiment, the IRES element is derived from DNA from organisms including, but not limited to, viruses, mammals, and Drosophila. Such viral DNA may be derived from, but is not limited to, picornaviral complementary DNA (cDNA) and encephalomyocarditis virus (EMCV) cDNA and poliovirus cDNA. In one embodiment, the Drosophila DNA from which the IRES element is derived includes, but is not limited to, the antennapedia gene from Drosophila melanogaster (Drosophila melanogaster).

In some embodiments, the IRES element is derived at least in part from a virus, e.g., it may be derived from a viral IRES element, e.g., ABPV_IGRpred、AEV、ALPV_IGRpred、BQCV_IGRpred、BVDV1_1-385、BVDV1_29-391、CrPV_5NCR、CrPV_IGR、crTMV_IREScp、crTMV_IRESmp75、crTMV_IRESmp228、crTMV_IREScp、crTMV_IREScp、CSFV、CVB3、DCV_IGR、EMCV-R、EoPV_5NTR、ERAV 245-961、ERBV 162-920、EV71_1-748、FeLV-Notch2、FMDV_type_C、GBV-A、GBV-B、GBV-C、gypsy_env、gypsyD5、gypsyD2、HAV_HM175、HCV_type_1a、HiPV_IGRpred、HIV-1、HoCV1_IGRpred、HRV-2、IAPV_IGRpred、idefix、KBV_IGRpred、LINE-1_ORF1_-101_to_-1、LINE-1_ORF1-302_to_-202、LINE-1_ORF2-138_to_-86、LINE-1_ORF1_-44to_-1、PSIV_IGR、PV_type1_Mahoney、PV_type3_Leon、REV-A、RhPV_5NCR、RhPV_IGR、SINV1_IGRpred、SV40_661-830、TMEV、TMV_UI_IRESmp228、TRV_5NTR、TrV_IGR or TSV IGR. In some embodiments, the IRES element is derived at least in part from a cellular IRES, such as AML1/RUNX1、Antp-D、Antp-DE、Antp-CDE、Apaf-1、Apaf-1、AQP4、AT1R_var1、AT1R_var2、AT1R_var3、AT1R_var4、BAG1_p36delta236 nt、BAG1_p36、BCL2、BiP_-222_-3、c-IAP1_285-1399、c-IAP1_1313-1462、c-jun、c-myc、Cat-1224、CCND1、DAPS、eIF4G、eIF4GI-ext、eIF4GII、eIF4GII- long, ELG1, ELH, FGF1A, FMR1, gtx-133-141, gtx-1-166, gtx-1-120, gtx-1-196, hairless (hairless), HAP4, HIF1a, hSNM1, hsp101, hsp70, hsp90, IGF 2_leader 2、Kv1.4_1.2、L-myc、LamB1_-335_-1、LEF1、MNT_75-267、MNT_36-160、MTG8a、MYB、MYT2_997-1152、n-MYC、NDST1、NDST2、NDST3、NDST4L、NDST4S、NRF_-653_-17、NtHSF1、ODC1、p27kip1、03_128-269、PDGF2/c-sis、Pim-1、PITSLRE_p58、Rbm3、reaper、Scamper、TFIID、TIF4631、Ubx_1-966、Ubx_373-961、UNR、Ure2、UtrA、VEGF-A-133-1、XIAP_5-464、XIAP_305-466, or YAP1.

Termination element

In some embodiments oRNA includes one or more cargo or payload sequences (also referred to as expression sequences), and each cargo or payload sequence may or may not have a termination element.

In some embodiments oRNA includes one or more cargo or payload sequences, and the sequences lack termination elements to allow for consistent translation of oRNA. The elimination of the termination element may result in rolling circle translation or continuous expression of the encoded peptide or polypeptide, as the ribosome will not stop or leave (fall-off). In such embodiments, rolling circle translation is expressed serially via each cargo or payload sequence expression.

In some embodiments, one or more of the cargo or payload sequences in oRNA comprise a termination element.

In some embodiments, oRNA not all cargo or payload sequences comprise termination elements. In such cases, the cargo or payload may leave (roll off) the ribosome when the ribosome encounters a termination element and translation is terminated. In some embodiments, translation is terminated while at least one region of the ribosome remains in contact with oRNA.

Rolling circle translation

In some embodiments, once translation of oRNA is initiated, the ribosome that binds to oRNA does not become detached oRNA until at least one round of translation of oRNA is completed. In some embodiments oRNA as described herein is adequate for rolling circle translation. In some embodiments, once translation of oRNA is initiated during rolling circle translation, the ribosome bound to oRNA does not break away oRNA before translation of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 500, at least 1000, at least 1500, at least 2000, at least 5000, at least 10000, at least 10.sup.5, or at least 10.sup.6 wheels oRNA is completed.

In some embodiments, the rolling circle translation of oRNA results in the production of a polypeptide translated from more than one round of oRNA translation. In some embodiments oRNA comprises staggered (stagger) elements, and rolling circle translation of oRNA results in the production of a polypeptide product resulting from single round oRNA translation or less than single round oRNA translation.

Cyclization

In one embodiment, the linear RNAs may be circularized or concatenated (concatemerized). In some embodiments, the linear RNA can be circularized in vitro prior to formulation and/or delivery. In some embodiments, the linear RNA can be circularized within the cell.

In some embodiments, the mechanism of cyclization or tandem can be via at least 3 different pathways: 1) chemistry, 2) enzymatic and 3) ribozyme catalysis. The newly formed 5'-/3' -linkages may be intramolecular or intermolecular.

In the first approach, the 5 'and 3' ends of the nucleic acid contain chemically reactive groups that, when brought together in close proximity, form new covalent linkages between the 5 'and 3' ends of the molecule. The 5 'end may contain a NHS-ester reactive group and the 3' end may contain a3 '-amino-terminated nucleotide, such that in an organic solvent, the 3' -amino-terminated nucleotide on the 3 'end of the synthetic mRNA molecule will undergo nucleophilic attack on the 5' -NHS-ester moiety, thereby forming a new 5'-/3' -amide bond.

In the second pathway, T4 RNA ligase can be used to enzymatically link a5 '-phosphorylated nucleic acid molecule to the 3' -hydroxyl group of a nucleic acid, thereby forming a novel phosphodiester linkage. In one example reaction, a ≡g nucleic acid molecule is incubated with 1-10 units of T4 RNA ligase (NEW ENGLAND Biolabs, ipswich, mass.) for 1 hour at 37℃according to the manufacturer's protocol. Ligation may be performed in the presence of a resolution oligonucleotide capable of base pairing with both juxtaposed 5 '-and 3' -regions of the auxiliary enzymatic ligation.

In a third approach, the 5 'or 3' end of the cDNA template encodes a ligase ribozyme sequence such that during in vitro transcription, the resulting nucleic acid molecule may contain an active ribozyme sequence capable of ligating the 5 'end of the nucleic acid molecule with the 3' end of the nucleic acid molecule. The ligase ribozyme may be derived from group I introns, hepatitis delta virus, hairpin ribozymes, or may be selected by evolution of the ligand index enrichment system (SYSTEMATIC EVOLUTION OF LIGANDS BY EXPONENTIAL ENRICHMENT; SELEX). The ribozyme ligase reaction may take 1 to 24 hours at a temperature between 0 and 37 ℃.

In some embodiments oRNA is prepared via circularization of linear RNA.

Extracellular ring system

In some embodiments, the linear RNAs are circularized or concatenated using chemical methods to form oRNA. In some chemical methods, the 5 'and 3' ends of a nucleic acid (e.g., linear RNA) include chemically reactive groups that, when brought together in close proximity, can form new covalent linkages between the 5 'and 3' ends of the molecule. The 5 'end may contain a NHS-ester reactive group and the 3' end may contain a3 '-amino-terminated nucleotide, such that in an organic solvent, the 3' -amino-terminated nucleotide on the 3 'end of the linear RNA will undergo nucleophilic attack on the 5' -NHS-ester moiety, thereby forming a new 5'-/3' -amide bond.

In one embodiment, a DNA or RNA ligase may be used to enzymatically join a 5 '-phosphorylated nucleic acid molecule (e.g., linear RNA) to the 3' -hydroxyl group of a nucleic acid (e.g., linear nucleic acid), thereby forming a novel phosphodiester linkage. In one example reaction, linear RNA is incubated with 1-10 units of T4 RNA ligase for 1 hour at 37℃according to the manufacturer's protocol. Ligation reactions can be performed in the presence of linear nucleic acids capable of base pairing with both juxtaposed 5 '-and 3' -regions that assist in enzymatic ligation reactions. In one embodiment, the ligation is a splint ligation (splint ligation) in which a single stranded polynucleotide (splint), such as single stranded RNA, may be designed to hybridize to both ends of a linear RNA such that the two ends may be juxtaposed after hybridization to the single stranded splint. Thus, the splint ligase catalyzes the ligation of the juxtaposed two ends of the linear RNA, resulting in oRNA.

In one embodiment, a DNA or RNA ligase may be used for the synthesis oRNA. As one non-limiting example, the ligase may be a circ ligase or a circular ligase.

In one embodiment, the 5 'or 3' end of the linear RNA may encode a ligase ribozyme sequence such that during in vitro transcription, the resulting linear RNA includes an active ribozyme sequence capable of linking the 5 'end of the linear RNA with the 3' end of the linear RNA. The ligase ribozyme may be derived from a group I intron, hepatitis delta virus, hairpin ribozyme, or may be selected by ligand index enrichment system evolution (SELEX).

In one embodiment, the linear RNA can be circularized or tandem through the use of at least one non-nucleic acid moiety. In one aspect, the at least one non-nucleic acid moiety can react with a region or feature near the 5 'end and/or near the 3' end of the linear RNA to circularize or tandem the linear RNA. In another aspect, the at least one non-nucleic acid moiety may be located in or linked to or located near the 5 'end and/or the 3' end of the linear RNA. The non-nucleic acid portions contemplated may be homologous or heterologous. As one non-limiting example, the non-nucleic acid moiety can be a linkage, such as a hydrophobic linkage, an ionic linkage, a biodegradable linkage, and/or a cleavable linkage. As another non-limiting example, the non-nucleic acid moiety is a linking moiety. As yet another non-limiting example, the non-nucleic acid moiety can be an oligonucleotide or peptide moiety, such as an aptamer or non-nucleic acid linker as described herein.

In one embodiment, the linear RNA can be circularized or tandem due to non-nucleic acid moieties that cause attraction between the surfaces of atoms, molecules at, near, or linked to the 5 'and 3' ends of the linear RNA. As one non-limiting example, one or more linear RNAs may be circularized or tandem by intermolecular or intramolecular forces. Non-limiting examples of intermolecular forces include dipole-dipole forces, dipole-induced dipole forces, induced dipole-induced dipole forces, van der Waals forces (VAN DER WAALS force), and London dispersion forces (London dispersion force). Non-limiting examples of intramolecular forces include covalent bonds, metallic bonds, ionic bonds, resonance bonds, agnostic bonds, dipole bonds, conjugation (hyperconjugation), super-conjugation (hyperconjugation), and anti-bonds.

In one embodiment, the linear RNA may comprise a ribozyme RNA sequence near the 5 'end and near the 3' end. The ribozyme RNA sequence can be covalently linked to the peptide when the sequence is exposed to the remainder of the ribozyme. In one aspect, peptides covalently linked to ribozyme RNA sequences near the 5 'end and the 3' end can associate with each other, thereby circularizing or concatenating linear RNAs. In another aspect, peptides covalently linked to ribozyme RNA near the 5 'and 3' ends can allow for linear RNA cyclization or tandem after undergoing ligation (e.g., but not limited to protein ligation) using various methods known in the art.

In some embodiments, the linear RNA can include a 5 'triphosphate of a nucleic acid that is converted to a 5' monophosphate, for example, by contacting the 5 'triphosphate with RNA 5' pyrophosphorohydrolase (RppH) or ATP diphosphate hydrolase (apyrase). Alternatively, conversion of the 5 'triphosphate of linear RNA to a 5' monophosphate can be performed by a two step reaction comprising: (a) Contacting the 5' nucleotide of the linear RNA with a phosphatase (e.g., antarctic phosphatase, shrimp alkaline phosphatase, or calf intestinal phosphatase) to remove all three phosphates; and (b) contacting the 5' nucleotide after step (a) with a kinase (e.g., a polynucleotide kinase) to which a single phosphate is added.

In some embodiments, the RNA can be circularized using the methods described in WO2017222911 and WO2016197121, the respective contents of which are incorporated herein by reference in their entirety.

In some embodiments, the RNA can be circularized, for example, by reverse splicing non-mammalian exogenous introns or splint ligation of the 5 'and 3' ends of the linear RNA. In one embodiment, the circular RNA is produced from a recombinant nucleic acid encoding the target RNA to be prepared as circular. As one non-limiting example, the method includes: a) Generating a recombinant nucleic acid encoding a target RNA to be prepared as a loop, wherein the recombinant nucleic acid comprises in 5 'to 3' order: i) A 3 'portion comprising an exogenous intron of a 3' splice site, ii) a nucleic acid sequence encoding a RNA of interest, and iii) a 5 'portion comprising an exogenous intron of a 5' splice site; b) Performing transcription, thereby producing RNA from the recombinant nucleic acid; and c) RNA splicing, whereby RNA circularization occurs to produce oRNA.

While not wanting to be bound by theory, the resulting circular RNAs with exogenous introns are recognized by the immune system as "non-self" and trigger an innate immune response. On the other hand, the resulting circular RNA with endogenous introns is recognized by the immune system as "self" and does not normally stimulate an innate immune response, even if carrying exons comprising foreign RNA.

Thus, circular RNAs with endogenous or exogenous introns can be generated as desired to control immunological self/non-self discrimination. Various intron sequences from a wide variety of organisms and viruses are known and include sequences derived from genes encoding proteins, ribosomal RNAs (rRNA) or transfer RNAs (tRNA).

Circular RNAs can be produced from linear RNAs in a variety of ways. In some embodiments, the circular RNA is produced from linear RNA by reverse splicing of a downstream 5 'splice site (splice donor) to an upstream 3' splice site (splice acceptor). The circular RNA can be produced in this manner by any non-mammalian splicing method. For example, linear RNAs containing various types of introns (including self-splicing group I introns, self-splicing group II introns, spliced-body introns, and tRNA introns) can be circularized. In particular, group I and group II introns are advantageous because they can be readily used to produce circular RNAs in vitro as well as in vivo because they are capable of self-splicing due to their autocatalytic ribozyme activity.

In some embodiments, the circular RNA can be produced in vitro from linear RNA by chemically or enzymatically ligating the 5 'and 3' ends of the RNA. Chemical ligation may be performed, for example, using cyanogen bromide (BrCN) or ethyl-3- (3' -dimethylaminopropyl) carbodiimide (EDC) for activating nucleotide phosphate monoester groups to allow phosphodiester bond formation. See, e.g., sokolova (1988) FEBS Lett 232:153-155; dolinnaya et al (1991)Nucleic Acids Res.,19:3067-3072;Fedorova(1996)Nucleosides Nucleotides Nucleic Acids 15:1 137-1 147; are incorporated herein by reference. Alternatively, enzymatic ligation may be used to circularize the RNA. Exemplary ligases that may be used include T4 DNA ligase (T4 Dnl), T4RNA ligase 1 (T4 Rnl 1), and T4RNA ligase 2 (T4 Rnl 2).

In some embodiments, splint ligation using oligonucleotide splint hybridized to both ends of the linear RNA may be used to ligate the ends of the linear RNA together. Hybridization of the splint (which may be DNA or RNA) orients the 5 '-phosphate and 3' -OH at the RNA end for ligation. Subsequent ligation may be performed using chemical or enzymatic techniques, as described above. Enzymatic ligation may be performed, for example, with T4 DNA ligase (DNA splint requirement), T4 RNA ligase 1 (RNA splint requirement) or T4 RNA ligase 2 (DNA or RNA splint). In some cases, chemical ligation, e.g., with BrCN or EDC, is more efficient than enzymatic ligation if the structure of the hybridized splint-RNA complex interferes with enzymatic activity.

In some embodiments oRNA may further comprise an Internal Ribosome Entry Site (IRES) operably linked to the RNA sequence encoding the polypeptide. Inclusion of IRES permits translation of one or more open reading frames from the circular RNA. IRES elements attract eukaryotic ribosomal translation initiation complexes and promote translation initiation. See, e.g., kaufman et al, nuc.acids res (1991) 19:4485-4490; gurtu et al biochem. Biophys. Res. Comm. (1996) 229:295-298; rees et al, bioTechniques (1996) 20:102-110; kobayashi et al, bioTechniques (1996) 21:399-402; and Mosser et al, bioTechniques 1997 22 150-161).

In some embodiments, the cyclization methods provided herein have a cyclization efficiency of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100%. In some embodiments, the cyclization methods provided herein have a cyclization efficiency of at least about 40%.

Splice element

In some embodiments oRNA comprises at least one splice element. The splice element may be a complete splice element that mediates splicing of oRNA, or the splice element may be a residual splice element from the completion of a splicing event. For example, in some cases, splice elements of linear RNAs may mediate splicing events that lead to cyclization of linear RNAs, such that the resulting oRNA comprises residual splice elements from such splice-mediated cyclization events. In some cases, the residual splice element is unable to mediate any splicing. In other cases, the residual splice element may still mediate splicing in some cases. In some embodiments, the splice element is adjacent to at least one expression sequence. In some embodiments oRNA comprises a splice element adjacent to each expressed sequence. In some embodiments, splice elements on one or both sides of each expressed sequence cause isolation of the expression products, e.g., peptides and/or polypeptides.

In some embodiments oRNA comprise internal splice elements that join together at the splicing end upon replication. Some examples may include a mini-intron (< 100 nt) with a splice site sequence and a short inverted repeat (30-40 nt) (e.g., aluSq, aluJr, and AluSz), an inverted sequence in a flanking intron, an Alu element in a flanking intron, and a motif found in a cis-sequence element near the reverse splicing event (suptable enriched motif), e.g., a sequence in 200bp before (upstream) or after (downstream) the reverse splice site of the flanking exon. In some embodiments oRNA comprises at least one repeated nucleotide sequence described elsewhere herein as an internal splice element. In such embodiments, the repetitive nucleotide sequence may include a repetitive sequence from an Alu family intron. See, for example, U.S. patent No. 11,058,706.

In some embodiments oRNA may include a typical splice site for head-to-tail joining of the side pieces oRNA.

In some embodiments oRNA may include a carina-helix-Long Tuji sequence comprising a 4 base pair stem flanked by two 3 nucleotide carina. Cleavage occurs at one site in the carina region, producing a characteristic fragment with terminal 5' -hydroxyl and 2',3' -cyclic phosphate. Cyclization is carried out by: nucleophilic attack of the 5' -OH group onto the 2',3' -cyclic phosphate of the same molecule, thereby forming a 3',5' -phosphodiester bridge.

In some embodiments oRNA may include sequences that mediate self-ligation. Non-limiting examples of sequences that can mediate self-ligation include self-circularizing introns (e.g., 5 'and 3' splice junctions) or self-circularizing catalytic introns (e.g., group I, group II, or group III introns). Non-limiting examples of group I intronic self-splicing sequences may include aligned intronic-exon sequences derived from self-splicing of T4 phage gene td, and the intermediate sequence (IVS) rRNA of Tetrahymena (Tetrahymena).

Other cyclization methods

In some embodiments, the linear RNA can include complementary sequences, including repeated or non-repeated nucleic acid sequences within individual introns or throughout flanking introns. In some embodiments oRNA comprises a repeat nucleic acid sequence. In some embodiments, the repetitive nucleotide sequence comprises a poly CA or poly UG sequence. In some embodiments, oRNA comprises at least one repeat nucleic acid sequence that hybridizes to a complementary repeat nucleic acid sequence in another segment of oRNA, wherein the hybridized segments form an internal double strand. In some embodiments, the repeat nucleic acid sequences from two separate oRNA and the complementary repeat nucleic acid sequence are hybridized to produce a single oRNA, wherein the hybridized segments form an internal double strand. In some embodiments, the complementary sequences are present at the 5 'and 3' ends of the linear RNA. In some embodiments, the complementary sequence comprises about 3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100 or more paired nucleotides.

In some embodiments, cyclization chemistry can be used to produce oRNA. Such methods may include, but are not limited to, click chemistry (e.g., alkyne and azide based methods or clickable bases), olefin metathesis, phosphoramidate ligation, semialdehyde-imine crosslinking, base modification, and any combination thereof.

In some embodiments, a cyclase enzymatic method may be used to produce oRNA. In some embodiments, a ligase, such as a DNA or RNA ligase, may be used to generate the oRNA or complementary sequence template, the complementary strand of oRNA, or oRNA.

Small interfering RNA (siRNA)

In some embodiments, the payload region may be or encode an RNA interference (RNAi) sequence that may be used to reduce or inhibit expression of a gene. RNAi (also known as post-transcriptional gene silencing (PTGS), suppression or co-suppression) is a post-transcriptional gene silencing approach in which RNA molecules reduce or inhibit gene expression in a sequence-specific manner, typically by disrupting specific mRNA molecules. The active component of RNAi is short/small double-stranded RNA (dsRNA), known as small interfering RNA (siRNA), which typically contains 15-30 nucleotides (e.g., 19 to 25, 19 to 24, or 19-21 nucleotides) and a2 nucleotide 3' overhang, and which matches the nucleic acid sequence of the target gene. These short RNA species can be naturally produced in vivo by Dicer-mediated cleavage of larger dsRNA, and are functional in mammalian cells.

Naturally expressed small RNA molecules, known as micrornas (mirnas), cause gene silencing by modulating the expression of mRNA. RNA-induced silencing complexes (RISC) containing mirnas target mrnas that exhibit perfect sequence complementarity to nucleotides 2-7 in the 5 'region of the miRNA, which is referred to as the seed region, and other bases pair with their 3' region. miRNA-mediated down-regulation of gene expression may be caused by cleavage of target mRNA, translational inhibition of target mRNA, or mRNA decomposition. The miRNA targeting sequence is typically located in the 3' -UTR of the target mRNA. A single miRNA can target more than 100 transcripts from various genes, and one mRNA can be targeted by a different miRNA.

SiRNA duplex or dsRNA targeting a specific mRNA can be designed and synthesized in vitro and introduced into cells for activating RNAi processes. It has been previously shown that 21-nucleotide siRNA duplex (termed small interfering RNA) is capable of achieving potent and specific gene knockdown in mammalian cells without inducing an immune response. Post-transcriptional gene silencing by siRNA has now been fast as a powerful tool for gene analysis in mammalian cells and has the potential to produce new therapeutic agents.

In vitro synthesized siRNA sequences can be introduced into cells to activate RNAi. Upon introduction into a cell, the exogenous siRNA duplex can be assembled to form an RNA-induced silencing complex (RISC), a multi-unit complex that interacts with an RNA sequence that is complementary to one of the two strands of the siRNA duplex (i.e., the antisense strand), similar to the endogenous dsRNA. During the process, the sense strand (or passenger strand) of the siRNA is lost from the complex, while the antisense strand (or guide strand) of the siRNA is matched to its complementary RNA. In particular, RISC complexes containing siRNA are targeted to mRNA exhibiting perfect sequence complementarity. siRNA mediated gene silencing then occurs by cleavage, release and degradation of the target.

SiRNA duplex consisting of a sense strand homologous to the target mRNA and an antisense strand complementary to the target mRNA offer much more advantages in terms of target RNA destruction efficiency than the use of single stranded (ss) -siRNA (e.g., antisense strand RNA or antisense oligonucleotides). In many cases, higher concentrations of ss-siRNA are required to achieve effective gene silencing efficacy for the corresponding duplex.

Design and sequence of siRNA duplex

Some guidelines for designing siRNA have been proposed in the art. These guidelines generally suggest the formation of a 19-nucleotide duplex region, a symmetrical 2-3 nucleotide 3' overhang, a 5' -phosphate, and a3' -hydroxyl group targeting the region to be silenced in the gene. Other rules governing siRNA sequence preferences include, but are not limited to: (i) an a/U at the 5' end of the antisense strand; (ii) G/C at the 5' end of the sense strand; (iii) At least five a/U residues in the 5' terminal third of the antisense strand; and (iv) there are no GC extensions longer than 9 nucleotides in length. Based on such considerations, along with the specific sequence of the target gene, highly efficient siRNA constructs necessary to inhibit expression of the mammalian target gene can be readily designed.

In some embodiments, siRNA constructs (e.g., siRNA duplex or encoded dsRNA) are designed that target a particular gene. In particular, such siRNA constructs can inhibit gene expression and protein production. In some aspects, the siRNA constructs are designed and used to selectively "knock out" gene variants in cells, i.e., mutant transcripts identified in patients or causative of various diseases and/or disorders. In some aspects, the siRNA constructs are designed and used to selectively "knock down" variants of genes in cells. In other aspects, the siRNA construct is capable of inhibiting or suppressing both wild-type and mutant forms of the gene.

In some embodiments, the siRNA sequence comprises a sense strand and a complementary antisense strand, wherein the two strands hybridize together to form a duplex structure. The antisense strand has sufficient complementarity to the mRNA sequence to direct targeted specific RNAi, i.e., the siRNA sequence has a sequence sufficient to trigger destruction of the targeted mRNA by the RNAi machinery or process.

In some embodiments, the siRNA sequence comprises a sense strand and a complementary antisense strand, wherein the two strands hybridize together to form a duplex structure, and wherein the initiation site for hybridization to the mRNA is between nucleotide 100 and 10,000 on the mRNA sequence. As a non-limiting example, the initiation site can be between nucleotides 100-150、150-200、200-250、250-300、300-350、350-400、400-450、450-500、500-550、550-600、600-650、650-700、700-70、750-800、800-850、850-900、900-950、950-1000、1000-1050、1050-1100、1100-1150、1150-1200、1200-1250、1250-1300、1300-1350、1350-1400、1400-1450、1450-1500、1500-1550、1550-1600、1600-1650、1650-1700、1700-1750、1750-1800、1800-1850、1850-1900、1900-1950、1950-2000、2000-2050、2050-2100、2100-2150、2150-2200、2200-2250、2250-2300、2300-2350、2350-2400、2400-2450、2450-2500、2500-2550、2550-2600、2600-2650、2650-2700、2700-2750、2750-2800、2800-2850、2850-2900、2900-2950、2950-3000、3000-3050、3050-3100、3100-3150、3150-3200、3200-3250、3250-3300、3300-3350、3350-3400、3400-3450、3450-3500、3500-3550、3550-3600、3600-3650、3650-3700、3700-3750、3750-3800、3800-3850、3850-3900、3900-3950、3950-4000、4000-4050、4050-4100、4100-4150、4150-4200、4200-4250、4250-4300、4300-4350、4350-4400、4400-4450、4450-4500、4500-4550、4550-4600、4600-4650、4650-4700、4700-4750、4750-4800、4800-4850、4850-4900、4900-4950、4950-5000、5000-5050、5050-5100、5100-5150、5150-5200、5200-5250、5250-5300、5300-5350、5350-5400、5400-5450、5450-5500、5500-5550、5550-5600、5600-5650、5650-5700、5700-5750、5750-5800、5800-5850、5850-5900、5900-5950、5950-6000、6000-6050、6050-6100、6100-6150、6150-6200、6200-6250、6250-6300、6300-6350、6350-6400、6400-6450、6450-6500、6500-6550、6550-6600、6600-6650、6650-6700、6700-6750、6750-6800、6800-6850、6850-6900、6900-6950、6950-7000、7000-7050、7050-7100、7100-7150、7150-7200、7200-7250、7250-7300、7300-7350、7350-7400、7400-7450、7450-7500、7500-7550、7550-7600、7600-7650、7650-7700、7700-7750、7750-7800、7800-7850、7850-7900、7900-7950、7950-8000、8000-8050、8050-8100、8100-8150、8150-8200、8200-8250、8250-8300、8300-8350、8350-8400、8400-8450、8450-8500、8500-8550、8550-8600、8600-8650、8650-8700、8700-8750、8750-8800、8800-8850、8850-8900、8900-8950、8950-9000、9000-9050、9050-9100、9100-9150、9150-9200、9200-9250、9250-9300、9300-9350、9350-9400、9400-9450、9450-9500、9500-9550、9550-9600、9600-9650、9650-9700、9700-9750、9750-9800、9800-9850、9850-9900、9900-9950、9950-10000 on the mRNA sequence.

In some embodiments, the antisense strand has 100% complementarity to the target mRNA sequence. The antisense strand may be complementary to any portion of the target mRNA sequence.

In other embodiments, the antisense strand and the target mRNA sequence comprise at least one mismatch. As one non-limiting example, the antisense strand and the target mRNA sequence have at least 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or at least 20-30％、20-40％、20-50％、20-60％、20-70％、20-80％、20-90％、20-95％、20-99％、30-40％、30-50％、30-60％、30-70％、30-80％、30-90％、30-95％、30-99％、40-50％、40-60％、40-70％、40-80％、40-90％、40-95％、40-99％、50-60％、50-70％、50-80％、50-90％、50-95％、50-99％、60-70％、60-80％、60-90％、60-95％、60-99％、70-80％、70-90％、70-95％、70-99％、80-90％、80-95％、80-99％、90-95％、90-99％ or 95-99% complementarity.

In some embodiments, the siRNA sequence is about 10 to 50 or more nucleotides in length, i.e., each strand comprises 10 to 50 nucleotides (or nucleotide analogs). Preferably, the siRNA sequence is about 15-30, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length in each strand, wherein one of the strands is substantially complementary to the region of interest. In some embodiments, the siRNA sequence is about 19 to 25, 19 to 24, or 19 to 21 nucleotides in length.

In some embodiments, the siRNA sequence may be a synthetic RNA duplex comprising about 19 nucleotides to about 25 nucleotides and two overhanging nucleotides at the 3' end. In some aspects, the siRNA construct can be an unmodified RNA molecule. In other aspects, the siRNA construct may contain at least one modified nucleotide, such as a base, sugar, or backbone modification.

In some embodiments, the siRNA sequence may be encoded in a plasmid vector, viral vector, or other nucleic acid expression vector for delivery to a cell. The DNA expression plasmid can be used to stably express siRNA duplex or dsRNA in a cell and achieve long-term inhibition of target gene expression. In one aspect, the sense and antisense strands of an siRNA duplex are typically joined by a short spacer sequence, thereby causing expression of a stem-loop structure known as a short hairpin RNA (shRNA). The hairpin is recognized by Dicer and cleaved, thereby generating the mature siRNA construct.

In some embodiments, the sense and antisense strands of the siRNA duplex may be linked by a short spacer sequence, which may optionally be linked to additional flanking sequences, thereby causing expression of a flanking arm-stem-loop structure known as primary microrna (pri-miRNA). pri-mirnas can be recognized and cleaved by Drosha and Dicer, and thus produce mature siRNA constructs.

In some embodiments, the siRNA duplex or encoded dsRNA inhibits (or degrades) the target mRNA. Thus, the siRNA duplex or encoded dsRNA can be used to substantially inhibit gene expression in a cell. In some aspects, inhibition of gene expression refers to inhibition of at least about 20%, preferably at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30％、20-40％、20-50％、20-60％、20-70％、20-80％、20-90％、20-95％、20-100％、30-40％、30-50％、30-60％、30-70％、30-80％、30-90％、30-95％、30-100％、40-50％、40-60％、40-70％、40-80％、40-90％、40-95％、40-100％、50-60％、50-70％、50-80％、50-90％、50-95％、50-100％、60-70％、60-80％、60-90％、60-95％、60-100％、70-80％、70-90％、70-95％、70-100％、80-90％、80-95％、80-100％、90-95％、90-100％ or 95-100%. Thus, the protein product of the targeted gene may be inhibited by at least about 20%, preferably at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30％、20-40％、20-50％、20-60％、20-70％、20-80％、20-90％、20-95％、20-100％、30-40％、30-50％、30-60％、30-70％、30-80％、30-90％、30-95％、30-100％、40-50％、40-60％、40-70％、40-80％、40-90％、40-95％、40-100％、50-60％、50-70％、50-80％、50-90％、50-95％、50-100％、60-70％、60-80％、60-90％、60-95％、60-100％、70-80％、70-90％、70-95％、70-100％、80-90％、80-95％、80-100％、90-95％、90-100％ or 95-100%.

In some embodiments, the siRNA construct comprises a miRNA seed match for a target located in the guide strand. In another embodiment, the siRNA construct comprises miRNA seed matching of a target located in the passenger strand. In yet another embodiment, the gene-targeted siRNA duplex or encoded dsRNA does not comprise seed matching of a target located in the guide or passenger strand.

In some embodiments, the siRNA duplex or encoded dsRNA of the targeted gene may have little significant full-length off-target for the guide strand. In another embodiment, the siRNA duplex or encoded dsRNA of the targeted gene may have little or no significant full length off-target effect on the passenger strand. The gene-targeted siRNA duplex or encoded dsRNA may have less than 1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、11％、12％、13％、14％、15％、20％、25％、30％、35％、40％、45％、50％、1-5％、2-6％、3-7％、4-8％、5-9％、5-10％、6-10％、5-15％、5-20％、5-25％、5-30％、10-20％、10-30％、10-40％、10-50％、15-30％、15-40％、15-45％、20-40％、20-50％、25-50％、30-40％、30-50％、35-50％、40-50％、45-50％ full-length off-target for the passenger strand. In yet another embodiment, the siRNA duplex or encoded dsRNA of the targeted gene may have little significant full-length off-target for the guide strand or passenger strand. The gene-targeted siRNA duplex or encoded dsRNA may have less than 1％、2％、3％、4％、5％、6％、7％、8％、9％、10％,11％、12％、13％、14％、15％、20％、25％、30％、35％、40％、45％、50％、1-5％、2-6％、3-7％、4-8％、5-9％、5-10％、6-10％、5-15％、5-20％、5-25％、5-30％、10-20％、10-30％、10-40％、10-50％、15-30％、15-40％、15-45％、20-40％、20-50％、25-50％、30-40％、30-50％、35-50％、40-50％、45-50％ full-length off-target for the guide or passenger strand.

In some embodiments, the gene-targeted siRNA duplex or encoded dsRNA may have high in vitro activity. In another embodiment, the siRNA construct may have low in vitro activity. In yet another embodiment, the gene-targeted siRNA duplex or dsRNA may have high guide strand activity and low passenger strand activity in vitro.

In some embodiments, the siRNA construct has high in vitro guide strand activity and low in vitro passenger strand activity. Target gene Knockdown (KD) by the guide strand can be at least 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5% or 100%. Target gene knockdown by the guide strand may be 40-50％、45-50％、50-55％、50-60％、60-65％、60-70％、60-75％、60-80％、60-85％、60-90％、60-95％、60-99％、60-99.5％、60-100％、65-70％、65-75％、65-80％、65-85％、65-90％、65-95％、65-99％、65-99.5％、65-100％、70-75％、70-80％、70-85％、70-90％、70-95％、70-99％、70-99.5％、70-100％、75-80％、75-85％、75-90％、75-95％、75-99％、75-99.5％、75-100％、80-85％、80-90％、80-95％、80-99％、80-99.5％、80-100％、85-90％、85-95％、85-99％、85-99.5％、85-100％、90-95％、90-99％、90-99.5％、90-100％、95-99％、95-99.5％、95-100％、99-99.5％、99-100％ or 99.5-100%. As a non-limiting example, the target gene Knockdown (KD) by the guide strand is greater than 70%. As a non-limiting example, the target gene Knockdown (KD) by the guide strand is greater than 60%.

In some embodiments, the ratio of guide/passenger (G: P) (also referred to as antisense/sense) strand expressed in vitro or in vivo is 1:10、1:9、1:8、1:7、1:6、1:5、1:4、1:3、1:2、1;1、2:10、2:9、2:8、2:7、2:6、2:5、2:4、2:3、2:2、2:1、3:10、3:9、3:8、3:7、3:6、3:5、3:4、3:3、3:2、3:1、4:10、4:9、4:8、4:7、4:6、4:5、4:4、4:3、4:2、4:1、5:10、5:9、5:8、5:7、5:6、5:5、5:4、5:3、5:2、5:1、6:10、6:9、6:8、6:7、6:6、6:5、6:4、6:3、6:2、6:1、7:10、7:9、7:8、7:7、7:6、7:5、7:4、7:3、7:2、7:1、8:10、8:9、8:8、8:7、8:6、8:5、8:4、8:3、8:2、8:1、9:10、9:9、9:8、9:7、9:6、9:5、9:4、9:3、9:2、9:1、10:10、10:9、10:8、10:7、10:6、10:5、10:4、10:3、10:2、10:1、1:99、5:95、10:90、15:85、20:80、25:75、30:70、35:65、40:60、45:55、50:50、55:45、60:40、65:35、70:30、75:25、80:20、85:15、90:10、95:5 or 99:1. The guide/passenger ratio refers to the ratio of guide strand to passenger strand after intracellular processing of the pri-microRNA. For example, a 80:20 guide/passenger ratio would have 8 guide chains per 2 passenger chains processed from the precursor. As one non-limiting example, the in vitro guidance/passenger chain ratio is 8:2. As one non-limiting example, the in-body guidance/passenger chain ratio is 8:2. As one non-limiting example, the in vitro guidance/passenger chain ratio is 9:1. As one non-limiting example, the in-body guidance/passenger chain ratio is 9:1.

In some embodiments, the expressed guide/passenger (G: P) (also referred to as antisense/sense) strand ratio is greater than 1. In some embodiments, the expressed guide/passenger (G: P) (also referred to as antisense/sense) strand ratio is greater than 2. In some embodiments, the expressed guide/passenger (G: P) (also referred to as antisense/sense) strand ratio is greater than 5. In some embodiments, the expressed guide/passenger (G: P) (also referred to as antisense/sense) strand ratio is greater than 10. In some embodiments, the expressed guide/passenger (G: P) (also referred to as antisense/sense) strand ratio is greater than 20. In some embodiments, the expressed guide/passenger (G: P) (also referred to as antisense/sense) strand ratio is greater than 50. In some embodiments, the expressed guide/passenger (G: P) (also referred to as antisense/sense) strand ratio is at least 3:1. In some embodiments, the expressed guide/passenger (G: P) (also referred to as antisense/sense) strand ratio is at least 5:1. In some embodiments, the expressed guide/passenger (G: P) (also referred to as antisense/sense) strand ratio is at least 10:1. In some embodiments, the expressed guide/passenger (G: P) (also referred to as antisense/sense) strand ratio is at least 20:1. In some embodiments, the expressed guide/passenger (G: P) (also referred to as antisense/sense) strand ratio is at least 50:1.

In some embodiments, the passenger/guide (P: G) (also referred to as sense/antisense) strand ratio expressed in vitro or in vivo is 1:10、1:9、1:8、1:7、1:6、1:5、1:4、1:3、1:2、1;1、2:10、2:9、2:8、2:7、2:6、2:5、2:4、2:3、2:2、2:1、3:10、3:9、3:8、3:7、3:6、3:5、3:4、3:3、3:2、3:1、4:10、4:9、4:8、4:7、4:6、4:5、4:4、4:3、4:2、4:1、5:10、5:9、5:8、5:7、5:6、5:5、5:4、5:3、5:2、5:1、6:10、6:9、6:8、6:7、6:6、6:5、6:4、6:3、6:2、6:1、7:10、7:9、7:8、7:7、7:6、7:5、7:4、7:3、7:2、7:1、8:10、8:9、8:8、8:7、8:6、8:5、8:4、8:3、8:2、8:1、9:10、9:9、9:8、9:7、9:6、9:5、9:4、9:3、9:2、9:1、10:10、10:9、10:8、10:7、10:6、10:5、10:4、10:3、10:2、10:1、1:99、5:95、10:90、15:85、20:80、25:75、30:70、35:65、40:60、45:55、50:50、55:45、60:40、65:35、70:30、75:25、80:20、85:15、90:10、95:5 or 99:1. The passenger/guidance ratio refers to the ratio of passenger strand to guidance strand after cutting the guidance strand. For example, an 80:20 passenger/guidance ratio would have 8 passenger chains per 2 guidance chains processed from the precursor. As one non-limiting example, the in vitro passenger/guide chain ratio is 80:20. As one non-limiting example, the in-vivo passenger/guide chain ratio is 80:20. As one non-limiting example, the in vitro passenger/guide chain ratio is 8:2. As one non-limiting example, the in-vivo passenger/guide chain ratio is 8:2. As one non-limiting example, the in vitro passenger/guide chain ratio is 9:1. As one non-limiting example, the in-vivo passenger/guide chain ratio is 9:1.

In some embodiments, the expressed passenger/guide (P: G) (also referred to as sense/antisense) strand ratio is greater than 1. In some embodiments, the expressed passenger/guide (P: G) (also referred to as sense/antisense) strand ratio is greater than 2. In some embodiments, the expressed passenger/guide (P: G) (also referred to as sense/antisense) strand ratio is greater than 5. In some embodiments, the expressed passenger/guide (P: G) (also referred to as sense/antisense) strand ratio is greater than 10. In some embodiments, the expressed passenger/guide (P: G) (also referred to as sense/antisense) strand ratio is greater than 20. In some embodiments, the expressed passenger/guide (P: G) (also referred to as sense/antisense) strand ratio is greater than 50. In some embodiments, the expressed passenger/guide (P: G) (also referred to as sense/antisense) strand ratio is at least 3:1. In some embodiments, the expressed passenger/guide (P: G) (also referred to as sense/antisense) strand ratio is at least 5:1. In some embodiments, the expressed passenger/guide (P: G) (also referred to as sense/antisense) strand ratio is at least 10:1. In some embodiments, the expressed passenger/guide (P: G) (also referred to as sense/antisense) strand ratio is at least 20:1. In some embodiments, the expressed passenger/guide (P: G) (also referred to as sense/antisense) strand ratio is at least 50:1.

In some embodiments, a passenger-guide strand duplex is considered effective when the pri-or pre-microRNA but the methods known in the art and described herein exhibit a guide/passenger strand ratio greater than 2-fold at the time of measurement processing. As one non-limiting example, when measured for processing, the pri-or pre-microRNA exhibits a guide/passenger strand ratio of greater than 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, or 2-to 5-fold, 2-to 10-fold, 2-to 15-fold, 3-to 5-fold, 3-to 10-fold, 3-to 15-fold, 4-to 5-fold, 4-to 10-fold, 4-to 15-fold, 5-to 10-fold, 5-to 15-fold, 6-to 10-fold, 6-to 15-fold, 7-to 10-fold, 7-to 15-fold, 8-to 10-fold, 8-to 15-fold, 9-to 10-fold, 9-to 15-fold, 10-to 15-fold, 11-to 15-fold, 12-to 15-fold, 13-to 15-fold, or 14-to 15-fold.

In some embodiments, the vector genome encoding the dsRNA comprises a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more than 99% of the full length of the construct. As one non-limiting example, the vector genome comprises a sequence that is at least 80% of the full-length sequence of the construct.

In some embodiments, the siRNA constructs can be used to silence a wild-type or mutant gene by targeting at least one exon on the sequence.

SiRNA modification

In some embodiments, the siRNA construct, when not delivered in precursor or DNA form, can be chemically modified to modulate some feature of the RNA molecule, such as, but not limited to, increasing the stability of the siRNA in vivo. Chemically modified siRNA constructs are useful for human therapeutic applications and are improved without compromising the RNAi activity of the siRNA construct. As a non-limiting example, siRNA constructs are modified at both the 3 'and 5' ends of the sense and antisense strands.

In some embodiments, the modified nucleotide may be on the sense strand only.

In some embodiments, the modified nucleotide may be on the antisense strand only.

In some embodiments, the modified nucleotide may be in both the sense and antisense strands.

In some embodiments, the chemically modified nucleotide does not affect the ability of the antisense strand to pair with the target mRNA sequence.

MicroRNA (miR) backbone

In some embodiments, the siRNA construct may be encoded in a polynucleotide sequence that also comprises a microrna (miR) backbone construct. As used herein, a "microrna (miR) backbone construct" is a framework or starting molecule that forms the basis of a sequence or structure for designing or manufacturing a subsequent molecule.

In some embodiments, the miR scaffold construct comprises at least one 5' flanking region. As one non-limiting example, the 5 'flanking region may comprise a 5' flanking sequence that may be of any length and may be derived entirely or in part from a wild-type microrna sequence or entirely artificial sequence.

In some embodiments, the miR scaffold construct comprises at least one 3' flanking region. As one non-limiting example, the 3 'flanking region may comprise a 3' flanking sequence that may be of any length and may be derived entirely or in part from a wild-type microrna sequence or entirely artificial sequence.

In some embodiments, the miR scaffold construct comprises at least one loop motif region. As one non-limiting example, a loop motif region can comprise a sequence that can have any length.

In some embodiments, the miR scaffold construct comprises a 5 'flanking region, a loop motif region, and/or a 3' flanking region.

In some embodiments, at least one payload (e.g., an siRNA, miRNA, or other RNAi agent described herein) can be encoded by a polynucleotide that can also comprise at least one miR backbone construct. The miR backbone construct can comprise 5' flanking sequences that can be of any length and can be derived entirely or partially from wild-type microrna sequences or entirely artificial. The 3' flanking sequences may reflect the 5' flanking sequences and/or the 3' flanking sequences in terms of size and origin. Either flanking sequence may not be present. The 3' flanking sequence may optionally contain one or more CNNC motifs, wherein "N" represents any nucleotide.

In some embodiments, the 5' arm of the stem-loop structure of a polynucleotide comprising or encoding a miR backbone construct comprises a sequence encoding a sense sequence.

In some embodiments, the 3' arm of the stem loop of a polynucleotide comprising or encoding a miR backbone construct comprises a sequence encoding an antisense sequence. In some cases, the antisense sequence comprises a "G" nucleotide at the 5' -most end.

In some embodiments, the sense sequence can reside on the 3 'arm of the stem-loop structure of a polynucleotide comprising or encoding a miR backbone construct, while the antisense sequence resides on its 5' arm.

In some embodiments, the sense and antisense sequences can be fully complementary over a majority of their length. In other embodiments, the sense and antisense sequences may have at least 70%, 80%, 90%, 95% or 99% complementarity in at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% of the length of the strand independently.

Neither the identity of the sense sequence nor the identity of the antisense sequence need be 100% complementary to the target sequence.

In some embodiments, separating the sense and antisense sequences of the stem-loop structure of the polynucleotide is a loop sequence (also referred to as a loop motif, linker, or linker motif). The loop sequence may have any length: between 4 and 30 nucleotides, between 4 and 20 nucleotides, between 4 and 15 nucleotides, between 5 and 15 nucleotides, between 6 and 12 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides and/or 15 nucleotides.

In some embodiments, the loop sequence comprises a nucleic acid sequence encoding at least one UGUG motif. In some embodiments, the nucleic acid sequence encoding the UGUG motif is located at the 5' end of the loop sequence.

In some embodiments, spacer regions can be present in the polynucleotide to separate one or more modules (e.g., 5 'flanking regions, loop motif regions, 3' flanking regions, sense sequences, antisense sequences) from each other. One or more such spacer regions may be present.

In some embodiments, spacer regions having between 8-20, i.e., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides may be present between the sense sequence and the flanking region sequences.

In some embodiments, the spacer region is 13 nucleotides in length and is located between the 5 'end of the sense sequence and the 3' end of the flanking sequence. In some embodiments, the spacer has a length sufficient to form approximately one helical turn of the sequence.

In some embodiments, a spacer region having between 8-20, i.e., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides may be present between the antisense sequence and the flanking sequence.

In some embodiments, the spacer sequence is between 10-13, i.e., 10, 11, 12, or 13 nucleotides, and is located between the 3 'end of the antisense sequence and the 5' end of the flanking sequence. In some embodiments, the spacer has a length sufficient to form approximately one helical turn of the sequence.

In some embodiments, the polynucleotide comprises a 5 'flanking sequence, a 5' arm, a loop motif, a 3 'arm, and a 3' flanking sequence in the 5 'to 3' direction. As one non-limiting example, the 5 'arm may comprise a sense sequence and the 3' arm comprises an antisense sequence. In another non-limiting example, the 5 'arm comprises an antisense sequence and the 3' arm comprises a sense sequence.

In some embodiments, the 5 'arm, payload (e.g., sense and/or antisense sequences), loop motif, and/or 3' arm sequence may be altered (e.g., by substitution of 1 or more nucleotides, addition of nucleotides, and/or deletion of nucleotides). Alterations may cause beneficial changes in the function of the construct (e.g., increased gene knockdown of the target sequence, reduced degradation of the construct, reduced off-target effects, increased efficiency of the payload, and reduced degradation of the payload).

In some embodiments, the miR backbone constructs of the polynucleotides are aligned such that the excision rate of the guide strand is greater than the excision rate of the passenger strand. The removal rate of the guide or passenger strand may be independently 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more than 99%. As a non-limiting example, the guide strand removal rate is at least 80%. As another non-limiting example, the guide strand excision rate is at least 90%.

In some embodiments, the guide strand is cut at a rate greater than the passenger strand. In one aspect, the guide strand may have a cut rate that is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more than 99% greater than the passenger strand.

In some embodiments, the guide strand excision efficiency is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more than 99%. As a non-limiting example, the guide strand excision efficiency is greater than 80%.

In some embodiments, the efficiency of excision of the guide strand is greater than the excision of the passenger strand from the miR backbone construct. The excision of the guide strand can be 2, 3, 4, 5, 6,7, 8, 9, 10, or more than 10-fold more efficient than the excision of the passenger strand from the miR backbone construct.

In some embodiments, the miR scaffold construct comprises a bifunctional targeting polynucleotide. As used herein, a "bifunctional targeting" polynucleotide is a polynucleotide that knocks down the same target for both the guide strand and the passenger strand or knockdown different targets for both the guide strand and the passenger strand.

In some embodiments, miR backbone constructs of polynucleotides described herein can comprise a 5 'flanking region, a loop motif region, and a 3' flanking region.

In some embodiments, the polynucleotide is designed using at least one of the following properties: loop variants, seed mismatch/carina/wobble variants, stem mismatch, loop and accessory (vassal) stem mismatch variants, seed mismatch and basal stem (basal stem) mismatch variants, stem mismatch and basal stem mismatch variants, seed wobble and basal stem wobble variants or stem sequence variants.

In some embodiments, the miR scaffold construct may be a native pri-miRNA scaffold.

In some embodiments, selection of the miR backbone construct is determined by a method of comparing polynucleotides in the pri-miRNA.

In some embodiments, selection of the miR scaffold construct is determined by comparing polynucleotides in natural pri-mirnas to synthetic pri-mirnas.

Transfer RNA (tRNA)

Transfer RNA (tRNA) is an RNA molecule that translates mRNA into protein. tRNA includes clover structures comprising a 3 'acceptor site, a 5' terminal phosphate, a D-arm, a T-arm, and an anticodon arm. the primary purpose of tRNA is to carry an amino acid at its 3' acceptor site to the ribosomal complex by means of an aminoacyl-tRNA synthetase (an enzyme that loads the appropriate amino acid onto the free tRNA to synthesize a protein). Once the amino acid binds to the tRNA, the tRNA is considered an aminoacyl-tRNA. the type of amino acid on the tRNA depends on the mRNA codon. the anticodon arm of a tRNA is the site of an anticodon that is complementary to the mRNA codon and specifies the amino acid to be carried. tRNA's are also known to have a role in regulating apoptosis by acting as cytochrome c scavengers.

In some embodiments, the initial construct and/or the reference construct comprises or encodes a tRNA.

Ribosomal RNA (rRNA)

Ribosomal RNA (rRNA) is RNA that forms a ribosome. Ribosomes are essential for protein synthesis and contain large and small ribosomal subunits. In prokaryotes, small 30S and large 50S ribosomal subunits constitute the 70S ribosomes. In eukaryotes, 40S and 60S subunits form 80S ribosomes. To bind aminoacyl-trnas and link amino acids together to produce a polypeptide, the ribosome contains 3 sites: leaving site (E), peptidyl site (P) and acceptor site (A).

In some embodiments, the initial construct and/or the reference construct comprises or encodes rRNA.

Micro RNA (miRNA)

Micrornas (or mirnas) are 19-25 nucleotide long non-coding RNAs that bind to the 3' utr of a nucleic acid molecule and down-regulate gene expression by either reducing nucleic acid molecule stability or inhibiting translation. The initial construct and/or the reference construct may comprise one or more microrna target sequences, microrna sequences, or microrna seeds.

The microrna sequence comprises a "seed" region, i.e., a sequence in the region of positions 2-8 of the mature microrna that has perfect Watson-Crick complementarity to the miRNA target sequence. The microRNA seed can comprise positions 2-8 or 2-7 of the mature microRNA. In some embodiments, the microrna seed can comprise 7 nucleotides (e.g., nucleotides 2-8 of the mature microrna), wherein the seed complementary site in the corresponding miRNA target is flanked by adenine (a) opposite microrna position 1. In some embodiments, the microrna seed can comprise 6 nucleotides (e.g., nucleotides 2-7 of the mature microrna), wherein the seed complementary site in the corresponding miRNA target is flanked by adenine (a) opposite microrna position 1. The bases of the microRNA seed are fully complementary to the target sequence. By engineering the microrna target sequence into the 3' utr of the mRNA, the molecule can be targeted for degradation or reduced translation, provided that the microrna in question is available. This process will reduce the risk of off-target effects upon delivery of the nucleic acid molecule.

As used herein, the term "microrna site" refers to a microrna target site or microrna recognition site, or any nucleotide sequence that binds to or associates with a microrna. It will be appreciated that "binding" may follow conventional Watson-Crick hybridization rules, or may reflect any stable association of the microRNA with the target sequence at or near the microRNA site.

Non-limiting examples of tissues known to have microRNAs modulating mRNA, and thus protein expression, include, but are not limited to, liver (miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells (miR-17-92, miR-126), bone marrow cells (miR-142-3 p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30 c), heart (miR-1 d, miR-149), kidney (miR-192, miR-194, miR-204) and lung epithelial cells (let-7, miR-133, miR-126). Micrornas can also modulate complex biological processes such as angiogenesis (miR-132).

For example, if the nucleic acid molecule is an mRNA and is not intended to be delivered to the liver, but eventually reaches there, miR-122 (a microrna abundant in the liver) can inhibit expression of the gene of interest when one or more sites of interest of miR-122 are engineered into the 3' utr of the mRNA. One or more binding sites for the introduction of different micrornas can be engineered to further reduce the lifetime, stability, and protein translation of the mRNA.

Instead, microrna binding sites can be engineered outside (i.e., removed from) the sequence in which they naturally occur in order to increase protein expression in a particular tissue. For example, the miR-122 binding site can be deleted to increase protein expression in the liver. Modulation of expression in multiple tissues may be achieved via the introduction or removal of one or several microrna binding sites.

Long non-coding RNA (lncRNA)

Long non-coding RNAs (lncrnas) are regulatory RNA molecules that do not code for proteins but affect a large number of biological processes. lncRNA names are generally limited to non-coding transcripts longer than about 200 nucleotides. Length designations distinguish lncRNA from small regulatory RNAs, such as short interfering RNAs (sirnas) and micrornas (mirnas). In vertebrates, the number of lncRNA species is believed to greatly exceed the number of protein-encoding species. It is also believed that lncRNA drives the biological complexity observed in vertebrates compared to invertebrates. Evidence of this complexity can be found in many cellular compartments of vertebrate organisms, such as the T lymphocyte compartment of the adaptive immune system. Differences in expression and function of lncRNA may be a major cause of human disease.

In some embodiments, the initial construct and/or the reference construct comprises lncRNA.

RNA modification

In some aspects, the initial construct or reference construct may contain one or more modified nucleotides, such as, but not limited to sugar modified nucleotides, nucleobase modifications, and/or backbone modifications. In some aspects, the initial construct or the reference construct may contain combined modifications, e.g., combined nucleobase and backbone modifications.

In some embodiments, the modified nucleotide may be a sugar modified nucleotide. Sugar modified nucleotides include, but are not limited to, 2 '-fluoro, 2' -amino, and 2 '-thio modified ribonucleotides, such as 2' -fluoro modified ribonucleotides. The modified nucleotide may be modified on the sugar moiety, as may a nucleotide having a sugar or an analog thereof that is not ribosyl. For example, the sugar moiety may be or be based on mannose, arabinose, glucopyranose, galactopyranose, 4' -thioribose, and other sugars, heterocycles or carbocycles.

In some embodiments, the modified nucleotide may be a nucleobase modified nucleotide.

In some embodiments, the modified nucleotide may be a backbone modified nucleotide. In some embodiments, the initial construct or reference construct may also comprise other modifications in the backbone. As used herein, the term "backbone" refers to repeated alternating sugar-phosphate sequences in a DNA or RNA molecule. Deoxyribose/ribose is attached to a phosphate group at the 3 '-hydroxyl and 5' -hydroxyl groups with ester linkages, also known as "phosphodiester" linkages/linkers (PO linkages). The PO backbone may be modified to a "phosphorothioate backbone (PS linkage)". In some cases, the natural phosphodiester linkage may be replaced with an amide linkage, but with four atoms between the two saccharide units maintained. Such amide modifications can facilitate solid phase synthesis of the oligonucleotide and increase the thermodynamic stability of the duplex formed with the siRNA complementary sequence.

Modified bases refer to nucleotide bases such as, but not limited to, adenine, guanine, cytosine, thymine, uracil, xanthine, inosine, and Q nucleoside that have been modified by substitution or addition of one or more atoms or groups. Some examples of modifications on nucleobase moieties include, but are not limited to, alkylated, halogenated, thiolated, aminated, amidated, or acetylated bases, alone or in combination. More specific examples include, for example, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N, -dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides with modifications at the 5-position, 5- (2-amino) propyluridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenine, 2-methyladenine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2-dimethylguanosine, 5-methylaminoethyl uridine, 5-methoxyuridine, deammonikonucleotides (e.g. 7-deaza-adenosine, 6-azo uridine, 6-azo cytidine), 5-methyl-2-thiouridine, other thiobases (e.g. 2-thiouridine and 4-thiouridine and 2-thiocytidine), dihydrouridine, pseudouridine, Q-nucleoside, purinyl and naphtalenyl and substituted 5-methoxy uridine (e.g. 5-oxo-pyridone, N-methyl-5-pyridone, N-oxo-5-oxo-pyridone) and any of 5-methylguanosine, phenyl and modified phenyl (e.g., aminophenol or 2,4, 6-trimethoxybenzene), modified cytosine (which acts as a G-clamp), 8-substituted adenine and guanine, 5-substituted uracil and thymine, azapyrimidine, carboxyhydroxyalkyl nucleotides, carboxyalkylamino nucleotides and alkylcarbonylalkylated nucleotides.

Included within the scope of the present disclosure are initial constructs and/or reference constructs that may include one or more substitutions, insertions and/or additions, deletions, and covalent modifications relative to a reference sequence (in particular, the parent RNA).

In some embodiments, the initial construct and/or the reference construct includes one or more post-transcriptional modifications (e.g., capping, cleavage, polyadenylation, splicing, poly a sequence, methylation, acylation, phosphorylation, methylation of lysine and arginine residues, acetylation, and nitrosylation of thiol and tyrosine residues, etc.). The one or more post-transcriptional modifications may be any post-transcriptional modification, such as any of more than one hundred different nucleoside modifications that have been identified in RNA (Rozenski, J, crain, P and McCloskey, J. (1999): the RNA Modification Database:1999update.Nucl Acids Res 27:196-197). In some embodiments, the first isolated nucleic acid comprises messenger RNA (mRNA). In some embodiments, the initial construct and/or the reference construct comprises at least one nucleoside selected from the group consisting of: pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl uridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine and 4-methoxy-2-thio-pseudouridine. In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of: 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, zebulin, 5-aza-zebulin, 5-methyl-zebulin, 5-aza-2-thio-zebulin, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine. in some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of: 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1-methyladenosine, N6-isopentenyl adenosine, N6- (cis-hydroxyisopentenyl) adenosine, 2-methylsulfanyl-N6- (cis-hydroxyisopentenyl) adenosine, N6-glycylcarbamoyladenosine, N6-threonyl carbamoyl adenosine, 2-methylsulfanyl-N6-threonyl carbamoyl adenosine, N6-dimethyl adenosine, 7-methyladenine, 2-methylsulfanyl-adenine and 2-methoxy-adenine. in some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of: inosine, 1-methyl-inosine, hurusoside, hurustin, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-7-deaza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine, N2-dimethyl-guanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, n2-methyl-6-thio-guanosine and N2, N2-dimethyl-6-thio-guanosine.

The initial construct and/or the reference construct may include, for example, any suitable modification of a sugar, nucleobase, or internucleoside linkage (e.g., linking a phosphate/phosphodiester linkage/phosphodiester backbone). One or more atoms of the pyrimidine nucleobase may be replaced or substituted with an optionally substituted amino group, an optionally substituted thiol, an optionally substituted alkyl group (e.g., methyl or ethyl) or a halo group (e.g., chloro or fluoro). In certain embodiments, modifications (e.g., one or more modifications) are present in each of the sugar and internucleoside linkages. The modification may be a ribonucleic acid (RNA) modification to deoxyribonucleic acid (DNA), threose Nucleic Acid (TNA), glycol Nucleic Acid (GNA), peptide Nucleic Acid (PNA), locked Nucleic Acid (LNA) or hybrids thereof. Additional modifications are described herein.

In some embodiments, the initial construct and/or the reference construct includes at least one N (6) methyl adenosine (m 6A) modification that increases translational efficiency. In some embodiments, the N (6) methyl adenosine (m 6A) modification can reduce the immunogenicity of the initial construct and/or the reference construct.

In some embodiments, the modification may include a chemical or cell-induced modification. For example, some non-limiting examples of intracellular RNA modifications are described by Lewis and Pan in "RNA modifications and structures cooperate to guide RNA-protein interactions",Nat.Reviews Mol.Cell Biol.,2017,18:202-210.

In some embodiments, chemical modification of RNA can enhance immune evasion. RNA can be synthesized and/or modified by methods well established in the art, such as those described in the following: "Current protocols in nucleic ACID CHEMISTRY", beaucage, s.l. et al (ed.), john Wiley & Sons, inc., new York, n.y., USA, incorporated herein by reference. Modifications include, for example, terminal modifications such as 5 'terminal modifications (phosphorylated (mono-, di-, and tri), conjugated, reverse-bonded, etc.), 3' terminal modifications (conjugated, DNA nucleotides, reverse-bonded, etc.), base modifications (e.g., substitution with a stabilized base, an unstable base, or a base with an enlarged base pair of a partner), abasic (abasic nucleotide), or conjugated bases. Modified ribonucleotide bases can also include 5-methylcytidine and pseudouridine. In some embodiments, the base modification may modulate RNA expression, immune response, stability, subcellular localization, to name a few functional roles. In some embodiments, the modification comprises a biorthogonal nucleotide, such as a non-natural base. See, for example, kimoto et al, chem Commun (Camb), 2017,53:12309, DOI:10.1039/c7cc06661a, which is incorporated herein by reference.

In some embodiments, sugar modifications (e.g., at the 2 'or 4' positions) or sugar substitutions as well as backbone modifications of one or more RNAs may include phosphodiester-linked modifications or substitutions. Specific examples of modifications include modified backbones or non-natural internucleoside linkages, such as internucleoside modifications, including modifications or substitutions of phosphodiester linkages. RNA having a modified backbone includes, inter alia, those having no phosphorus atoms in the backbone. For the purposes of the present application, and as sometimes referred to in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered oligonucleotides. In particular embodiments, the RNA will include ribonucleotides with a phosphorus atom in their internucleoside backbone.

Modified RNA backbones can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkylphosphonates (e.g., 3 '-alkylene phosphonates), and chiral phosphonates, phosphonites, phosphoramidates (e.g., 3' -phosphoramidates and aminoalkyl phosphoramidates), thiocarbonyl phosphoramidates, thiocarbonylalkyl phosphonates, thiocarbonylalkyl phosphotriesters, and borane phosphates with normal 3'-5' linkages, 2'-5' linked analogs of these, and those with reversed polarity (where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5 '-2'). Also included are various salts, mixed salts and free acid forms. In some embodiments, the RNA may be negatively or positively charged.

The modified nucleotides may be modified on internucleoside linkages (e.g., phosphate backbones). Herein, the phrases "phosphate" and "phosphodiester" are used interchangeably in the context of a polynucleotide backbone. The backbone phosphate group may be modified by replacing one or more oxygen atoms with different substituents. In addition, modified nucleosides and nucleotides can include batch substitution of the unmodified phosphate moiety with another internucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioates, phosphoroselenos, boranophosphates (boranophosphates), boranophosphates (boranophosphate ester), hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Both non-linking oxygens of the dithiophosphate are replaced by sulfur. Phosphate linkers can also be modified by replacing the linking oxygen with nitrogen (bridged amino phosphate), sulfur (bridged phosphorothioate), and carbon (bridged methylene-phosphonate).

The a-thio substituted phosphate moieties are provided to impart stability to RNA and DNA polymers via non-natural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently have a longer half-life in the cellular environment. Phosphorothioates linked to RNA are expected to reduce the innate immune response via weaker binding/activation of cellular innate immune molecules.

In particular embodiments, the modified nucleoside comprises an α -thio-nucleoside (e.g., 5' -O- (1-phosphorothioate) -adenosine, 5' -O- (1-phosphorothioate) -cytidine (a-thio-cytidine), 5' -O- (1-phosphorothioate) -guanosine, 5' -O- (1-phosphorothioate) -uridine, or 5' -O- (1-phosphorothioate) -pseudouridine).

Other internucleoside linkages, including internucleoside linkages that do not contain a phosphorus atom, that can be employed in accordance with the present disclosure are described herein.

In some embodiments, the RNA can include one or more cytotoxic nucleosides. For example, a cytotoxic nucleoside may be incorporated into an RNA, such as a bifunctional modification. Cytotoxic nucleosides can include, but are not limited to, arabinoside, 5-azacytidine, 4' -thio-arabinoside, cyclopentylcytosine, cladribine (cladribine), clofarabine (clofarabine), cytarabine, cytosine arabinoside, 1- (2-C-cyano-2-deoxy- β -D-arabino-pentofuranosyl) -cytosine, decitabine (decitabine), 5-fluorouracil (5-fluoroucil), fludarabine (fludarabine), fluorouridine (floxuridine), gemcitabine (gemcitabine), tegafur (tegafur) in combination with uracil, tegafur ((RS) -5-fluoro-1- (tetrahydrofuran-2-yl) pyrimidine-2, 4 (1 h,3 h) -dione), troxacitabine (xacitabine), tizacitabine (tezacitabine), 2' -deoxy-2 ' -methylene cytidine (DMDC), and 6-mercapto (mercaptopurine). Additional examples include fludarabine phosphate (fludarabine phosphate), N4-behenoyl-1- β -D-arabinofuranosyl cytosine, N4-octadecyl-1- β -D-arabinofuranosyl cytosine, N4-palmitoyl-1- (2-C-cyano-2-deoxy- β -D-arabino-pentofuranosyl) cytosine, and P-4055 (cytarabine 5' -elaidite).

In some embodiments, the RNA sequence includes or comprises a natural nucleoside (e.g., adenosine, guanosine, cytidine, uridine), nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deadenosine, 7-deazaguanosine, 8-oxo-guanosine, O (6) -methylguanosine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), inserted bases, modified sugars (e.g., 2 '-fluororibose, ribose, 2' -deoxyribose, arabinose, and hexose), and/or modified phosphate groups (e.g., N-phosphorothioamide). In one embodiment, the RNA sequence comprises or comprises the incorporation of pseudouridine (y). In another embodiment, the RNA sequence comprises or comprises 5-methylcytosine (m 5C).

The RNA may be modified uniformly along the entire length of the molecule or may be modified unevenly. For example, one or more or all types of nucleotides (e.g., naturally occurring nucleotides, purines or pyrimidines, or any or more or all of A, G, U, C, I, pU) may be modified uniformly or non-uniformly in the RNA or in a given predetermined sequence region thereof. In some embodiments, the RNA comprises pseudouridine. In some embodiments, the RNA includes inosine, which may help the immune system to characterize the RNA as endogenous RNA and viral RNA. The incorporation of inosine may also mediate improved RNA stability/reduced degradation.

In some embodiments, all nucleotides in the RNA (or a given sequence region thereof) are modified. In some embodiments, modifications may include m6A (which may increase expression), inosine (which may attenuate immune responses), pseudouridine (which may increase RNA stability or translational readthrough (staggered elements)), m5C (which may increase stability), and 2, 7-trimethylguanosine (which assists subcellular translocation (e.g., nuclear localization)).

Different sugar modifications, nucleotide modifications, and/or internucleoside linkages (e.g., backbone structures) may be present at multiple positions in the RNA. It will be appreciated by those of ordinary skill in the art that nucleotide analogs or other modifications may be located at any position of the RNA such that the function of the RNA is not substantially reduced. The modification may also be a non-coding region modification. The RNA can include about 1% to about 100% modified nucleotides (relative to the total nucleotide content, or relative to any one or more of the types of nucleotides, i.e., A, G, U or C) or any intermediate percentage (e.g., 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 80%, 80% to 95%, and 80% to 95% to 100%).

Codon optimization

The nucleotide sequences of the initial construct and/or the reference construct may be codon optimized. Codon optimization methods are known in the art and may be adapted to strive to achieve one or more of several goals. These targets include matching codon frequencies in the target organism and the host organism to ensure proper folding; deviation from GC content to increase mRNA stability or decrease secondary structure; minimizing tandem repeat codon or base runs that may impair gene construction or expression; custom transcription and translation control regions; inserting or removing protein transport sequences; removal/addition of post-translational modification sites (e.g., glycosylation sites) in the encoded protein; adding, removing or shuffling protein domains; insertion or deletion of restriction sites; modifying the ribosome binding site and the mRNA degradation site; regulating the rate of translation to allow proper folding of the individual domains of the protein; or to reduce or eliminate problematic secondary structures within the mRNA. Codon optimization tools, algorithms and services are known in the art, non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and/or proprietary methods. In some embodiments, the ORF sequence is optimized using an optimization algorithm.

III. lipids

The present disclosure provides ionizable lipids that exhibit high efficacy in related tissues as well as low toxicity, low sustained lipid levels, and for local delivery to various tissues. The ionizable lipid may be a cationic lipid.

(CY)

The present disclosure provides compounds of formula (CY)

Or a pharmaceutically acceptable salt thereof,

Wherein:

R ¹ is selected from the group consisting of: -OH, -OAc, R ^1a,

Z ¹ is optionally substituted C ₁-C₆ alkyl;

X ¹ is optionally substituted C ₂-C₆ alkylene;

x ² is selected from the group consisting of bond, -CH ₂ -, and-CH ₂CH₂ -;

X ²' is selected from the group consisting of a bond, -CH ₂ -, and-CH ₂CH₂ -;

X ³ is selected from the group consisting of bond, -CH ₂ -, and-CH ₂CH₂ -;

X ³' is selected from the group consisting of a bond, -CH ₂ -, and-CH ₂CH₂ -;

X ⁴ and X ⁵ are independently optionally substituted C ₂-C₁₄ alkylene or optionally substituted C ₂-C₁₄ alkenylene;

y ¹ and Y ² are independently selected from the group consisting of:

Wherein the bond labeled with "+" is attached to X ⁴ or X ⁵;

Each Z ² is independently H or optionally substituted C ₁-C₈ alkyl;

Each Z ³ is independently optionally substituted C ₁-C₆ alkylene;

R ² is selected from the group consisting of optionally substituted C ₄-C₂₀ alkyl, optionally substituted C ₂-C₁₄ alkenyl, and- (CH ₂)_pCH(OR⁶)(OR⁷);

r ³ is selected from the group consisting of optionally substituted C ₄-C₂₀ alkyl, optionally substituted C ₂-C₁₄ alkenyl, or- (CH ₂)_qCH(OR⁸)(OR⁹);

R ^1a is:

R ^2a、R^2b and R ^2c are independently hydrogen and C ₁-C₆ alkyl;

r ^3a、R^3b and R ^3c are independently hydrogen and C ₁-C₆ alkyl;

R ^4a、R^4b and R ^4c are independently hydrogen and C ₁-C₆ alkyl;

R ^5a、R^5b and R ^5c are independently hydrogen and C ₁-C₆ alkyl;

R ⁶、R⁷、R⁸ and R ⁹ are independently optionally substituted C ₁-C₁₄ alkyl, optionally substituted C ₂-C₁₄ alkenyl or- (CH ₂)_m-A-(CH₂)_n H;

Each a is independently C ₃-C₈ cycloalkylene;

Each m is independently 0, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, or 12;

Each n is independently 0, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, or 12;

p is selected from the group consisting of 0,1,2,3,4,5,6 and 7; and

Q is selected from the group consisting of 0,1, 2, 3, 4, 5, 6 and 7.

In some embodiments, the present disclosure includes compounds of formula (CY-I), (CY-II), (CY-III), (CY-IV), or (CY-V):

Or a pharmaceutically acceptable salt thereof,

Wherein X¹、X²、X²'、X³、X³'、X⁴、X⁵、Y¹、Y²、R¹、R² and R ³ are defined herein.

In some embodiments, the present disclosure includes compounds of formula (CY-VI) or (CY-VII):

Or a pharmaceutically acceptable salt thereof,

Wherein X ¹、X⁴、X⁵、R¹、R² and R ³ are defined herein.

In some embodiments, the present disclosure includes compounds of formula (CY-VIII) or (CY-IX):

Or a pharmaceutically acceptable salt thereof.

Wherein X ¹、X⁴、X⁵、R¹、R² and R ³ are defined herein.

In some embodiments, the present disclosure includes compounds of formula (CY-IV-a), (CY-IV-b), or (CY-IV-c)

Or a pharmaceutically acceptable salt thereof.

Wherein X ¹、X⁴、X⁵、R² and R ³ are defined herein.

In some embodiments, the present disclosure includes compounds of formula (CY-IV-d), (CY-IV-e), or (CY-IV-f)

Or a pharmaceutically acceptable salt thereof.

Wherein X ¹、X⁴、X⁵、R² and R ³ are defined herein.

R¹

In some embodiments, R ¹ is selected from the group consisting of-OH, -OAc, R ^1a, A group of groups. In some embodiments, R ¹ is-OH or-OAc. In some embodiments, R ¹ is OH. In some embodiments, R ¹ is —oac. In some embodiments, R ¹ is R ^1a. In some embodiments, R ¹ is imidazolyl. In some embodiments, R ¹ is

R²

In some embodiments, R ² is selected from the group consisting of optionally substituted C ₄-C₂₀ alkyl, optionally substituted C ₂-C₁₄ alkenyl, and- (CH ₂)_pCH(OR⁶)(OR⁷).

In some embodiments, R ² is optionally substituted C ₄-C₂₀ alkyl. In some embodiments, R ² is optionally substituted C ₈-C₁₇ alkyl. In some embodiments, R ² is optionally substituted C ₉-C₁₆ alkyl. In some embodiments, R ² is optionally substituted C ₈-C₁₀ alkyl. In some embodiments, R ² is optionally substituted C ₁₁-C₁₃ alkyl. In some embodiments, R ² is optionally substituted C ₁₄-C₁₆ alkyl. In some embodiments, R ² is optionally substituted C ₉ alkyl. In some embodiments, R ² is optionally substituted C ₁₀ alkyl. In some embodiments, R ² is optionally substituted C ₁₁ alkyl. In some embodiments, R ² is optionally substituted C ₁₂ alkyl. in some embodiments, R ² is optionally substituted C ₁₃ alkyl. In some embodiments, R ² is optionally substituted C ₁₄ alkyl. in some embodiments, R ² is optionally substituted C ₁₅ alkyl. In some embodiments, R ² is optionally substituted C ₁₆ alkyl.

In some embodiments, R ² is optionally substituted C ₂-C₁₄ alkenyl. In some embodiments, R ² is optionally substituted C ₅-C₁₄ alkenyl. In some embodiments, R ² is optionally substituted C ₇-C₁₄ alkenyl. In some embodiments, R ² is optionally substituted C ₉-C₁₄ alkenyl. In some embodiments, R ² is optionally substituted C ₁₀-C₁₄ alkenyl. In some embodiments, R ² is optionally substituted C ₁₂-C₁₄ alkenyl.

In some embodiments, R ² is- (CH ₂)_pCH(OR⁶)(OR⁷). In some embodiments, R ² is-CH (OR ⁶)(OR⁷). In some embodiments, R ² is-CH ₂CH(OR⁶)(OR⁷). In some embodiments, R ² is- (CH 2) ₂CH(OR⁶)(OR⁷). In some embodiments, R ² is- (CH ₂)₃CH(OR⁶)(OR⁷). In some embodiments, R ² is- (CH ₂)₄CH(OR⁶)(OR⁷).

In some embodiments, R ² is selected from the group consisting of:

R³

In some embodiments, R ³ is selected from the group consisting of optionally substituted C ₄-C₂₀ alkyl, optionally substituted C ₂-C₁₄ alkenyl, and- (CH ₂)_qCH(OR⁶)(OR⁷).

In some embodiments, R ³ is optionally substituted C ₄-C₂₀ alkyl. In some embodiments, R ³ is optionally substituted C ₈-C₁₇ alkyl. in some embodiments, R ³ is optionally substituted C ₉-C₁₆ alkyl. In some embodiments, R ³ is optionally substituted C ₈-C₁₀ alkyl. In some embodiments, R ³ is optionally substituted C ₁₁-C₁₃ alkyl. In some embodiments, R ³ is optionally substituted C ₁₄-C₁₆ alkyl. In some embodiments, R ³ is optionally substituted C ₉ alkyl. In some embodiments, R ³ is optionally substituted C ₁₀ alkyl. In some embodiments, R ³ is optionally substituted C ₁₁ alkyl. In some embodiments, R ³ is optionally substituted C ₁₂ alkyl. In some embodiments, R ³ is optionally substituted C ₁₃ alkyl. In some embodiments, R ³ is optionally substituted C ₁₄ alkyl. In some embodiments, R ³ is optionally substituted C ₁₅ alkyl. In some embodiments, R ³ is optionally substituted C ₁₆ alkyl.

In some embodiments, R ³ is optionally substituted C ₂-C₁₄ alkenyl. In some embodiments, R ³ is optionally substituted C ₅-C₁₄ alkenyl. In some embodiments, R ³ is optionally substituted C ₇-C₁₄ alkenyl. In some embodiments, R ³ is optionally substituted C ₉-C₁₄ alkenyl. In some embodiments, R ³ is optionally substituted C ₁₀-C₁₄ alkenyl. In some embodiments, R ³ is optionally substituted C ₁₂-C₁₄ alkenyl.

In some embodiments, R ³ is- (CH ₂)_qCH(OR⁸)(OR⁹). In some embodiments, R ³ is-CH (OR ⁸)(OR⁹). In some embodiments, R ³ is-CH ₂CH(OR⁸)(OR⁹). In some embodiments, R ³ is- (CH ₂)₂CH(OR⁸)(OR⁹). In some embodiments, R ³ is- (CH ₂)₃CH(OR⁸)(OR⁹). In some embodiments, R ³ is- (CH ₂)₄CH(OR⁸)(OR⁹).

In some embodiments, R ³ is selected from the group consisting of:

R⁶、R⁷、R⁸、R⁹

In some embodiments, R ⁶、R⁷、R⁸ and R ⁹ are independently optionally substituted C ₁-C₁₄ alkyl, optionally substituted C ₂-C₁₄ alkenyl, or- (CH ₂)_m-A-(CH₂)_n H. In some embodiments, R ⁶、R⁷、R⁸ and R ⁹ are independently optionally substituted C ₁-C₁₄ alkyl.

In some embodiments, R ₁-C₁₄ is optionally substituted C ₁-C₁₄ alkyl, optionally substituted C ₂-C₁₄ alkenyl, or- (CH ₂)_m-A-(CH₂)_n H. In some embodiments, R ⁶ is optionally substituted C ₃-C₁₀ alkyl, in some embodiments, R ⁶ is optionally substituted C ₄-C₁₀ alkyl, in some embodiments, R ⁶ is independently optionally substituted C ₅-C₁₀ alkyl, in some embodiments, R ⁶ is optionally substituted C ₉-C₁₀ alkyl, in some embodiments, R ⁶ is optionally substituted C ₁-C₁₄ alkyl, in some embodiments, R3836 is optionally substituted C ₂-C₁₄ alkenyl, in some embodiments, R ⁶ is- (CH ₂)_m-A-(CH₂)_n H.

In some embodiments, R ₁-C₁₄ is optionally substituted C ₁-C₁₄ alkyl, optionally substituted C ₂-C₁₄ alkenyl, or- (CH ₂)_m-A-(CH₂)_n H. In some embodiments, R ⁷ is optionally substituted C ₃-C₁₀ alkyl, in some embodiments, R ⁷ is optionally substituted C ₄-C₁₀ alkyl, in some embodiments, R ⁷ is optionally substituted C ₅-C₁₀ alkyl, in some embodiments, R ⁷ is optionally substituted C ₉-C₁₀ alkyl, in some embodiments, R ⁷ is optionally substituted C ₁-C₁₄ alkyl, in some embodiments, R ⁷ is optionally substituted C ₂-C₁₄ alkenyl.

In some embodiments, R ₁-C₁₄ is optionally substituted C ₁-C₁₄ alkyl, optionally substituted C ₂-C₁₄ alkenyl, or- (CH ₂)_m-A-(CH₂)_n H. In some embodiments, R ⁸ is optionally substituted C ₃-C₁₀ alkyl, in some embodiments, R ⁸ is optionally substituted C ₄-C₁₀ alkyl, in some embodiments, R ⁸ is optionally substituted C ₅-C₁₀ alkyl, in some embodiments, R ⁸ is optionally substituted C ₉-C₁₀ alkyl, in some embodiments, R ⁸ is optionally substituted C ₁-C₁₄ alkyl, in some embodiments, R ⁸ is optionally substituted C ₂-C₁₄ alkenyl.

In some embodiments, R ₁-C₁₄ is optionally substituted C ₁-C₁₄ alkyl, optionally substituted C ₂-C₁₄ alkenyl, or- (CH ₂)_m-A-(CH₂)_n H. In some embodiments, R ⁹ is optionally substituted C ₃-C₁₀ alkyl, in some embodiments, R ⁹ is optionally substituted C ₄-C₁₀ alkyl, in some embodiments, R ⁹ is optionally substituted C ₅-C₁₀ alkyl, in some embodiments, R ⁹ is optionally substituted C ₉-C₁₀ alkyl, in some embodiments, R ⁹ is optionally substituted C ₁-C₁₄ alkyl, in some embodiments, R ⁹ is optionally substituted C ₂-C₁₄ alkenyl.

In some embodiments, each m is independently 0, 1,2, 3, 4,5, 6,7, 8, 9, 10, 11, or 12. In some embodiments, each m is 0. In some embodiments, each m is 1. In some embodiments, each m is 2. In some embodiments, each m is 3. In some embodiments, each m is 4. In some embodiments, each m is 5. In some embodiments, each m is 6. In some embodiments, each m is 7. In some embodiments, each m is 8. In some embodiments, each m is 9. In some embodiments, each m is 10. In some embodiments, each m is 11. In some embodiments, each m is 12.

In some embodiments, each n is independently 0, 1,2, 3, 4,5, 6,7, 8, 9, 10, 11, or 12. In some embodiments, each n is 0. In some embodiments, each n is 1. In some embodiments, each n is 2. In some embodiments, each n is 3. In some embodiments, each n is 4. In some embodiments, each n is 5. In some embodiments, each n is 6. In some embodiments, each n is 7. In some embodiments, each n is 8. In some embodiments, each n is 9. In some embodiments, each n is 10. In some embodiments, each n is 11. In some embodiments, each n is 12.

In some embodiments, each a is independently C ₃-C₈ cycloalkylene. In some embodiments, each a is cyclopropenyl.

X¹

In some embodiments, X ¹ is optionally substituted C ₂-C₆ alkylene. In some embodiments, X ¹ is optionally substituted C ₂-C₅ alkylene. In some embodiments, X ¹ is optionally substituted C ₂-C₄ alkylene. In some embodiments, X ¹ is optionally substituted C ₂-C₃ alkylene. In some embodiments, X ¹ is optionally substituted C ₂ alkylene. In some embodiments, X ¹ is optionally substituted C ₃ alkylene. in some embodiments, X ¹ is optionally substituted C ₄ alkylene. In some embodiments, X ¹ is optionally substituted C ₅ alkylene. In some embodiments, X ¹ is optionally substituted C ₆ alkylene. In some embodiments, X ¹ is optionally substituted- (CH ₂)₂ -. In some embodiments, X ¹ is optionally substituted- (CH ₂)₃ -. In some embodiments, X ¹ is optionally substituted- (CH ₂)₄ -). In some embodiments, X ¹ is optionally substituted- (CH ₂)₅ -. In some embodiments, X ¹ is optionally substituted- (CH ₂)₆ -).

X²

In some embodiments, X ² is selected from the group consisting of a bond, -CH ₂ -, and-CH ₂CH₂ -. In some embodiments, X ² is a bond. In some embodiments, X ² is-CH ₂ -. In some embodiments, X ² is-CH ₂CH₂ -.

X^2'

In some embodiments, X ^2' is selected from the group consisting of a bond, -CH ₂ -, and-CH ₂CH₂ -. In some embodiments, X ^2' is a bond. In some embodiments, X ^2' is-CH ₂ -. In some embodiments, X ^2' is-CH ₂CH₂ -.

X³

In some embodiments, X ³ is selected from the group consisting of a bond, -CH ₂ -, and-CH ₂CH₂ -. In some embodiments, X ³ is a bond. In some embodiments, X ³ is-CH ₂ -. In some embodiments, X ³ is-CH ₂CH₂ -.

X^3'

In some embodiments, X ^3' is selected from the group consisting of a bond, -CH ₂ -, and-CH ₂CH₂ -. In some embodiments, X ^3' is a bond. In some embodiments, X ^3' is-CH ₂ -. In some embodiments, X ^3' is-CH ₂CH₂ -.

X⁴

In some embodiments, X ⁴ is selected from the group consisting of optionally substituted C ₂-C₁₄ alkylene and optionally substituted C ₂-C₁₄ alkenylene. In some embodiments, X ⁴ is optionally substituted C ₂-C₁₄ alkylene. In some embodiments, X ⁴ is optionally substituted C ₂-C₁₀ alkylene. In some embodiments, X ⁴ is optionally substituted C ₂-C₈ alkylene. In some embodiments, X ⁴ is optionally substituted C ₂-C₆ alkylene. In some embodiments, X ⁴ is optionally substituted C ₃-C₆ alkylene. In some embodiments, X ⁴ is optionally substituted C ₃ alkylene. In some embodiments, X ⁴ is optionally substituted C ₄ alkylene. In some embodiments, X ⁴ is optionally substituted C ₅ alkylene. In some embodiments, X ⁴ is optionally substituted C ₆ alkylene. in some embodiments, X ⁴ is optionally substituted- (CH ₂)₂ -. In some embodiments, X ⁴ is optionally substituted- (CH ₂)₃ -). In some embodiments, X ⁴ is optionally substituted- (CH ₂)₄ -. In some embodiments, X ⁴ is optionally substituted- (CH ₂)₅ -). In some embodiments, X ⁴ is optionally substituted- (CH ₂)₆ -.

X⁵

In some embodiments, X ⁵ is selected from the group consisting of optionally substituted C ₂-C₁₄ alkylene and optionally substituted C ₂-C₁₄ alkenylene. In some embodiments, X ⁵ is optionally substituted C ₂-C₁₄ alkylene. In some embodiments, X ⁵ is optionally substituted C ₂-C₁₀ alkylene. In some embodiments, X ⁵ is optionally substituted C ₂-C₈ alkylene. in some embodiments, X ⁵ is optionally substituted C ₂-C₆ alkylene. In some embodiments, X ⁵ is optionally substituted C ₃-C₆ alkylene. In some embodiments, X ⁵ is optionally substituted C ₃ alkylene. In some embodiments, X ⁵ is optionally substituted C ₄ alkylene. In some embodiments, X ⁵ is optionally substituted C ₅ alkylene. In some embodiments, X ⁵ is optionally substituted C ₆ alkylene. In some embodiments, X ⁵ is optionally substituted- (CH ₂)₂ -. In some embodiments, X ⁵ is optionally substituted- (CH ₂)₃ -). In some embodiments, X ⁵ is optionally substituted- (CH ₂)₄ -. In some embodiments, X ⁵ is optionally substituted- (CH ₂)₅ -). In some embodiments, X ⁵ is optionally substituted- (CH ₂)₆ -.

Y¹

In some embodiments, Y ¹ is selected from the group consisting of:

in some embodiments, Y ¹ is selected from the group consisting of:

in some embodiments, Y ¹ is

In some embodiments, Y ¹ is

Y²

In some embodiments, Y ² is selected from the group consisting of:

in some embodiments, Y ² is selected from the group consisting of:

In some embodiments, Y ² is

Formula (CY-I')

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-I'):

Or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is-OH, R ^1a,

Z ¹ is optionally substituted C ₁-C₆ alkyl;

X ¹ is optionally substituted C ₂-C₆ alkylene;

X ² and X ³ are independently a bond, -CH ₂ -or-CH ₂CH₂ -;

y ¹ and Y ² are independently

Wherein the bond labeled with "+" is attached to X ⁴ or X ⁵;

Each Z ² is independently H or optionally substituted C ₁-C₈ alkyl;

Each Z ³ is independently optionally substituted C ₁-C₆ alkylene;

R ² is optionally substituted C ₄-C₂₀ alkyl, optionally substituted C ₂-C₁₄ alkenyl OR-CH (OR ⁶)(OR⁷);

R ³ is optionally substituted C ₄-C₂₀ alkyl, optionally substituted C ₂-C₁₄ alkenyl OR-CH (OR ⁸)(OR⁹);

R ^1a is:

R ^2a、R^2b and R ^2c are independently hydrogen and C ₁-C₆ alkyl;

r ^3a、R^3b and R ^3c are independently hydrogen and C ₁-C₆ alkyl;

R ^4a、R^4b and R ^4c are independently hydrogen and C ₁-C₆ alkyl;

R ^5a、R^5b and R ^5c are independently hydrogen and C ₁-C₆ alkyl;

A is C ₃-C₈ cycloalkylene;

Each m is independently 0,1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; and

Each n is independently 0, 1,2,3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, the lipids of the present disclosure have the structure of formula (CY-I'), wherein:

R ¹ is-OH, R ^1a,

Wherein Z ¹ is optionally substituted C ₁-C₆ alkyl;

X ¹ is optionally substituted C ₂-C₆ alkylene;

X ² and X ³ are independently a bond, -CH ₂ -or-CH ₂CH₂ -;

x ⁴ and X ⁵ are independently optionally substituted C ₂-C₁₄ alkylene;

y ¹ and Y ² are independently

R ² and R ³ are independently optionally substituted C ₄-C₂₀ alkyl;

R ^1a is:

R ^2a、R^2b and R ^2c are independently hydrogen and C ₁-C₆ alkyl;

r ^3a、R^3b and R ^3c are independently hydrogen and C ₁-C₆ alkyl;

R ^4a、R^4b and R ^4c are independently hydrogen and C ₁-C₆ alkyl; and

R ^5a、R^5b and R ^5c are independently hydrogen and C ₁-C₆ alkyl.

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-II'), wherein:

R ¹ is-OH, R ^1a,

Wherein Z ¹ is optionally substituted C ₁-C₆ alkyl;

X ¹ is optionally substituted C ₂-C₆ alkylene;

X ² and X ³ are independently a bond, -CH ₂ -or-CH ₂CH₂ -;

y ¹ and Y ² are independently

R ² and R ³ are independently optionally substituted C ₄-C₂₀ alkyl;

R ^1a is:

R ^2a、R^2b and R ^2c are independently hydrogen and C ₁-C₆ alkyl;

r ^3a、R^3b and R ^3c are independently hydrogen and C ₁-C₆ alkyl;

R ^4a、R^4b and R ^4c are independently hydrogen and C ₁-C₆ alkyl; and

R ^5a、R^5b and R ^5c are independently hydrogen and C ₁-C₆ alkyl.

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-I'), wherein R ¹ is-OH,

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-I'), wherein Y ¹ and Y ² are independently:

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-I'), wherein R ² is-CH (OR ⁶)(OR⁷).

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-I'), wherein R ³ is-CH (OR ⁸)(OR⁹).

Non-limiting examples of lipids having the structure of formula (CY-I') include compounds CY1, CY2, CY3, CY9, CY10, CY11, CY12, CY22, CY23, CY24, CY30, CY31, CY32, CY33, CY43, CY44, CY45, CY50, CY51, CY52, and CY53.

Formula (CY-II')

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-II'):

Or a pharmaceutically acceptable salt thereof, wherein R ¹、R²、R³、X¹、X²、X³、X⁴、X⁵、Y¹ and Y ² are as defined for formula (CY-I').

R ¹ is-OH, R ^1a,

Wherein Z ¹ is optionally substituted C ₁-C₆ alkyl;

X ¹ is optionally substituted C ₂-C₆ alkylene;

X ² and X ³ are independently a bond, -CH ₂ -or-CH ₂CH₂ -;

y ¹ and Y ² are independently

R ² and R ³ are independently optionally substituted C ₄-C₂₀ alkyl;

R ^1a is:

R ^2a、R^2b and R ^2c are independently hydrogen and C ₁-C₆ alkyl;

r ^3a、R^3b and R ^3c are independently hydrogen and C ₁-C₆ alkyl;

R ^4a、R^4b and R ^4c are independently hydrogen and C ₁-C₆ alkyl; and

R ^5a、R^5b and R ^5c are independently hydrogen and C ₁-C₆ alkyl.

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-II'), wherein R ¹ is-OH,

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-II'), wherein Y ¹ and Y ² are independently:

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-II'), wherein R ² is-CH (OR ⁶)(OR⁷).

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-II'), wherein R ³ is-CH (OR ⁸)(OR⁹).

Non-limiting examples of lipids having the structure of formula (CY-II') include compounds CY4, CY5, CY16, CY17, CY18, CY25, CY26, CY37, CY38, CY39, CY46, CY56, and CY57.

Formula (CY-III')

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-III'):

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-III'), wherein

R ¹ is-OH, R ^1a,

Wherein Z ¹ is optionally substituted C ₁-C₆ alkyl;

X ¹ is optionally substituted C ₂-C₆ alkylene;

X ² and X ³ are independently a bond, -CH ₂ -or-CH ₂CH₂ -;

y ¹ and Y ² are independently

R ² and R ³ are independently optionally substituted C ₄-C₂₀ alkyl;

R ^1a is:

R ^2a、R^2b and R ^2c are independently hydrogen and C ₁-C₆ alkyl;

r ^3a、R^3b and R ^3c are independently hydrogen and C ₁-C₆ alkyl;

R ^4a、R^4b and R ^4c are independently hydrogen and C ₁-C₆ alkyl; and

R ^5a、R^5b and R ^5c are independently hydrogen and C ₁-C₆ alkyl.

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-III'), wherein R ¹ is-OH,

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-III'), wherein Y ¹ and Y ² are independently:

in some embodiments, the lipids of the present disclosure have the structure of formula (CY-III'), wherein R ² is-CH (OR ⁶)(OR⁷).

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-III'), wherein R ³ is-CH (OR ⁸)(OR⁹).

Non-limiting examples of lipids having the structure of formula (CY-III') include CY6, CY14, CY27, CY35, CY47, and CY55.

Formula (CY-IV)

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-IV'):

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-IV'), wherein:

R ¹ is-OH, R ^1a,

Wherein Z ¹ is optionally substituted C ₁-C₆ alkyl;

X ¹ is optionally substituted C ₂-C₆ alkylene;

X ² and X ³ are independently a bond, -CH ₂ -or-CH ₂CH₂ -;

y ¹ and Y ² are independently

R ² and R ³ are independently optionally substituted C ₄-C₂₀ alkyl;

R ^1a is:

R ^2a、R^2b and R ^2c are independently hydrogen and C ₁-C₆ alkyl;

r ^3a、R^3b and R ^3c are independently hydrogen and C ₁-C₆ alkyl;

R ^4a、R^4b and R ^4c are independently hydrogen and C ₁-C₆ alkyl; and

R ^5a、R^5b and R ^5c are independently hydrogen and C ₁-C₆ alkyl

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-IV'), wherein R ¹ is-OH,

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-IV'), wherein Y ¹ and Y ² are independently:

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-IV'), wherein R ² is-CH (OR ⁶)(OR⁷).

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-IV'), wherein R ³ is-CH (OR ⁸)(OR⁹).

Non-limiting examples of lipids having the structure of formula (CY-IV') include compounds CY7, CY8, CY19, CY20, CY21, CY28, CY29, CY40, CY41, CY42, CY48, CY49, CY58, CY59, and CY60.

(CY-V')

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-V):

Or a pharmaceutically acceptable salt thereof, wherein:

X ⁶ and X ⁷ are independently-CH ₂ -or-CH ₂CH₂ -; and

R ¹、R²、R³、X¹、X⁴、X⁵、Y¹ and Y ² are as defined for formula (CY-I').

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-V'), wherein:

R ¹ is-OH, R ^1a,

Wherein Z ¹ is optionally substituted C ₁-C₆ alkyl;

X ¹ is optionally substituted C ₂-C₆ alkylene;

X ² and X ³ are independently a bond, -CH ₂ -or-CH ₂CH₂ -;

y ¹ and Y ² are independently

R ² and R ³ are independently optionally substituted C ₄-C₂₀ alkyl;

R ^1a is:

R ^2a、R^2b and R ^2c are independently hydrogen and C ₁-C₆ alkyl;

r ^3a、R^3b and R ^3c are independently hydrogen and C ₁-C₆ alkyl;

R ^4a、R^4b and R ^4c are independently hydrogen and C ₁-C₆ alkyl; and

R ^5a、R^5b and R ^5c are independently hydrogen and C ₁-C₆ alkyl

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-V'), wherein Y ¹ and Y ² are independently:

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-V'), wherein R ² is-CH (OR ⁶)(OR⁷).

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-V'), wherein R ³ is-CH (OR ⁸)(OR⁹).

Non-limiting examples of lipids having the structure of formula (CY-V') include compounds CY13, CY15, CY34, CY36, and CY54.

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-VI'):

or a pharmaceutically acceptable salt thereof, wherein R¹、R⁶、R⁷、R⁸、R⁹、X¹、X²、X³、X⁴、X⁵、Y¹ and Y ² are as defined for formula (CY-I').

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein R ¹ is-OH.

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein X ¹ is C ₂-C₆ alkylene.

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein X ² is-CH ₂CH₂ -.

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein X ⁴ is C ₂-C₆ alkylene.

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein X ⁵ is C ₂-C₆ alkylene.

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein Y ¹ is:

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein Y ² is:

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein each Z ³ is independently optionally substituted C ₁-C₆ alkylene.

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein each Z ³ is-CH ₂CH₂ -.

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein R ⁶ is C ₅-C₁₄ alkyl.

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein R ⁷ is C ₅-C₁₄ alkyl.

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein R ⁶ is C ₆-C₁₄ alkenyl.

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein R ⁷ is C ₆-C₁₄ alkenyl.

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein R ⁸ is C ₅-C₁₆ alkyl.

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein R ⁹ is C ₅-C₁₄ alkyl.

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein R ⁸ is C ₆-C₁₄ alkenyl.

In some embodiments, the lipids of the present disclosure have the structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein R ⁹ is C ₆-C₁₄ alkenyl.

In some embodiments, the lipids of the present disclosure comprise a heterocyclic core, wherein the heteroatom is nitrogen. In some embodiments, the heterocyclic core comprises pyrrolidine or a derivative thereof. In some embodiments, the heterocyclic core comprises piperidine or a derivative thereof. In some embodiments, the lipid of the present disclosure is selected from any of the lipids in table (I) below, or a pharmaceutically acceptable salt thereof:

Table (I) non-limiting examples of ionizable lipids with cyclic cores

Delivery vehicle and tracking system

The initial constructs and reference constructs described herein may be formulated in a delivery vehicle. Non-limiting examples of delivery vehicles include lipid nanoparticles, non-lipid nanoparticles, exosomes, liposomes, micelles, virosomes and polymeric delivery technologies.

In some embodiments, the delivery vehicle comprises at least one lipid in table (I).

In some embodiments, the delivery vehicle comprises at least two lipids in table (I).

In some embodiments, the delivery vehicle comprises at least three lipids in table (I).

In some embodiments, the delivery vehicle comprises at least four lipids in table (I).

The total weight percentage of lipids in table (I) in the delivery vehicle is between about 10% and about 95%, such as between about 10% and about 20%, between about 21% and about 30%, between about 31% and about 40%, between about 41% and about 50%, between about 51% and about 60%, between about 61% and about 70%, between about 71% and about 80%, between about 81% and about 90%, or between about 91% and about 95%.

The total mole percent of lipids in table (I) in the delivery vehicle is between about 10% and about 95%, such as between about 10% and about 20%, between about 21% and about 30%, between about 31% and about 40%, between about 41% and about 50%, between about 51% and about 60%, between about 61% and about 70%, between about 71% and about 80%, between about 81% and about 90%, or between about 91% and about 95%.

In some embodiments, at least one lipid in the delivery vehicle has the structure of formula (CY-I), (CY-II), (CY-III), or (CY-IV).

In some embodiments, at least two lipids in the delivery vehicle have the structure of formula (CY-I), (CY-II), (CY-III), or (CY-IV).

In some embodiments, at least three lipids in the delivery vehicle have the structure of formula (CY-I), (CY-II), (CY-III), or (CY-IV).

In some embodiments, at least four lipids in the delivery vehicle have the structure of formula (CY-I), (CY-II), (CY-III), or (CY-IV).

The total weight percentage of lipids having the structure of formula (CY-I), (CY-II), (CY-III), or (CY-IV) in the delivery vehicle is between 10% -95%, such as between about 10% and about 20%, between about 21% and about 30%, between about 31% and about 40%, between about 41% and about 50%, between about 51% and about 60%, between about 61% and about 70%, between about 71% and about 80%, between about 81% and about 90%, or between about 91% and about 95%.

The total mole percent of lipids having the structure of formula (CY-I), (CY-II), (CY-III), or (CY-IV) in the delivery vehicle is between 10% -95%, such as between about 10% and about 20%, between about 21% and about 30%, between about 31% and about 40%, between about 41% and about 50%, between about 51% and about 60%, between about 61% and about 70%, between about 71% and about 80%, between about 81% and about 90%, or between about 91% and about 95%.

In some embodiments, the delivery vehicle further comprises at least one additional lipid. Non-limiting examples include additional cationic lipids, neutral lipids, anionic lipids, helper lipids, stealth lipids, or polyethylene glycol (PEG) lipids.

"Helper lipids" are lipids that enhance transfection, for example, transfection of delivery vehicles including payloads and cargo. Mechanisms by which helper lipids enhance transfection may include enhanced particle stability and/or enhanced membrane fusion. Helper lipids include steroids and alkyl resorcinol. Auxiliary lipids suitable for use in the present disclosure include, but are not limited to, cholesterol, 5-heptadecyl resorcinol, and cholesterol hemisuccinate.

A "stealth lipid" is a lipid that extends the length of time a delivery vehicle can be present in the body (e.g., in blood). Stealth lipids suitable for use in the lipid compositions of the present disclosure include, but are not limited to, stealth lipids having a hydrophilic head group attached to a lipid moiety.

Non-limiting examples of cationic lipids suitable for use in the delivery vehicle of the present disclosure include, but are not limited to, N-dioleyl-N, N-dimethylammonium chloride (DODAC), N-distearyl-N, N-dimethylammonium bromide (DDAB), N- (1- (2, 3-dioleoyloxy) propyl) -N, N-trimethylammonium chloride (DOTAP), 1, 2-dioleoyl3-dimethylammonium-propane (DOTAP), N- (1- (2, 3-dioleyloxy) propyl) -N, N-trimethylammonium (DOTMA), 1, 2-dioleoylcarbamoyl-3-dimethylammonium-propane (DOCDAP), 1, 2-dioleoyl3-dimethylammonium-propane (DLINDAP), dilauryl (C12: 0) trimethylammonium propane (DLTAP), dioctadecylaminoyl spermine (DOTAP), DC-Choi, trifluoroyloxy-3-dimethylammonium-propane (DOTAP), 2- [ 2-dioleyloxy ] -N, N-trimethylammonium chloride (DOTMA), 1, 2-dioleoylcarbamoyl-3-dimethylammonium-propane (DLINDAP), dilauryl- (2-dioleyloxy) -2- [ 2-dioleyloxy ] -N-2-dimethylammonium-propane (d-2-d-methyl-3-carbamide-2- [ 2-dioleyloxy ] -2-d-methyl ] -2-carbamide (d) methyl-ethyl ] -2- [ 2-dioleyloxy ] -2-methyl ] -2-amine, cis-9, 12-octadecadienyloxy) propane (CLinDMA), N-dimethyl-2, 3-dioleyloxy) propylamine (DODMA), 2- [5' - (cholesterol-5-en-3 [ beta ] -oxy) -3' -oxapentenyloxy) -3-dimethyl-1- (cis, cis-9 ',12' -octadecadienyloxy) propane (CpLinDMA) and N, N-dimethyl-3, 4-dioleyloxy benzylamine (DMOBA) and 1,2-N, N ' -dioleylcarbamoyl-3-dimethylaminopropane (DOcarbDAP).

Non-limiting examples of neutral lipids suitable for use in the delivery vehicle of the present disclosure include a variety of neutral, uncharged or zwitterionic lipids. Examples of neutral phospholipids suitable for use in the present disclosure include, but are not limited to: 5-heptadecylphenyl-1, 3-diol (resorcinol), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), phosphocholine (DOPC), dimyristoyl phosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1, 2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), lecithin phosphatidylcholine (EPC), dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylcholine (MPPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1, 2-distearoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1, 2-dicacosenoyl-sn-glycero-3-Phosphocholine (PE), dimyristoyl phosphatidylcholine (DPPC), ditolyphosphatidylcholine (DPPC), stearoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyl Oleoyl Phosphatidylethanolamine (POPE), lysophosphatidylethanolamine, and combinations thereof.

Non-limiting examples of anionic lipids suitable for use in the delivery vehicle of the present disclosure include, but are not limited to, phosphatidylglycerol, cardiolipin, diacyl phosphatidylserine, diacyl phosphatidic acid, N-dodecyl phosphatidylethanolamine, N-succinyl phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine Cholesterol Hemisuccinate (CHEMS), and lysyl phosphatidylglycerol.

In some embodiments, the weight ratio of delivery vehicle (including all lipids) to payload is between about 100:1 and about 1:1, such as between about 100:1 and about 90:1, between about 89:1 and about 80:1, between about 79:1 and about 70:1, between about 69:1 and about 60:1, between about 59:1 and about 50:1, between about 49:1 and about 40:1, between about 39:1 and about 30:1, between about 29:1 and about 20:1, between about 19:1 and about 10:1, and between about 9:1 and about 1:1.

In some embodiments, the delivery vehicle comprises an initial construct or reference construct having at least one cargo or payload. The cargo or payload may be any DNA, RNA or polypeptide described herein.

In some embodiments, the delivery vehicle comprises an initial construct or reference construct having at least one cargo or payload that is a coding RNA.

In some embodiments, the delivery vehicle comprises an initial construct or reference construct with at least one cargo or payload that is a non-coding RNA.

In some embodiments, the delivery vehicle comprises an initial construct or reference construct having at least one cargo or payload of oRNA.

In some embodiments, the delivery vehicle comprises an initial construct or reference construct having at least one cargo or payload that is mRNA.

In some embodiments, the at least one RNA compound consists of a functional RNA, wherein the RNA causes a change in at least one cell, tissue, organ, and/or organism. The state change may include, but is not limited to: altering the expression level of the polypeptide; altering the level of translation of the nucleic acid; altering the expression level of the nucleic acid; altering the amount of polypeptide present in a cell, tissue, organ and/or organism; altering the gene sequence of a cell, tissue, organ and/or organism; adding nucleic acid to the target genome; subtracting the nucleic acid from the genome of interest; altering physiological activity in cells, tissues, organs and/or organisms; or any combination thereof.

In some embodiments, the delivery vehicle comprises an initial construct or reference construct having at least one cargo or payload that is DNA.

In some embodiments, the delivery vehicle comprises an initial construct or reference construct having two cargo or payloads that are DNA. The DNA may be the same DNA or different DNA. As one non-limiting example, DNA is identical. As one non-limiting example, DNA is different. As one non-limiting example, DNA is different but encodes the same payload or cargo. As one non-limiting example, DNA is a larger payload or different fragments of cargo (e.g., heavy or light chain of an antibody) that can be brought together using natural systems or synthetic methods known in the art for producing functional polypeptides (e.g., antibodies).

In some embodiments, the delivery vehicle comprises an initial construct or reference construct with three cargo or payloads that are DNA. The DNA may be the same DNA or different DNA. As one non-limiting example, DNA is identical. As one non-limiting example, DNA is different. As a non-limiting example, two DNAs are identical and one is different. As one non-limiting example, the first DNA is different from the second DNA and the third DNA. As one non-limiting example, the first DNA, the second DNA, and the third DNA are all different. As one non-limiting example, the first DNA is different from the second DNA and the third DNA, but all encode the same payload or cargo. As one non-limiting example, the first DNA is different from the second DNA and the third DNA, but the second DNA and the third DNA encode the same payload or cargo.

In some embodiments, the delivery vehicle comprises an initial construct or reference construct having at least one cargo or payload that is a polypeptide.

In some embodiments, the delivery vehicle comprises an initial construct or reference construct having two cargo or payloads that are polypeptides. The polypeptides may be the same polypeptide or different polypeptides. As one non-limiting example, the polypeptides are identical. As one non-limiting example, polypeptides are different. As one non-limiting example, polypeptides are distinct fragments of a larger payload or cargo (e.g., heavy or light chain of an antibody) that can be brought together using natural systems or synthetic methods known in the art for producing functional polypeptides (e.g., antibodies).

In some embodiments, the delivery vehicle comprises an initial construct or reference construct having three cargo or payloads that are polypeptides. The polypeptides may be the same polypeptide or different polypeptides. As one non-limiting example, the polypeptides are identical. As one non-limiting example, polypeptides are different. As a non-limiting example, two polypeptides are identical and one is different. As one non-limiting example, the first polypeptide is different from the second polypeptide and the third polypeptide. As one non-limiting example, the first polypeptide, the second polypeptide, and the third polypeptide are all different. As one non-limiting example, the first polypeptide is different from the second polypeptide and the third polypeptide, but all encode the same payload or cargo. As one non-limiting example, the first polypeptide is different from the second polypeptide and the third polypeptide, but the second polypeptide and the third polypeptide encode the same payload or cargo.

In some embodiments, the delivery vehicle comprises an initial construct or reference construct having at least one cargo or payload that is a peptide.

In some embodiments, the delivery vehicle comprises an initial construct or reference construct with two cargo or payloads that are peptides. The peptides may be the same peptide or different peptides. As one non-limiting example, the peptides are identical. As one non-limiting example, peptides are different. As one non-limiting example, peptides are different fragments of a larger payload or cargo (e.g., heavy or light chain of an antibody) that can be brought together using natural systems or synthetic methods known in the art for producing functional polypeptides (e.g., antibodies).

In some embodiments, the delivery vehicle comprises an initial construct or reference construct with three cargo or payloads that are peptides. The peptides may be the same peptide or different peptides. As one non-limiting example, the peptides are identical. As one non-limiting example, peptides are different. As one non-limiting example, two peptides are identical and one is different. As one non-limiting example, the first peptide is different from the second peptide and the third peptide. As one non-limiting example, the first peptide, the second peptide, and the third peptide are all different. As one non-limiting example, the first peptide is different from the second peptide and the third peptide, but all encode the same payload or cargo. As one non-limiting example, the first peptide is different from the second peptide and the third peptide, but the second peptide and the third peptide encode the same payload or cargo.

In some embodiments, the delivery vehicle comprises an initial construct or reference construct having at least one cargo or payload that is RNA.

In some embodiments, the delivery vehicle comprises an initial construct or reference construct with two cargo or payloads that are RNAs. The RNAs may be the same RNA or different RNAs. As one non-limiting example, RNAs are identical. As one non-limiting example, RNAs are different. As one non-limiting example, RNAs are different but encode the same payload or cargo. As one non-limiting example, RNAs are distinct fragments of a larger payload or cargo (e.g., heavy or light chain of an antibody), which may be brought together using natural systems or synthetic methods known in the art for producing functional polypeptides (e.g., antibodies).

In some embodiments, the delivery vehicle comprises an initial construct or reference construct with three cargo or payloads that are RNAs. The RNAs may be the same RNA or different RNAs. As one non-limiting example, RNAs are identical. As one non-limiting example, RNAs are different. As a non-limiting example, two RNAs are identical and one is different. As one non-limiting example, the first RNA is different from the second RNA and the third RNA. As one non-limiting example, the first RNA, the second RNA, and the third RNA are all different. As one non-limiting example, the first RNA is different from the second RNA and the third RNA, but all encode the same payload or cargo. As one non-limiting example, the first RNA is different from the second RNA and the third RNA, but the second RNA and the third RNA encode the same payload or cargo.

In some embodiments, the delivery vehicle comprises an initial construct or reference construct with two cargo or payloads, one of which is RNA and one of which is DNA. The RNA and DNA may encode the same peptide or polypeptide, or may encode different peptides or polypeptides. As one non-limiting example, RNA and DNA may encode the same peptide or polypeptide. As one non-limiting example, RNA and DNA may encode different peptides or polypeptides. As one non-limiting example, RNA and DNA are different fragments of a larger payload or cargo (e.g., heavy or light chain of an antibody) that can be brought together using natural systems or synthetic methods known in the art for producing functional polypeptides (e.g., antibodies).

In some embodiments, the delivery vehicle comprises an initial construct or reference construct with two cargo or payloads, one of which is RNA and one of which is a peptide. The RNA can encode the same peptide as the peptide cargo/payload and the RNA can encode a different peptide. As a non-limiting example, RNA encodes the same peptide. As one non-limiting example, RNAs encode different peptides. As one non-limiting example, RNA and peptides are distinct fragments of a larger payload or cargo (e.g., heavy or light chain of an antibody) that can be brought together using natural systems or synthetic methods known in the art for producing functional polypeptides (e.g., antibodies).

In some embodiments, the delivery vehicle comprises an initial construct or reference construct with two cargo or payloads, one of which is RNA and one of which is a polypeptide. The RNA can encode the same polypeptide as the polypeptide cargo/payload and the RNA can encode a different polypeptide. As one non-limiting example, RNA encodes the same polypeptide. As one non-limiting example, RNAs encode different polypeptides. As one non-limiting example, RNA and polypeptides are distinct fragments of a larger payload or cargo (e.g., heavy or light chain of an antibody) that can be brought together using natural systems or synthetic methods known in the art for producing functional polypeptides (e.g., antibodies).

In some embodiments, the delivery vehicle comprises an initial construct or reference construct with two cargo or payloads, one of which is DNA and one of which is a peptide. DNA may encode the same peptide as the peptide cargo/payload and DNA may encode a different peptide. As a non-limiting example, DNA encodes the same peptide. As one non-limiting example, DNA encodes different peptides. As one non-limiting example, DNA and peptides are distinct fragments of a larger payload or cargo (e.g., heavy or light chain of an antibody) that can be brought together using natural systems or synthetic methods known in the art for producing functional polypeptides (e.g., antibodies).

In some embodiments, the delivery vehicle comprises an initial construct or reference construct with two cargo or payloads, one of which is DNA and one of which is a polypeptide. The DNA may encode the same polypeptide as the polypeptide cargo/payload and the DNA may encode a different polypeptide. As a non-limiting example, DNA encodes the same polypeptide. As one non-limiting example, DNA encodes different polypeptides. As one non-limiting example, DNA and polypeptides are distinct fragments of a larger payload or cargo (e.g., heavy or light chain of an antibody) that may be brought together using natural systems or synthetic methods known in the art for producing functional polypeptides (e.g., antibodies).

Delivery vehicle

Nanoparticles

In some embodiments, the delivery vehicle is a nanoparticle. As used herein, the term "nanoparticle" refers to any particle having a size in the range of 10-1000 nm. The nanoparticle may be 10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、105、110、115、120、125、130、135、140、145、150、155、160、165、170、175、180、185、190、195、200、205、210、215、220、225、230、235、240、245、250、255、260、265、270、275、280、285、290、295、300、305、310、315、320、325、330、335、340、345、350、355、360、365、370、375、380、385、390、395、400、405、410、415、420、425、430、435、440、445、450、455、460、465、470、475、480、485、490、495、500、505、510、515、520、525、530、535、540、545、550、555、560、565、570、575、580、585、590、595、600、605、610、615、620、625、630、635、640、645、650、655、660、665、670、675、680、685、690、695、700、705、710、715、720、725、730、735、740、745、750、755、760、765、770、775、780、785、790、795、800、805、810、815、820、825、830、835、840、845、850、855、860、865、870、875、880、885、890、895、900、905、910、915、920、925、930、935、940、945、950、955、960、965、970、975、980、985、990、995 or 1000nm.

Lipid nanoparticles

In some embodiments, the nanoparticle may be a Lipid Nanoparticle (LNP). In general, LNP can be characterized as small solid or semi-solid particles having an outer lipid layer with a hydrophilic outer surface exposed to a non-LNP environment; an internal space that may be aqueous (vesicle-like) or non-aqueous (microcellular); and at least one hydrophobic membrane-to-membrane space. The LNP film may be layered or non-layered and may be composed of 1,2, 3, 4,5 or more layers. In some embodiments, the LNP may comprise cargo or payload into its interior space, into the inter-membrane space, into its exterior surface, or any combination thereof.

LNPs suitable for use herein are known in the art and generally comprise cholesterol (which aids stability and promotes membrane fusion), phospholipids (which provide structure to the LNP bilayer and can also aid endosomal escape), polyethylene glycol (PEG) derivatives (which reduce LNP aggregation and "protect" LNP from non-specific endocytosis by immune cells), and ionizable lipids (which complex with negatively charged RNAs and enhance endosomal escape), which form LNP-forming compositions.

The composition of the LNP may be selected based on the desired target, cargo, size, etc. As a non-limiting example, previous studies have shown that polymeric nanoparticles composed of low molecular weight polyamines and lipids can deliver nucleic acids to endothelial cells with high potency. (Dahlman et al, ,In vivo endothelial siRNA delivery using polymeric nanoparticles with low molecular weight,Nat Nanotechnol.2014, month 8; 9 (8): 648-655; the contents of which are incorporated herein by reference in their entirety).

In some embodiments, the initial construct and the reference construct of the present disclosure may be incorporated into a Lipid Nanoparticle (LNP). In some embodiments, the lipid nanoparticle may be comprised of at least one cationic lipid, at least one non-cationic lipid, at least one sterol, at least one particle activity modulator, or any combination thereof. In some embodiments, the lipid nanoparticle may be comprised of at least one cationic lipid, at least one non-cationic lipid, at least one sterol, and at least one particle activity modulator. In some embodiments, the LNP may be comprised of at least one cationic lipid, at least one non-cationic lipid, and at least one sterol. In some embodiments, the LNP may be comprised of at least one cationic lipid, at least one non-cationic lipid, and at least one particulate activity modulator. In some embodiments, the LNP may be comprised of at least one non-cationic lipid, at least one sterol, and at least one particulate activity modulator. In some embodiments, the LNP may be comprised of at least one cationic lipid and at least one non-cationic lipid. In some embodiments, the LNP may be comprised of at least one cationic lipid and at least one sterol. In some embodiments, the LNP may be comprised of at least one cationic lipid and at least one particulate activity modulator. In some embodiments, the LNP may be comprised of at least one non-cationic lipid and at least one sterol. In some embodiments, the LNP may be comprised of at least one non-cationic lipid and at least one particulate activity modulator. In some embodiments, the LNP may be comprised of at least one sterol and at least one particulate activity modulator. In some embodiments, the LNP may be comprised of at least one cationic lipid. In some embodiments, the LNP may be comprised of at least one non-cationic lipid. In some embodiments, the LNP may be comprised of sterols. In some embodiments, the LNP may be comprised of a particulate activity modulator.

In some embodiments, the at least one cationic lipid may comprise any one of the following: at least one ionizable cationic lipid, at least one amino lipid, at least one saturated cationic lipid, at least one unsaturated cationic lipid, at least one zwitterionic lipid, at least one multivalent cationic lipid, or any combination thereof. In some embodiments, the LNP may be substantially free of at least one cationic lipid. In some embodiments, the LNP may contain an amount of at least one cationic lipid of zero.

In some embodiments, the at least one cationic lipid may be selected from, but is not limited to, at least one of the following: 1, 3-bis (1, 2-bis-tetradecyloxy-propyl-3-dimethylethoxyammonium bromide) -propan-2-ol ((R) -PLC-2), 2- (dinonylamino) ethan-1-ol (17-10), 2- (didodecylamino) ethan-1-ol (17-11), 3- (didodecylamino) propan-1-ol (17-12), 4- (didodecylamino) butan-1-ol (17-13), 2- (hexyl ((9Z, 12Z) -octadeca-9, 12-dien-1-yl) amino) ethan-1-ol (17-2), 2- (nonyl ((9Z, 12Z) -octadec-9, 12-dien-1-yl) amino) ethan-1-ol (17-3), 2- (dodecyl ((9Z, 12Z) -octadec-9, 12-dien-1-yl) amino) ethan-1-ol (17-4), 2- (((9Z, 12Z) -octadec-9, 12-dien-1-yl) (tetradecyl) amino) ethan-1-ol (17-5), 2- (((9Z, 12Z) -octadec-9, 12-dien-1-yl) (octadecyl) amino) ethan-1-ol (17-6), 2- (bistetradecylamino) ethan-1-ol (17-7), 2- (di ((Z) -octadec-9-en-1-yl) amino) ethan-1-ol (17-8), (9Z, 12Z) -N- (2-methoxyethyl) -N- ((9Z, 12Z) -octadec-9, 12-dien-1-yl) octadec-9, 12-dien-1-amine (17-9), N-nonyl-N- (2- (piperazin-1-yl) ethyl) non-1-amine (19-1), N-dodecyl-N- (2- (piperazin-1-yl) ethyl) dodecan-1-amine (19-2), (9Z, 12Z) -N- ((9Z, 12Z) -octadec-9, 12-dien-1-yl) -N- (2- (piperazin-1-yl) ethyl) octadec-9, 12-dien-1-amine (19-3), N-dodecyl-N- (2- (4-methylpiperazin-1-yl) ethyl) dodecane-1-amine intermediate 1:2- (bisdodecylamino) ethan-1-ol (19-4), N-dodecyl-N- (2- (4-methoxyphenylmethyl) piperazin-1-yl) ethyl) dodecane-1-amine (19-5), (9Z, 12Z) -N- (2- (4-dodecylpiperazin-1-yl) ethyl) -N- ((9Z, 12Z) -octadeca-9, 12-dien-1-yl) octadeca-9, 12-dien-1-amine (19-6), (3- ((6Z, 9Z,28Z, 31Z) -trisheptadec-6,9,28,31-tetraen-19-yloxy) -N, N-dimethylpropan-1-amine) (1-Bl 1), N- (2- (Bidodecylamino) ethyl) -N-dodecylglycine (20-1), dinonyl 8,8' - ((2- (dodecyl (2-hydroxyethyl) amino) ethyl) azodiyl) dioctanoate (20-10), 3- ((2- (Bidodecylamino) ethyl) (dodecyl) amino) propan-1-ol (20-11), 2- ((2- (Bidodecylamino) ethyl) (tetradecyl) amino) ethan-1-ol (20-12), 2- ((2- (di ((9Z, 12Z) -octadeca-9, 12-dien-1-yl) amino) ethyl) (dodecyl) amino) ethan-1-ol (20-13), 2- ((2- (di ((9Z, 12Z) -octadeca-9, 12-dien-1-yl) amino) ethyl) ((9Z, 12Z) -octadeca-9, 12-dien-1-yl) amino) ethan-1-ol (20-14), 2- ((2- (didodecylamino) ethyl) (hexyl) amino) ethan-1-ol (20-15), 2- ((2- (dinonylamino) ethyl) (nonylamino) ethan-1-ol (20-16), 2- ((2- (didodecylamino) ethyl) (nonylamino) ethan-1-ol (20-17), 2- ((2- (dinonylamino) ethyl) (dodecyl) amino) ethan-1-ol (20-18), 2- ((2- (Didodecylamino) ethyl) amino) ethan-1-ol (20-19), amyl 6- (dodecyl (2-hydroxyethyl) amino) ethyl) amino) hexanoate (20-2), 2- ((2- (didodecylamino) ethyl) (dodecyl) amino) ethan-1-ol (20-20), 3- ((2- (didodecylamino) ethyl) (dodecyl) amino) propan-1-ol (20-21), 4- ((2- (didodecylamino) ethyl) (dodecyl) amino) butan-1-ol (20-22), (Z) -2- ((2- (didodecylamino) ethyl) (dodecyl-6-en-1-yl) amino) ethan-1-ol (20-23), 2- ((2- (Didodecylamino) ethyl) (tetradecylamino) ethan-1-ol (20-24), 2- ((2- (Didodecylamino) ethyl) ((9Z, 12Z) -octadeca-9, 12-dien-1-yl) amino) ethan-1-ol (20-25), amyl 6- ((2- (Didodecylamino) ethyl) (2-hydroxyethyl) amino) hexanoate (20-3), dipentyl 6,6'- ((2- (dodecyl (2-hydroxyethyl) amino) ethyl) azodiyl) dihexanoate (20-4), diheptyl 6,6' - ((2- ((6- (heptyloxy) -6-oxohexyl) (2-hydroxyethyl) amino) ethyl) azodiyl) dihexanoate (20-5), Amyl 6- ((2- (dinonylamino) ethyl) (2-hydroxyethyl) amino) hexanoate (20-6), heptyl 6- (dodecyl (2-hydroxyethyl) amino) ethyl) amino) hexanoate (20-7), nonyl 8- ((2- (didodecylamino) ethyl) (2-hydroxyethyl) amino) octanoate (20-8), heptadec-9-yl 8- ((2- (didodecylamino) ethyl) (2-hydroxyethyl) amino) octanoate (20-9), 1- (2, 2-di ((9Z, 12Z) -octadeca-9, 12-dien-1-yl) cyclopropyl) -N, N-dimethylamine (21-1), 3, 3-bis ((9Z, 12Z) -octadeca-9, 12-dien-1-yl) cyclobutyl 4- (dimethylamino) butanoate (21-2), 3-bis ((9Z, 12Z) -octadeca-9, 12-dien-1-yl) cyclopentyl 3- (dimethylamino) propanoate (21-3), 3-bis ((9Z, 12Z) -octadeca-9, 12-dien-1-yl) cyclopentyl 4- (dimethylamino) butanoate (21-4), 1- (2, 3-bis ((8Z, 11Z) -heptadeca-8, 11-dien-1-yl) cyclopropyl) -N, N-dimethylamine (21-6), Unknown (75-016B), poly {4- ((2- (dimethylamino) ethyl) thio) tetrahydro-2H-pyran-2-one } -r-poly {4- (octylthio) tetrahydro-2H-pyran-2-one } (A7), (3 aR5s,6 aS) -N, N-dimethyl-2, 2-bis ((9Z, 12Z) -octadeca-9, 12-dienyl) tetrahydro-3 aH-cyclopentad 1,3 dioxol-5-amine (ALN 100), (3 aR,5s,6 aS) -N, N-dimethyl-2, 2-bis ((9Z, 12Z) -octadeca-9, 12-dienyl) tetrahydro-3 aH-cyclopenta [ d ] [1,3] dioxol-5-amine (ALN 1001), ((3 aR,5s,6 aS) -N, N-dimethyl-2, 2-bis ((9Z, 12Z) -octadeca-9, 12-dienyl) tetrahydro-3 aH-cyclopenta [ d ] [1,3] dioxol-5-amine)) (ALNY-100), dimyristoyltrimethyl-ammoniumpropane (amino lipid 6), benzamide-dialkyl-carboxylic acid (BADACA), N-dihydroxyethyl methyl-N-2- (cholesteryloxycarbonylamino) ethyl ammonium bromide (BHEM-Chol), N-bis- (2-hydroxyethyl) -N-methyl-N- (2-cholesteryloxycarbonylamino-ethyl) ammonium bromide (BHEM-Chol 1), 2- {4- [ (3β) -cholest-5-en-3-yloxy ] butoxy } -iVN-dimethyl-3- [ (9Z, 12Z) -octadeca-9 ]! 12-dien-1-yloxy ] propan-1-amine (butyl-CLinDMA), (2R) -2- {4- [ (3β) -cholesterol-5-en-3-yloxy ] butoxy } - Λ/-dimethyl-3- [ (9Z, 12Z) -octadeca-9-! 12-dien-1-yloxyprop-1-amine (butyl-CLinDMA (2R)), (25) -2- {4- [ (3β) -cholesterol-5-en-3-yloxy ] butoxy } -iVy/V-dimethyl-3- [ (9Z, 12Z) -octadeca-9, 12-dien-1-yloxy ] prop-1-amine (butyl-CLinDMA (2S)), 1' - (2- (4- (2- ((2-hydroxydodecyl) amino) ethyl) piperazin-1-yl) ethylazadiyl) didodecan-2-ol (C12-200), 1,1' - ((2- (4- (2- ((2-hydroxydodecyl) amino) ethyl) (2-hydroxydodecyl) amino) piperazin-1-yl) ethyl) azetidin-2-ol) (C12-200), cholesteryl-succinylsilane (C2), (9 z,9' z,12' z) -2- ((4- (((3- (dimethylamino) propoxy) carbonyl) oxy) hexadecyl) oxy) propane-1, 3-diylbis (octadeca-9, 12-dienoate) (cationic lipid A2), 9z,12 z) -3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- (((((3- (diethylamino) propoxy) carbonyl) oxy) methyl) propyloctadeca-9, 12-dienoate (cationic lipid A3), 1- (3-cholesteryl) -oxycarbonyl-aminomethylimidazole (CHIM), [ (2-morpholin-4-yl-ethylcarbamoyl) methyl ] -carbamic acid cholesterol ester (Chol-C3N-Mo 2), [ (2-morpholin-4-yl-ethylcarbamoyl) -ethyl ] -carbamic acid cholesterol ester Chol-DMC3N-Mo2[ 1-methyl-2- (2-morpholin-4-yl-ethylcarbamoyl) -propyl ] -carbamic acid cholesterol ester (Chol-C4N-Mo 2), 1, 17-bis (2-octylcyclopropyl) heptadec-9-yl 4- (dimethylamino) butanoate (CL), Triheptadeca-6,9,28,31-tetraen-19-yl-4- (dimethylamino) -butyrate (CL 01), cholesteryl 3- (dimethylamino) propionate (CL 06), cholesteryl 2- (dimethylamino) acetate (CL 08), N-dimethyl-2, 3-bis (((9Z, 12Z) -octadeca-9, 12-dien-1-yl) oxy) propan-1-amine (CL-1), N-methyl-2- (((9Z, 12Z) -octadeca-9, 12-dien-1-yl) oxy) -N- (2- ((((9Z, 12Z) -octadeca-9, 12-dien-1-yl) oxy) ethyl) ethan-1-amine (CL-11), (3R, 4R) -3, 4-bis (((Z) -hexadec-9-en-1-yl) oxy) -1-methylpyrrolidine (compound CL-12) (CL-12), 2- (dimethylamino) -N- ((6Z, 9Z,28Z, 31Z) -heptadeca-6,9,28,31-tetraen-19-yl) acetamide (CL-13), 3- (dimethylamino) propane-1, 2-diyl (9Z, 9'Z, 12' Z) -bis (octadeca-9, 12-dienoate) (CL-14), (9Z, 12Z) -bis ((9Z, 12Z) -octadeca-9, 12-dien-1-yl) amine (CL-15), 7-hydroxy 7- (4- ((1-methylpiperidine-4-carbonyl) oxy) butyl) tridecane-1, 13-diyl-didodecanoate (CL 15B 6), 7-hydroxy 7- (4- ((1-methylpiperidine-4-carbonyl) oxy) butyl) tridecane-1, 13-diyl-dimetradecanoate (CL 15C 6), 7-hydroxy 7- (4- ((1-methylpiperidine-4-carbonyl) oxy) butyl) tridecane-1, 13-diyl-dipalmitate (CL 15D 6), 7-hydroxy 7- (4- ((1-methylpiperidine-4-carbonyl) oxy) butyl) tridecane-1, 13-diyl dioleate (CL 15H 6), Bis (2- (((9Z, 12Z) -octadeca-9, 12-dien-1-yl) oxy) ethyl) amine (CL-16), (9Z, 12Z) -N-methyl-N- (2- (((9Z, 12Z) -octadeca-9, 12-dien-1-yl) oxy) ethyl) octadeca-9, 12-dien-1-amine (CL-17), (9Z, 12Z) -N- (3- (((9Z, 12Z) -octadeca-9, 12-dien-1-yl) oxy) propyl) octadeca-9, 12-dien-1-amine (CL-18), (1-methylpiperidin-3-yl) methyldi ((11Z, 14Z) -eicosa-11, 14-dien-1-yl) carbamate (CL-19), N-methyl-N, N-bis (2- ((Z) -hexadec-9-enyloxy) ethyl) amine (CL-2), (13Z, 16Z) -N, N-dimethyl-4- ((9Z, 12Z) -octadec-9, 12-dien-1-yl) docosa-3,13,16-trien-1-amine (CL-20), (S) -2-amino-3-hydroxy-N, N-bis (2- (((Z) -octadec-9-en-1-yl) oxy) ethyl) propionamide (CL-21), C2:N, N-diacetyl-N' - (3-triethoxysilylpropyl) butanediamide (CL 3), Trans-1-methyl-3, 4-bis (((Z) -octadeca-9-en-1-yl) oxy) methyl) pyrrolidine (CL-3), trans-1-methylpyrrolidine-3, 4-diyl) bis (methylene) (9Z, 9'Z, 12' Z) -bis (octadeca-9, 12-dienoate) (CL-4), 7- (4- (diisopropylamino) butyl) -7-hydroxytrideca-1, 13-diyl ditetradecanoate (CL 4C 6), 7- (4- (diisopropylamino) butyl) -7-hydroxytrideca-1, 13-diyl dipalmitate (CL 4D 6), 11- (4- (diisopropylamino) butyl) -11-hydroxy-di-undecane-1, 21-diyldioleate (CL 4H 10), 7- (4- (diisopropylamino) butyl) -7-hydroxy-tridecane-1, 13-diyldioleate (CL 4H 6), 9- (4- (diisopropylamino) butyl) -7-hydroxy-heptadecane-1, 17-diyldioleate (CL 4H 8), (6Z, 9Z, 28Z) -heptadeca-6,9,28,31-tetraen-19-yl 4- (dimethylamino) butanoate (CL-5), 2- (dimethylamino) -N- (2- (((Z) -octadeca-9-en-1-yl) oxy) ethyl) -N- ((9Z, 12Z) -octadeca-9, 12-dien-1-yl) acetamide (CL-53), 3- ((2- (((Z) -octadec-9-en-1-yl) oxy) ethyl) ((9Z, 12Z) -octadec-9, 12-dien-1-yl) amino) propane-1-All (CL-54), 1-methyl-3, 3-bis ((((9Z, 12Z) -octadec-9, 12-dien-1-yl) oxy) methyl) azetidine (CL-55), 1-methyl-3, 3-bis (2- (((9Z, 12Z) -octadec-9, 12-dien-1-yl) oxy) ethyl) azetidine (CL-56), 1-methyl-3, 3-bis (2- (((9Z, 12Z) -octadec-9, 12-dien-1-yl) oxy) propyl) azetidine (CL-57), 2- (3, 3-bis ((9Z, 12Z) -octadeca-9, 12-dien-1-yl) azetidin-1-yl) ethan-1-ol (CL-58), 2- (3, 3-bis ((9Z, 12Z) -octadeca-9, 12-dien-1-yl) azetidin-1-ol (CL-59), 3- (bis ((9Z, 12Z) -octadeca-9, 12-dien-1-yl) amino) propan-1-ol (CL-6), 3- (dimethylamino) propyl 3, 3-bis ((9Z, 12Z) -octadeca-9, 12-dien-1-yl) azetidin-1-carboxylate (CL-60), 2- (di ((Z) -octadec-9-en-1-yl) amino) ethan-1-ol (CL-61), 3- (di ((Z) -octadec-9-en-1-yl) amino) propan-1-ol (CL-62), (11Z, 14Z) -2- ((dimethylamino) methyl) -2- ((9Z, 12Z) -octadec-9, 12-dien-1-yl) eicos-11, 14-dien-1-ol (CL-63), (11Z, 14Z) -2- (dimethylamino) -2- ((9Z, 12Z) -octadec-9, 12-dien-1-yl) eicos-11, 14-dien-1-ol (CL-64), 3- (dimethylamino) -2, 2-bis (((9Z, 12Z) -octadeca-9, 12-dien-1-yl) oxy) methyl) propan-1-ol (CL-65), (9Z, 12Z) -N- (2- (((Z) -octadeca-9-en-1-yl) oxy) ethyl) octadeca-9, 12-dien-1-amine (CL-7), 1-methyl-3, 3-bis ((9Z, 12Z) -octadeca-9, 12-dien-1-yl) azetidine (CL-8), N, 2-dimethyl-1, 3-bis (((9Z, 12Z) -octadeca-9, 12-dien-1-yl) oxy) propan-2-amine (CL-9), 3-dimethylamino-2- (cholesterol-5-en-3B-oxybutynin-4-oxy) -1- (cis, cis-9, 12-octadecadienyloxy) propane (CLinDMA), 2- [5' - (cholesterol-5-en-3-oxy) -3' -oxapentenyloxy) -3-dimethyl-1- (cis, cis-9 ',12' -octadecadienyloxy) propane (CpLinDMA), cetyltrimethylammonium bromide (CTAB), l-two arachidonyloxy- ] TV-dimethyl-propyl-S-amine (DAraDMA), 0' -bitetradecanoyl-N- (. Alpha. -trimethylaminoacetyl) diethanolamine chloride (DC-6-14), 3β - [ N- (N ', N' -dimethylaminoethane) carbamoyl ] cholesterol (DC-Chol), dimethyl Dioctadecyl Ammonium (DDA), dimethyl dioctadecyl ammonium bromide (DDA), N-distearyl-N, N-dimethyl ammonium bromide (DDAB), 1, 2-bis-docosahexenoxy- (7 v, N-dimethyl) -propyl-3-amine (DDocDMA), N- (2- (dimethylamino) ethyl) -4, 5-bis (dodecylthio) pentanamide (DEDPA), 3-dimethylamino-2- (cholesterol-5-ene-3β -oxopentan-3-oxa-an-5-oxy) -1- (cis, cis-9, 12-octadecadienoxy) propane (DEG-CLinDMA), 1, 6-Dioleoyl triethylenetetraamide (dio-TETA), N1, N19-bis ((S, 23E,25E,27E, 29E) -16- ((2E, 4E,6E, 8E) -3, 7-dimethyl-9- (2, 6-trimethylcyclohex-1-en-1-yl) non-2, 4,6, 8-tetraenamido) -24, 28-dimethyl-15, 22-dioxo-30- (2, 6-trimethylcyclohex-1-en-1-yl) -4,7, 10-trioxa-14, 21-diazatriaconta-23, 25,27, 29-tetraen-1-yl) -4,7,10,13,16-pentaoxanonadec-1, 19-diamide (diVA-PEG-diVA), DiLin-N-methylpiperazine (DL-033), diLin-N, N-dimethylglycine (DL-036), dioleyl-N, N-dimethylglycine (DL-048), 3- ((1, 3-bis (((9Z, 12Z) -octadeca-9, 12-dienoyl) oxy) propan-2-yl) amino) propanoic acid (DLAPA), 1, 2-secondary linolenoxy-3-dimethylaminopropane (DLenDMA), 1-linolenoyl-2-linolenoxy-3-dimethylaminopropane (DLin-2-DMAP), 3- (N, N-diileylamino) -1, 2-propanediol (DLinAP), 1,2-N, N' -Dioleoylcarbamoyl-3-dimethylaminopropane (DLincarbDAP), 1, 2-dioleylcarbamoyl-3-dimethylaminopropane (DLinCDAP), 1, 2-dioleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1, 2-dioleyloxy-3- (dimethylamino) acetoxypropane (DLin-DAC), 1, 2-dioleoyl-3-dimethylaminopropane (DLinDAP), 1, 2-dioleyloxy-N, N-dimethylaminopropane (DLinDMA), 1, 2-Dioleoyloxy-3-dimethylaminopropane (DLinDMA 1), 1, 2-Dioleoyloxy-3- (2-N, N-dimethylamino) ethoxypropane (DLin-EG-DMA), diionoyl-4-aminobutyric acid (DLinFAB), 2-diionoyl-4- (2-dimethylaminoethyl) - [1,3] -dioxolane (DLin-K-C2-DMA), 2-diionoyl-4-dimethylaminomethyl- [1,3] -dioxolane (DLin-K-DMA), 1, 2-diionooxy-3- (N-morpholinyl) propane (DLin-MA), (6Z, 9Z,28Z, 31Z) -Triseventeen-6,9,28,31-tetralin-19-yl 4- (dimethylamino) butanoate (DLin-MC 3-DMA), 1, 2-dioleyloxy-3- (N-methylpiperazinyl) propane (DLinMPZ), 1, 2-dioleyloxy-3- (N-methylpiperazinyl) propane (DLin-MPZ), dioleyloxy 3-piperidylpropylamine (DLinPip), 1.2 dioleyloxy 3- (3 '-hydroxypiperidinyl) -propylamine (DLinPip-3 OH), 1,2 dioleyloxy 3- (4' -hydroxypiperidinyl) -propylamine (DLinPip-4 OH), 1, 2-Dioleoyloxy-3-hydroxypropane (DLinPO), 1, 2-Dioleoylsulfanyl-3-dimethylaminopropane (DLin-S-DMA), 1, 2-Dioleoyl-3-trimethylaminopropane (DLinTAP), 1, 2-Dioleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1, 2-Dioleoyloxy-3-trimethylaminopropane (DLinTMA), 1, 2-Dioleoyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 3- ((1, 3-bis (((9Z, 12 Z.15Z) -octadeca-9, 12, 15-trienoyl) oxy) propan-2-yl) amino) propionic acid (DLLAPA), 1,2 Dioleoyloxy 3- (N, N-dimethyl D-propylamine (DLmDEA), 1, 2-dilauroyl-sn-glycero-3-phosphate ethanolamine (DLPE), 1, 2-dilauroyl-sn-glycero-3-glycerol (DLPG), N-dimethyl-3, 4-dioleyloxy benzylamine (DMOBA), dimyristoyl phosphatidylserine (DMPS), N- [1- (2, 3-dimyristoyloxy) propyl ] -N, N-dimethyl-N- (2-hydroxyethyl) ammonium bromide (DMRIE), 1, 2-dimyristoyloxy propyl-3-dimethyl-hydroxyethyl ammonium bromide (DMRIE 1), 1, 2-dimyristoyl-3-trimethylammoniopropane (DMTAP), 3- (N, N-dioleylamino) -1, 2-propanediol (DOAP), 3- ((1, 3-bis (oleoyloxy) propan-2-yl) amino) propionic acid (DOAPA), 1,2-N, N' -dioleylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 1, 2-dioleylcarbamoyl-3-dimethylammonium-propane (DOCDAP), N-dioleyl-N, N-dimethylammonium chloride (DODAC), 1, 2-dioleoyl-3-dimethylammonium-propane (DODAP), n, N-dihydroxyethyl N, N-dioctadecyl ammonium chloride (DODEAC), N-dimethyl-2, 3-dioleyloxypropylamine (DODMA), dioleoyl-4-aminobutyric acid (DOFAB), dioctadecyl amidoglycyl spermine (DOGS), 1, 2-dioleoyl-3-methyl- (methoxycarbonyl-ethyl) ammonium-propane (DOMCAP), 1, 2-dioleoyl-3-N-pyrrolidine-propane (DOP 5P), 1, 2-dioleoyl-3-N-pyridinium-propane, bromide salt (DOP 6P), 1, 2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide (DORI), 1, 2-dioleoxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), 1, 2-dioleoxypropyl-3-dimethyl-hydroxybutyl ammonium bromide (DORIE-HB), 1, 2-dioleoxypropyl-3-dimethyl-hydroxypropyl ammonium bromide (DORIE-HP), 1, 2-dioleoxypropyl-3-dimethyl-hydroxypentyl ammonium bromide (DORIE-Hpe), 2, 3-dioleoxy-N- [2 (spermine-carboxamido) ethyl ] -N, N-dimethyl-1-propylamine onium trifluoroacetate (DOSPA), 1, 3-dioleoyloxy-2- (6-carboxy-spermine) -propionamide (DOSPER), N- (1- (2, 3-dioleoyloxy) propyl) -N, N, N-trimethylammonium chloride (DOTAP), 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP 1), N- [5'- (2', 3 '-dioleoyl) uridine ] -N', N ', N' -trimethylammonium tosylate (DOTAU), 1- [2- (9 (Z) -octadecenyloxy) ethyl ] -2- (8 (Z) -heptadecenyl-3- (2-hydroxyethyl) imidazolinium chloride (DOTIM), N- (1- (2, 3-dioleyloxy) propyl) -N, N, N-trimethylammonium chloride (DOTMA), Dioleyl phosphatidyluridine phosphatidylcholine (DOUPC), 1, 2-di-phytyloxy-N, N-dimethyl) -butyl-4-amine (DPan-C2-DMA), 1, 2-di-phytyloxy-3- (iV, 7V-dimethyl) -propylamine (DPanDMA), 2, 3-bis (dodecylthio) propyl (2- (dimethylamino) ethyl) carbamate (DPDEC), dipalmitoyl-4-aminobutyric acid (DPFAB), 1, 2-dipalmitoyloxy propyl-3-dimethyl-hydroxyethylammonium bromide (DPRIE), 1, 2-dipalmitoyl-3-trimethylammoniopropane (DPTAP), 1- [2- (hexadecyloxy) ethyl ] -2-pentadecyl-3- (2-hydroxyethyl) imidazolinium chloride (DPTIM), 3- ((1, 3-bis (stearyloxy) propan-2-yl) amino) propanoic acid (DSAPA), distearyldimethylammonium (DSDMA), 1, 2-distearyloxy-N, N-dimethylaminopropane (DSDMA 1), 1, 2-dioxido (disteryloxy) propyl-3-dimethyl-hydroxyethylammonium bromide (DSRIE), 1, 2-dioxido-3-trimethylammonium propane (DSTAP), ditetradecyltrimethylammonium (DTDTMA), 1, 2-dioleoyl-sn-glycero-3-Ethylphosphocholine (EDOPC), N2- [ N2, N5-bis (3-aminopropyl) -L-guanyl (ormithyl) ] -N, N-dioctadecyl-L-glutamyltetrahydrotrifluoroacetate (GC 33), cholest-5-en-3-ol (3P) -,3- [ (3-aminopropyl) [4- [ (3-aminopropyl) amino ] butyl ] carbamate ] (GL 67), glyceryl Monooleate (GMO), guanidino-dialkyl-carboxylic acid (GUADACA), 2- (bis (2- (tetradecanoyloxy) ethyl) amino) -N- (2-hydroxyethyl) -N, N-dimethyl-2-oxoethyl-ammonium bromide (HEDC), 2,2' - (tert-Butoxycarbonylazodiyl) bis (ethane-2, 1-diyl) ditetradecanoate (HEDC-BOC-TN), 1- (2- (((3S, 10R, 13R) -10, 13-dimethyl-17- ((R) -6-methylheptan-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16,17-decatetrahydro-1H-cyclopenta [ a ] phenanthren-3-yldihydrothio) ethyl) guanidine (HGT 4002), (15Z, 18Z) -N, N-dimethyl-6- (9Z, 12Z) -octadeca-9, 12-dien-1-yl) tetracos-15, 18-dien-1-amine (HGT 5000), (15Z, 18Z) -N, N-dimethyl-6- ((9Z, 12Z) -octadeca-9, 12-dien-1-yl) tetracos-4,15,18-trien-1-amine (HGT 5001), histidino-cholesterol hemisuccinate (HisChol), histidylcholesterol hemisuccinate (Hist-Chol), hydroSoyPC (HSPC), imidazolyl Cholesterol Ester (ICE), 3- (didodecylamino) -N1, N1, 4-tris (dodecyl) -1-piperazineethylamine (KL 10), N1- [2- (didodecylamino) ethyl ] -N1, N4, N4-tris (dodecyl) -1, 4-piperazinedieethylamine (KL 22), 14, 25-ditridecyl-15,18,21,24-tetraaza-trioctadecyl (KL 25), N-di-N-tetradecyl, N-methyl-N- (2-guanidino) ethylammonium (lipid 1), N-di-N-octadecyl chloride, N-methyl-N- (2-guanidino) ethylammonium (lipid 2), 3- ((4, 4-bis (octyloxy) butyryl) oxy) -2- (((((3- (diethylamino) propoxy) carbonyl) oxy) methyl) propyl (9Z, 12Z) -octadeca-9, 12-dienoate (lipid A), (9Z, 12Z) -3- ((4, 4-bis (octyloxy) butyryl) oxy) -2- (((((3- (diethylamino) propoxy) carbonyl) oxy) methyl) propyloctadeca-9, 12-dienoate (lipid A1), 2, 2-Di-lino-4-dimethylaminoethyl- [1,3] -dioxolane (lipid A2), ((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (oxy)) bis (octane-8, 1-diyl) bis (decanoate) (lipid B), 2- ((4- (((3- (dimethylamino) propoxy) carbonyl) oxy) hexadecyl) oxy) propane-1, 3-diyl 9Z,9'Z, 12' Z) -bis (octadeca-9, 12-dienoate) (lipid C), 3- (((3- (dimethylamino) propoxy) carbonyl) -13- (octanoyloxy) tridecyl 3-octyl undecanoate (lipid D), (6Z, 16Z) -12- ((Z) -dec-4-en-1-yl) docosa-6, 16-dien-11-yl 5- (dimethylamino) pentanoate (lipid I), dioctadecyl- (2-hydroxy-3-propylamino) aminopolylysine (lipid T), (3- ((6Z, 9Z,28Z, Z) -tric-6,9,28,31-tetraen-19-yloxy) -N, N-dimethylpropan-1-amine (MC 3 ether), described in U.S. provisional application Ser. No. 61/384,050 (MC 3 Thioester), (4- ((6Z, 9Z,28Z, 31Z) -tricyclodecan-6,9,28,31-tetraen-19-yloxy) -N, N-dimethylbutyl-1-amine (MC 4 ether), 3- ((2- (((9 z,12 z) -octadeca-9, 12-dienoyl) oxy) ethyl) amino) propanoic acid (MLAPA), 3- ((2- (((9 z,12z,15 z) -octadeca-9, 12, 15-trienoyl) oxy) ethylamino) propanoic acid (MLLAPA), mono-branched acylglycerol (monomycolylglycerol; MMG), 3- ((2- (oleoyloxy) ethyl) amino) propionic acid (MOAPA), 4- (2-aminoethyl) - (N-morpholino) -cholesterol hemisuccinate (MoChol), 1, 2-dioleoyl-3-N-morpholino-propane (MoDO), picolyl-dialkyl-carboxylic acid (MPDACA), monopalmitoyl phosphatidylcholine (MPPC), 3- ((2- (stearyloxy) ethyl) amino) propionic acid (MSAPA), N1- [2- ((1S) -1- [ (3-aminopropyl) amino ] -4- [ bis (3-amino-propyl) amino ] butylcarboxamido) ethyl ] -3, 4-di [ oleoyl ] -benzamide (MVL 5), 2- ({ 8- [ (3β) -cholest-5-en-3-yloxy ] octyl } oxy) -N, N-dimethyl-3- [ (9 z,12 z) -octadec-9, 12-dien-1-yloxy ] propan-1-amine (octyl-CLinDMA), (2R) -2- ({ 8- [ (3β) -cholest-5-en-3-yloxy ] octyl } oxy) -N, N-dimethyl-3- [ (9 z,12 z) -octadec-9, 12-dien-1-yloxy ] propan-1-amine (octyl-CLinDMA (2R)), phosphatidylcholine (PC), 1, 3-bis- (1, 2-bis-tetradecyloxy-propyl-3-dimethylethoxyammonium bromide) -propan-2-ol (PCL-2), Palmitoyl-oleoyl-nor-arginine (PONA), stearylamine (STA), 2- (((tert-butyldimethylsilyl) oxy) methyl) -2- (hydroxymethyl) propane-1, 3-diol) (synthesis example 1 (a)), 3- ((tert-butyl (dimethyl) silyl) oxy) -2, 2-bis (((9Z) -tetradeca-9-enoyloxy) methyl) propyl (9Z) -tetradeca-9-enoic acid ester) (synthesis example 1 (B)), 3-hydroxy-2, 2-bis (((9Z) -tetradeca-9-enoyloxy) methyl) propyl (9Z) -tetradeca-9-enoic acid ester) (synthesis example 1 (C)) 3- ((4- (dimethylamino) butyryloxy) -2, 2-bis (((9Z) -tetradec-9-enoyloxy) methyl) propyl (9Z) -tetradec-9-enoate) (synthetic example 1 (D)), 3- (5- (bis (2-hydroxydodecyl) amino) pent-2-yl) -6- (5- ((2-hydroxydodecyl) (2-hydroxyundecyl) amino) pent-2-yl) -1, 4-dioxane-2, 5-dione) (target 24), trehalose-6 '-dibehenate (TDB), 1' - (2- (4- ((2- (bis (2-hydroxydodecyl) amino) ethyl) piperazin-1-yl) ethylazadiyl) didodecan-2-ol (Tech G1), 3- ((1, 3-bis (((9Z, 12Z) -octadeca-9, 12-dienoyl) oxy) -2- ((((9Z, 12Z) -octadeca-9, 12-dienoyl) oxy) methyl) propan-2-yl) amino) propanoic acid (TLAPA), (1- (2, 3-oleoylpropoxy) -2- (oleoyloxy) - (7V, Λ/-dimethyl) -propyl-3-amine) (TLinDMA), 3- ((1, 3-bis (((9 Z.12 Z.15Z) -octadeca-9.12.15-trienoyl) oxy) -2- (((((9 Z.12 Z.15E) -octadeca-9, 12, 15-trienoyl) oxy) methyl) propan-2-yl) amino) propanoic acid (TLLAPA), Chlorinated N- (α -trimethylaminoacetyl) -didodecyl-D-glutamate (TMAG), 3- ((1, 3-bis (((Z) -octadec-9-enoyl) oxy) -2- ((((Z) -octadec-9-enoyl) oxy) methyl) propan-2-yl) amino) propanoic acid (TOAPA), 3- ((1, 3-bis (stearoyloxy) -2- ((stearoyloxy) methyl) propan-2-yl) amino) propanoic acid (TSAPA), 1, N19-bis ((16 e,18e,20e,22 e) -17, 21-dimethyl-15-oxo-23- (2, 6-trimethylcyclohex-1-en-1-yl) -4,7, 10-trioxa-14-azaditridec-16, 18,20, 22-tetraen-1-yl) -4,7,10,13,16-pentaoxanonadecane-1, 19-diamide (VA-PEG-VA), 2, 2-Di-lino-4-dimethylaminoethyl- [1,3] -dioxolane (XTC), disclosed in non-patent document 11 (YSK 05), 1, 2-di-gamma-linolenyloxy-N, N-dimethylaminopropane (gamma-DLenDMA), a-D-tocopheryl hemisuccinyl, (9Z, 9, Z,12, Z) -2- ((2- (((3- (dimethylamino) propoxy) carbonyl) oxy) tetradecanoyl) oxy) propane-1, 3-diylbis (octadeca-9, 12-dienoate), 2- (((13Z, 16Z) -4- (((3- (diethylamino) propoxy) carbonyl) oxy) docosyl-13, 16-dienoyl) oxy) propane-1, 3-diyldioctoate, 2- (((13Z, 16Z) -4- (((3- (dimethylamino) propoxy) carbonyl) oxy) docosyl-13, 16-dienoyl) oxy) propane-1, 3-diyldioctanoate, 2- ((4- (((3- (ethyl (methyl) amino) propoxy) carbonyl) oxy) hexadecyl) oxy) propane-1, 3-diylbis (decanoate), 2- ((4- (((3- (diethylamino) propoxy) carbonyl) oxy) hexadecyl) propane-1, 3-diylbis (decanoate), and, 2- (10-dodecyl-3-ethyl-8, 14-dioxo-7, 9, 13-trioxa-3-azaeicos-20-yl) propane-1, 3-diyldioctanoate, 2- (((4- (dimethylamino) butanoyl) oxy) methyl) -2- ((octanoyloxy) methyl) propane-1, 3-diyl (9Z, 9' Z) bis-tetradeca-9-enoic acid ester, (9Z, 9' Z,12' Z) -2- (((1- (cyclopropylmethyl) piperidine-4-carbonyl) oxy) methyl) propane-1, 3-diylbis (octadeca-9, 12-dienoate), ((2- (((1-isopropylpiperidine-4-carbonyl) oxy) methyl) -1, 4-phenylene) bis (octane-8, 1-diyl) bis (decanoate), 2- ((4- (((3- (ethyl (methyl) amino) propoxy) carbonyl) oxy) hexadecyl) oxy) propane-1, 3-diyl didodecanoate, 2- ((4- (((3- (diethylamino) propoxy) carbonyl) oxy) hexadecyl) oxy) propane-1, 3-diyl didodecanoate, 2- ((4- (((3- (dimethylamino) propoxy) carbonyl) oxy) hexadecyl) propane-1, 3-diyl didodecanoate, 2- ((4- (((3- (ethyl (methyl) amino) propoxy) carbonyl) oxy) hexadecyloxy) propane-1, 3-diyl ditetradecanoate, 2- ((4- (((3- (dimethylamino) propoxy) carbonyl) oxy) hexadecyloxy) propane-1, 3-diyl ditetradecanoate, 2- ((4- (((3- (diethylamino) propoxy) carbonyl) oxy) hexadecyloxy) propane-1, 3-diyl ditetradecanoate, (Z) -2- ((4- (((3- (dimethylamino) propoxy) carbonyl) oxy) hexadecyloxy) propane-1, 3-diyl dioleate, (9Z, 9, Z,12Z, 15Z) -2- ((4- (((3- (dimethylamino) propoxy) carbonyl) oxy) hexadecyl) oxy) propane-1, 3-diylbis (octadeca-9, 12, 15-trienoate), (9Z, 9, Z,12, Z) -2- ((4- (((3- (diethylamino) propoxy) carbonyl) oxy) hexadecyl) oxy) propane-1, 3-diylbis (octadeca-9, 12-dienoate), (9Z, 9, Z,12, Z) -2- ((4- (((3- (dimethylamino) propoxy) carbonyl) oxy) hexadecyl) oxy) propane-1, 3-diylbis (octadeca-9, 12-dienoate), N, N, N-trimethyl-5-oxo-5- (3- ((3-pentyloxy) oxy) -2, 2-bis (((3-pentyloxy) oxy) methyl) propoxy) pentane-1-ammonium iodide 3- ((5- (dimethylamino) pentanoyl) oxy) -2, 2-bis (((3-pentyloxy) methyl) propyl 3-pentylcactanoate, 3-dimethylaminopropyl carbonate (9Z, 12Z) -dioctadec-19, 22-dien-11-yl, 2- (((N, N-dimethyl- β -alanyl) oxy) methyl } -2- [ (octanoyloxy) methyl) propane-1, 3-diyl (9Z, 9' Z) bis-tetradec-9-enoic acid ester, O 'I, O1- (2- (7-dodecyl-14-methyl-3, 9-dioxo-2, 4,8, 10-tetraoxa-14-aza-pentadecyl) propane-1, 3-diyl) 8-dimethyldioctanedioate, 8-dimethylO' I,01- (2- (((1-methylpyrrolidine-3-carbonyl) oxy) methyl) propane-1, 3-diyl) dioctanedioate, 1- (3- ((6, 6-bis ((2-propylpentyl) oxy) hexanoyl) oxy) -2- (((1, 4-dimethylpiperidine-4-carbonyl) oxy) methyl) propyl) 8-methyloctanedioate, (9Z, 12Z) -5- (((3- (dimethylamino) propoxy) carbonyl) oxy) -7-octylpentadecyl octadeca-9, 12-dienoate, 5- (((3- (dimethylamino) propoxy) carbonyl) oxy) -7-octylpentadecyl octanoate, 1- (3- ((6, 6-bis ((2-propylpentyl) oxy) hexanoyl) oxy) -2- (((1, 4-dimethylpiperidine-4-carbonyl) oxy) methyl) propyl) 10-octylsebacate, 3- (((3- (dimethylamino) propoxy) carbonyl) oxy) -5-octyltridecyl decanoate, 1- (16- (((4, 4-bis (octyloxy) butanoyloxy) methyl) -9-dodecyl-2-methyl-7, 13-dioxo-6,8,12,14-tetraoxa-2-aza-heptadec-17-yl) 8-methyloctanedioate, 3- ((5- (dimethylamino) pentanoyl) oxy) -2, 2-bis (((9Z) -tetradeca-9-enyloxy) methyl) propyl (9Z, 12Z) -octadeca-9, 12-dienoate, 3- ((5- (dimethylamino) pentanoyl) oxy) -2, 2-bis (((3-pentyloxy) methyl) propyl 3-pentyloxy) octanoate, (9Z, 9'Z, 12' Z) -2- (((3- (diethylamino) propionyl) oxy) methyl) propane-1, 3-diylbis (octadeca-9, 12-dienoate), ((2- (((4- (dimethylamino) butanoyl) oxy) methyl) -1, 4-phenylene) bis (oxy)) bis (octane-8, 1-diyl) bis (decanoate), 1- (3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- (((1-methylpyrrolidin-3-carbonyl) oxy) methyl) propyl) 8-methyl octane dioate, 3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- ((palmitoyloxy) methyl) propyl 1-methylpyrrolidine-3-carboxylate, 3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- ((tetradecanoyloxy) methyl) propyl 1-methylpyrrolidine-3-carboxylate, 3- (((3- (dimethylamino) propoxy) carbonyl) oxy) -13- (octanoyloxy) tridecyl 9-pentylmethyl myristate, 3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- ((dodecanoyloxy) methyl) propyl 1-methylpyrrolidine-3-carboxylate, 3- (((3- (dimethylamino) propoxy) carbonyl) oxy) -13-hydroxytridecyl 9-pentylmethyl myristate, 3- (((3- (dimethylamino) propoxy) carbonyl) oxy) -13- (octanoyloxy) tridecyl 7-hexyltridecyl ester, 3- (((dimethylamino) propoxy) carbonyl) oxy) -tridecyl 7-hexyltridecyl ester, 2- (5- (3- ((1-methylpyrrolidine-3-carbonyl) oxy) -2- ((tetradecanoyloxy) methyl) propoxy) -5-oxopentyl) propane-1, 3-diyldioctanoate, 3- (((3- (dimethylamino) propoxy) carbonyl) oxy) -13- (octanoyloxy) tridecyl 5-heptyldodecanoate, 2- (5- (3- ((1-methylpyrrolidine-3-carbonyl) oxy) -2- ((palmitoyloxy) methyl) propoxy) -5-oxopentyl) propane-1, 3-diyldioctanoate, 3- (((3- (dimethylamino) propoxy) carbonyl) oxy) -13-hydroxytridecyl 5-heptyldodecanoate, 2- (((1-methylpyrrolidine-3-carbonyl) oxy) methyl) propane-1, 3-diylbis (6, 6-bis (octyloxy) hexanoate), (9Z, 12Z) -3- (((3-dimethylamino) propoxy) carbonyl) oxy) -13- (octanoyloxy) tridecyl octadeca-9, 12-dienoate, 3- ((5- (dimethylamino) pentanoyl) oxy) -2, 2-bis (((9Z) -tetradeca-9-enoyloxy) methyl) propyl (9Z) -octadeca-9-enoate, 2- (10-dodecyl-3-ethyl-8, 14-dioxo-7, 9, 13-trioxa-3-azanonadec-19-yl) propane-1, 3-diyldioctoate, ((2- (((1-methylpiperidine-4-carbonyl) oxy) methyl) -1, 4-phenylene) bis (oxy)) bis (octane-8, 1-diyl) bis (decanoate), 2- (((3- (dimethylamino) propionyl) oxy) methyl) propane-1, 3-diylbis (4, 4-bis (octoxy) butanoate), (9Z, 12Z) -2- (((11Z, 14Z) -2- ((3- (dimethylamino) propionyl) oxy) eicosa-11, 14-dien-1-yl) oxy) ethyl octadeca-9, 12-dienoate, 2- (((1, 3-dimethylpyrrolidine-3-carbonyl) oxy) methyl) propane-1, 3-diylbis (4, 4-bis (octoxy) butanoate), (13Z, 16Z) -4- (((3- (dimethylamino) propoxy) carbonyl) oxy) docosyl-13, 16-dien-1-yl heptadec-9-yl succinate, 2-bis (heptyloxy) ethyl 3- ((3-ethyl-10- ((9Z, 12Z) -octadec-9, 12-dien-1-yl) -8, 15-dioxo-7,9,14-trioxa-3-azepan-17-yl) dihydrothio) propionate, 2- (((1-methylpyrrolidine-3-carbonyl) oxy) methyl) propane-1, 3-diylbis (4, 4-bis (octyloxy) butane, 1- (3- ((1, 3-dimethylpyrrolidine-3-carbonyl) oxy) -2- (((9Z, 12Z) -octadeca-9, 12-dienoyloxy) methyl) propyl) 10-octyl sebacate, (13Z, 16Z) -4- (((3- (diethylamino) propoxy) carbonyl) oxy) docosa-13, 16-dien-1-yl 2, 2-bis (heptyloxy) acetate, (13Z, 16Z) -4- (((2- (dimethylamino) ethoxy) carbonyl) oxy) docosa-13, 16-dien-1-yl 2, 2-bis (heptyloxy) acetate, Acetic acid (20,23R) -2-methyl-9- [ (9Z, 12Z) -octadeca-9, 12-dien-1-yl ] -7-oxo-6, 8, 11-trioxa-2-aza-icosa-20-en-23-yl 3- (dimethylamino) propylcarbonate (11Z, 14Z) -1- { [ (9Z, 12R) -12-hydroxyoctadeca-9-en-1-yl ], (12Z, 15Z) -1- ((((9Z, 12Z) -octadeca-9, 12-dien-1-yloxy) carbonyl) oxy) heneicosa-12, 15-dien-3-yl 3- (dimethylamino) propionate, (9Z, 12Z) -3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- (((((3- (dimethylamino) propyl) carbamoyl) oxy) methyl) propyloctadeca-9, 12-dienoate, (12Z, 15Z) -3- ((4- (dimethylamino) butanoyl) oxy) heneicosa-12, 15-dien-1-yl 9-pentylmercanoate, (9Z, 12Z) -3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- (((((((1, 2, 6-pentamethylpiperidin-4-yl) oxy) carbonyl) oxy) methyl) propyloctadeca-9, 12-dienoate, (12Z, 15Z) -3- ((4- (dimethylamino) butyryl) oxy) heneicosyl-12, 15-dien-1-yl 7-hexyltridecanoate, (9Z, 12Z) -3- ((4, 4-bis (octyloxy) butyryl) oxy) -2- (((((((1-methylpiperidin-4-yl) methoxy) carbonyl) oxy) methyl) propyloctadeca-9, 12-dienoate, (12Z, 15Z) -3- ((4- (dimethylamino) butyryl) oxy) heneicosyl-12, 15-dien-1-yl 5-heptyldodecanoate, (9Z, 12Z) -3- ((4, 4-bis (octyloxy) butyryl) oxy) -2- ((((((1-ethylpiperidin-4-yl) oxy) carbonyl) oxy) methyl) propyloctadeca-9, 12-dienoate, (12Z, 15Z) -3- ((4- (dimethylamino) butyryl) oxy) heneicosyl-12, 15-dien-1-yl 3-octylundecanoate, formate salt, 3- ((5- (dimethylamino) pentanoyl) oxy) -2, 2-bis (((9Z) -tetradeca-9-enoyloxy) methyl) propyl (9Z) -hexadeca-9-enoate, (9Z, 12Z) -3- ((4, 4-bis (octyloxy) butyryl) oxy) -2- (((((((1-methylazetidin-3-yl) oxy) carbonyl) oxy) methyl) propyloctadeca-9, 12-dienoate, (9Z, 12Z) - (12Z, 15Z) -3- ((3- (dimethylamino) propionyl) oxy) heneicosa-12, 15-dien-1-yl octadeca-9, 12-dienoate, 2- (((3- (diethylamino) propoxy) carbonyl) oxy) tetradecyl 4, 4-bis ((2-ethylhexyl) oxy) butanoate, (9Z, 12Z) -3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- (((((((1-methylpiperidin-4-yl) oxy) carbonyl) oxy) methyl) propyloctadeca-9, 12-dienoate, (9Z, 12Z) -3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- (((((((1-methylpyrrolidin-3-yl) oxy) carbonyl) oxy) methyl) propyloctadeca-9, 12-dienoate, (9Z, 12Z) -3- (((2- (dimethylamino) ethoxy) carbonyl) pentadecyl octadeca-9, 12-dienoate, (9Z, 12Z) -3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- (((((3- (4-methylpiperazin-1-yl) propoxy) carbonyl) oxy) methyl) propyl octadeca-9, 12-dienoate, 3- (dimethylamino) propyl triacontan-11-ylcarbonate triacontan-11-ol, (9Z, 12Z) -3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- ((((((3- (pyrrolidin-1-yl) propoxy) carbonyl) methyl) propyl octadeca-9, 12-dienoate, (9Z, 12Z) -3- (((3- (ethyl (methyl) amino) propoxy) carbonyl) oxy) pentadecyl octadeca-9, 12-dienoate, 3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- (((9Z, 12Z) -octadeca-9, 12-dienoyloxy) methyl) propyl 4- ((diethylamino) methyl) benzoate, (9Z, 12Z) -3- (((3- (diethylamino) propoxy) carbonyl) oxy) pentadecyl octadeca-9, 12-dienoate, 3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- (((9Z, 12Z) -octadeca-9, 12-dienoyloxy) methyl) propyl 3- ((dimethylamino) methyl) benzoate, (9Z, 12Z) -3- (((3- (dimethylamino) propoxy) carbonyl) oxy) pentadecyl octadeca-9, 12-dienoate, 3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- (((9Z, 12Z) -octadeca-9, 12-dienoyloxy) methyl) propyl 1-methylpiperidine-3-carboxylate, 3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- (((9Z, 12Z) -octadeca-9, 12-dienoyloxy) methyl) propyl 1-methylpiperidine-4-carboxylate, 3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- (((9Z, 12Z) -octadeca-9, 12-dienoyloxy) methyl) propyl 1, 4-dimethylpiperidine-4-carboxylate, 3- ((4- (dimethylamino) butanoyl) oxy) -2, 2-bis (((9Z) -tetradeca-9-enoyloxy) methyl) propyl (9Z) -hexadeca-9-enoate, 2- (10-dodecyl-3-ethyl-8, 14-dioxo-7, 9, 13-trioxa-3-azahexadecan-16-yl) propane-1, 3-diyldioctanoate, (9Z, 9'Z, 12' Z) -2- (((4- (piperidin-1-yl) butanoyl) oxy) methyl) propane-1, 3-diylbis (octadeca-9, 12-dienoate), 3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- (((9Z, 12Z) -octadeca-9, 12-dienyloxy) methyl) propyl 4-methylmorpholine-2-carboxylate, (2R) -3- ((4, 4-bis (octyloxy) butyryloxy) oxy) -2- (((9Z, 12Z) -octadeca-9, 12-dienoyloxy) methyl) propyl 1-methylpyrrolidine-2-carboxylate, (2S) -3- ((4, 4-bis (octyloxy) butyryloxy) oxy) -2- (((9Z, 12Z) -octadeca-9, 12-dienoyloxy) methyl) propyl 1-methylpyrrolidine-2-carboxylate, (9Z, 9'Z, 12' Z) -2- ((((3- (diethylamino) propoxy) carbonyl) oxy) methyl) -2- (((9Z, 12Z) -octadeca-9, 12-dienoyloxy) methyl) propane-1, 3-diylbis (octadeca-9, 12-dienoate), (9Z, 12Z) -3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- (((((1-ethylpiperidin-3-yl) methoxy) oxy) methyl) propyloctadeca-9, 12-dienoate, 3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- (((9Z, 12Z) -octadeca-9, 12-dienoyloxy) methyl) propyl 1- (cyclopropylmethyl) piperidine-4-carboxylate, 3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- (((9Z, 12Z) -octadeca-9, 12-dienoyloxy) methyl) propyl 1-isopropylpiperidine-4-carboxylate, (9Z, 12Z) -3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- (((3- (dimethylamino) propionyl) oxy) methyl) propyloctadeca-9, 12-dienoate, 4- (dimethylamino) butylcarbonate (6Z, 9Z,26Z, 29Z) -tricyclopentadec-6,9,26,29-tetraen-18-yl, 3- ((6- (dimethylamino) hexanoyl) oxy) -2, 2-bis (((9Z) -tetradeca-9-enoyloxy) methyl) propyl (9Z) -tetradeca-9-enoate, 2, 5-bis ((9Z, 12Z) -octadeca-9, 12-dienyloxy) benzyl 3- (dimethylamino) propylcarbonate, (9Z, 9'Z, 12' Z) -2- (((4- (pyrrolidin-1-yl) butanoyl) oxy) methyl) propane-1, 3-diylbis (octadeca-9, 12-dienoate), 3- (((3- (dimethylamino) propoxy) carbonyl) oxy) pentadecyl 5-heptyldodecanoate, acetic acid (7R, 9Z) -18- ({ [3- (dimethylamino) propoxy ] carbonyl } oxy) octacosa-9-en-7-yl, 3- (((3- (dimethylamino) propoxy) carbonyl) oxy) pentadecyl 9-pentylmercanoate, (9Z, 12Z) -3- ((6, 6-bis (octoxy) hexanoyl) oxy) -2- (((((3- (diethylamino) propoxy) methyl) propylcarbon-9, 12-dienoate, 3- (((3- (dimethylamino) propoxy) carbonyl) oxy) pentadecyl 7-hexyltridec-6-enoate, (9Z, 12Z) -3- (2, 2-bis (heptyloxy) acetoxy) -2- ((((2- (dimethylamino) ethoxy) carbonyl) oxy) methyl) propyloctadeca-9, 12-dienoate, 3- (((3- (dimethylamino) propoxy) carbonyl) oxy) pentadecyl 3-octylundec-2-enoate, (9Z, 12Z) -3- (((3- (diethylamino) propoxy) carbonyl) oxy) -2- (((5-heptyldodecanoyl) oxy) methyl) propyloctadeca-9, 12-dienoate, 3- (((3-dimethylamino) propoxy) carbonyl) oxy) pentadecyl 3 octyl undecanoate, (9Z, 12Z) -3- (((3- (diethylamino) propoxy) carbonyl) oxy) -2- (((9-pentylmethyl) oxy) methyl) propyloctadeca-9, 12-dienoate, diacetic acid (7R, 9Z,26Z, 29R) -18- ({ [3- (dimethylamino) propoxy ] carbonyl } oxy) thirty-five carbon-9, 26-diene-7, 29-diyl, 3- (((3- (dimethylamino) propoxy) carbonyl) oxy) pentadecyl 8, 8-bis ((2-propylpentyl) oxy) octanoate, (9Z, 12Z) -3- (((3- (diethylamino) propoxy) carbonyl) oxy) -2- (((7-hexyltridecyl) oxy) methyl) propyloctadeca-9, 12-dienoate, 3- (((3- (ethyl (methyl) amino) propoxy) carbonyl) oxy) pentadecyl 8, 8-bis ((2-propylpentyl) oxy) octanoate, (9Z, 12Z) -3- (((3- (diethylamino) propoxy) carbonyl) oxy) -2- (((3-octylundecyl) oxy) methyl) propyloctadeca-9, 12-dienoate, 3- (((3- (diethylamino) propoxy) carbonyl) pentadecyl 8, 8-bis ((2-propylpentyl) oxy) octanoate, 3- (((3- (diethylamino) propoxy) carbonyl) oxy) pentadecyl 8, 8-dibutoxyoctanoate, 3- ((5- (dimethylamino) pentanoyl) oxy) -2, 2-bis (((9Z) -tetradec-9-enoyloxy) methyl) propyl (9Z) -tetradec-9-enoate, 3- (dimethylamino) propylcarbonate (6Z, 9Z,26Z, 29Z) -tricyclopentadecan-6,9,26,29-tetraen-18-yl, 2, 5-bis ((9Z, 12Z) -octadeca-9, 12-dien-1-yloxy) benzyl 3- (dimethylamino) propionate, (9Z, 9'Z, 12' Z) -2- (((3- (4-methylpiperazin-1-yl) propanoyl) oxy) methyl) propane-1, 3-diylbis (octadeca-9, 12-dienoate), 3- (((3- (diethylamino) propoxy) carbonyl) oxy) pentadecyl 8, 8-bis (octoxy) octanoate, 3- (dimethylamino) propyl octan-11-ylcarbonate, 2, 4-bis ((9Z, 12Z) -octadeca-9, 12-dienyloxy) benzyl 4- (dimethylamino) butanoate, (9Z, 12Z) -3- (((3- (diethylamino) propoxy) carbonyl) oxy) -2- (((2-heptylundecanoyl) oxy) methyl) propyl octadeca-9, 12-dienoate, 3- (((3- (diethylamino) propoxy) carbonyl) oxy) pentadecyl 6, 6-bis ((2-ethylhexyl) oxy) hexanoate, 2- (((((3- (dimethylamino) propoxy) carbonyl) oxy) methyl) propane-1, 3-diylbis (2-heptylundecanoate), 3- (((3- (diethylamino) propoxy) carbonyl) oxy) pentadecyl 6, 6-bis (hexyloxy) hexanoate, 4-methyl-2, 5-bis ((9Z, 12Z) -octadeca-9, 12-dien-1-yloxy) benzyl 4- (dimethylamino) butanoate, 3- (((3- (diethylamino) propoxy) carbonyl) oxy) pentadecyl 6, 6-bis (octyloxy) hexanoate, 4- (dimethylamino) butyl 4-methyl-2, 5-bis ((9Z, 12Z) -octadeca-9, 12-dienyloxy) benzyl carbonate, 3- (((3- (dimethylamino) propoxy) carbonyl) oxy) pentadecyl 4, 4-bis ((2-propylpentyl) oxy) butanoate, 2- (12-dodecyl-3-ethyl-8, 14-dioxo-7, 9, 13-trioxa-3-azaoctadeca-18-yl) propane-1, 3-diyldioctanoate, 2- (5-oxo-5- ((3- (((3- (piperidin-1-yl) propoxy) carbonyl) oxy) pentadecyl) propane-1, 3-diyldioctoate, 3- (dimethylamino) propyl 4-methyl-2, 5-bis ((9Z, 12Z) -octadeca-9, 12-dien-1-yloxy) benzyl carbonate, 3- (((3- (ethyl (methyl) amino) propoxy) carbonyl) oxy) pentadecyl 4, 4-bis ((2-propylpentyl) oxy) butanoate, 2- (11-dodecyl-3-ethyl-9, 15-dioxo-8, 10, 14-trioxa-3-azanonadec-19-yl) propane-1, 3-diyldioctanoate, 2- (10-dodecyl-3-ethyl-8, 15-dioxo-7,9,14-trioxa-3-azanonadec-19-yl) propane-1, 3-diyldioctanoate, 2- (5- ((4- (((((1-methylpiperidin-4-yl) oxy) carbonyl) oxy) hexadecyl) oxy) -5-oxopentyl) propane-1, 3-diyldioctanoate, 2- (5- ((4- (((((1-ethylpiperidin-3-yl) methoxy) carbonyl) oxy) hexadecyl) oxy) -5-oxopentyl) propane-1, 3-diyldioctanoate, 2- (5- ((4- ((((((((R) -1-methylpyrrolidin-3-yl) oxy) carbonyl) oxy) cetyl) oxy) hexadecyl) oxy) -5-oxopentyl) propane-1, 3-diyldioctanoate, 2- (5- ((4- ((((((S) -1-methylpyrrolidin-3-yl) oxy) oxo) hexadecyl) oxy) -5-oxopentyl) propane-1, 3-diyldioctanoate, 2- (5-oxo-5- ((4- (((S) -pyrrolidine-2-carbonyl) oxy) hexadecyl) oxy) pentyl) propane-1, 3-diyldioctanoate, 2- (5- ((4- ((1, 3-dimethylpyrrolidine-3-carbonyl) oxy) hexadecyl) oxy) -5-oxopentyl) propane-1, 3-diyldioctanoate, 2- (5- ((4- ((1, 4-dimethylpiperidine-4-carbonyl) oxy) hexadecyl) oxy) -5-oxopentyl) propane-1, 3-diyldioctanoate, 4-bis (octyloxy) butyl (3- (diethylamino) propyl) pentadecane-1, 3-diyldicarbonate, 3- (((3- (diethylamino) propoxy) carbonyl) oxy) pentadecyl 4, 4-bis ((2-propylpentyl) oxy) butanoate, ((2- ((((3- (diethylamino) propoxy) carbonyl) oxy) methyl) -1, 4-phenylene) bis (oxy)) bis (octane-8, 1-diyl) bis (decanoate), 4-bis (octyloxy) butyl 5- (((3- (diethylamino) propoxy) carbonyl) oxy) heptadecanoate, 6- ((6, 6-bis (octyloxy) hexanoyl) oxy) -4- (((3- (diethylamino) propoxy) carbonyl) hexyl octanoate, (12Z, 15Z) -3- (((3- (diethylamino) propoxy) carbonyl) oxy) heneicosyl-12, 15-dien-1-yl 6, 6-bis (octyloxy) hexanoate, 3- (((3- (diethylamino) propoxy) carbonyl) oxy) tridecyl 6, 6-bis (octyloxy) hexanoate, 3- (((3- (diethylamino) propoxy) carbonyl) oxy) undecyl 6, 6-bis (octyloxy) hexanoate, 3- (((3- (diethylamino) propoxy) carbonyl) oxy) pentadecyl 5- (4, 6-diheptyl-1, 3-dioxan-2-yl) pentanoate, 3- ((5- (diethylamino) pentanoyl) oxy) pentadecyl 6, 6-bis (octyloxy) hexanoate, 1- ((6, 6-bis (octyloxy) hexanoyl) oxy) pentadecyl-3-yl 1, 4-dimethylpiperidin-4-carboxylate, 3- ((3- (1-methylpiperidin-4-yl) propionyl) oxy) pentadecyl 6, 6-bis (octyloxy) hexanoate, 1- ((6, 6-bis (octyloxy) hexanoyl) oxy) pentadecyl-3-yl 1, 3-dimethylpyrrolidine-3-carboxylate, 3- (((3- (diethylamino) propoxy) carbonyl) oxy) pentadecyl 4, 4-bis ((2-ethylhexyl) oxy) butyrate, 2- (((1, 3-dimethylpyrrolidine-3-carbonyl) oxy) methyl) propane-1, 3-diylbis (8- (octanoyloxy) octanoate), ((2- (((((3- (dimethylamino) propoxy) carbonyl) oxy) methyl) -1, 4-phenylene) bis (oxy)) bis (octane-8, 1-diyl) bis (decanoate), (2R) -1- ((6, 6-bis (octyloxy) hexanoyl) oxy) pentadec-3-ylpyrrolidine-2-carboxylate, (2S) -1- ((6, 6-bis (octyloxy) hexanoyl) oxy) pentadec-3-yl 1-methylpyrrolidine-2-carboxylate, (2R) -1- ((6, 6-bis (octyloxy) hexanoyl) oxy) pentadec-3-yl 1-methylpyrrolidine-2-carboxylate, 3- (((3- (dimethylamino) propoxy) carbonyl) oxy) pentadec-6, 6-bis ((3-ethylpentyl) oxy) hexanoate, 3- (((3- (dimethylamino) propoxy) carbonyl) oxy) pentadec-6, 6-bis ((2-propylpentyl) oxy) hexanoate, 3- (((3- (diethylamino) propoxy) carbonyl) oxy) pentadec-6, 6-bis ((2-propylpentyl) oxy) hexanoate, 3- (((2- (diethylamino) ethoxy) carbonyl) oxy) pentadec-6, 6-bis (octyloxy) hexanoate, 3- (((3- (N-morpholinyl) propoxy) carbonyl) oxy) pentadecyl 6, 6-bis (octyloxy) hexanoate, 3- (((((1-methylpiperidin-4-yl) methoxy) carbonyl) oxy) pentadecyl 6, 6-bis (octyloxy) hexanoate, 3- (((3- (4-methylpiperidin-1-yl) propoxy) carbonyl) oxy) pentadecyl 6, 6-bis (octyloxy) hexanoate, 3- (((3- (diethylamino) propoxy) carbonyl) oxy) pentadecyl 4, 4-bis (octyloxy) butanoate, 2- (((4- (dimethylamino) butanoyl) oxy) methyl) -2- ((dodecanoyloxy) methyl) propane-1, 3-diyl (9Z, 9' Z) bis-tetradecane-9-enoate, (9Z, 9'Z, 12' Z) -2- (((4- (dimethylamino) butanoyl) oxy) methyl) propane-1, 3-diylbis (octadeca-9, 12-dienoate), 3- (((4- (diethylamino) butoxy) carbonyl) pentadecyl 6, 6-bis (octyloxy) hexanoate, 3- (((3- (piperazin-1-yl) propoxy) carbonyl) oxy) pentadecyl 6, 6-bis (octyloxy) hexanoate, 3- (((3-piperidin-1-yl) propoxy) carbonyl) oxy) pentadecyl 6, 6-bis (octyloxy) hexanoate, 3- (((3- (dimethylamino) propoxy) carbonyl) oxy) pentadecyl 4, 4-bis (octyloxy) butanoate, (9Z, 9'Z, 12' Z) -2- (9-dodecyl-2-methyl-7, 12-dioxo-6,8,13-trioxa-2-azatetradecan-14-yl) propane-1, 3-diylbis (octadeca-9, 12-dienoate), (9Z, 12Z) -10-dodecyl-3-ethyl-14- (2- ((9Z, 12Z) -octadeca-9, 12-dienoyloxy) ethyl) -8, 13-dioxo-7, 9-dioxa-3, 14-diazahexadecan-16-yl octadeca-9, 12-dienoate, 2- ((2- (((3- (diethylamino) propoxy) carbonyl) oxy) tetradecanoyl) oxy) propane-1, 3-diyldioctoate, 2- (9-dodecyl-2-methyl-7, 13-dioxo-6, 8, 12-trioxa-2-azanonadec-19-yl) propane-1, 3-diyldioctanoate, 2- ((decanoyloxy) methyl) -2- (((4- (dimethylamino) butanoyl) oxy) methyl) propane-1, 3-diyl (9Z, 9' Z) bis-tetradeca-9-enoic acid ester, (9Z, 9' Z,12' Z) -2- (((3- (N-morpholinyl) propionyl) oxy) methyl) propane-1, 3-diylbis (octadeca-9, 12-dienoate), 3- (dimethylamino) propyl carbonate (6Z, 9Z,28Z, 31Z) -heptadecan-6,9,28,31-tetraen-19-yl, 2, 5-bis ((9Z, 12Z) -octadeca-9, 12-dien-1-yloxy) benzyl 4- (dimethylamino) butyrate, 2- (10-dodecyl-3-ethyl-8, 14-dioxo-7, 9, 13-trioxa-3-azaoctadeca-18-yl) propane-1, 3-diyldioctanoate, (9Z, 9'Z, 12' Z) -2- (((1, 3-dimethylpyrrolidine-3-carbonyl) oxy) methyl) propane-1, 3-diylbis (octadeca-9, 12-dienoate), ((5- ((dimethylamino) methyl) benzene-1, 2, 3-tri-yl) tris (oxy)) decane-10, 1-diyl) trioctoate, 0',0- (((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (oxy)) bis (propane-3, 1-diyl)) 9-dioctyl-dinonedioate, (9Z, 12Z) -3- (3- ((dimethylamino) methyl) -5- (3- ((3-octylundecanoyl) oxy) propoxy) phenoxy) propyloctadeca-9, 12-dienoate, (((((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (oxy)) bis (propane-3, 1-diyl)) bis (oxy)) bis (4-oxobutane-4, 1-diyl) bis (decanoate), (R) -4- (3- ((R) -3, 4-bis (octanoyloxy) butoxy) -5- ((dimethylamino) methyl) phenoxy) butane-1, 2-diyldioctanoate, (S) -4- (3- ((S) -3, 4-bis (octanoyloxy) butoxy) -5- ((dimethylamino) methyl) phenoxy) butane-1, 2-diyldioctanoate, (R) -4- (3- ((S) -3, 4-bis (octanoyloxy) butoxy) -5- ((dimethylamino) methyl) phenoxy) butane-1, 2-diyldioctanoate, 4' - ((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (butane 1, 2-diyl) tetraoctanoate, Bisdodecyl 6,6'- ((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (oxy)) dihexanoate, di ((9 z,12 z) -octadeca-9, 12-dien-1-yl) 5,5' - ((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (oxy)) bisvalerate, (((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (methylene)) bis (oxy)) bis (6-oxohexane-6, 1-diyl) bis (decanoate), (5- ((dimethylamino) methyl) -1, 3-phenylene) bis (8- (octanoyloxy) octanoate), (5- ((dimethylamino) methyl) -1, 3-phenylene) bis (10- (octanoyloxy) decanoate), ((((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (methylene)) bis (oxy)) bis (6-oxohexane-6, 1-diyl) dioctanoate, (((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (methylene)) bis (oxy)) bis (8-oxooctane-8, 1-diyl) bis (decanoate), (9Z, 9'Z, 12' Z) - (((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (methylene)) bis (oxy)) bis (4-oxobutane-4, 1-diyl) bis (octadeca-9, 12-dienoate), 0',0- ((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (methylene)) 8-dinonyldioctanedioate, 0' - ((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (methylene)) bis (10- (octanoyloxy) decyl) disuccinate, 0' - ((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (methylene)) bis ((9Z, 12Z) -octadeca-9, 12-dien-1-yl) disuccinate, (9Z, 9' Z,12' Z) - (5- (((((3- (diethylamino) propoxy) carbonyl) oxy) methyl) -1, 3-phenylene) bis (octadeca-9, 12-dienoate), (9Z, 12Z) -4- (3- ((dimethylamino) methyl) -5- (4- (oleoyloxy) butoxy) phenoxy) butyl octadeca-9, 12-dienoate, (9Z, 9' Z,12' Z,15' Z) - ((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (oxy)) bis (butane-4, 1-diyl) bis (octadeca-9, 12, 15-trienoate), ((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (butane-4, 1-diyl) ditetradecanoate, (Z) - ((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (oxy)) bis (butane-4, 1-diyl) dioleate, ((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (oxy)) bis (hexane-6, 1-diyl) bisdodecanoate, (9Z, 9' Z,12' Z) - (((((5- ((diethylamino) methyl) -1, 3-phenylene) bis (oxy)) bis (ethane-2, 1-diyl)) bis (oxy)) bis (ethane-2, 1-diyl) bis (octadeca-9, 12-dienoate), didecyl 8,8' - ((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (oxy)) dioctanoate, ((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (oxy)) bis (propane-3, 1-diyl) bis (3-octylundecanoate), (9Z.9 'Z.12Z.12' Z) - ((5- ((diethylamino) methyl-2-methyl-1.3-phenylene) bis (oxy)) bis (butane-4, 1-diyl) bis (octadeca-9, 12-dienoate), ((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (oxy)) bis (octane-8, 1-diyl) didodecanoate, ((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (oxy)) bis (octane-8, 1-diyl) bis (decanoate), (9Z.9 'Z.12Z.12' Z) - ((5- ((dimethylamino) methyl-2-methyl-1.3-phenylene) bis (oxy)) bis (butane-4, 1-diyl) bis (octadeca-9, 12-dienoate), (8Z, 8' Z) - ((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (oxy)) bis (hexane-bis (dodeca-8-enoate), (9Z, 9' Z,12' Z) - ((5- ((3-hydroxyazetidin-1-yl) methyl) -1, 3-phenylene) bis (oxy)) bis (butane-4, 1-diyl) bis (octadeca-9, 12-dienoate), ((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (oxy)) bis (hexane-6, 1-diyl) dioctoate, ((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (oxy)) bis (hexane-6, 1-diyl) bis (decanoate), (9Z.9 'Z.12Z.12' Z) - ((5- ((dimethylamino) methyl-1.3-phenylene) bis (oxy)) bis (octane-8, 1-diyl) bis (octadeca-9, 12-dienoate), (9Z, 9'Z, 12' Z) - ((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (oxy)) bis (hexane-6, 1-diyl) bis (octadeca-9, 12-dienoate), ((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (oxy)) bis (decane-10, 1-diyl) dihexanoate, ((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (oxy)) bis (decane-10, 1-diyl) dioctanoate, ((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (oxy)) bis (octane-8, 1-diyl) dihexanoate, (9Z, 9'Z, 12' Z) - ((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (oxy)) bis (ethane-2, 1-diyl) bis (octadeca-9, 12-dienoate), (9Z, 9'Z, 12' Z) - ((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (oxy)) bis (propane-3, 1-diyl) bis (octadeca-9, 12-dienoate), (9Z, 9'Z, 12' Z) - ((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (oxy)) bis (butane-4, 1-diyl) bis (octadeca-9, 12-dienoate), (5- ((dimethylamino) methyl) -1, 3-phenylene) bis (methylene) ditridecanoate, (9Z, 9'Z, 12' Z) - (5- ((dimethylamino) methyl) -1, 3-phenylene) bis (methylene) bis (octadeca-9, 12-dienoate), (2, 6-bis ((9Z, 12Z) -octadeca-9, 12-dien-1-yloxy) pyridin-4-yl) methyl 3- (dimethylamino) propionate, (9Z, 9'Z, 12' Z) -5- (((3- (dimethylamino) propionyl) oxy) methyl) -1, 3-phenylenebis (octadeca-9, 12-dienoate), 1- (3, 5-bis ((9Z, 12Z) -octadeca-9, 12-dien-1-yloxy) phenyl) -N, N-dimethylamine, 3, 5-bis ((9Z, 12Z) -octadeca-9, 12-dien-1-yloxy) benzyl 3- (dimethylamino) propionate, 1- (3, 5-bis (4, 4-bis (octyloxy) butoxy) phenyl) -N, N-dimethylamine, ((((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (oxy)) bis (butane-4, 1-diyl)) bis (oxy)) bis (propane-3, 2, 1-diyl) tetraoctanoate, ((5- (((4- (dimethylamino) butyryl) oxy) methyl) -1, 3-phenylene) bis (oxy)) bis (octane-8, 1-diyl) bis (decanoate), ((5- (((3- (dimethylamino) propionyl) oxy) methyl) -1, 3-phenylene) bis (oxy)) bis (octane-8, 1-diyl) bis (decanoate), and, (9Z, 9'Z, 12' Z) - ((5- (3- (N-morpholino) propyl) -1, 3-phenylene) bis (oxy)) bis (butane 4, 1-diyl) bis (octadeca-9, 12-dienoate), (9Z, 9'Z, 12' Z) - ((5- (3- (dimethylamino) propyl) -1, 3-phenylene) bis (oxy)) bis (butane-9, 1-diyl) bis (octadeca-9, 12-dienoate), (9Z, 9'Z, 12' Z) - ((5- (3- (piperidin-1-yl) propyl) -1, 3-phenylene) bis (oxy)) bis (butane-4, 1-diyl) bis (octadeca-9, 12-dienoate), (5- ((dimethylamino) methyl) -1, 3-phenylene) bis (9-pentylmethyl myristate), (5- ((dimethylamino) methyl) -1, 3-phenylene) bis (7-hexyltridecanoate), (5- ((dimethylamino) methyl) -1, 3-phenylene) bis (methylene) bis (5-heptyldodecanoate), ((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (oxy)) bis (butane-4, 1-diyl) bis (3-octylundecanoate), ((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (5-heptyldodecanoate), ((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (oxy)) bis (butane-4, 1-diyl) bis (9-pentylmethyl myristate), ((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (oxy)) bis (butane-4, 1-diyl) bis (7-hexyltridecanoate), (9Z, 9'Z, 12' Z) - ((5- (pyrrolidin-1-ylmethyl) -1, 3-phenylene) bis (oxy)) bis (butane-4, 1-diyl) bis (octadeca-9, 12-dienoate), ((((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (oxy)) bis (methylene)) bis (propane-3, 2, 1-diyl) tetraoctanoate, (((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (butane-4, 1-diyl)) bis (propane-3, 2, 1-diyl) tetraoctanoate, (9z.12z) -4- (3- ((dimethylamino) methyl-5- (4- ((3-octylundecanoyl) oxy) butoxy) phenoxy) butyl octadeca-9, 12-dienoate, bis (1, 3-bis (octanoyloxy) propan-2-yl) 0,0' - ((5- ((dimethylamino) methyl) -1, 3-phenylene) bis (methylene)) bis (propane-3, 2-diyl) tetraoctanoate, (5- ((dimethylamino) methyl) -1, 3-phenylene) bis (methylene) bis (6- (((nonyloxy) carbonyl) oxy) hexanoate), and, 2- (3- (4- (5- ((dimethylamino) methyl) -2-methyl-3- ((9Z, 12Z) -octadeca-9, 12-dien-1-yloxy) phenoxy) butoxy) -3-oxopropyl) propane-1, 3-diyl dihexanoate, 3- ((dimethylamino) methyl) -5- (((8- (octanoyloxy) octanoyl) oxy) methyl) benzyl 3-octyl undecanoate, ((5- ((diethylamino) methyl) benzene-1, 2, 3-triyl) tris (oxy)) tris (decane-10, 1-diyl) trioctanoate, 1- (3, 5-bis ((Z) -octadeca-9-en-1-yloxy) phenyl) -N, N-dimethylmethylamine, n ' -methyl-N ', N ' -tris ((2E.6E) -3.7.11-trimethyldodeca-2.6.10-trien-1-ylpropane-1, 3-diamine, 1, 17-bis (2- ((2-pentylcyclopropyl) methyl) cyclopropyl) heptadec-9-yl 4- (dimethylamino) butyrate, ethyl (7Z) -17- { [4- (dimethylamino) butyryl ] oxy } hexa-2-dienoic acid ester, (Z) -methyl 6- (2- (dimethylamino) -3- (octadeca-9-en-1-yloxy) propoxy) hexanoate, 2- (didodecylamino) -1- (4- (N- (2- (dinonylamino) ethyl) -N-dodecylglycyl) piperazin-1-yl) ethan-1-one, 3- ((3- (1- (3- ((2- (dinonylamino) ethyl) (nonyl) amino) propan-4-yl) piperidin-4-yl) propyl) (nonyl) amino) propylhexanoate, 3- ((3- (4- (3- ((2- (dinonylamino) ethyl) (nonyl) amino) propan-1-yl) -3-oxopropyl) (nonyl) amino) propylhexanoate, 3- ((2- (dinonylamino) ethyl) (nonyl) amino) -1- (4- (3- (dinonylamino) propyl) piperidin-1-yl) propan-1-one, pentyl 4- ((3- (1- (3- ((2- (dinonylamino) ethyl) (nonyl) amino) propan-4-yl) propan-yl) piperidin-4-yl) butanols, Amyl 4- ((2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperidin-4-yl) ethyl) (nonyl) amino) butyrate, amyl 4- (((1- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) pyrrolidin-3-yl) methyl) (nonyl) amino) butyrate, amyl 4- ((2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) pyrrolidin-3-yl) ethyl) (nonyl) amino) butyrate, amyl 4- ((2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperidin-3-yl) ethyl) (nonyl) amino) butyrate, 2- (Didodecylamino) -1- (4- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperazin-1-yl) ethan-1-one, 2- ((2- (dinonylamino) ethyl) (nonylamino) -1- (3- (2- (dinonylamino) ethyl) piperidin-1-yl) ethan-1-one, dipentyl 4,4' - ((2- (4- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperazin-1-yl) -2-oxoethyl) azetidine) dibutyrate, amyl 4- (nonyl (2- (4- (N-nonyl-N- (2- (nonyl (4-oxo-4- (pentyloxy) butyl) amino) ethyl) glycinyl) piperazin-1-yl) -2-oxoethyl) amino) butyrate, 2- ((2- (dinonylamino) ethyl) (nonyl) amino) -1- (3- ((dinonylamino) methyl) pyrrolidin-1-yl) ethan-1-one, 2- ((2- (didodecylamino) ethyl) (dodecyl) amino) -1- (4- (dinonylglycyl) piperazin-1-yl) ethan-1-one, 2- ((2- (dinonylamino) ethyl) (nonyl) amino) -1- (3- (2- (dinonylamino) ethyl) pyrrolidin-1-yl) ethan-1-one, pentyl 4- ((3- (4- (3- ((2- (dinonylamino) ethyl) (nonyl) amino) propionyl) piperazin-1-yl) -3-oxopropyl) (nonyl) amino) butanoate, 3- ((2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperidin-4-yl) ethyl) (nonyl) amino) propylhexanoate, butyl 5- ((2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperidin-4-yl) ethyl) (nonyl) amino) pentanoate, 2- ((2- (didodecylamino) ethyl) (nonyl) amino) -1- (4- (dinonylglycyl) piperazin-1-yl) ethyl-1-one, propyl 6- ((2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperidin-4-yl) ethyl) (nonyl) amino) hexanoate, Ethyl 7- ((2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperidin-4-yl) ethyl) (nonyl) amino) heptanoate, methyl 8- ((2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperidin-4-yl) ethyl) (nonyl) amino) octanoate, 3- ((2- (4- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperazin-1-yl) -2-oxoethyl) (nonyl) amino) propylhexanoate, butyl 5- ((2- (4- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperazin-1-yl) -2-oxoethyl) (nonyl) amino) pentanoate, Propyl 6- ((2- (4- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperazine-2-oxoethyl) (nonyl) amino) hexanoate, ethyl 7- ((2- (4- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperazin-1-yl) -2-oxoethyl) (nonyl) amino) heptanoate, 3- (dinonylamino) -1- (4- (3- ((2- (dinonylamino) ethyl) (nonyl) amino) propionyl) piperazin-1-yl) propan-1-one, 2- ((2- (dinonylamino) ethyl) (nonyl) amino) -1- (4- (bitetradecylglycinyl) piperazin-1-yl) ethan-1-one, 2- (dinonylamino) -1- (4- (2- ((2- (dinonylamino) ethyl) (nonyl) amino) ethyl) piperidin-1-yl) ethan-1-one, 2- (dinonylamino) -1- (4- (N- (2- (dinonylamino) ethyl) -N-dodecylglycyl) piperazin-1-yl) ethan-1-one, 2- ((2- (dinonylamino) ethyl) (nonyl) amino) -1- (4- (2- (dinonylamino) ethyl) piperidin-1-yl) ethan-1-one, methyl 8- ((2- (4- (dinonylamino) piperazin-1-yl) -2-oxoethyl) (2- ((8-methoxy-8-oxooctyl) (nonyl) amino) ethyl) amino) octanoate, methyl 8- ((2- (dinonylamino) ethyl) (2- (4- (dinonylamino) piperazin-1-yl) -2-oxoethyl) amino) octanoate, methyl 8- ((2- ((2- (4- (dinonylamino) piperazin-1-yl) -2-oxoethyl) (nonyl) amino) ethyl) (nonyl) amino) octanoate, pentyl 4- ((2- (4- (N- (2- (dinonylamino) ethyl) -N-nonylalanyl) piperazin-2-oxoethyl) (nonyl) amino) butanoate, methyl 8- ((2- (4- (N- (2- (dinonylamino) ethyl) -N-nonylamino) piperazin-1-yl) -2-oxoethyl) (nonyl) amino) octanoate, 2- ((2- (Didodecylamino) ethyl) (dodecylamino) -1- (5- (dinonylglycyl) -2, 5-diazabicyclo [2.2.1] heptan-2-yl) ethan-1-one 3, 2- (dinonylamino) -1- (5- (N- (2- (dinonylamino) ethyl) -N-nonylglycyl) -2, 5-diazabicyclo [2.2.1] heptan-2-yl) ethan-1-one, N1, N2-tris ((9Z, 12Z) -octadeca-9, 12-dien-1-yl) -N2- (2- (piperazin-1-yl) ethyl) ethan-2-1, 2-diamine, N1, N1, N2-tris ((Z) -octadec-9-en-1-yl) -N2- (2- (piperazin-1-yl) ethyl) ethane-1, 2-diamine, 2- (dinonylamino) -1- (4- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperazin-1-yl) ethan-1-one, N1, N2-tris (dodecyl) -N2- (2- (piperazin-1-yl) ethyl) ethane-1, 2-diamine, N1, N2-trisnonyl-N2- (2- (piperazin-1-yl) ethyl) ethane-1, 2-diamine, N1, N2-trihexyl-N2- (2- (piperazin-1-yl) ethyl) ethane-1, 2-diamine, N1- (2- (4- (2- (didodecylamino) ethyl) piperazin-1-yl) ethyl) -N1, N2, N2-tris ((9Z, 12Z) -octadeca-9, 12-dien-1-yl) ethane-1, 2-diamine, N1- (2- (4- (2- (didodecylamino) ethyl) piperazin-1-yl) ethyl) -N1, N2, N2-tris ((Z) -octadeca-9-en-1-yl) ethane-1, 2-diamine, N1- (2- (4- (2- (didodecylamino) ethyl) piperazin-1-yl) ethyl) -N1, N2, N2-tris (tetradecyl) ethane-1, 2-diamine, N1- (2- (4- (2- (didodecylamino) ethyl) piperazin-1-yl) ethyl) -N1, N2, N2-tris (tetradecyl) ethane-1, 2-diamine, N1- (2- (4- (2- (dinonylamino) ethyl) piperazin-1-yl) ethyl) -N1, N2, N2-tris (tetradecyl) ethane-1, 2-diamine, 2- (didodecylamino) -1- (4- (2- ((2- (didodecylamino) ethyl) (dodecylamino) ethyl) amino) piperazin-1-yl) ethyl) ethan-1-one, N1- (2- (di ((9Z, 12Z) -octadeca-9, 12-dien-1-yl) amino) ethyl) piperazin-1-yl) ethyl) -N1, N2, N2-tris (dodecyl) ethane-1, 2-diamine, N1- (2- (4- (2- (di ((Z) -octadeca-9-en-1-yl) amino) ethyl) piperazin-1-yl) ethyl) -N1, N2, N2-tris (dodecyl) ethane-1, 2-diamine, N1, N2-tris (dodecyl) -N2- (2- (dodecyl ((9Z, 12Z) -octadeca-9, 12-dien-1-yl) amino) ethyl) piperazin-1-yl) ethyl) ethane-1, 2-diamine, N1- (2- (4- (2- (bitetradecylamino) ethyl) piperazin-1-yl) ethyl) -N1, N2, N2-tris (dodecyl) ethane-1, 2-diamine, N1- (2- (4- (2- (di- (Z) -dodeca-6-en-1-yl) amino) ethyl) piperazin-1-yl) ethyl) -N1, N2, N-tris (dodecyl) ethane-1, 2-diamine, (Z) -N1- (2- (4- (2- (dodeca-6-en-1-yl) amino) ethyl) piperazin-1-yl) ethyl) -N, N2, N2-tris (dodecyl) ethane-1, 2-diamine, N1- (2- (4- (2- (dinonylamino) ethyl) piperazin-1-yl) ethyl) -N1, N2, N2-tris (dodecyl) ethane-1, 2-diamine, N1- (2- (4- (2- (dioctylamino) ethyl) piperazin-1-yl) ethyl) -N1, N2, N2-tris (dodecyl) ethane-1, 2-diamine, N1- (2- (4- (2- (dihexylamino) ethyl) piperazin-1-yl) ethyl) -N1, N2, N2-tris (dodecyl) ethane-1, 2-diamine, N1- (2- (4- (2- (ditetradecylamino) ethyl) piperazin-1-yl) ethyl) -N1, N2, N2-trisnonylethane-1, 2-diamine, 2- ((2- (didodecylamino) ethyl) (dodecylamino) -1- (4- (2- (didodecylamino) ethyl) piperazin-1-yl) ethane-1-one, N1- (2- (4- (2- (didodecylamino) ethyl) piperazin-1-yl) ethyl) -N1, N2, N2-trisnonylethane-1, 2-diamine, N1- (2- (4- (2- (dinonylamino) ethyl) piperazin-1-yl) ethyl) -N1, N2, N2-trisnonylethane-1, 2-diamine, N1- (2- (4- (2- (didodecylamino) ethyl) piperazin-1-yl) ethyl) -N1, N2, N2-trihexylethane-1, 2-diamine, dimethyl 12,12' - ((2- (4- (2- ((2- (didodecylamino) ethyl) (dodecylamino) ethyl) piperazin-1-yl) ethyl) azetidine, Methyl 12- ((2- (4- (2- ((2- (didodecylamino) ethyl) (dodecyl) amino) ethyl) piperazin-1-yl) ethyl) (dodecyl) amino) dodecanoate, dipentyl 6,6' - ((2- (4- (2- ((2- (didodecylamino) ethyl) (dodecyl) amino) ethyl) piperazin-1-yl) ethyl) azetidine) dihexanoate, amyl 6- ((2- (4- (2- ((2- (didodecylamino) ethyl) (tetradecyl) amino) ethyl) piperazin-1-yl) ethyl) (dodecyl) amino) hexanoate, amyl 6- ((2- (4- (2- ((2- (didodecylamino) ethyl) (dodecyl) amino) ethyl) piperazin-1-yl) ethyl) (dodecyl) amino) hexanoate, 2- (didodecylamino) -1- (4- (N- (2- (didodecylamino) ethyl) -N-dodecylglycyl) piperazin-1-yl) ethan-1-one, 2- (didodecylamino) -1- (4- (N- (2- (didodecylamino) ethyl) -N-nonylglycinyl) piperazin-1-yl) ethan-1-one, 2- (didodecylamino) -N- (2- (4- (2- (didodecylamino) ethyl) piperazin-1-yl) ethyl) -N-dodecylacetamide, ((2- ((3, S ', 4R) -3, 4-dihydroxypyrrolidin-1-yl) acetyl) azodiyl) bis (ethane-2, 1-diyl) (9Z, 9' Z,12' Z) -bis (octadeca-9, 12-dienoate), 2-amino-N, N-Dihexadecyl-3- (1H-imidazol-5-yl) propanamide, (2-amino-N, N-Dihexadecyl-3- (1H-imidazol-5-yl) propanamide, methyl (9Z) -19- [2- (dimethylamino) ethyl ] heptadecan-9-enoic acid ester, methyl 8- (2- {9- [2- (dimethylamino) ethyl ] octadecyl } cyclopropyl) octanoate, methyl (9Z) -19- [2- (dimethylamino) ethyl ] octan-9-enoic acid ester, ethyl 8- (2- {11- [ (dimethylamino) methyl ] heptadecan-yl } cyclopropyl) octanoate, Ethyl 8- (2- {11- [ (dimethylamino) methyl ] octadecyl } cyclopropyl) octanoate, di ((9 z,12 z) -octadeca-9, 12-dien-1-yl) 3- (((2- (dimethylamino) ethoxy) carbonyl) amino) pentanedioate, heptyl 6- ((2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperidin-4-yl) ethyl) (tetradecyl) amino) hexanoate, ethyl 8- (2- {11- [ (dimethylamino) methyl ] nonadecyl } cyclopropyl) octanoate, pentyl 8- ((2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperidin-4-yl) ethyl) (tetradecyl) amino) octanoate, Ethyl 8- (2- {11- [ (dimethylamino) methyl ] eicosyl } cyclopropyl) octanoate, ethyl 8- (2- {9- [ (dimethylamino) methyl ] pentadecyl } cyclopropyl) octanoate, 3- ((2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperidin-4-yl) ethyl) (tetradecyl) amino) propyldecanoate, heptyl 6- ((2- (4- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperazin-1-yl) -2-oxoethyl) (tetradecyl) amino) hexanoate, ethyl 8- (2- {9- [ (dimethylamino) methyl ] hexadecyl } cyclopropyl) octanoate, Amyl 8- ((2- (4- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperazin-2-oxoethyl) (tetradecyl) amino) octanoate, ethyl 8- (2- {9- [ (dimethylamino) methyl ] heptadecyl } cyclopropyl) octanoate, methyl 6- (2- (8- (2- (dimethylamino) -3- (nonyloxy) propoxy) octyl) cyclopropyl) hexanoate, methyl (9Z) -21- (dimethylamino) heptadecan-9-enoate, methyl (9Z) -21- { [4- (dimethylamino) butyryl ] oxy } heptadecan-9-enoate, (2R) -N, N-dimethyl-1- [ (9Z, 12Z) -octadec-9, 12-dien-1-yloxy ] dodecane-2-amine, (15Z, 18Z) -N, N-dimethyltetracosane (tetracoda) -15, 18-dien-5-amine, ethyl 8- (2- {9- [ (dimethylamino) methyl ] octadecyl } cyclopropyl) octanoate, 3- ((2- (4- (N- (2- (dinonylamino) ethyl) -N-nonylglycoyl) piperazin-1-yl) -2-oxoethyl) (tetradecyl) amino) propyldecanoate, ethyl 4- (2- {11- [ (dimethylamino) methyl ] eicosyl } cyclopropyl) butanoate, Ethyl 8- (2- {7- [ (dimethylamino) methyl ] hexadecyl } cyclopropyl) octanoate, 3- ((3- (1- (3- ((2- (dinonylamino) ethyl) (nonyl) amino) propionyl) piperidin-4-yl) propyl) (nonyl) amino) propylhexanoate, ethyl 6- (2- {9- [ (dimethylamino) methyl ] pentadecyl } cyclopropyl) hexanoate, 3- ((3- (4- (3- ((2- (dinonylamino) ethyl) (nonyl) amino) propionyl) piperazin-1-yl) -3-oxopropyl) (nonyl) amino) propylhexanoate, ethyl 6- (2- {9- [ (dimethylamino) methyl ] hexadecyl } cyclopropyl) hexanoate, 3- ((2- (dinonylamino) ethyl) (nonyl) amino) -1- (4- (3- (dinonylamino) propyl) piperidin-1-yl) propan-1-one, pentyl 4- ((3- (1- (3- ((2- (dinonylamino) ethyl) (nonyl) amino) propan-4-yl) propyl) (nonyl) amino) butanol, ethyl 6- (2- {9- [ (dimethylamino) methyl ] heptadecyl } cyclopropyl) hexanoate, pentyl 4- ((2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylanilino) piperidin-4-yl) ethyl) (nonyl) amino) butanoate, Ethyl 6- (2- {9- [ (dimethylamino) methyl ] octadecyl } cyclopropyl) hexanoate, amyl 4- (((1- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) pyrrolidin-3-yl) methyl) (nonyl) amino) butanoate, ethyl (9Z) -21- [ (dimethylamino) methyl ] heptadecan-9-enoic acid ester, amyl 4- ((2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) pyrrolidin-3-yl) ethyl) (nonyl) amino) butanoate, ethyl (9Z) -21- [ (dimethylamino) methyl ] octacosa-9-enoic acid ester, ((2- ((3, S ', 4R) -3, 4-dihydroxypyrrolidin-1-yl) acetyl) azodiyl) bis (ethane-2, 1-diyl) (9Z, 9' Z,12' Z) -bis (octadeca-9, 12-dienoate), pentyl 4- ((2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperidin-3-yl) ethyl) (nonyl) amino) butyrate, ethyl (9Z) -21- [ (dimethylamino) methyl ] icosac-9-enoate, methyl 6- (2- (8- (2- (dimethylamino) -3- (heptyloxy) propoxy) octyl) cyclopropyl) hexanoate, Methyl (9Z) -21- { [4- (dimethylamino) butyryl ] oxy } octacosa-9-enoic acid ester, methyl (9Z) -21- (dimethylamino) octacosa-9-enoic acid ester, 2- (didodecylamino) -1- (4- (N- (2- (dinonylamino) ethyl) -N-nonylglycol) piperazin-1-yl) ethan-1-, (2S) -N.N-dimethyl-1- [ (9Z, 12Z) -octadeca-9, 12-dien-1-yloxy ] non-2-amine, (18Z, 21Z) -N, N-dimethylheptadecan-18, 21-dien-10-amine, Ethyl (9Z) -21- [ (dimethylamino) methyl ] triacont-9-enoic acid ester, ethyl (9Z) -19- [ (dimethylamino) methyl ] pentadecan-9-enoic acid ester, ethyl (9Z) -19- [ (dimethylamino) methyl ] hexa-dec-9-enoic acid ester, ethyl (9Z) -19- [ (dimethylamino) methyl ] hepta-dec-9-enoic acid ester, ethyl (9Z) -19- [ (dimethylamino) methyl ] octan-9-enoic acid ester, ethyl (5Z) -17- [ (dimethylamino) methyl ] hexa-5-enoic acid ester, ethyl (9Z) -17- [ (dimethylamino) methyl ] hexa-9-enoic acid ester, 2- ((2- (dinonylamino) ethyl) (nonyl) amino) -1- (3- (2- (dinonylamino) ethyl) piperidin-1-yl) ethan-1-one, ethyl (7Z) -17- [ (dimethylamino) methyl ] ditridec-7-enoic acid ester, dipentyl 4,4' - ((2- (4- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperazin-1-yl) -2-oxoethyl) azetidine) dibutyrate, amyl 4- (nonyl (2- (4- (N-nonyl-N- (2- (nonyl (4-oxo-4- (pentoxy) butyl) amino) ethyl) glycinyl) piperazin-1-yl) -2-oxoethyl) amino) butyrate, Ethyl (7Z) -17- [ (dimethylamino) methyl ] tetracos-7-enoic acid ester, ethyl (7Z) -17- [ (dimethylamino) methyl ] pentadecan-7-enoic acid ester, 2- ((2- (dinonylamino) ethyl) (nonyl) amino) -1- (3- ((dinonylamino) methyl) pyrrolidin-1-yl) ethan-1-one, trans-3- [ (3}7-dimethyloctyl) oxy ] -1-methyl-4- [ (9Z, 12Z) -octadec-9512-dien-1-yloxy-pyrrolidine, methyl 6- (2- (8- (2- (dimethylamino) -3- (hexyloxy) propoxy) octyl) cyclopropyl) hexanoate, Methyl (9Z) -21- { [4- (dimethylamino) butyryl ] oxy } icosacarbon-9-enoate, methyl (9Z) -21- (dimethylamino) icosacarbon-9-enoate, (2S) -N, N-dimethyl-1- [ (9Z, 12Z) -octadeca-9, 12-dien-1-yloxy ] tridecan-2-amine, (15Z, 18Z) -N, N-dimethyltetracos-15, 18-dien-7-amine, ethyl (7Z) -17- [ (dimethylamino) methyl ] hexa-dien-7-enoate, 2- ((2- (dinonylamino) ethyl) (nonylamino) ethyl) -1- (3- (2- (dinonylamino) ethyl) pyrrolidin-1-yl) ethan-1-one, Methyl 6- (2- {11- [ (dimethylamino) methyl ] eicosyl } cyclopropyl) hexanoate, methyl 10- (2- {7- [ (dimethylamino) methyl ] hexadecyl } cyclopropyl) decanoate, methyl 8- (2- {11- [ (dimethylamino) methyl ] heptadecyl } cyclopropyl) octanoate, methyl 8- (2- {11- [ (dimethylamino) methyl ] octadecyl } cyclopropyl) octanoate, methyl 8- (2- {11- [ (dimethylamino) methyl ] nonadecyl } cyclopropyl) octanoate, methyl 8- (2- {11- [ (dimethylamino) methyl ] eicosyl } cyclopropyl) octanoate, and, Amyl 4- ((3- (4- (3- ((2- (dinonylamino) ethyl) (nonyl) amino) propionyl) piperazin-1-yl) -3-oxopropyl) (nonyl) amino) butanoate, methyl 8- (2- {9- [ (dimethylamino) methyl ] pentadecyl } cyclopropyl) octanoate, methyl 8- (2- {9- [ (dimethylamino) methyl ] hexadecyl } cyclopropyl) octanoate, 3- ((2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperidin-4-yl) ethyl) (nonyl) amino) propylhexanoate, methyl 8- (2- {9- [ (dimethylamino) methyl ] heptadecyl } cyclopropyl) octanoate, Methyl 8- (2- (dimethylamino) -3- ((6- ((2-octylcyclopropyl) methoxy) -6-oxohexyl) oxy) propoxy) octanoate, butyl 5- ((2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperidin-4-yl) ethyl) (nonyl) amino) pentanoate, trans-1-methyl-3- [ (12Z) -octadeca-12-en-1-yloxy ] -4- (octyloxy) pyrrolidine, methyl (9Z) -21- { [4- (dimethylamino) butanoyl ] oxy } triacontan-9-enoate, methyl (9Z) -21- (dimethylamino) triacontan-9-enoate, 2- ((2- (Didodecylamino) ethyl) (nonylamino) -1- (4- (dinonylglycyl) piperazin-1-yl) ethyl-1-one methyl N- (2- (didodecylamino) ethyl) -N-nonylglycine ester, 1- ((2R, 3S, 5R) -3- (bis (hexadecyloxy) methoxy) -5- (5-methyl-2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) tetrahydrofuranmethanesulfonate, (Z) -methyl 16- (3- (decyloxy) -2- (dimethylamino) propoxy) hexadeca-7-enoate, (2S) -1- [ (9Z, 12Z) -octadeca-9, 12-dien-1-yloxy ] non-2-amine, (14Z, 17Z) -N, N-dimethylditridec-14, 17-dien-6-amine, propyl 6- ((2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperidin-4-yl) ethyl) (nonyl) amino) hexanoate, methyl 7- (2- (dimethylamino) -3- ((6- ((2-octylcyclopropyl) methoxy) -6-oxohexyl) oxy) propoxy) heptanoate, methyl (7Z) -19- [ (dimethylamino) methyl ] dioctadec-7-enoate, methyl (HZ) -19- [ (dimethylamino) methyl ] octacos-11-enoate, Ethyl 7- ((2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperidin-4-yl) ethyl) (nonyl) amino) heptanoate, (2-octylcyclopropyl) methyl 6- (2- (dimethylamino) -3- ((5-methoxy-5-oxopentyl) oxy) propoxy) hexanoate, methyl 8- ((2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperidin-4-yl) ethyl) (nonyl) amino) octanoate, methyl (9Z) -21- [ (dimethylamino) methyl ] heptadecan-9-enoic acid ester, (2-octylcyclopropyl) methyl 6- (2- (dimethylamino) -3- (4-methoxy-4-oxobutoxy) propoxy) hexanoate, Methyl (9Z) -21- [ (dimethylamino) methyl ] octacosa-9-enoic acid ester, 3- ((2- (4- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperazin-1-yl) -2-oxoethyl) (nonyl) amino) propylhexanoate, (Z) -methyl 8- (2- (dimethylamino) -3- ((6-oxo-6- (undec-2-en-1-yloxy) hexyl) oxy) propoxy) octanoate, methyl (9Z) -21- [ (dimethylamino) methyl ] octacosa-9-enoic acid ester, butyl 5- ((2- (4- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperazin-1-yl) -2-oxoethyl) (nonyl) amino) pentanoate, (Z) -methyl 7- (2- (dimethylamino) -3- ((6-oxo-6- (undec-2-en-1-yloxy) hexyl) oxy) propoxy) heptanoate, propyl 6- ((2- (4- (N- (2- (dinonylamino) ethyl) -N-nonylgeranyl) piperazin-1-yl) -2-oxoethyl) (nonyl) amino) hexanoate, methyl (9Z) -21- [ (dimethylamino) methyl ] triacont-9-enoic acid ester, (Z) -undec-2-en-1-yl 6- (2- (dimethylamino) -3- ((5-methoxy-5-oxopentyl) oxy) propoxy) hexanoate, methyl (9Z) -19- [ (dimethylamino) methyl ] pentadecan-9-enoate, ethyl 7- ((2- (4- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperazin-1-yl) -2-oxoethyl) (nonyl) amino) heptanoate, (Z) -undec-2-en-1-yl 6- (2- (dimethylamino) -3- (4-methoxy-4-oxobutoxy) propoxy) hexanoate, methyl 6- (2- (dimethylamino) -3- ((6- ((2-octylcyclopropyl) methoxy) -6-oxohexyl) oxy) propoxy) hexanoate, Methyl (9Z) -19- [ (dimethylamino) methyl ] hexa-2-enoate, 3- (dinonylamino) -1- (4- (3- ((2- (dinonylamino) ethyl) (nonyl) amino) propan-1-yl) piperazin-1-one, methyl (9Z) -19- [ (dimethylamino) methyl ] hepta-9-enoate, 2- ((2- (dinonylamino) ethyl) (nonyl) amino) -1- (4- (ditetradecylglycinyl) piperazin-1-yl) ethan-1-one, (Z) -methyl 6- (2- (dimethylamino) -3- ((6-oxo-6- (undec-2-en-1-yloxy) hexyl) oxy) propanoxy) hexanoate, Methyl 8- (2- (dimethylamino) -3- ((8- (2- (6-methoxy-6-oxohexyl) cyclopropyl) oxy) propoxy) octanoate, methyl 8- (2- {9- [ (dimethylamino) methyl ] octadecyl } cyclopropyl) octanoate, 2- (dinonylamino) -1- (4- (2- ((2- (dinonylamino) ethyl) (nonyl) amino) ethyl) piperidin-1-yl) ethan-1-one, trans-1-methyl-3- [ (9Z) -octadec-9-en-1-yloxy ] -4- (octyloxy) pyrrolidine, methyl (9Z) -19- { [4- (dimethylamino) butanoyl ] oxy } cyclopentadec-9-enoic acid ester, methyl (9Z) -19- (dimethylamino) cyclopentadec-9-enoate, (Z) -methyl 16- (2- (dimethylamino) -3- (nonyloxy) propoxy) hexadeca-7-enoate, (2S) -1- [ (9Z, 12Z) -octadec-9, 12-dien-1-yloxy ] dec-2-amine, (12Z, 15Z) -N, N-dimethyldi-undeca-12, 15-dien-4-amine, methyl 7- (2- (dimethylamino) -3- ((8- (2- (6-methoxy-6-oxohexyl) cyclopropyl) oxy) octyl) propoxy) heptanoate, Methyl (9Z) -19- [ (dimethylamino) methyl ] octacosa-9-enoic acid ester, 2- ((2- (dinonylamino) ethyl) (nonyl) amino) -1- (4- (2- (dinonylamino) ethyl) piperidin-1-yl) ethan-1-one, methyl 8- ((2- (4- (dinonylamino) piperazin-1-yl) -2-oxoethyl) (2- ((8-methoxy-8-oxooctyl) (nonyl) amino) ethyl) amino) octanoate, methyl 6- (2- (8- (2- (dimethylamino) -3- ((5-methoxy-5-oxopentyl) oxy) propoxy) octyl) cyclopropyl) hexanoate, Ethyl 8- {2- [11- (dimethylamino) heptadecyl ] cyclopropyl } caprylate, methyl 8- ((2- (dinonylamino) ethyl) (2- (4- (dinonylamino) piperazin-1-yl) -2-oxoethyl) amino) caprylate, methyl 6- (2- (8- (2- (dimethylamino) -3- (4-methoxy-4-oxobutoxy) propoxy) octyl) cyclopropyl) hexanoate, ethyl 8- {2- [11- (dimethylamino) octadecyl ] cyclopropyl } caprylate, methyl 8- ((2- ((2- (4- (dinonylamino) piperazin-1-yl) -2-oxoethyl) (nonyl) amino) ethyl) (nonyl) amino) caprylate, Ethyl 8- {2- [11- (dimethylamino) nonadecyl ] cyclopropyl } octanoate, (Z) -methyl 16- (2- (dimethylamino) -3- ((8-methoxy-8-oxooctyl) oxy) propoxy) hexadeca-7-enoate, pentyl 4- ((2- (4- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperazin-1-yl) -2-oxoethyl) (nonyl) amino) butanoate, ethyl 8- {2- [11- (dimethylamino) eicosyl ] cyclopropyl } octanoate, (Z) -methyl 16- (2- (dimethylamino) -3- ((7-methoxy-7-oxoheptyl) oxy) propoxy) hexadeca-7-enoate, Methyl 8- ((2- (4- (N- (2- (dinonylamino) ethyl) -N-nonylglycol) piperazin-1-yl) -2-oxoethyl) (nonyl) amino) octanoate, ethyl 8- {2- [9- (dimethylamino) pentadecyl ] cyclopropyl } octanoate, (Z) -methyl 16- (2- (dimethylamino) -3- ((5-methoxy-5-oxopentyl) oxy) propoxy) hexadeca-7-enoic acid ester, (11E, 20Z, 23Z) -N, N-dimethyl icosacarbon-11,20,23-trien-10-amine, N-dimethyl-1- [ (1S, 2R) -2-octylcyclopropyl ] pentadecyl-8-amine, Ethyl 8- {2- [9- (dimethylamino) hexadecyl ] cyclopropyl } octanoate, 2- ((2- (didodecylamino) ethyl) (dodecyl) amino) -1- (5- (dinonylglycyl) -2, 5-diazabicyclo [2.2.1] heptan-2-yl) ethan-1-one 3, (Z) -methyl 16- (2- (dimethylamino) -3- (4-methoxy-4-oxobutoxy) propoxy) hexadeca-7-enoic acid ester, methyl 6- (2- (8- (2- (dimethylamino) -3- ((6-methoxy-6-oxohexyl) oxy) propoxy) octyl) cyclopropyl) hexanoate, Ethyl 8- {2- [9- (dimethylamino) heptadecyl ] cyclopropyl } caprylate, 2- (dinonylamino) -1- (5- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) -2, 5-diazabicyclo [2.2.1] heptan-2-yl) ethan-1-one, 1- [ (1 s,2 r) -2-decylcyclopropyl ] -N, N-dimethylpentadecan-6-amine, N1, N2-tris ((9 z,12 z) -octadeca-9, 12-dien-1-yl) -N2- (2- (piperazin-1-yl) ethyl) ethane-1, 2-diamine, ethyl 8- {2- [9- (dimethylamino) octadecyl ] cyclopropyl } caprylate, 1- [ (1R, 2S) -2-heptylcyclopropyl ] -N, N-dimethyloctadecan-9-amine, (Z) -methyl 16- (2- (dimethylamino) -3- ((6-methoxy-6-oxohexyl) oxy) propoxy) hexadeca-7-enoic acid ester, N1, N2-tris ((Z) -octadeca-9-en-1-yl) -N2- (2- (piperazin-1-yl) ethyl) ethane-1, 2-diamine, N-dimethyl-3- {7- [ (1S, 2R) -2-octylcyclopropyl ] heptyl } dodecyl-1-amine, methyl 8- (2- (dimethylamino) -3- ((8- (2- ((2-pentylcyclopropyl) methyl) cyclopropyl) oxy) octyl) oxy) propoxy) octanoate, Ethyl 4- {2- [11- (dimethylamino) eicosyl ] cyclopropyl } butanoate, trans-1-methyl-3- [ ((9Z, 12Z) -octadeca-9, 12-dienyl) oxy ] -4-octoxy-pyrrolidine, methyl (9Z) -19- (dimethylamino) dicetyl-9-enoate, methyl (9Z) -19- { [4- (dimethylamino) butanoyl ] oxy } dicetyl-9-enoate, (Z) -methyl 16- (2- (dimethylamino) -3- (heptyloxy) propoxy) hexadeca-7-enoate, (2R) -1- [ (9Z, 12Z) -octadeca-9, 12-dien-1-yloxy ] dodec-2-amine, (13Z, 16Z) -N, N-dimethylbehenyl-13, 16-dien-5-amine, N-dimethyl-1- [ (1R, 2S) -2-undecylcyclopropyl ] tetradecan-5-amine, methyl 7- (2- (dimethylamino) -3- ((8- (2- ((2-pentylcyclopropyl) methyl) cyclopropyl) octyl) oxy) propoxy) heptanoate, ethyl 8- {2- [7- (dimethylamino) hexadecyl ] cyclopropyl } octanoate, 2- (didodecylamino) -N-dodecyl-N- (2- (piperazin-1-yl) ethyl) acetamide, N-dimethyl-1- [ (1S, 2R) -2-octylcyclopropyl ] hexadecan-8-amine, N1- (2- (piperazin-1-yl) ethyl) -N1, N2, N2-tris (tetradecyl) ethane-1, 2-diamine, methyl 6- (2- (dimethylamino) -3- ((8- (2- ((2-pentylcyclopropyl) methyl) cyclopropyl) oxy) propoxy) hexanoate, ethyl 6- {2- [9- (dimethylamino) pentadecyl ] cyclopropyl } hexanoate, N-dimethyl-1- [ (1S, 2S) -2- { [ (1R, 2R) -2-pentylcyclopropyl ] methyl } cyclopropyl ] nona-ne-10-amine, NN1, N2-tris (dodecyl) -N2- (2- (piperazin-1-yl) ethyl) ethane-1, 2-diamine, Methyl 5- (2- (dimethylamino) -3- ((8- (2- ((2-pentylmethyl) cyclopropyl) octyl) oxy) propoxy) pentanoate, ethyl 6- {2- [9- (dimethylamino) hexadecyl ] cyclopropyl } hexanoate, N-dimethyl-21- [ (1S, 2R) -2-octylcyclopropyl ] heneicosane-10-amine, NNN 2-trisnonyl-N2- (2- (piperazin-1-yl) ethyl) ethane-1, 2-diamine, methyl 4- (2- (dimethylamino) -3- ((8- (2- ((2-pentylmethyl) cyclopropyl) octyl) oxy) propoxy) butanoate, Ethyl 6- {2- [9- (dimethylamino) heptadecyl ] cyclopropyl } hexanoate, N-dimethyl-1- [ (1 s,2 r) -2-octylcyclopropyl ] nonadecan-10-amine, N1, N2-trihexyl-N2- (2- (piperazin-1-yl) ethyl) ethane-1, 2-diamine, methyl 8- (2- (dimethylamino) -3- ((9 z,12 z) -octadeca-9, 12-dien-1-yloxy) propoxy) octanoate, ethyl 6- {2- [9- (dimethylamino) octadecyl ] cyclopropyl } hexanoate, N1- (2- (4- (2- (didodecyl amino) ethyl) piperazin-1-yl) ethyl) -N1, N2-tris ((9 z,12 z) -octadeca-9, 12-dien-1-yl) ethane-1, 2-diamine, Methyl 7- (2- (dimethylamino) -3- ((9Z, 12Z) -octadeca-9, 12-dien-1-yloxy) propoxy) heptanoate, ethyl (9Z) -21- (dimethylamino) heptadecan-9-enoic acid ester, 1- [ (1S, 2R) -2-hexylcyclopropyl ] -N, N-dimethylnonadecan-10-amine, 1-methyl 18- [ (2Z) -non-2-en-1-yl ]9- { [4- (dimethylamino) butanoyl ] oxy } octadecanedioate, N1- (2- (4- (2- (didodecylamino) ethyl) piperazin-1-yl) ethyl) -N1, N2, N2-tris ((Z) -octadeca-9-en-1-yl) ethane-1, 2-diamine, n, N-dimethyl-1- [ (1S, 2R) -2-octylcyclopropyl ] heptadec-8-amine, methyl 6- (2- (dimethylamino) -3- ((9Z, 12Z) -octadeca-9, 12-dien-1-yloxy) propoxy) hexanoate, ethyl (9Z) -21- (dimethylamino) octacosa-9-enoate, dimethyl (9Z) -19- { [4- (dimethylamino) butanoyl ] oxy } heptadecan-9-enedioate, N1- (2- (4- (2- (ditetradecylamino) ethyl) piperazin-1-yl) ethyl) -N1, N2, N2-tri (tetradecyl) ethane-1, 2-diamine, Methyl 5- (2- (dimethylamino) -3- ((9Z, 12Z) -octadeca-9, 12-dien-1-yloxy) propoxy) pentanoate, ethyl 8- { [4- (dimethylamino) butanoyl ] oxy } -15- (2-octylcyclopropyl) pentadecanoate, ethyl (9Z) -21- (dimethylamino) icosa-9-enoic acid ester, (13Z, 16Z) -N, N-dimethyl-3-nonylbehenyl-13, 16-dien-1-amine, N1- (2- (4- (2- (didodecylamino) ethyl) piperazin-1-yl) ethyl) -N1, N2, N2-tri (tetradecyl) ethane-1, 2-diamine, Methyl 9- { [4- (dimethylamino) butanoyl ] oxy } -16- (2-octylcyclopropyl) hexadecanoate, methyl 4- (2- (dimethylamino) -3- ((9Z, 12Z) -octadeca-9, 12-dien-1-yloxy) propoxy) butanoate, ethyl (9Z) -21- (dimethylamino) triacontan-9-enoate, (12Z, 15Z) -N, N-dimethyl-2-nonyldi-undecan-12, 15-dien-1-amine, methyl 8- (2- (dimethylamino) -3- ((8- (2-octylcyclopropyl) oxy) propoxy) propanoyl) octanoate, Ethyl (9Z) -19- (dimethylamino) cyclopentadec-9-enoate, ethyl (18Z, 21Z) -8- { [4- (dimethylamino) butanoyl ] oxy } heptacosyl-18, 21-dienoate, (16Z) -N, N-dimethylcyclopentadec-16-en-8-amine, methyl (9Z) -19- { [4- (dimethylamino) butanoyl ] oxy } heptadec-9-enoate, methyl (9Z) -19- (dimethylamino) heptadec-9-enoate, 2- (didodecylamino) -1- (4- (2- ((2- (didodecylamino) ethyl) (dodecyl) amino) ethyl) piperazin-1-yl) ethan-1-one, (Z) -methyl 16- (2- (dimethylamino) -3- (hexyloxy) propoxy) hexadeca-7-enoate, (2S) -1- [ (9Z, 12Z) -octadeca-9, 12-dien-1-yloxy ] dodecane-2-amine, (16Z, 19Z) -N, N-dimethylcyclopentadec-16, 19-dien-8-amine, N1- (2- (4- (2- (dinonylamino) ethyl) piperazin-1-yl) ethyl) -N1, N2V 2-tri (tetradecyl) ethane-1, 2-diamine, methyl 7- (2- (dimethylamino) -3- ((8- (2-octylcyclopropyl) oxy) propoxy) heptanoate, Methyl (19Z, 22Z) -9- { [4- (dimethylamino) butanoyl ] oxy } octacosa-19, 22-dienoate, ethyl (9Z) -19- (dimethylamino) hexacosa-9-enoic acid ester, (22Z) -N, N-dimethyl hen-triacontan-22-en-10-amine, N1- (2- (4- (2- (di ((Z) -octadeca-9-en-1-yl) amino) ethyl) piperazin-1-yl) ethyl) -N-! a process for preparing (E) -tris (dodecyl) ethane-1, 2-diamine, methyl 5- (2- (dimethylamino) -3- ((8- (2-octylcyclopropyl) oxy) propoxy) pentanoate, ethyl (9Z) -19- (dimethylamino) twenty-7 carbon-9-enoate, (2-butylcyclopropyl) methyl 12- { [4- (dimethylamino) butanoyl ] oxy } twenty-one carboxylate, (20Z) -N, N-dimethyl-twenty-nine carbon-20-en-10-amine, N1, N2-tris (dodecyl) -N2- (2- (4- (2- (dodecyl ((9Z, 12Z) -octadeca-9, 12-dien-yl) amino) ethyl) piperazin-1-yl) ethyl) ethane-1, 2-diamine, Methyl 4- (2- (dimethylamino) -3- ((8- (2-octylcyclopropyl) oxy) propoxy) butanoate, ethyl (9Z) -19- (dimethylamino) octacosa-9-enoate, (2-octylcyclopropyl) methyl 8- { [4- (dimethylamino) butanoyl ] oxy } heptadecanoate, (24Z) -N, N-dimethyltridec-24-en-10-amine, N1- (2- (4- (2- (bistetradecylamino) ethyl) piperazin-1-yl) ethyl) -N1, N2, N2-tris (dodecyl) ethane-1, 2-diamine, ethyl (5Z) -17- (dimethylamino) hexacosa-5-enoate, (Z) -methyl 8- (2- (dimethylamino) -3- (octadeca-9-en-1-yloxy) propoxy) octanoate, (2Z) -hept-2-en-1-yl 12- { [4- (dimethylamino) butanoyl ] oxy } di-undecanoate, (17Z) -N, N-dimethyl-icosazin-17-en-10-amine, N1- (2- (4- (2- (di- (Z) -dodeca-6-en-1-yl) amino) ethyl) piperazin-1-yl) ethyl) -N1, N2, N2-tri (dodecyl) ethane-1, 2-diamine, ethyl (9Z) -17- (dimethylamino) hexa-dec-9-enoate, (Z) -methyl 7- (2- (dimethylamino) -3- (octadeca-9-en-1-yloxy) propoxy) heptanoate, (2Z) -undec-2-en-1-yl 8- { [4- (dimethylamino) butanoyl ] oxy } heptadecanoate, (14Z) -N, N-dimethylicosadecan-14-en-10-amine, ethyl (7Z) -17- (dimethylamino) ditridec-7-enoate, (Z) -N1- (2- (4- (2- (dodeca-6-en-1-yl (dodecyl) amino) ethyl) piperazine-N-! The preparation of a pharmaceutical composition comprising ≡tris (dodecyl) ethane-1, 2-diamine, (Z) -methyl 5- (2- (dimethylamino) -3- (octadecyl-9-en-1-yloxy) propoxy) pentanoate, (2-hexylcyclopropyl) methyl 10- { [4- (dimethylamino) butyryl ] oxy } nonadecanoate, (15Z) -N, N-dimethylheptadecan-15-en-10-amine, ethyl (7Z) -17- (dimethylamino) tetracos-7-enoic acid ester, (Z) -methyl 4- (2- (dimethylamino) -3- (octadecyl-9-en-1-yloxy) propoxy) butanoate, (2Z) -non-2-en-1-yl 10- { [4- (dimethylamino) butanoyl ] oxy } nonadecanoate, (20Z) -N, N-dimethylheptadecan-20-en-10-amine, N1- (2- (4- (2- (dioctylamino) ethyl) piperazin-1-yl) ethyl) -N1, N2 ≡V 2-tri (dodecyl) ethane-1, 2-diamine, methyl 6- (2- (dimethylamino) -3- ((8- (2-octylcyclopropyl) octyl) oxy) propoxy) hexanoate, ethyl 6- [2- (9- { [4- (dimethylamino) butanoyl ] oxy } octadecyl) cyclopropyl ] hexanoate, Ethyl (7Z) -17- (dimethylamino) cyclopentadec-7-enoate, 1- [ (11Z, 14Z) -1-nonyleicosa-11, 14-dien-1-yl ] pyrrolidine, ethyl (7Z) -17- (dimethylamino) hexadec-7-enoate, (20Z, 23Z) -N-ethyl-N-methyl-nonadec-20, 23-dien-10-amine, N-dimethylheptadec-10-amine, methyl 6- {2- [11- (dimethylamino) eicosyl ] cyclopropyl } hexanoate, methyl 6- [2- (11- { [4- (dimethylamino) butanoyl ] oxy } eicosyl) cyclopropyl ] hexanoate, and, (2-octylcyclopropyl) methyl 6- (3- (decyloxy) -2- (dimethylamino) propoxy) hexanoate, methyl 8- {2- [9- (dimethylamino) octadecyl ] cyclopropyl } octanoate, methyl 8- [2- (9- { [4- (dimethylamino) butanoyl ] oxy } octadecyl) cyclopropyl ] octanoate, methyl 7- (2- (8- (2- (dimethylamino) -3- (octyloxy) propoxy) octyl) cyclopropyl) heptanoate, heptadec-9-yl 8- ((2-hydroxyethyl) (tetradecyl) amino) octanoate, 2- ((2- (didodecylamino) ethyl) (dodecyl) amino) -1- (4- (2- (didodecylamino) ethyl) piperazin-1-yl) ethan-1-one, (2S) -1- [ (9Z, 12Z) -octadec-9, 12-dien-1-yloxy ] undecan-2-amine, (17Z, 20Z) -N, N-dimethylhexa-dec-17, 20-dien-9-amine, (18Z) -heptadec-18-en-10-yl 4- (dimethylamino) butanoate, (2S) -1- ({ 6- [ 3B)) -cholesterol-5-en-3-yloxy ] hexyl } oxy) -N, N-dimethyl-3- [ (9Z) -octadec-9-en-1-yloxy ] propan-2-amine, methyl 10- {2- [7- (dimethylamino) hexadecyl ] cyclopropyl } decanoate, Methyl 10- [2- (7- { [4- (dimethylamino) butyryl ] oxy } hexadecyl) cyclopropyl ] decanoate, (2S) -N, N-dimethyl-1- ({ 8- [ (1R, 2R) -2- { [ (1S, 2S) -2-pentylcyclopropyl ] methyl } cyclopropyl ] octyl } oxy) tridecan-2-amine, (2-octylcyclopropyl) methyl 6- (2- (dimethylamino) -3- (nonyloxy) propoxy) hexanoate, (19Z, 22Z) -N, N-dimethyldioctadec-19, 22-diene-7-amine, 4- ((N- (2- (dinonylamino) ethyl) -N-nonylglycinoyl) oxy) penta-2-yldinonylglycine ester, 3-hydroxybut-2-yl N- (2- (dinonylamino) ethyl) -N-nonyl, di (heptadec-9-yl) 8,8'- (26, 28-dimethyl-11,24,30,43-tetraoxo-10,25,29,44-tetraoxa-19,35-diazapentatridec-19,35-diyl) dioctanoate, di (heptadec-9-yl) 8,8' - (26, 27-dimethyl-11,24,29,42-tetraoxo-10,25,28,43-tetraoxa-19,34-diazapentadodecane-19,34-diyl) dioctanoate, Di (heptadec-9-yl) 8,8'- (11,24,29,42-tetraoxo-10,25,28,43-tetraoxa-19,34-diazapentadodecane-19,34-diyl) dioctanoate, di (heptadec-9-yl) 8,8' - ((piperazine-1, 4-diylbis (5-oxopentan-5, 1-diyl)) bis ((8- (nonyloxy) -8-oxooctyl) azodiyl)) dioctanoate, di (heptadec-9-yl) 15, 18-dimethyl-9, 24-bis (8- (nonyloxy) -8-oxooctyl) -14, 19-dioxo-9,15,18,24-tetraazatridecanedioate, Di (heptadec-9-yl) 15, 19-dimethyl-9, 25-bis (8- (nonyloxy) -8-oxooctyl) -14, 20-dioxo-9,15,19,25-tetraazatridecanedioate, di (heptadec-9-yl) 15, 18-diethyl-9, 24-bis (8- (nonyloxy) -8-oxooctyl) -14, 19-dioxo-9,15,18,24-tetraazatridecanedioate, N-dimethyl-3- { [ (9Z, 12Z) -octadeca-9, 12-dien-1-yloxy ] methyl } dodecyl-1-amine, methyl 8- [2- (11- { [4- (dimethylamino) butanoyl ] oxy } octadecyl) cyclopropyl ] octanoate, Methyl 8- {2- [11- (dimethylamino) heptadecyl ] cyclopropyl } caprylate (compound 18), heptadec-9-yl 8- ((2-hydroxyethyl) (8- (nonyloxy) -8-oxooctyl) amino) caprylate, (2-octylcyclopropyl) methyl 6- (2- (dimethylamino) -3- (heptyloxy) propoxy) hexanoate, (17Z) -N, N-dimethylhexadec-17-en-9-amine, N1- (2- (4- (2- (didodecylamino) ethyl) piperazin-1-yl) ethyl) -N1V 2, N2-trihexylethane-1, 2-diamine, N, N-dimethyl-2- { [ (9Z, 12Z) -octadeca-9, 12-dien-1-yloxy ] methyl } undecan-1-amine, methyl 8- {2- [11- (dimethylamino) octadecyl ] cyclopropyl } octanoate, (2-octylcyclopropyl) methyl 6- (2- (dimethylamino) -3- (hexyloxy) propoxy) hexanoate, (18Z) -N, N-dimethylheptadecan-18-en-10-amine, 2- ((2- (dinonylamino) ethyl) (nonyl) amino) ethyl tetradecanoate, 2- ((2- (dinonylamino) ethyl) (nonyl) amino) ethyl nonanoate, Tetradecyl N- (2- (dinonylamino) ethyl) -N-nonylglycine ester, nonylan- (2- (dinonylamino) ethyl) -N-nonylglycine ester, 4- (2- ((2- (dinonylamino) ethyl) (nonylamino) acetamido) butyl valerate ester, 1' - (piperazine-1, 4-diyl) bis (5- (didecylamino) pentan-1-one, 2- ((2- (dinonylamino) ethyl) (nonylamino) -N-tetradecylacetamide, N-decyl-2- ((2- (dinonylamino) ethyl) (nonylamino) amino), N1- (3- (3- (dinonylamino) propoxy) propyl) -N1, N2, N2-trisnonylethane-1, 2-diamine, n1- (2- (dinonylamino) ethyl) -N\N8, N8-trisnonyloctane-1, 8-diamine, methyl 8- [2- (11- { [4- (dimethylamino) butanoyl ] oxy } nonadecyl) cyclopropyl ] octanoate, methyl 8- {2- [11- (dimethylamino) nonadecyl ] cyclopropyl } octanoate, (Z) -undec-2-en-1-yl 6- (3- (decyloxy) -2- (dimethylamino) propoxy) hexanoate, (2R, 12Z, 15Z) -N, N-dimethyl-1- (undecyloxy) heneicosa-12, 15-dien-2-amine, (21Z, 24Z) -N, N-dimethylthirty-carbon-21, 24-dien-9-amine, 2- (dinonylamino) -N- (4- (2- ((2- (dinonylamino) ethyl) (nonyl) amino) -N-methylacetamido) butyl) -N-methylacetamide, 7, 10-dimethyl-13, 16-dinonyl-6, 11-dioxo-4-tetradecyl-4,7,10,13,16-pentaazapentacosyl decanoate, 2- (dinonylamino) -N- (2- (2- ((2- (dinonylamino) ethyl) (nonyl) amino) -N-ethylacetamido) ethyl) -N-ethylacetamide, 2- (dinonylamino) -N- (3- (2- ((2- (dinonylamino) ethyl) (nonyl) amino) -N-methylacetamide propyl) -N-methylacetamide, 2- ((2- (di ((Z) -non-3-en-1-yl) amino) ethyl) ((Z) -non-3-en-1-yl) amino) -N- (2- (2- (dinonylamino) -N-methylacetamide ethyl) -N-methylacetamide, 2- (dinonylamino) -N- (2- (2- ((2- (dinonylamino) ethyl) (nonyl) amino) acetamido) ethyl) acetamide, pentyl 8, 11-dimethyl-5,14,17-trisnonyl-7, 12-dioxo-5, 8,11,14, 17-pentaazahexa-dicetyl acid ester 2- ((2- (dinonylamino) ethyl) (nonyl) amino) -N-methyl-N- (2- (methylamino) ethyl) acetamido, 2- (dinonylamino) -N- (2- (2- ((2- (dinonylamino) ethyl) (nonyl) amino) -N-methylacetamido) ethyl) -N-methylacetamido 2- (dinonylamino) -N-methyl-N- (2- (methylamino) ethyl) acetamide, 2- ((N- (2- (dinonylamino) ethyl) -N-nonylglycoyl) oxy) ethyldinonylglycinate 2-hydroxyethyl dinonylglycinate, methyl 8- [2- (11- { [4- (dimethylamino) butanoyl ] oxy } eicosyl) cyclopropyl ] octanoate, methyl 8- {2- [11- (dimethylamino) eicosyl ] cyclopropyl } octanoate, (Z) -undec-2-en-1-yl 6- (2- (dimethylamino) -3- (nonyloxy) propoxy) hexanoate, (2R, 12Z, 15Z) -1- (hexadecyloxy) -N, N-dimethyldi-undec-12, 15-dien-2-amine, (22Z, 25Z) -N, N-dimethyltri-undec-22, 25-dien-10-amine, 1- (piperazine-1, 4-diyl) bis (4- (didecylamino) butan-1-one) tert-butyl 4- (didecylamino butyrate, heptyl 5- (4- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperazin-1-yl) -5-oxopentanoate 5- (heptyloxy) -5-oxopentanoic acid, Heptyl 5- (4- (N- (2- (dinonylamino) ethyl) -N-nonylglycoyl) piperazin-1-yl) -5-oxopentanoate 5- (heptyloxy) -5-oxopentanoic acid, (Z) -4- ((2- (4- (N- (2- (dinonylamino) ethyl) -N-nonylglycoyl) piperazin-1-yl) -2-oxoethyl) (tetradecyl) amino) but-2-en-1-ylnonanoate (Z) -4-hydroxybut-2-en-1-ylnonanoate, (Z) -3- ((2- (4- (N- (2- (dinonylamino) ethyl) -N-nonylglycoyl) piperazin-1-yl) -2-oxoethyl) (tetradec-9-en-1-yl) amino) propyldecanoate (Z) -tetradec-9-en-1-ylmethane sulfonate, Methyl 8- [2- (9- { [4- (dimethylamino) butyryl ] oxy } pentadecyl) cyclopropyl ] octanoate, methyl 8- {2- [9- (dimethylamino) pentadecyl ] cyclopropyl } octanoate, (Z) -undec-2-en-1-yl 6- (2- (dimethylamino) -3- (heptyloxy) propoxy) hexanoate, (2R, 12Z, 15Z) -1- (hexyloxy) -N, N-dimethyldi-undec-12, 15-dien-2-amine, (16Z, 19Z) -N, N-dimethyldi-pentadecan-16, 19-dien-6-amine, methyl 8- ((2- (4- (N- (2- (di ((Z) -non-3-en-1-yl) amino) ethyl) -N- ((Z) -non-3-en-1-yl) glycyl) piperazin-1-yl) -2-oxoethyl) (nonyl) amino) tert-butyl 4- (nonylglycinyl) piperazine-1-carboxylate, 3- ((2- (4- (N- (2- (dinonylamino) ethyl) -N-nonylglycoyl) piperazin-1-yl) -2-oxoethyl) (tetradecyl) amino) propyl (Z) -dec-3-enoate (Z) -dec-3-en-1-ol, 2- ((2- (di ((Z) -non-3-en-1-yl) amino) ethyl) ((Z) -non-3-en-1-yl) amino) -1- (4- (dinonylglycyl) piperazin-1-yl) ethyl-1-one (Z) -1-bromonon-4-ene, 3- ((2- (4- (N- (2- (dinonylamino) ethyl) -N-nonylglycoyl) piperazine-oxoethyl) (dodecyl) amino) propyloctanoate tert-butyldodecylglycinate, S-pentyl 4- ((2- (4- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperazin-1-yl) -2-oxoethyl) (nonyl) amino) thiobutyrate, 3- ((2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperidin-) -yl) ethyl) (nonyl) amino) propyl 3-methylhexanoate tert-butyl 4- (2- ((3- ((3-methylhexanoyl) oxy) propyl) (nonyl) amino) ethyl) piperidin-1-, 3- ((2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperidin-4-yl) ethyl) (nonyl) amino) -2-methylpropyl hexanoate, 3- ((2- (4- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperazine-oxoethyl) (nonyl) amino) propyl 3-methylhexanoate, 3- ((2- (4- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperazine-oxoethyl) (nonyl) amino) -2-methylpropyl hexanoate, methyl 8- [2- (9- { [4- (dimethylamino) butyryl ] oxy } hexadecyl) cyclopropyl ] octanoate, methyl 8- {2- [9- (dimethylamino) hexadecyl ] cyclopropyl } octanoate, (Z) -undec-2-en-1-yl 6- (2- (dimethylamino) -3- (hexyloxy) propoxy) hexanoate, (2R, 12Z, 15Z) -1- (decyloxy) -N, N-dimethyldi-undec-12, 15-dien-2-amine, (17Z, 20Z) -N, N-dimethyldi-hexadeca-17, 20-dien-7-amine, 2- ((2- (dinonylamino) ethyl) (nonyl) amino) ethyl 1- (dinonylamino) piperidine-4-carboxylate, 1- (2- (dinonylamino) ethyl) 4- (2- ((2- (dinonylamino) ethyl) (nonyl) amino) ethyl) cyclohexane-1, 4-dicarboxylate 2- (dinonylamino) ethyl-1-ol, methyl 12- ((2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylamyl) pyrrolidin-3-yl) ethyl) (tetradecyl) amino) dodecanoate tert-butyl 3- (2- ((12-methoxy-12-oxododecyl) (tetradecyl) amino) ethyl) pyrrolidine-1-carboxylate, 3- ((2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycol-3-yl) ethyl) (tetradecyl) amino) propyldecanoate tert-butyl 3- (2- ((3- (decanoyloxy) propyl) (tetradecyl) amino) ethyl) pyrrolidine-1-carboxylate, "heptyl 6- ((2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycol) pyrrolidin-3-yl) ethyl) (tetradecyl) amino) hexanoate tert-butyl 3- (2- ((6- (heptyloxy) -6-oxohexyl) (tetradecyl) amino) ethyl) pyrrolidine-1-carboxylate" Amyl 8- ((2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycol-3-yl) ethyl) (tetradecyl) amino) octanoate/-butyl 3- (2- (tetradecylamino) ethyl) pyrrolidine-1-carboxylate, methyl 12- ((2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycol) piperidin-3-yl) ethyl) (tetradecyl) amino) dodecanoate-butyl 3- (2- ((12-methoxy-12-oxododecyl) (tetradecyl) amino) ethyl) piperidine-1-carboxylate, 3- ((2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperidin-3-yl) ethyl) (tetradecyl) amino) propyl decanoate, heptyl 6- ((2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperidin-3-yl) ethyl) (tetradecyl) amino) hexanoate, pentyl 8- ((2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperidin-3-yl) ethyl) (tetradecyl) amino) octanoate, pentyl 6- ((2- (4- (2- ((2- (didodecylamino) ethyl) (dodecyl) amino) ethyl) piperazin-1-yl) ethyl) (dodecyl) amino) hexanoate pentyl 6-bromohexanoate, Methyl 8- [2- (9- { [4- (dimethylamino) butyryl ] oxy } heptadecyl) cyclopropyl ] octanoate, methyl 8- {2- [9- (dimethylamino) heptadecyl ] cyclopropyl } octanoate, (2S, 12Z, 15Z) -N, N-dimethyl-1- (octyloxy) heneicosane-12, 15-dien-2-amine, (2-octylcyclopropyl) methyl 6- (2- (dimethylamino) -3- (octyloxy) propoxy) hexanoate, (18Z, 21Z) -N, N-dimethylheptadecan-18, 21-dien-8-amine, trans-1-methyl-3, 4-bis (((Z) -hexadeca-9-enoyloxy) methyl) pyrrolidine, (Z) -non-2-en-1-yl 4- ((2- (4- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperazin-1-yl) -2-oxoethyl) (tetradecyl) amino) butanoate, trans-1-methyl-3, 4-bis (((9Z, 12Z) -octadeca-9, 12-dienoyloxy) methyl) pyrrolidine, methyl 12- ((2- (4- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperazin-1-yl) -2-oxoethyl) (tetradecyl) amino) dodecanoate, ethyl (7Z) -17- [2- (dimethylamino) ethyl ] diacetyl-7-enoate, Trans-1-methyl-3, 4-bis (((Z) -octadeca-9-enoyloxy) methyl) pyrrolidine, methyl 6- (2- { ]11- ] 2- (dimethylamino) ethyl ] eicosyl } cyclopropyl) hexanoate, methyl 12- ((2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperidin-4-yl) ethyl) (tetradecyl) amino) dodecanoate, methyl 10- (2-V- ] 2- (dimethylamino) ethyl ] hexadecyl } cyclopropyl) decanoate, methyl 8- (2- {111-; 2- (dimethylamino) ethyl ] heptadecyl } cyclopropyl) octanoate, 2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperidin-4-yl) ethyldinonylglycinate t-butyl 4- (2- ((dinonylglycyl) oxy) ethyl) piperidine-1-carboxylate, methyl 8- (2- { lLl-; 2- (dimethylamino) ethyl ] octadecyl } cyclopropyl) octanoate, methyl 8- (2- { l11- "2- (dimethylamino) ethyl ] nonadecyl } cyclopropyl) octanoate, 1, - (piperazine-1, 4-diyl) bis (2- (dinonylamino) ethan-1-one), methyl 8- [2- { ]11- ] 2- (dimethylamino) ethyl ] eicosyl } cyclopropyl) octanoate, methyl 8- (2- {9- [2- (dimethylamino) ethyl ] pentadecyl } cyclopropyl) octanoate, methyl (7Z) -19- { [4- (dimethylamino) butyryl ] oxy } octacosa-7-enoic acid ester, Methyl (7Z) -19- (dimethylamino) octacosa-7-enoic acid ester, cis-1-methyl-3- [ (9Z, 12Z) -octadec-9, 12-dien-1-yloxy ] -4- (octyloxy) pyrrolidine, 2- (didodecylamino) -1- (4- (N- (2- (didodecylamino) ethyl) -N-dodecylglycyl) piperazin-1-yl) ethan-1-one, (Z) -undec-2-en-1-yl 6- (2- (dimethylamino) -3- (octyloxy) propoxy) hexanoate, (2 SN, N-dimethyl-1- [ (9Z, 12Z) -octadec-9, 12-dien-1-yloxy ] dec-2-amine (compound 11), (19Z, 22Z) -N, N-dimethyloctacosa-19, 22-dien-9-amine, methyl 8- (2- {9- [2- (dimethylamino) ethyl ] hexadecyl } cyclopropyl) octanoate, 5- ((2- (4- (N- (2- (dinonylamino) ethyl) -N-nonylglycinoyl) piperazine-oxoethyl) (nonyl) amino) pentylmethyl carbonate, methyl 8- (2- {9- [2- (dimethylamino) ethyl ] heptadecyl } cyclopropyl) octanoate, methyl (7Z) -19- [2- (dimethylamino) ethyl ] octacosa-7-enoic acid ester, (Z) -pent-2-en-1-yl 4- ((2- (4- (N- (2- (dinonylamino) ethyl) -N-nonylglycinyl) piperazine-1-yl) -2-oxoethyl) (nonyl) amino) butanoate, Methyl (11Z) -19- [2- (dimethylamino) ethyl ] octacosa-11-enoic acid ester, methyl (9Z) -21- [2- (dimethylamino) ethyl ] heptadecan-9-enoic acid ester, methyl (9Z) -21- [2- (dimethylamino) ethyl ] octacosa-9-enoic acid ester, methyl (9Z) -21- [2- (dimethylamino) ethyl ] nonadecan-9-enoic acid ester, 2- (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycol-3-yl) ethyl dinonylglycine ester, methyl (9Z) -21- [2- (dimethylamino) ethyl ] triacont-9-enoic acid ester, (1- (N- (2- (dinonylamino) ethyl) -N-nonylglycol-3-yl) methyldinonylglycine ester, methyl (9Z) -19- [2- (dimethylamino) ethyl ] pentadecan-9-enoic acid ester, methyl (9Z) -19- [2- (dimethylamino) ethyl ] hexa-dec-9-enoic acid ester, methyl 6- (2- (8- (3- (decyloxy) -2- (dimethylamino) propoxy) octyl) cyclopropyl) hexanoate, methyl (11Z) -19- { [4- (dimethylamino) butanoyl ] oxy } octa-eicosa-11-enoic acid ester, methyl (11Z) -19- (dimethylamino) octa-11-enoic acid ester, (2S) -N, N-dimethyl-1- [ (9Z, 12Z) -octadeca-9, 12-dien-1-yloxy ] dodecane-2-amine, (14Z, 17Z) -N, N-dimethylditridec-14, 17-dien-4-amine, methyldi ((9Z, 12Z) -octadeca-9, 12-dienyl) amine, methyl (9Z) -19- { [4- (dimethylamino) butanoyl ] oxy } octa-2-enoic acid ester, methyl (9Z) -19- (dimethylamino) octa-9-enoic acid ester, (Z) -methyl 17- (2- (dimethylamino) -3- (octyloxy) propoxy) heptadecan-8-enoic acid ester, (3R, 4R) -3, 4-bis ((Z) -hexadec-9-enyloxy) -1-methylpyrrolidine, (2S) -N, N-dimethyl-1- [ (9Z, 12Z) -octadec-9, 12-dien-1-yloxy ] undecan-2-amine, (20Z, 23Z) -icosacarbon-20, 23-dien-10-yl 4- (dimethylamino) butanoate, (20Z, 23Z) -N, N-dimethyl-icosacarbon-20, 23-dien-10-amine, 3- ((6Z, 9Z,28Z, 31Z) -heptadecan-6,9,28,31-tetraen-19-yloxy) -N, N-dimethylpropan-1-amine, 3- ((6Z, 9Z,28Z, 31Z) -heptadeca-6,9,28,31-tetraen-19-yloxy) -N, N-dimethylpropan-1-amine, (6Z, 9Z,28Z, 31Z) -heptadeca-6,9,28,31-tetraen-19-yl 4- (dimethylamino) butanoate), (6Z, 16Z) -12- ((Z) -dec-4-enyl) docosa-6, 16-dien-11-yl 5- (dimethylamino) pentanoate, (6Z, 16Z) -12- ((Z) -dec-4-enyl) docosa-6, 16-dien-11-yl 5- (dimethylamino) pentanoate, L-arginine-alpha- (2, 3-dilauroyl) propanamide, L-lysine-alpha- (2, 3-dilauroyl) propanamide, 2, 3-dioleoxypropylamine, 2, 3-distearoxypropylamine, 2, 3-dilauroyl propylamine, dioleylmeth-4- (dimethylamino) propyl ether, dioleylmeth-4- (dimethylamino) butyl ether) and 2, 2-dioleyl-4- (2-dimethylaminoethyl) - [1,3] -dioxolane.

In some embodiments, the at least one non-cationic lipid comprises at least one phospholipid, at least one fusogenic lipid, at least one anionic lipid, at least one helper lipid, at least one neutral lipid, or any combination thereof. In some embodiments, the LNP may be substantially free of at least one non-cationic lipid. In some embodiments, the LNP may contain zero amount of at least one non-cationic lipid.

In some embodiments, the at least one non-cationic lipid may be selected from, but is not limited to, at least one of the following: 1, 2-di-O-octadecenyl-sn-glycero-3-phosphorylcholine (18:0 diether PC), DSPC (18:3 PC) with 3 unsaturated double bonds per tail (pertail), acyl Carnosine (AC), 1-hexadecyl-sn-glycero-3-phosphorylcholine (C16 Lyso PC), N-oleoyl-SPM (C18:l), N-tetracosyl SPM (C24:0), N-neuroyl C (C24:l), Carbamoyl ] cholesterol (Cet-P), cholesterol Hemisuccinate (CHEMS) (Chol), cholesterol hemisuccinate (Chol-C12), 12-cholesteryl oxycarbonylaminododecanoic acid (Chol-C13N), cholesterol hemistearate (Chol-C2), cholesterol hemisalonate (Chol-C3), N- (cholesteryl-oxycarbonyl) glycine (Chol-C3N), cholesterol hemisalonate (Chol-C5), cholesterol hemisalonate (Chol-C6), cholesterol hemisalonate (Chol-C7), Cholesterol half suberate (Chol-C8), cardiolipin (Cardiolipid; CL), 1, 2-bis (tricosanol-10, 12-diacetylenoyl) -sn-glycero-3-phosphorylcholine (DC 8-9 PC), dicetyl phosphate (DCP 1), 1, 2-dipalmitoyl glycerol-3-hemisuccinate (DGSucc), short-chain bis-n-heptadecanoyl phosphatidylcholine (DHPC), dicetyl phosphoethanolamine (DHPE), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DLPC), 1, 2-dilauroyl-sn-glycero-3-PE (DLPE), Dimyristoyl glycerol hemisuccinate (DMGS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoyl phosphatidylglycerol (DMPG), dioleyloxy benzyl alcohol (DOBA), 1, 2-dioleoyl glycerol-3-hemisuccinate (DOGHEMS), N- [2- (2- {2- [2- (2, 3-bis-octadeca-9-alkenyloxy-propoxy) -ethoxy ] -ethoxy } -ethoxy) -ethyl ] -3- (3, 4, 5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yl-hydrothio) -propionamide (DOGP. Alpha. Man), di-oleoyl phosphatidylcholine (DOPC), di-oleoyl phosphatidylethanolamine (DOPE), di-oleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal), di-oleoyl phosphatidylglycerol (DOPG), 1, 2-dioleoyl-sn-glycerol-3- (phospho-L-serine) (DOPS), cell fusion phospholipid (DPhPE), di-palmitoyl phosphatidylcholine (DPPC), di-palmitoyl phosphatidylethanolamine (DPPE), di-palmitoyl phosphatidylglycerol (DPPG), di-palmitoyl phosphatidylserine (DPPS), cell fusion phospholipid (DPhPE), Distearoyl phosphatidylcholine (DSPC), distearoyl-phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamine imidazole (DSPEI), 1, 2-bis-undecanoyl-sn-glycero-phosphocholine (DUPC), lecithin (EPC), N-histidylcholesteryl carbamate (HCChol), histidinol distearoyl glycerol (HDSG), N-histidylcholesteryl hemisuccinate (HistChol), 1, 2-dipalmitoyl glycerol hemisuccinate-na-histidyl hemisuccinate (HistSuccDG), N- (5 '-hydroxy-3' -oxopentyl) -10-12-dipentadecyldiacetylamide (h-Pegi-PCDA), 2- [ 1-hexyloxyethyl ] -2-deglylmethapsid chlorophyllin-a (HPPH), hydrogenated Soybean Phosphatidylcholine (HSPC), 1, 2-dipalmitoyl glycerol-O.alpha. -histidyl-N.alpha. -hemisuccinate (IsohistsuccDG), mannosylated (mannosialized) dipalmitoyl phosphatidylethanolamine (ManDOG), 1, 2-dioleoyl-sn-glycerol-3-phosphoethanolamine-N- [4- (p-maleimidomethyl) cyclohexane-carboxamide ] (MCC-PE), 1, 2-Diphytoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1-myristoyl-2-hydroxy-sn-glycero-phosphorylcholine (MHPC), thiol-reactive maleimido-headlipids, such as 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- [4- (p-maleimidophenyl) butyramide (MPB-PE), nervonic Acid (NA), sodium cholate (NaChol), 1, 2-dioleoyl-sn-glycero-3- [ phosphoethanolamine-N-dodecanoyl (NC 12-DOPE), are defined by the synthesis examples in WO2008042973A2 (ND 98), "N-glutaryl phosphatidylethanolamine" (NG-PE) of formulation 1, N-hydroxysulfosuccinimide (NHS- 'x'), "N- (co) -dicarboxylic acid-derivatized phosphatidylethanolamine" (NωPE- 'x'), oleic Acid (OA), 1-oleoyl-2-cholesterol hemisuccinyl-sn-glycerol-3-phosphorylcholine (OChemsPC), phosphatidic Acid (PA), phosphatidylethanolamine lipid (PE), PE lipid conjugated with polyethylene glycol (PEG) covered by formulation 1. one example of a PEG-PE may be polyethylene glycol distearoyl phosphatidylethanolamine lipid (PEG-PE), phosphatidylglycerol (PG), partially Hydrogenated Soybean Phosphatidylcholine (PHSPC), inositol phosphatidate lipid (PI), phosphatidylinositol-4-phosphate (PIP), palmitoyl Oleoyl Phosphatidylcholine (POPC), phosphatidylethanolamine (POPE), palmitoyl base oil acyl phosphatidylglycerol (POPG), phosphatidylserine (PS), lissamine rhodamine B-phosphatidylethanolamine lipid (Rh-PE), purified soybean-derived phospholipid mixture (SIOO), Phosphatidylcholine (SM), 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), soybean Phosphatidylcholine (SPC), sphingomyelin (SPM), alpha '-trehalose-6, 6' -dibehenate (TDB), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (transDOPE), ((23S, 5R) -3- (bis (hexadecyloxy) methoxy) -5- (5-methyl-2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) tetrahydrofuran-2-yl) methyl phosphate, 1, 2-Diarachidonoyl-sn-glycero-3-phosphorylcholine, 1, 2-Diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1, 2-didodecylhexaenoyl-sn-glycero-3-phosphocholine, 1, 2-didodecylhexaenoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphocholine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-distearoyl-glycero-3-phosphoethanolamine, 16-O-monomethyl PE, 16-O-dimethyl PE and dioleyl phosphatidylethanolamine.

In some embodiments, the LNP comprises an ionizable lipid or lipid-like material. As one non-limiting example, the ionizable lipid may be C12-200, CKK-E12, 5A2-SC8, BAMEA-016B, or 7C1. Other ionizable lipids are known in the art and are suitable for use herein.

In some embodiments, the LNP comprises a phospholipid. As one non-limiting example, the phospholipid (assist) may be DOPE, DSPC, DOTAP or DOTMA.

In some embodiments, the LNP comprises a PEG derivative. As one non-limiting example, the PEG derivative may be lipid anchored, for example PEG C14-PEG2000、C14-PEG1000、C14-PEG3000、C14-PEG5000、C12-PEG1000、C12-PEG2000、C12-PEG3000、C12-PEG5000、C16-PEG1000、C16-PEG2000、C16-PEG3000、C16-PEG5000、C18-PEG1000、C18-PEG2000、C18-PEG3000 or C18-PEG5000.

In some embodiments, the at least one sterol comprises at least one cholesterol or cholesterol derivative. In some embodiments, the LNP may be substantially free of at least one sterol. In some embodiments, the LNP may contain zero amount of at least one sterol.

In some embodiments, the at least one particle activity modulator comprises: at least one component that reduces aggregation of particles, at least one component that reduces circulatory clearance of LNP from the subject, at least one component that increases the ability of LNP to cross a mucus layer, at least one component that reduces an immune response of a subject to LNP administration, at least one component that modulates membrane fluidity of LNP, at least one component that contributes to the stability of LNP, or any combination thereof. In some embodiments, the LNP may be substantially free of at least one modulator of particle activity. In some embodiments, the LNP may contain zero amount of at least one particulate activity modulator.

In some embodiments, the particle activity modulator may be comprised of a polymer. In some embodiments, the polymer comprising the particle activity modulator may consist of: at least one polyethylene glycol (PEG), at least one polypropylene glycol (PPG), poly (2-oxazoline) (POZ), at least one polyamide (ATTA), at least one cationic polymer, or any combination thereof.

In some embodiments, the average molecular weight of the polymer portion (e.g., PEG) may be between 500 and 20,000 daltons. In some embodiments, the molecular weight of the polymer may be about 500 to 20,000, 1,000 to 20,000, 1,500 to 20,000, 2,000 to 20,000, 2,500 to 20,000, 3,000 to 20,000, 3,500 to 20,000, 4,000 to 20,000, 4,500 to 20,000, 5,000 to 20,000, 5,500 to 20,000, 6,000 to 20,000, 6,500 to 20,000, 7,000 to 20,000, 7,500 to 20,000, and, 8,000 to 20,000, 8,500 to 20,000, 9,000 to 20,000, 9,500 to 20,000, 10,000 to 20,000, 10,500 to 20,000, 11,000 to 20,000, 11,500 to 20,000, 12,000 to 20,000, 12,500 to 20,000, 13,000 to 20,000, 13,500 to 20,000, 14,000 to 20,000, 14,500 to 20,000, 15,000 to 20,000, 15,500 to 20,000, 16,000 to 20,000, 16,500 to 20,000, 17,000 to 20,000, 17,500 to 20,000, 18,000 to 20,000, 18,500 to 20,000, 19,000 to 20,000, 19,500 to 20,000, 500 to 19,500, 1,000 to 19,500, 1,500 to 19,500, 2,000 to 19,500, 2,500 to 19,500, 3,000 to 19,500, 3,500 to 19,500, 4,000 to 19,500, 4,500 to 19,500, 5,000 to 19,500, 5,500 to 19,500, 6,000 to 19,500, 6,500 to 19,500, 7,000 to 19,500, 7,500 to 19,500, 8,000 to 19,500, 8,500 to 19,500, 9,000 to 19,500, 9,500 to 19,500, 10,000 to 19,500, 10,500 to 19,500, 11,000 to 19,500, 11,500 to 19,500, 12,000 to 19,500, 12,500 to 19,500, 13,000 to 19,500, 13,500 to 19,500, 14,000 to 19,500, 14,500 to 19,500, 15,000 to 19,500, 15,500 to 19,500, 16,000 to 19,500, 16,500 to 19,500, 17,000 to 19,500, 17,500 to 19,500, 18,000 to 19,500, 18,500 to 19,500, 19,000 to 19,500, 1,500 to 19,000, 2,000 to 19,000, 2,500 to 19,000, 3,000 to 19,000, 3,500 to 19,000, 4,000 to 19,000, 4,500 to 19,000, 5,000 to 19,000, 5,500 to 19,000, 6,000 to 19,000, 6,500 to 19,000, 7,000 to 19,000, 7,500 to 19,000, 8,000 to 19,000, 8,500 to 19,000, and, 9,000 to 19,000, 9,500 to 19,000, 10,000 to 19,000, 10,500 to 19,000, 11,000 to 19,000, 11,500 to 19,000, 12,000 to 19,000, 12,500 to 19,000, 13,000 to 19,000, 13,500 to 19,000, 14,000 to 19,000, 14,500 to 19,000, 15,000 to 19,000, 15,500 to 19,000, 16,000 to 19,000, 16,500 to 19,000, 17,000 to 19,000, 17,500 to 19,000, 18,000 to 19,000, 18,500 to 19,000, 1,500 to 18,500, 2,000 to 18,500, 2,500 to 18,500, 3,000 to 18,500, 3,500 to 18,500, 4,000 to 18,500, 4,500 to 18,500, 5,000 to 18,500, 5,500 to 18,500, 6,000 to 18,500, 6,500 to 18,500, 7,000 to 18,500, 7,500 to 18,500, 8,000 to 18,500, 8,500 to 18,500, 9,000 to 18,500, 9,500 to 18,500, 10,000 to 18,500, 10,500 to 18,500, 11,000 to 18,500, 11,500 to 18,500, 12,000 to 18,500, 12,500 to 18,500, 13,000 to 18,500, 13,500 to 18,500, 14,000 to 18,500, 14,500 to 18,500, 15,000 to 18,500, 15,500 to 18,500, 16,000 to 18,500, 16,500 to 18,500, 17,000 to 18,500, 17,500 to 18,500, 18,000 to 18,500, 1,500 to 18,000, 2,000 to 18,000, 2,500 to 18,000, 3,000 to 18,000, 3,500 to 18,000, 4,000 to 18,000, 4,500 to 18,000, 5,000 to 18,000, 5,500 to 18,000, 6,000 to 18,000, 6,500 to 18,000, 7,000 to 18,000, 7,500 to 18,000, 8,000 to 18,000, 8,500 to 18,000, 9,000 to 18,000, 9,500 to 18,000, 10,000 to 18,000, 10,500 to 18,000, 11,000 to 18,000, 11,500 to 18,000, 12,000 to 18,000, 12,500 to 18,000, and, 13,000 to 18,000, 13,500 to 18,000, 14,000 to 18,000, 14,500 to 18,000, 15,000 to 18,000, 15,500 to 18,000, 16,000 to 18,000, 16,500 to 18,000, 17,000 to 18,000, 17,500 to 18,000, 1,500 to 17,500, 2,000 to 17,500, 2,500 to 17,500, 3,000 to 17,500, 3,500 to 17,500, and, 4,000 to 17,500, 4,500 to 17,500, 5,000 to 17,500, 5,500 to 17,500, 6,000 to 17,500, 6,500 to 17,500, 7,000 to 17,500, 7,500 to 17,500, 8,000 to 17,500, 8,500 to 17,500, 9,000 to 17,500, 9,500 to 17,500, 10,000 to 17,500, 10,500 to 17,500, 11,000 to 17,500, 11,500 to 17,500, and, 12,000 to 17,500, 12,500 to 17,500, 13,000 to 17,500, 13,500 to 17,500, 14,000 to 17,500, 14,500 to 17,500, 15,000 to 17,500, 15,500 to 17,500, 16,000 to 17,500, 16,500 to 17,500, 17,000 to 17,500, 1,500 to 17,000, 2,000 to 17,000, 2,500 to 17,000, 3,000 to 17,000, 3,500 to 17,000, 4,000 to 17,000, 4,500 to 17,000, 5,000 to 17,000, 5,500 to 17,000, 6,000 to 17,000, 6,500 to 17,000, 7,000 to 17,000, 7,500 to 17,000, 8,000 to 17,000, 8,500 to 17,000, 9,000 to 17,000, 9,500 to 17,000, 10,000 to 17,000, 10,500 to 17,000, 11,000 to 17,000, and, 11,500 to 17,000, 12,000 to 17,000, 12,500 to 17,000, 13,000 to 17,000, 13,500 to 17,000, 14,000 to 17,000, 14,500 to 17,000, 15,000 to 17,000, 15,500 to 17,000, 16,000 to 17,000, 16,500 to 17,000, 1,500 to 16,500, 2,000 to 16,500, 2,500 to 16,500, 3,000 to 16,500, 3,500 to 16,500, 4,000 to 16,500, 4,500 to 16,500, 5,000 to 16,500, 5,500 to 16,500, 6,000 to 16,500, 6,500 to 16,500, 7,000 to 16,500, 7,500 to 16,500, 8,000 to 16,500, 8,500 to 16,500, 9,000 to 16,500, 9,500 to 16,500, 10,000 to 16,500, 10,500 to 16,500, 11,000 to 16,500, 11,500 to 16,500, 12,000 to 16,500, 12,500 to 16,500, 13,000 to 16,500, 13,500 to 16,500, 14,000 to 16,500, 14,500 to 16,500, 15,000 to 16,500, 15,500 to 16,500, 16,000 to 16,500, 1,500 to 16,000, 2,000 to 16,000, 2,500 to 16,000, 3,000 to 16,000, 3,500 to 16,000, 4,000 to 16,000, 4,500 to 16,000, 5,000 to 16,000, 5,500 to 16,000, 6,000 to 16,000, 6,500 to 16,000, 7,000 to 16,000, 7,500 to 16,000, 8,000 to 16,000, 8,500 to 16,000, 9,000 to 16,000, 9,500 to 16,000, 10,000 to 16,000, 10,500 to 16,000, 11,000 to 16,000, 11,500 to 16,000, and, 12,000 to 16,000, 12,500 to 16,000, 13,000 to 16,000, 13,500 to 16,000, 14,000 to 16,000, 14,500 to 16,000, 15,000 to 16,000, 15,500 to 16,000, 1,500 to 15,500, 2,000 to 15,500, 2,500 to 15,500, 3,000 to 15,500, 3,500 to 15,500, 4,000 to 15,500, 4,500 to 15,500, and, 5,000 to 15,500, 5,500 to 15,500, 6,000 to 15,500, 6,500 to 15,500, 7,000 to 15,500, 7,500 to 15,500, 8,000 to 15,500, 8,500 to 15,500, 9,000 to 15,500, 9,500 to 15,500, 10,000 to 15,500, 10,500 to 15,500, 11,000 to 15,500, 11,500 to 15,500, 12,000 to 15,500, 12,500 to 15,500, and, 13,000 to 15,500, 13,500 to 15,500, 14,000 to 15,500, 14,500 to 15,500, 15,000 to 15,500, 1,500 to 15,000, 2,000 to 15,000, 2,500 to 15,000, 3,000 to 15,000, 3,500 to 15,000, 4,000 to 15,000, 4,500 to 15,000, 5,000 to 15,000, 5,500 to 15,000, 6,000 to 15,000, 6,500 to 15,000, and, 7,000 to 15,000, 7,500 to 15,000, 8,000 to 15,000, 8,500 to 15,000, 9,000 to 15,000, 9,500 to 15,000, 10,000 to 15,000, 10,500 to 15,000, 11,000 to 15,000, 11,500 to 15,000, 12,000 to 15,000, 12,500 to 15,000, 13,000 to 15,000, 13,500 to 15,000, 14,000 to 15,000, 14,500 to 15,000, 1,500 to 14,500, 2,000 to 14,500, 2,500 to 14,500, 3,000 to 14,500, 3,500 to 14,500, 4,000 to 14,500, 4,500 to 14,500, 5,000 to 14,500, 5,500 to 14,500, 6,000 to 14,500, 6,500 to 14,500, 7,000 to 14,500, 7,500 to 14,500, 8,000 to 14,500, 8,500 to 14,500, and, 9,000 to 14,500, 9,500 to 14,500, 10,000 to 14,500, 10,500 to 14,500, 11,000 to 14,500, 11,500 to 14,500, 12,000 to 14,500, 12,500 to 14,500, 13,000 to 14,500, 13,500 to 14,500, 14,000 to 14,500, 1,500 to 14,000, 2,000 to 14,000, 2,500 to 14,000, 3,000 to 14,000, 3,500 to 14,000, 4,000 to 14,000, 4,500 to 14,000, 5,000 to 14,000, 5,500 to 14,000, 6,000 to 14,000, 6,500 to 14,000, 7,000 to 14,000, 7,500 to 14,000, 8,000 to 14,000, 8,500 to 14,000, 9,000 to 14,000, 9,500 to 14,000, 10,000 to 14,000, 10,500 to 14,000, 11,000 to 14,000, and, 11,500 to 14,000, 12,000 to 14,000, 12,500 to 14,000, 13,000 to 14,000, 13,500 to 14,000, 1,500 to 13,500, 2,000 to 13,500, 2,500 to 13,500, 3,000 to 13,500, 3,500 to 13,500, 4,000 to 13,500, 4,500 to 13,500, 5,000 to 13,500, 5,500 to 13,500, 6,000 to 13,500, 6,500 to 13,500, and, 7,000 to 13,500, 7,500 to 13,500, 8,000 to 13,500, 8,500 to 13,500, 9,000 to 13,500, 9,500 to 13,500, 10,000 to 13,500, 10,500 to 13,500, 11,000 to 13,500, 11,500 to 13,500, 12,000 to 13,500, 12,500 to 13,500, 13,000 to 13,500, 1,500 to 13,000, 2,000 to 13,000, 2,500 to 13,000, 3,000 to 13,000, 3,500 to 13,000, 4,000 to 13,000, 4,500 to 13,000, 5,000 to 13,000, 5,500 to 13,000, 6,000 to 13,000, 6,500 to 13,000, 7,000 to 13,000, 7,500 to 13,000, 8,000 to 13,000, 8,500 to 13,000, 9,000 to 13,000, 9,500 to 13,000, 10,000 to 13,000, 10,500 to 13,000, 11,000 to 13,000, 11,500 to 13,000, 12,000 to 13,000, 12,500 to 13,000, 1,500 to 12,500, 2,000 to 12,500, 2,500 to 12,500, 3,000 to 12,500, 3,500 to 12,500, 4,000 to 12,500, 4,500 to 12,500, 5,000 to 12,500, 5,500 to 12,500, 6,000 to 12,500, 6,500 to 12,500, and, 7,000 to 12,500, 7,500 to 12,500, 8,000 to 12,500, 8,500 to 12,500, 9,000 to 12,500, 9,500 to 12,500, 10,000 to 12,500, 10,500 to 12,500, 11,000 to 12,500, 11,500 to 12,500, 12,000 to 12,500, 1,500 to 12,000, 2,000 to 12,000, 2,500 to 12,000, 3,000 to 12,000, 3,500 to 12,000, and, 4,000 to 12,000, 4,500 to 12,000, 5,000 to 12,000, 5,500 to 12,000, 6,000 to 12,000, 6,500 to 12,000, 7,000 to 12,000, 7,500 to 12,000, 8,000 to 12,000, 8,500 to 12,000, 9,000 to 12,000, 9,500 to 12,000, 10,000 to 12,000, 10,500 to 12,000, 11,000 to 12,000, 11,500 to 12,000, and, 1,500 to 11,500, 2,000 to 11,500, 2,500 to 11,500, 3,000 to 11,500, 3,500 to 11,500, 4,000 to 11,500, 4,500 to 11,500, 5,000 to 11,500, 5,500 to 11,500, 6,000 to 11,500, 6,500 to 11,500, 7,000 to 11,500, 7,500 to 11,500, 8,000 to 11,500, 8,500 to 11,500, 9,000 to 11,500, 9,500 to 11,500, 10,000 to 11,500, 10,500 to 11,500, 11,000 to 11,500, 1,500 to 11,000, 2,000 to 11,000, 2,500 to 11,000, 3,000 to 11,000, 3,500 to 11,000, 4,000 to 11,000, 4,500 to 11,000, 5,000 to 11,000, 5,500 to 11,000, 6,000 to 11,000, 6,500 to 11,000, 7,000 to 11,000, 7,500 to 11,000, 8,000 to 11,000, 8,500 to 11,000, 9,000 to 11,000, 9,500 to 11,000, 10,000 to 11,000, 10,500 to 11,000, 1,500 to 10,500, 2,000 to 10,500, 2,500 to 10,500, 3,000 to 10,500, 3,500 to 10,500, 4,000 to 10,500, 4,500 to 10,500, 5,000 to 10,500, 5,500 to 10,500, and, 6,000 to 10,500, 6,500 to 10,500, 7,000 to 10,500, 7,500 to 10,500, 8,000 to 10,500, 8,500 to 10,500, 9,000 to 10,500, 9,500 to 10,500, 10,000 to 10,500, 1,500 to 10,000, 2,000 to 10,000, 2,500 to 10,000, 3,000 to 10,000, 3,500 to 10,000, 4,000 to 10,000, 4,500 to 10,000, and, 5,000 to 10,000, 5,500 to 10,000, 6,000 to 10,000, 6,500 to 10,000, 7,000 to 10,000, 7,500 to 10,000, 8,000 to 10,000, 8,500 to 10,000, 9,000 to 10,000, 9,500 to 10,000, 1,500 to 9,500, 2,000 to 9,500, 2,500 to 9,500, 3,000 to 9,500, 3,500 to 9,500, 4,000 to 9,500, 4,500 to 9,500, 5,000 to 9,500, 5,500 to 9,500, 6,000 to 9,500, 6,500 to 9,500, 7,000 to 9,500, 7,500 to 9,500, 8,000 to 9,500, 8,500 to 9,500, 9,000 to 9,500, 1,500 to 9,000, 2,000 to 9,000, 2,500 to 9,000, 3,000 to 9,000, 3,500 to 9,000, 4,000 to 9,000, 4,500 to 9,000, and, 5,000 to 9,000, 5,500 to 9,000, 6,000 to 9,000, 6,500 to 9,000, 7,000 to 9,000, 7,500 to 9,000, 8,000 to 9,000, 8,500 to 9,000, 1,500 to 8,500, 2,000 to 8,500, 2,500 to 8,500, 3,000 to 8,500, 3,500 to 8,500, 4,000 to 8,500, 4,500 to 8,500, 5,000 to 8,500, 5,500 to 8,500, 6,000 to 8,500, 6,500 to 8,500, 7,000 to 8,500, 7,500 to 8,500, 8,000 to 8,500, 1,500 to 8,000, 2,000 to 8,000, 2,500 to 8,000, 3,000 to 8,000, 3,500 to 8,000, 4,000 to 8,000, 4,500 to 8,000, 5,000 to 8,000, 5,500 to 8,000, 6,000 to 8,000, 6,500 to 8,000, 7,000 to 8,000, 7,500 to 8,000, 1,500 to 7,500, 2,000 to 7,500, 2,500 to 7,500, 3,000 to 7,500, 3,500 to 7,500, 4,000 to 7,500, 4,500 to 7,500, 5,000 to 7,500, 5,500 to 7,500, 6,000 to 7,500, 6,500 to 7,500, 7,000 to 7,500, 1,500 to 7,000, 2,000 to 7,000, 2,500 to 7,000, 3,000 to 7,000, and, 3,500 to 7,000, 4,000 to 7,000, 4,500 to 7,000, 5,000 to 7,000, 5,500 to 7,000, 6,000 to 7,000, 6,500 to 7,000, 1,500 to 6,500, 2,000 to 6,500, 2,500 to 6,500, 3,000 to 6,500, 3,500 to 6,500, 4,000 to 6,500, 4,500 to 6,500, 5,000 to 6,500, 5,500 to 6,500, 6,000 to 6,500, 1,500 to 6,000, 2,000 to 6,000, 2,500 to 6,000, 3,000 to 6,000, 3,500 to 6,000, 4,000 to 6,000, 4,500 to 6,000, 5,000 to 6,000, 5,500 to 6,000, 1,500 to 5,500, 2,000 to 5,500, 2,500 to 5,500, 3,000 to 5,500, 3,500 to 5,500, 4,000 to 5,500, 4,500 to 5,500, 5,000 to 5,500, 1,500 to 5,000, 2,000 to 5,000, 2,500 to 5,000, 3,000 to 5,000, 3,500 to 5,000, 4,000 to 5,000, 4,500 to 5,000, 1,500 to 4,500, 2,000 to 4,500, 2,500 to 4,500, 3,000 to 4,500, 3,500 to 4,500, 4,000 to 4,500, 1,500 to 4,000, 2,000 to 4,000, 2,500 to 4,000, 3,000 to 4,000, and, 3,500 to 4,000, 1,500 to 3,500, 2,000 to 3,500, 2,500 to 3,500, 3,000 to 3,500, 1,500 to 3,000, 2,000 to 3,000, 2,500 to 3,000, 1,500 to 2,500, 2,000 to 2,500, and 1,500 to 2,000 daltons.

In some embodiments, the polymer (e.g., PEG) is conjugated to at least one lipid. In some embodiments, the lipid conjugated to the polymer consists of: at least one neutral lipid, at least one phospholipid, at least one anionic lipid, at least one cationic lipid, at least one cholesterol derivative, or any combination thereof.

In some embodiments, the polymer conjugated lipid may be selected from, but is not limited to, at least one of the following: the cationic lipids, non-cationic lipids or sterol lipids previously listed.

In some embodiments, the at least one PEG-lipid conjugate may be selected from, but is not limited to, at least one of the following: siglec-1L-PEG-DSPE, (R) -2, 3-bis (octadecyloxy) propyl-1- (methoxypoly (ethylene glycol) 2000) propylcarbamate 、PEG-S-DSG、PEG-S-DMG、PEG-PE、PEG-PAA、PEG-OH DSPE C18、PEG-DSPE、PEG-DSG、PEG-DPG、PEG-DOMG、PEG-DMPE Na、PEG-DMPE、PEG-DMG2000、PEG-DMG C14、PEG-DMG 2000、PEG-DMG、PEG-DMA、PEG- ceramide C16、PEG-C-DOMG、PEG-c-DMOG、PEG-c-DMA、PEG-cDMA、PEGA、PEG750-C-DMA、PEG400、PEG2k-DMG、PEG2k-C11、PEG2000-PE、PEG2000P、PEG2000-DSPE、PEG2000-DOMG、PEG2000-DMG、PEG2000-C-DMA、PEG2000、PEG200、PEG(2k)-DMG、PEG DSPE C18、PEG DMPE C14、PEG DLPE C12、PEG clicks DMG C14, PEG clicks C12, PEG clicks C10, N (carbonyl-methoxypolyethylene glycol-2000) -1, 2-distearoyl-sn-glycero-3-phosphoethanolamine 、Myrj52、mPEG-PLA、MPEG-DSPE、mPEG3000-DMPE、MPEG-2000-DSPE、MPEG2000-DSPE、mPEG2000-DPPE、mPEG2000-DMPE、mPEG2000-DMG、mDPPE-PEG2000、1,2- distearoyl-sn-glycero-3-phosphoethanolamine-PEG 2000, HPEG-2K-LIPD, folic acid PEG-DSPE, DSPE-PEGMA 500, DSPE-PEGMA, DSPE-PEG6000, DSPE-PEG5000, DSPE-PEG2K-NAG, DSPE-PEG2K, DSPE-PEG2000 maleimido 、DSPE-PEG2000、DSPE-PEG、DSG-PEGMA、DSG-PEG5000、DPPE-PEG-2K、DPPE-PEG、DPPE-mPEG2000、DPPE-mPEG、DPG-PEGMA、DOPE-PEG2000、DMPE-PEGMA、DMPE-PEG2000、DMPE-Peg、DMPE-mPEG2000、DMG-PEGMA、DMG-PEG2000、DMG-PEG、 distearoyl-glycerol-polyethylene glycol 、Cl8PEG750、CI8PEG5000、CI8PEG3000、CI8PEG2000、CI6PEG2000、CI4PEG2000、C18-PEG5000、C18PEG、C16PEG、C16 mPEG( polyethylene glycol) 2000 ceramide, C14-PEG200, C14-PEG2000, C14-PEG, KPE 2, DSPE-2K-5, DSPE-2K-PEG 6000, (DSPE-2K-methoxypolyethylene glycol) and (DSPE-35) 2K-NAG, DSPE-35.

In some embodiments, the LNP comprises a lipid of the present disclosure, distearyl phosphatidylcholine (DSPC), cholesterol, and 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000).

In some embodiments, the LNP comprises a lipid of the present disclosure, distearyl phosphatidylcholine (DSPC), cholesterol, and 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000) in a molar ratio of about 48.5:10:40:1.5, respectively.

In some embodiments, the LNP comprises a lipid of the present disclosure, distearyl phosphatidylcholine (DSPC), cholesterol, and 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000) in a molar ratio of about 48.5:10:39:2.5, respectively.

The amount and ratio of LNP components can vary by any amount depending on the desired form, structure, function, cargo, target, or any combination thereof. The amount of each component may be expressed in various embodiments as a percentage (mol%) of the indicated component to the total molar mass of all lipid or lipid-conjugated components. The amount of each component can be expressed in various embodiments in terms of the relative ratio (molar ratio) of each component based on molar mass. The amount of each component can be expressed in various embodiments in terms of the weight (mg or equivalent) of each component used to formulate the LNP prior to manufacture. The amount of each component may be expressed in various embodiments by any other method known in the art. Any formulation given in one representation of the amount of a component ("unit") is expressly intended to encompass any formulation in which the amount of the component in a different unit is expressed, where those representations are effectively equivalent when converted to the same unit. In some embodiments, "effective equivalent (EFFECTIVELY EQUIVALENT)" means that two or more values are within about 10% of each other.

In some embodiments, the LNP comprises at least one cationic lipid in an amount of about 0.1mol% to 100 mol%. In some embodiments, the LNP comprises at least one cationic lipid in an amount of about 20mol% to 60 mol%. In some embodiments, the LNP comprises at least one cationic lipid in an amount of about 50mol% to 85 mol%. In some embodiments, the LNP comprises at least one cationic lipid in an amount of less than about 20 mol%. In some embodiments, the LNP comprises at least one cationic lipid in an amount of greater than about 60mol% or about 85 mol%. in some embodiments, the LNP comprises at least one cationic lipid in an amount of about 95mol% or less. In some embodiments, the LNP comprises cationic lipids in the following amounts: less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 mole percent. In some embodiments, the LNP comprises at least one cationic lipid in the following amounts: greater than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95 mole percent. In some embodiments, the LNP comprises at least one cationic lipid in the following amounts: about 20mol% to 30mol%, 20mol% to 35mol%, 20mol% to 40mol%, 20mol% to 45mol%, 20mol% to 50mol%, 20mol% to 55mol%, 20mol% to 60mol%, 20mol% to 65mol%, 20mol% to 70mol%, 20mol% to 75mol%, 20mol% to 80mol%, 20mol% to 85mol%, 20mol% to 90mol%, and, 25mol% to 35mol%, 25mol% to 40mol%, 25mol% to 45mol%, 25mol% to 50mol%, 25mol% to 55mol%, 25mol% to 60mol%, 25mol% to 65mol%, 25mol% to 70mol%, 25mol% to 75mol%, 25mol% to 80mol%, 25mol% to 85mol%, 25mol% to 90mol%, 30mol% to 40mol%, 30mol% to 45mol%, 30mol% to 50mol%, and, 30mol% to 55mol%, 30mol% to 60mol%, 30mol% to 65mol%, 30mol% to 70mol%, 30mol% to 75mol%, 30mol% to 80mol%, 30mol% to 85mol%, 30mol% to 90mol%, 35mol% to 40mol%, 35mol% to 45mol%, 35mol% to 50mol%, 35mol% to 55mol%, 35mol% to 60mol%, 35mol% to 65mol%, 35mol% to 70mol%, and, 35 to 75mol%, 35 to 80mol%, 35 to 85mol%, 35 to 90mol%, 40 to 45mol%, 40 to 50mol%, 40 to 55mol%, 40 to 60mol%, 40 to 65mol%, 40 to 70mol%, 40 to 75mol%, 40 to 80mol%, 40 to 85mol%, 40 to 90mol%, 45 to 55mol%, 45mol% to 60mol%, 45mol% to 65mol%, 45mol% to 70mol%, 45mol% to 75mol%, 45mol% to 80mol%, 45mol% to 85mol%, 45mol% to 90mol%, 50mol% to 60mol%, 50mol% to 65mol%, 50mol% to 70mol%, 50mol% to 75mol%, 50mol% to 80mol%, 50mol% to 85mol%, 50mol% to 90mol%, 55mol% to 65mol%, and, 55mol% to 70mol%, 55mol% to 75mol%, 55mol% to 80mol%, 55mol% to 85mol%, 55mol% to 90mol%, 60mol% to 70mol%, 60mol% to 75mol%, 60mol% to 80mol%, 60mol% to 85mol%, 60mol% to 90mol%, 65mol% to 75mol%, 65mol% to 80mol%, 65mol% to 85mol%, 65mol% to 90mol%, 70mol% to 80mol%, and, 70mol% to 85mol%, 70mol% to 90mol%, 75mol% to 85mol%, 75mol% to 90mol%, 80mol% to 90mol%, or 85mol% to 95mol%.

In some embodiments, the LNP comprises at least one non-cationic lipid in an amount of about 0.1mol% to 100 mol%. In some embodiments, the LNP comprises at least one non-cationic lipid in an amount of about 5mol% to 35 mol%. In some embodiments, the LNP comprises at least one non-cationic lipid in an amount of about 5mol% to 25 mol%. In some embodiments, the LNP comprises at least one non-cationic lipid in an amount of less than about 5mol%. In some embodiments, the LNP comprises at least one non-cationic lipid in an amount of greater than about 25mol% or about 35 mol%. In some embodiments, the LNP comprises at least one non-cationic lipid in an amount of about 95mol% or less. In some embodiments, the LNP comprises at least one non-cationic lipid in the following amounts: less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 mole percent. In some embodiments, the LNP comprises at least one non-cationic lipid in the following amounts: greater than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95 mole percent. In some embodiments, the LNP comprises at least one non-cationic lipid in the following amounts: about 5mol% to 15mol%, 5mol% to 25mol%, 5mol% to 35mol%, 5mol% to 45mol%, 5mol% to 55mol%, 10mol% to 20mol%, 10mol% to 30mol%, 10mol% to 40mol%, 10mol% to 50mol%, 15mol% to 25mol%, 15mol% to 35mol%, 15mol% to 45mol%, 20mol% to 30mol%, 20mol% to 40mol%, 20mol% to 50mol%, 25mol% to 35mol%, 25mol% to 45mol%, 30mol% to 40mol%, 30mol% to 50mol%, and 35mol% to 45mol%.

In some embodiments, the LNP comprises at least one sterol in an amount of about 0.1mol% to 100 mol%. In some embodiments, the LNP comprises at least one sterol in an amount of about 20mol% to 45 mol%. In some embodiments, the LNP comprises at least one sterol in an amount of about 25mol% to 55 mol%. In some embodiments, the LNP comprises at least one sterol in an amount of less than about 20 mol%. In some embodiments, the LNP comprises at least one sterol in an amount of greater than about 45mol% or about 55 mol%. In some embodiments, the LNP comprises at least one sterol in an amount of about 95mol% or less. In some embodiments, the LNP comprises at least one sterol in the following amounts: less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 mole percent. In some embodiments, the LNP comprises at least one sterol in the following amounts: greater than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95 mole percent. In some embodiments, the LNP comprises at least one sterol in the following amounts: about 10 to 20mol%, 10 to 30mol%, 10 to 40mol%, 10 to 50mol%, 10 to 60mol%, 15 to 25mol%, 15 to 35mol%, 15 to 45mol%, 15 to 55mol%, 15 to 65mol%, 20 to 30mol%, 20 to 40mol%, 20 to 50mol%, 20 to 60mol%, 25 to 35mol%, 25 to 45mol%, 25 to 55mol%, 25 to 65mol%, 30 to 40mol%, 30 to 50mol%, 30 to 60mol%, 35 to 45mol%, 35 to 55mol%, 35 to 65mol%, 40 to 50mol%, 40 to 60mol%, 45 to 55mol%, 45 to 65mol%, 50 to 60mol%, and 55 to 65mol%.

In some embodiments, the LNP comprises at least one particulate activity modulator in an amount of about 0.1mol% to 100 mol%. In some embodiments, the LNP comprises at least one particulate activity modulator in an amount of about 0.5mol% to 15 mol%. In some embodiments, the LNP comprises at least one particulate activity modulator in an amount of about 15mol% to 40 mol%. In some embodiments, the LNP comprises at least one particulate activity modulator in an amount of less than about 0.1 mol%. In some embodiments, the LNP comprises at least one particulate activity modulator in an amount of greater than about 15mol% or about 40 mol%. In some embodiments, the LNP comprises at least one particulate activity modulator in an amount of about 95mol% or less. In some embodiments, the LNP comprises at least one particle activity modulator in the following amounts: less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 mole percent. In some embodiments, the LNP comprises at least one particle activity modulator in the following amounts: greater than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95 mole percent. In some embodiments, the LNP comprises at least one particle activity modulator in the following amounts: about 0.1mol% to 1mol%, 0.1mol% to 2mol%, 0.1mol% to 3mol%, 0.1mol% to 4mol%, 0.1mol% to 5mol%, 0.1mol% to 6mol%, 0.1mol% to 7mol%, 0.1mol% to 8mol%, 0.1mol% to 9mol%, 0.1mol% to 10mol%, 0.1mol% to 15mol%, 0.1mol% to 20mol% >, 0.1mol% to 25mol%, 1mol% to 2mol%, 1mol% to 3mol%, 1mol% to 4mol%, 1mol% to 5mol%, 1mol% to 6mol%, 1mol% to 7mol%, 1mol% to 8mol%, 1mol% to 9mol%, 1mol% to 10mol%, 1mol% to 15mol%, 1mol% to 20mol%, 1mol% to 25mol%, 2mol% to 3mol%, 2mol% to 4mol%, 2mol% to 5mol%, 2mol% to 6mol%, and, 2mol% to 7mol%, 2mol% to 8mol%, 2mol% to 9mol%, 2mol% to 10mol%, 2mol% to 15mol%, 2mol% to 25mol%, 3mol% to 4mol%, 3mol% to 5mol%, 3mol% to 6mol%, 3mol% to 7mol%, 3mol% to 8mol%, 3mol% to 9mol%, 3mol% to 10mol%, 3mol% to 15mol%, 3mol% to 20mol%, 3mol% to 25mol%, 4mol% to 5mol%, and, 4mol% to 6mol%, 4mol% to 7mol%, 4mol% to 8mol%, 4mol% to 9mol%, 4mol% to 10mol%, 4mol% to 15mol%, 4mol% to 20mol%, 4mol% to 25mol%, 5mol% to 10mol%, 5mol% to 15mol%, 5mol% to 20mol%, 5mol% to 25mol%, 10mol% to 15mol%, 10mol% to 20mol%, 10mol% to 25mol%, 15mol% to 20mol%, and, 15mol% to 25mol% and 20mol% to 25mol%.

In some embodiments, the LNP consists of: about 30-60 mole% of at least one cationic lipid, about 0-30 mole% of at least one non-cationic lipid (e.g., a phospholipid), about 18.5-48.5 mole% of at least one sterol (e.g., cholesterol), and about 0-10 mole% of at least one particulate activity modulator (e.g., a pegylated lipid).

In some embodiments, the LNP consists of: about 35 to 55 mole% of at least one cationic lipid, about 5 to 25 mole% of at least one non-cationic lipid (e.g., a phospholipid), about 30 to 40 mole% of at least one sterol (e.g., cholesterol), and about 0 to 10 mole% of at least one particulate activity modulator (e.g., a pegylated lipid).

In some embodiments, the LNP consists of: about 35 to 45 mole% of at least one cationic lipid, about 25 to 35 mole% of at least one non-cationic lipid (e.g., a phospholipid), about 20 to 30 mole% of at least one sterol (e.g., cholesterol), and about 0 to 10 mole% of at least one particulate activity modulator (e.g., a pegylated lipid).

In some embodiments, the LNP consists of: about 45-65 mole% of at least one cationic lipid, about 5-10 mole% of at least one non-cationic lipid (e.g., a phospholipid), about 25-40 mole% of at least one sterol (e.g., cholesterol), and about 0.5-10 mole% of at least one particulate activity modulator (e.g., a pegylated lipid).

In some embodiments, the LNP consists of: about 40-60 mole% of at least one cationic lipid, about 5-15 mole% of at least one non-cationic lipid (e.g., a phospholipid), about 35-45 mole% of at least one sterol (e.g., cholesterol), and about 0.5-3 mole% of at least one particulate activity modulator (e.g., a pegylated lipid).

In some embodiments, the LNP consists of: about 30-60 mole% of at least one cationic lipid, about 0-30 mole% of at least one non-cationic lipid (e.g., a phospholipid), about 15-50 mole% of at least one sterol (e.g., cholesterol), and about 0.01-10 mole% of at least one particulate activity modulator (e.g., a pegylated lipid).

In some embodiments, the LNP consists of: about 10-75 mole% of at least one cationic lipid, about 0.5-50 mole% of at least one non-cationic lipid (e.g., a phospholipid), about 5-60 mole% of at least one sterol (e.g., cholesterol), and about 0.1-20 mole% of at least one particulate activity modulator (e.g., a pegylated lipid).

In some embodiments, the LNP consists of: about 50-65 mole% of at least one cationic lipid, about 3-15 mole% of at least one non-cationic lipid (e.g., a phospholipid), about 30-40 mole% of at least one sterol (e.g., cholesterol), and about 0.5-2 mole% of at least one particulate activity modulator (e.g., a pegylated lipid).

In some embodiments, the LNP consists of: about 50-85 mole% of at least one cationic lipid, about 3-15 mole% of at least one non-cationic lipid (e.g., a phospholipid), about 30-40 mole% of at least one sterol (e.g., cholesterol), and about 0.5-2 mole% of at least one particulate activity modulator (e.g., a pegylated lipid).

In some embodiments, the LNP consists of: about 25 to 75 mole% of at least one cationic lipid, about 0.1 to 15 mole% of at least one non-cationic lipid (e.g., a phospholipid), about 5 to 50 mole% of at least one sterol (e.g., cholesterol), and about 0.5 to 20 mole% of at least one particulate activity modulator (e.g., a pegylated lipid).

In some embodiments, the LNP consists of: about 50-65 mole% of at least one cationic lipid, about 5-10 mole% of at least one non-cationic lipid (e.g., a phospholipid), about 25-35 mole% of at least one sterol (e.g., cholesterol), and about 5-10 mole% of at least one particulate activity modulator (e.g., a pegylated lipid).

In some embodiments, the LNP consists of: about 20-60 mole% of at least one cationic lipid, about 5-25 mole% of at least one non-cationic lipid (e.g., a phospholipid), about 25-55 mole% of at least one sterol (e.g., cholesterol), and about 0.5-15 mole% of at least one particulate activity modulator (e.g., a pegylated lipid).

In some embodiments, the LNP may be characterized by its shape. In some embodiments, the LNP is substantially spherical. In some embodiments, the LNP is substantially rod-shaped (i.e., cylindrical). In some embodiments, the LNP is substantially disc-shaped.

In some embodiments, the LNP may be characterized by its size. In some embodiments, the size of the LNP may be defined as the diameter of its largest circular cross section, referred to herein simply as its diameter. In some embodiments, the LNP may have a diameter between 30nm and about 150 nm. In some embodiments, the diameter of the LNP may be in a range between: about 40 to 150nm, 50 to 150nm, 60 to 150nm, about 70 to 150nm, or 80 to 150nm, 90 to 150nm, 100 to nm, 110 to 150nm, 120 to 150nm, 130 to 150nm, 140 to 150nm, 30mol% to 140mol%, 40mol% to 140mol%, 50mol% to 140mol%, 60mol% to 140mol%, 70mol% to 140mol%, and, 80mol% to 140mol%, 90mol% to 140mol%, 100mol% to 140mol%, 110mol% to 140mol%, 120mol% to 140mol%, 130mol% to 140mol%, 140mol% to 140mol%, 30mol% to 140mol%, 40mol% to 130mol%, 50mol% to 130mol%, 60mol% to 130mol%, 70mol% to 130mol%, 80mol% to 130mol%, 90mol% to 130mol%, and, 100mol% to 130mol%, 110mol% to 130mol%, 120mol% to 130mol%, 30mol% to 120mol%, 40mol% to 120mol%, 50mol% to 120mol%, 60mol% to 120mol%, 70mol% to 120mol%, 80mol% to 120mol%, 90mol% to 120mol%, 100mol% to 120mol%, 110mol% to 120mol%, 30mol% to 110mol%, 40mol% to 110mol%, and, 50mol% to 110mol%, 60mol% to 110mol%, 70mol% to 110mol%, 80mol% to 110mol%, 90mol% to 110mol%, 100mol% to 110mol%, 30mol% to 100mol%, 40mol% to 100mol%, 50mol% to 100mol%, 60mol% to 100mol%, 70mol% to 100mol%, 80mol% to 100mol%, 90mol% to 100mol%, 30mol% to 90mol%, and, 40mol% to 90mol%, 50mol% to 90mol%, 60mol% to 90mol%, 70mol% to 90mol%, 80mol% to 90mol%, 30mol% to 80mol%, 40mol% to 80mol%, 50mol% to 80mol%, 60mol% to 80mol%, 70mol% to 80mol%, 30mol% to 70mol%, 40mol% to 70mol%, 50mol% to 70mol%, 60mol% to 70mol%, 30mol% to 60mol%, and, 40mol% to 60mol%, 50mol% to 60mol%, 30mol% to 50mol%, 40mol% to 50mol%, and 30mol% to 40mol%.

In some embodiments, populations of LNPs (e.g., those produced by the same formulation) can be characterized by measuring the uniformity of size, shape, or mass of particles in the population. Homogeneity may be expressed in some embodiments as the Polydispersity Index (PI) of the population. In some embodiments, the uniformity may in some embodiments be a population inequalityAnd (5) formal expression. The terms "polydispersity index" and "inequality" are understood herein to be equivalent and used interchangeably. In some embodiments, the PI of the LNP population produced by a given formulation will be between about 0.1 and 1. In some embodiments, the PI of the LNP population produced by a given formulation will be less than about 1, less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1. In some embodiments, the PI of the LNP population produced by a given formulation will be between: about 0.1 to 1, 0.1 to 0.8, 0.1 to 0.6, 0.1 to 0.4, 0.1 to 0.2, 0.2 to 1, 0.2 to 0.8, 0.2 to 0.6, 0.2 to 0.4, 0.4 to 1, 0.4 to 0.8, 0.4 to 0.6, 0.6 to 1, 0.6 to 0.8, and 0.8 to 1.

In some embodiments, the LNP may encapsulate cargo, such as the initial construct and the reference construct of the present disclosure, in whole or in part. In some embodiments, substantially 0% of the cargo present in the final formulation is exposed to the environment outside the LNP (i.e., the cargo is fully encapsulated). In some embodiments, the cargo is associated with the LNP but is at least partially exposed to the environment outside the LNP. In some embodiments, the LNP may be characterized by cargo% that is not exposed to the environment outside the LNP, such as encapsulation efficiency. For clarity, an encapsulation efficiency of about 100% refers to an LNP formulation in which substantially all cargo is completely encapsulated by the LNP; whereas an encapsulation efficiency of about 0% refers to an LNP in which substantially no cargo is encapsulated in the LNP, such as in the case of an LNP in which cargo is bound to the outer surface of the LNP. In some embodiments, the LNP can have an encapsulation efficiency of less than about 100%, less than about 95%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than 5%. In some embodiments, the encapsulation efficiency of the LNP may be between: about 90% to 100%, 80% to 100%, 70% to 100%, 60% to 100%, 50% to 100%, 40% to 100%, 30% to 100%, 20% to 100%, 10% to 100%, 80% to 90%, 70% to 90%, 60% to 90%, 50% to 90%, 40% to 90%, 30% to 90%, 20% to 90%, 10% to 90%, 70% to 80%, 60% to 80%, 50% to 80%, 40% to 80%, 30% to 80%, 20% to 80%, 10% to 80%, 60% to 70%, 50% to 70%, 40% to 70%, 30% to 70%, 20% to 70%, 10% to 70%, 40% to 50%, 30% to 50%, 20% to 50%, 30% to 40%, 20% to 40%, 10% to 40%, 20% to 30%, 10% to 30%, and 10% to 20%.

In some embodiments, the LNP may include at least one identifier portion, as shown in fig. 5. Non-limiting examples of identifier moieties include glycans, antibodies, peptides, small molecules, and any combination thereof. In some embodiments, at least one targeting agent may be incorporated into the lipid membrane of the lipid-based nanoparticle. In some embodiments, at least one targeting agent may be present on the outer surface of the nanoparticle. In some embodiments, at least one targeting agent may be conjugated to the lipid component of the nanoparticle. In some embodiments, at least one targeting agent may be conjugated to the polymer component of the nanoparticle. In some embodiments, the at least one targeting agent may be anchored to the nanoparticle via hydrophobic and hydrophilic interactions in an aqueous environment internal or external to the at least one targeting agent, nanoparticle film, and nanoparticle. In some embodiments, at least one targeting agent is conjugated to the peptide/protein component of the nanoparticle film. In some embodiments, at least one targeting agent is conjugated to a suitable linker moiety conjugated to a component of the nanoparticle membrane. In some embodiments, any combination of forces and bonds may result in the association of the targeting agent with the nanoparticle.

The LNPs described herein may be formed using techniques known in the art. As one non-limiting example, an organic solution containing lipids is mixed in a microfluidic channel along with an acidic aqueous solution containing the initial or reference construct, resulting in the formation of a targeting system (delivery vehicle and reference construct).

In some embodiments, each LNP formulation includes a reference construct with a unique identifiable nucleotide identifier sequence (e.g., a barcode). The unique identifier sequence provides the ability to identify the particular LNP that produces the desired result. LNP formulations may also differ from LNP-forming compositions used to produce LNP. For example, the molar amount and/or structure of the ionizable lipid, the molar amount and/or structure of the helper lipid, the molar amount and/or structure of PEG, and/or the molar amount of cholesterol of the LNP-forming composition can vary. Alternatively or additionally, the LNP formulation may comprise reference constructs with different coding sequences for the bioactive molecules. Alternatively or additionally, the LNP formulation may comprise a reference construct with different modifications to the nucleic acid sequence.

In some embodiments, the lipid composition is described in terms of the corresponding molar ratio of the component lipids in the formulation. As one non-limiting example, the mole% of the ionizable lipid can be from about 10 mole% to about 80 mole%. As one non-limiting example, the mole% of the ionizable lipid can be from about 20 mole% to about 70 mole%. As one non-limiting example, the mole% of the ionizable lipid can be from about 30 mole% to about 60 mole%. As one non-limiting example, the mole% of the ionizable lipid can be from about 35 mole% to about 55 mole%. As one non-limiting example, the mole% of the ionizable lipid can be from about 40 mole% to about 50 mole%. As one non-limiting example, the ionizable lipid mole% of the transfer vehicle batch will be ± 30%, ±25%, ±20%, ±15%, ±10%, ±5% or ± 2.5% of the target mole%. In some embodiments, the transfer vehicle variability from lot to lot will be less than 15%, less than 10%, or less than 5%.

In some embodiments, the mole% of the helper lipid may be from about 1 mole% to about 50 mole%. In some embodiments, the mole% of the helper lipid may be from about 2 mole% to about 45 mole%. In some embodiments, the mole% of the helper lipid may be from about 3 mole% to about 40 mole%. In some embodiments, the mole% of the helper lipid may be from about 4 mole% to about 35 mole%. In some embodiments, the mole% of the helper lipid may be from about 5 mole% to about 30 mole%. In some embodiments, the mole% of the helper lipid may be from about 10 mole% to about 20 mole%. In some embodiments, the helper lipid mole% of the transfer vehicle batch will be ± 30%, ±25%, ±20%, ±15%, ±10%, ±5% or ± 2.5% of the target mole%.

In some embodiments, the mole% of the structural lipid may be about 10 mole% to about 80 mole%. In some embodiments, the mole% of the structural lipid may be from about 20 mole% to about 70 mole%. In some embodiments, the mole% of the structural lipid may be about 30 mole% to about 60 mole%. In some embodiments, the mole% of the structural lipid may be about 35 mole% to about 55 mole%. In some embodiments, the mole% of the structural lipid may be about 40 mole% to about 50 mole%. In some embodiments, the structural lipid mole% of the transfer vehicle batch will be ± 30%, ±25%, ±20%, ±15%, ±10%, ±5% or ± 2.5% of the target mole%.

In some embodiments, the mole% of PEG-modified lipids may be about 0.1 mole% to about 10 mole%. In some embodiments, the mole% of PEG-modified lipids may be about 0.2 mole% to about 5 mole%. In some embodiments, the mole% of PEG-modified lipids may be about 0.5 mole% to about 3 mole%. In some embodiments, the mole% of PEG-modified lipids can be about 1 mole% to about 2 mole%. In some embodiments, the mole% of PEG-modified lipid may be about 1.5 mole%. In some embodiments, the PEG-modified lipid mole% of the transfer vehicle batch will be ± 30%, ±25%, ±20%, ±15%, ±10%, ±5% or ± 2.5% of the target mole%.

In some embodiments, the delivery vehicle may be any of the lipid nanoparticles described in WO2021113777, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the delivery vehicle is a lipid nanoparticle comprising any of the ionizable lipids (e.g., amine lipids), PEG lipids, non-cationic (helper) lipids, or structural lipids in WO2021113777 (the contents of which are incorporated herein by reference in their entirety).

In some embodiments, lipid nanoparticle formulations can be prepared by the methods described in international publication No. WO2011127255 or No. W02008103276, the respective contents of each of which are incorporated herein by reference in their entirety. In some embodiments, the lipid nanoparticle formulation may be as described in international publication No. W02019131770, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, lipid nanoparticle formulations can be prepared by the methods described in International publication No. WO2020237227, the contents of each of which are incorporated herein by reference in their entirety. In some embodiments, the lipid nanoparticle formulation may be as described in international publication No. WO2020237227, the contents of which are incorporated herein by reference in their entirety.

Non-lipid nanoparticles

In some embodiments, the nanoparticle is a non-lipid based nanoparticle. Non-lipid based nanoparticles include, but are not limited to, silicon-based nanoparticles (i.e., porous silicon nanoparticles), gold nanoparticles, polypeptide-based nanoparticles, nucleotide-based nanoparticles, and carbon-based nanoparticles.

Exosome

In some embodiments, the delivery vehicle comprises at least one exosome. As used herein, "exosomes" refer to membrane-bound vesicles having endocytic origin. Without wishing to be bound by theory, exosomes are typically released from the host/progenitor cells into the extracellular environment after the multivesicular body fuses the cytoplasmic membrane. Thus, exosomes will tend to include components of the progenitor cell membrane in addition to the designed components and cargo. The exosome membrane is typically lamellar, consisting of a lipid bilayer and an aqueous nanoparticle space.

In some embodiments, the exosomes may include at least one identifier portion, as shown in fig. 5. Non-limiting examples of identifier moieties include glycans, antibodies, peptides, small molecules, and any combination thereof.

Liposome

In some embodiments, the delivery vehicle comprises at least one liposome. As used herein, a "liposome" is a vesicle composed of at least one lipid bilayer membrane surrounding an aqueous nanoparticle interior space that is not typically derived from a progenitor/host cell. The liposome can be: (large) multilamellar vesicles (MLVs), possibly hundreds of nanometers in diameter, comprising a series of concentric bilayers separated by narrow aqueous spaces; single cell vesicles (SUVs), possibly less than 50nm in diameter; and unilamellar large vesicles (LUVs), possibly between 50 and 500nm in diameter. In some embodiments, the liposome may be composed of any or all of the same components and amounts of the same components as the lipid nanoparticle, except for the method of its manufacture.

Microcells

In some embodiments, the delivery vehicle comprises at least one micelle. As used herein, "micelle" refers to a small particle that does not have an aqueous particle interior space. Without wishing to be bound by theory, the particle interior space of the microcell is occupied by the hydrophobic tail of the lipid comprising the microcell membrane and possibly associated cargo, rather than any additional lipid head groups. In some embodiments, the micelles may be composed of any or all of the same components as the lipid nanoparticle, differing primarily in the method of their manufacture.

In some embodiments, the microcell may include at least one identifier portion, as shown in fig. 5. Non-limiting examples of identifier moieties include glycans, antibodies, peptides, small molecules, and any combination thereof.

Virus particles

In some embodiments, the delivery vehicle comprises at least one virus-like particle. As used herein, "virus-like particle" refers to a vesicle that is predominantly a viral-derived protein capsid, sheath, shell or housing (all used interchangeably herein) that can be loaded with a cargo moiety. In general, virus-like particles are non-infectious and can be synthesized using cellular mechanisms that express viral capsid protein sequences, then self-assemble and incorporate associated cargo moieties, but it is possible to form virus-like particles by providing capsid and cargo components without expressing the relevant cellular mechanisms and allowing for self-assembly.

In some embodiments, the virus-like particle may be derived from, for example, but not limited to, at least one of the following virus species: the picoviridae, retrovirus, flaviviridae, paramyxoviridae and phage families. In some embodiments, the virus-like particle may be derived from adeno-associated virus, HIV, hepatitis c virus, HPV, or any combination thereof.

In some embodiments, the virus-like particle is an AAV particle, and the AAV particle may include at least one identifier portion, as shown in fig. 5. Non-limiting examples of identifier moieties include glycans, antibodies, peptides, small molecules, and any combination thereof.

Polymer delivery technology

In some embodiments, the delivery vehicle may comprise at least one polymeric delivery agent. As used herein, "polymeric delivery agent" refers to a non-aggregating delivery agent comprised of a soluble polymer conjugated to a cargo moiety via various linking groups. In some embodiments, the polymer delivery agent may comprise any of the polymers described herein.

Tracking system

The isotropic discovery platform disclosed herein may use a variety of tracking systems including identifier sequences and portions (also referred to as "barcodes") to allow for the post-administration inspection of delivery vehicles and/or fiducial constructs, cargo, and payloads.

In some embodiments, the tracking system is a single identifier sequence or portion. The identifier sequence or portion may be located in a delivery vehicle, a reference construct, a cargo or payload region, a 5'utr, a 3' utr, a promoter region, or a tailed region. As one non-limiting example, the identifier sequence or portion is located in or on the delivery vehicle. As one non-limiting example, the identifier sequence or portion is located in or on the reference construct. As one non-limiting example, the identifier sequence or portion is located in or on the 5' utr. As one non-limiting example, the identifier sequence or portion is located in or on the 3' utr. As one non-limiting example, the identifier sequence or portion is located in or on the promoter region. As one non-limiting example, the identifier sequence or portion is located in or on the payload area. As one non-limiting example, the identifier sequence or portion is located in or on the tailed region.

In some embodiments, the tracking system is a set of identifier sequences or portions having a first identifier sequence or portion for the delivery vehicle and a second identifier sequence or portion for the reference construct, cargo, and payload. The first and second identifier sequences or portions may be the same or different. If additional fiducial constructs, cargoes, and payloads are present in the delivery vehicle, each fiducial construct, cargo, and payload may have its own identifier sequence or portion, or it may be the same at a second identifier sequence or portion.

In some embodiments, the isotropic exploration platform is comprised of multiple tracking systems, where each tracking system allows detection of delivery vehicles and/or reference constructs, cargo, and payloads at different resolution levels.

In some embodiments, the tracking system comprises at least one barcode sequence. As used herein, a "barcode" or "barcode sequence" is any sequence or any sequence administered that can be detected using methods known in the art and that is different from sequences in cells, tissues, organs, and/or organisms. The barcode sequences may be included in or attached to the delivery vehicle and/or included in the reference construct, cargo, and payload. As one non-limiting example, the delivery vehicle comprises a barcode sequence. As one non-limiting example, the cargo or payload comprises a barcode sequence. As one non-limiting example, the reference construct comprises a barcode sequence.

In some embodiments, the location of the identifier sequence or portion in the targeting system is random. As one non-limiting example, the identifier sequence or portion is in a delivery vehicle. As one non-limiting example, the identifier sequence or portion is in a reference construct. As one non-limiting example, the identifier sequence or portion is in the cargo or payload. As one non-limiting example, the identifier sequence or portion is in the delivery vehicle and the reference construct. As one non-limiting example, the identifier sequence or portion is in a delivery vehicle and cargo or payload. As one non-limiting example, the identifier sequence or portion is in a reference construct and a cargo or payload. As one non-limiting example, the identifier sequence or portion is in the delivery vehicle, the reference construct, and the cargo or payload.

In some embodiments, the location of the identifier sequence or portion in the targeting system is predetermined. As one non-limiting example, the identifier sequence or portion is in a delivery vehicle. As one non-limiting example, the identifier sequence or portion is in a reference construct. As one non-limiting example, the identifier sequence or portion is in the cargo or payload. As one non-limiting example, the identifier sequence or portion is in the delivery vehicle and the reference construct. As one non-limiting example, the identifier sequence or portion is in a delivery vehicle and cargo or payload. As one non-limiting example, the identifier sequence or portion is in a reference construct and a cargo or payload. As one non-limiting example, the identifier sequence or portion is in the delivery vehicle, the reference construct, and the cargo or payload.

In some embodiments, the position of the identifier sequence or portion in the targeting system is inverted. As one non-limiting example, the identifier sequence or portion is in a delivery vehicle. As one non-limiting example, the identifier sequence or portion is in a reference construct. As one non-limiting example, the identifier sequence or portion is in the cargo or payload. As one non-limiting example, the identifier sequence or portion is in the delivery vehicle and the reference construct. As one non-limiting example, the identifier sequence or portion is in a delivery vehicle and cargo or payload. As one non-limiting example, the identifier sequence or portion is in a reference construct and a cargo or payload. As one non-limiting example, the identifier sequence or portion is in the delivery vehicle, the reference construct, and the cargo or payload.

In some embodiments, the identifier sequence is a randomly generated sequence for avoiding replication during deep sequencing. In some embodiments, the identifier sequence is a repeated sequence of nucleotides or amino acids. In some embodiments, the identifier sequence is a fragment of a larger sequence (such as, but not limited to, a cargo or payload). The identifier sequences may be designed to any useful length using synthetic techniques (see Clement et al, volume ,AmpUMI:design and analysis of unique molecular identifiers for deep amplicon sequencing,Bioinformatics,, volume 34, 13, month 7, 01, 2018, pages i202-i 210; the contents of which are incorporated herein by reference in their entirety).

In some embodiments, the length of the identifier sequence is between 2 and 1000 nucleotides. For example, the identifier sequence may be 2、3、4、5、6、7、8、9、10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190、200、210、220、230、240、250、260、270、280、290、300、310、320、330、340、350、360、370、380、390、400、410、420、430、440、450、460、470、480、490、500、510、520、530、540、550、560、570、580、590、600、610、620、630、640、650、660、670、680、690、700、710、720、730、740、750、760、770、780、790、800、810、820、830、840、850、860、870、880、890、900、910、920、930、940、950、960、970、980、990、1000 or more than 1000 nucleotides in length. The length of the identifier sequence may be between ：2-5、2-10、2-15、2-20、2-30、2-50、2-70、2-90、2-100、2-250、2-300、2-350、2-400、2-450、2-500、2-550、2-600、2-650、2-700、2-750、2-800、2-850、2-900、2-950、2-1000、5-10、5-15、5-20、5-30、5-50、5-70、5-90、5-100、5-250、5-300、5-350、5-400、5-450、5-500、5-550、5-600、5-650、5-700、5-750、5-800、5-850、5-900、5-950、5-1000、10-30、10-50、10-70、10-90、10-100、10-250、10-300、10-350、10-400、10-450、10-500、10-550、10-600、10-650、10-700、10-750、10-800、10-850、10-900、10-950、10-1000、20-30、20-50、20-70、20-90、20-100、20-250、20-300、20-350、20-400、20-450、20-500、20-550、20-600、20-650、20-700、20-750、20-800、20-850、20-900、20-950、20-1000、30-50、30-70、30-90、30-100、30-250、30-300、30-350、30-400、30-450、30-500、30-550、30-600、30-650、30-700、30-750、30-800、30-850、30-900、30-950、30-1000、40-50、40-70、40-90、40-100、40-250、40-300、40-350、40-400、40-450、40-500、40-550、40-600、40-650、40-700、40-750、40-800、40-850、40-900、40-950、40-1000、50-70、50-90、50-100、50-250、50-300、50-350、50-400、50-450、50-500、50-550、50-600、50-650、50-700、50-750、50-800、50-850、50-900、50-950、50-1000、60-70、60-90、60-100、60-250、60-300、60-350、60-400、60-450、60-500、60-550、60-600、60-650、60-700、60-750、60-800、60-850、60-900、60-950、60-1000、70-90、70-100、70-250、70-300、70-350、70-400、70-450、70-500、70-550、70-600、70-650、70-700、70-750、70-800、70-850、70-900、70-950、70-1000、80-90、80-100、80-250、80-300、80-350、80-400、80-450、80-500、80-550、80-600、80-650、80-700、80-750、80-800、80-850、80-900、80-950、80-1000、90-100、90-250、90-300、90-350、90-400、90-450、90-500、90-550、90-600、90-650、90-700、90-750、90-800、90-850、90-900、90-950、90-1000、100-250、100-300、100-350、100-400、100-450、100-500、100-550、100-600、100-650、100-700、100-750、100-800、100-850、100-900、100-950、100-1000、150-250、150-300、150-350、150-400、150-450、150-500、150-550、150-600、150-650、150-700、150-750、150-800、150-850、150-900、150-950、150-1000、200-250、200-300、200-350、200-400、200-450、200-500、200-550、200-600、200-650、200-700、200-750、200-800、200-850、200-900、200-950、200-1000、250-300、250-350、250-400、250-450、250-500、250-550、250-600、250-650、250-700、250-750、250-800、250-850、250-900、250-950、250-1000、300-350、300-400、300-450、300-500、300-550、300-600、300-650、300-700、300-750、300-800、300-850、300-900、300-950、300-1000、350-400、350-450、350-500、350-550、350-600、350-650、350-700、350-750、350-800、350-850、350-900、350-950、350-1000、400-450、400-500、400-550、400-600、400-650、400-700、400-750、400-800、400-850、400-900、400-950、400-1000、450-500、450-550、450-600、450-650、450-700、450-750、450-800、450-850、450-900、450-950、450-1000、500-550、500-600、500-650、500-700、500-750、500-800、500-850、500-900、500-950、500-1000、550-600、550-650、550-700、550-750、550-800、550-850、550-900、550-950、550-1000、600-650、600-700、600-750、600-800、600-850、600-900、600-950、600-1000、650-700、650-750、650-800、650-850、650-900、650-950、650-1000、700-750、700-800、700-850、700-900、700-950、700-1000、750-800、750-850、750-900、750-950、750-1000、800-850、800-900、800-950、800-1000、850-900、850-950、850-1000、900-950、900-1000、950-1000 or more than 1000 nucleotides.

In some embodiments, the identifier sequence or portion may generate a signal that is detectable immediately after administration. In some embodiments, the identifier sequence or portion can produce a signal that is detectable within an unlimited amount of time after administration. In some embodiments, the identifier sequence or portion can produce a signal detectable more than 1,2, 3, 4, 5, 6, or 7 days after administration. In some embodiments, the identifier sequence or portion may produce a signal that is detectable within about 1 to 24 hours. As one non-limiting example, the signal may be detectable within about 1 to 6, 1 to 12, 1 to 18, 6 to 12, 6 to 18, 6 to 24, or 18 to 24 hours, or 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments, the identifier sequence or portion may generate a signal that is detectable in about 1-60 minutes, such as, but not limited to 1-5、1-10、1-20、1-30、1-40、1-50、10-20、10-30、10-40、10-50、10-60、20-30、20-40、20-50、20-60、30-40、30-50、30-60、40-50、40-60、 or 50-60 minutes, or 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59 or 60 minutes. In some embodiments, the identifier sequence or portion can produce a signal that is detectable in less than 1 minute after administration.

In some embodiments, the identifier sequence or portion may produce a signal that is detectable outside the body of the subject. In some embodiments, the identifier sequence or portion may generate a signal that is detectable via non-invasive imaging techniques, e.g., outside of an organ or tissue of the subject but within the body of the subject. In some embodiments, the identifier sequence or portion may produce a signal that is detectable on a macro-level. In some embodiments, the identifier sequence or portion may produce a signal that is detectable at the microscopic level. In some embodiments, the identifier sequence or portion may produce a signal detectable at the nanoscale level. In some embodiments, the identifier sequence or portion may generate a detectable signal only after the target cells are collected and assayed, via mass spectrometry, electrophoresis, flow cytometry, or deep sequencing, for non-limiting examples.

In some embodiments, the delivery vehicle comprises or is operably linked to an identifier moiety.

In some embodiments, the delivery vehicle comprises or is operably linked to an identifier moiety that binds to an immune cell antigen. As one non-limiting example, the immune cell antigen may be a T cell antigen, such as CD2, CD3, CD5, CD7, CD8, CD4, beta 7 integrin, beta 2 integrin, and C1q. As one non-limiting example, the immune cell antigen may be an NK cell, NKT cell, macrophage or neutrophil. As one non-limiting example, the immune cell antigen may be a macrophage antigen, such as mannose receptor, CD206, and C1q.

In some embodiments, the delivery vehicle comprises or is operably linked to an identifier moiety that is a small molecule that binds to an extracellular enzyme on an immune cell. Extracellular enzymes may be, but are not limited to, CD38, CD73, adenosine 2a receptor and adenosine 2b receptor.

In some embodiments, the delivery vehicle comprises or is operably linked to an identifier moiety that is a small molecule, such as, but not limited to, mannose, lectin, acitretin (acivicin), biotin, or digoxin (digoxigenin).

In some embodiments, the delivery vehicle comprises or is operably linked to an identifier moiety that is a single chain Fv (scFv) fragment, a nanobody, a peptide-based macrocycle, a minibody, a small molecule ligand such as folic acid, argininoglycaemic acid (RGD), or a phenol-soluble regulatory protein α1 peptide (PSMA 1), a heavy chain variable region, a light chain variable region, or a fragment thereof.

Tracking system: fluorescence

In some embodiments, the at least one tracking system comprises an identifier sequence or portion that is detectable by fluorescence.

In some embodiments, fluorescence is achieved via the inclusion of at least one fluorescent dye in the delivery vehicle. In some embodiments, the at least one fluorescent dye may be selected from, but is not limited to, fluorescein, TAMRA (carboxytetramethyl rhodamine), cy dye, texas red (Texas red), HEX, JOE, oregon green (Oregon green), rhodamine 6G, coumarin (coumarin), pyrene, and DiOC6 (3, 3' -dihexyloxycarbonyl cyanine iodide).

In some embodiments, fluorescence is achieved via the inclusion of at least one fluorescent protein in or associated with the delivery vehicle. In some embodiments, at least one fluorescent protein is encoded in a reference construct or reference construct comprising a fluorescent protein. Non-limiting examples of fluorescent proteins include Green Fluorescent Protein (GFP), yellow Fluorescent Protein (YFP), red Fluorescent Protein (RFP), sirius, excitable blue fluorescent protein (EBFP 2), cyan Fluorescent Protein (CFP), cerulean, excitable Green Fluorescent Protein (EGFP), excitable yellow fluorescent protein (EYFP)、mOrange、mCherry、mPlum、NIR、iRFP、EosFP、PamCherry、Dronpa、Dreiklang、asFP595、mMaple、mGeo、mEos2、Dendra2、psCFP2, and 2,3,5, 6-tetracarbazole-4-cyano-pyridine (CPy).

In some embodiments, fluorescence is achieved via the inclusion of at least one fluorescent nanoparticle associated with a delivery vehicle or a reference construct. In some embodiments, the fluorescent nanoparticle may be, but is not limited to, a carbon dot, graphene quantum dot, gold nanorod, polymer-based nanoparticle, aggregation-induced emission dot, conjugated polymer nanoparticle (CP-dot), gold nanosphere, gold nanoshell, gold nanocage, and AIE feromone (pheromone).

In some embodiments, fluorescence is achieved via a delivery system comprising at least one fluorescent lipid associated with or contained in a delivery vehicle. In some embodiments, the fluorescent lipid may be, but is not limited to DiR, diD, diO and DiI, other members of the Di series of phospholipids, bodipy, and FL-sphingomyelin.

In some embodiments, fluorescence is achieved via the inclusion of at least one luciferase in the delivery vehicle or at least one luciferase associated therewith. In some embodiments, at least one luciferase protein is encoded in a reference construct or reference construct comprising a luciferase. Non-limiting examples of types of luciferases that may be used include Renilla luciferase (Renilla luciferase), metridia luciferase (Gaussia luciferase), nanoluc luciferase, firefly luciferase (Firefly luciferase), and kowtow luciferase (Click Beetle luciferase).

In some embodiments, fluorescence is achieved via a method comprising associating or containing β -galactosidase (β -gal) with a delivery vehicle. In some embodiments, at least one β -galactosidase (β -gal) protein is encoded in a reference construct or reference construct comprising β -galactosidase (β -gal).

In some embodiments, fluorescence is achieved via a delivery vehicle comprising at least one quencher molecule associated with or contained within the delivery vehicle. In some embodiments, fluorescence is achieved via the inclusion of at least one quencher molecule associated with or encoded by the baseline construct. Non-limiting examples of quencher molecules include dimethylaminophenyl azobenzoic acid (DABCYL), QSY 7, cu (II) ions, dabcyl, QSY 35, BHQ-0, eclipse, BHQ-1, QSY 9, BHQ-2, elleQuencher, iowa Black, QSY 21, and BHQ-3.

Tracking system: fluorophores and radiophosphates

In some embodiments, the at least one tracking system comprises an identifier sequence or portion that is a fluorophore or a radioactive phosphate.

In some embodiments, the at least one tracking system comprises at least one fluorophore comprising an association with or contained in a delivery vehicle. In some embodiments, the at least one tracking system comprises at least one fluorophore comprising an association with, encoding therein or contained therein, with a reference construct. Non-limiting examples of fluorophores include quantum dots and small organic molecules.

In some embodiments, the at least one tracking system comprises at least one quantum dot comprising an association with or contained in a delivery vehicle. In some embodiments, the at least one tracking system comprises at least one quantum dot comprising an association, encoding therein, or contained therein, with a reference construct. Non-limiting examples of quantum dots include CdSe/ZnS、CdTe/ZnS、CdTe/CdSe、CdSe/ZnTe、CdSe/CdTe/ZnSe、nAs/ZnSe、InAs/CdSe、InAs/InP、Cu:InP/ZnSe、InAsxP1-x/InP/ZnSe、CdS/CdSe、ZnSe/CdSe、ZnSe/InP/ZnS、ZnSe/InP/ZnS、CdTe/ZnSe、QD585 and QD655.

In some embodiments, the at least one tracking system comprises at least one small organic molecule comprising an amino acid associated with or contained in a delivery vehicle. In some embodiments, the at least one tracking system comprises at least one small organic molecule comprising an association, encoding therein, or contained therein, with a reference construct. Non-limiting examples of small organic molecules include the following classes: coumarin, naphthalimide, fluorescein and rhodamine derivatives, BODIPY, cyanine, dibenzopyran, oxazine, oligothiophenes and phthalocyanine derivatives (PcDer). In some embodiments, the at least one small organic molecule may be selected from, but is not limited to, 7-dialkyl-amino-4-trifluoromethylcoumarin, rhodamine B, coumarin 314, fluorescein CH, fluorescein, rhodamine 123, BODIPY FL NHS ester, cy5, rhodamine 6G, silicon-rhodamine (SiR), cy3, cy5.5, cy7, cy2, ATTO655, ATTO680, ATTO700, nitrobenzoxadiazole (NBD), 1, 6-diphenyl-1, 3, 5-hexanetriene (DPH)、ABBERIOR^TM、ALEXA FLUOR^TM、ATTO^TM、DYLIGHT FLUOR^TM、ALEXA FLUOR 647^TM, and TOPFLUOR ^TM.

In some embodiments, the at least one tracking system comprises at least one imaging contrast agent comprising an agent associated with or contained in a delivery vehicle. In some embodiments, the at least one tracking system comprises at least one imaging contrast agent comprising an agent associated with, encoded in, or contained in a reference construct. Non-limiting examples of imaging contrast agents include gadolinium-based small molecules, gadolinium-encapsulated liposomes, manganese-based small molecules, and iron oxide nanoparticles.

In some embodiments, the at least one tracking system comprises a device comprising at least one radiolabel associated with or contained in a delivery vehicle. In some embodiments, the at least one tracking system comprises at least one radiolabel comprising an association, encoding, or inclusion therein, with a reference construct. Non-limiting examples of radiolabels include ¹¹¹In、^99mTc、¹³N、⁶⁸Ga、¹⁸F、⁶⁴Cu、⁸⁶Y、⁷⁶Br、⁸⁹Zr、⁷²As、¹²⁴I、⁷⁴As、 fluoro-18, gallium-68, nitrogen-13, copper-64, bromo-76, iodo-125, arsenic-74, carbon-11, iodo-131, ¹⁵³Sm、¹⁷⁷Lu、¹⁸⁶Re、¹⁸⁸Re、¹⁹⁸ Au, and ²²⁵ Ac.

In some embodiments, the at least one tracking system comprises at least one biotin comprising an agent associated with or contained in a delivery vehicle.

In some embodiments, the at least one tracking system comprises at least one digoxigenin (digoxygenin) associated with or contained in the delivery vehicle.

In some embodiments, the at least one tracking system comprises at least one Dinitrophenyl (DNP) comprising an agent associated with or contained in a delivery vehicle.

In some embodiments, the at least one tracking system comprises at least one fluorescein, including associated with or contained in the delivery vehicle.

In some embodiments, the at least one tracking system comprises at least one fucose comprising in association with or contained in a delivery vehicle.

In some embodiments, the at least one tracking system comprises at least one amine comprising an amino group associated with or contained in the delivery vehicle.

In some embodiments, the at least one tracking system comprises at least one Texas including an agent associated with or contained in a delivery vehicle

In some embodiments, the at least one tracking system comprises at least one biotin comprising an association with, encoded in, or contained in a reference construct.

In some embodiments, the at least one tracking system comprises at least one digoxigenin comprising, being associated with, encoded in, or contained in the reference construct.

In some embodiments, the at least one tracking system comprises at least one Dinitrophenyl (DNP) comprising an association with, encoded in, or contained in a reference construct.

In some embodiments, the at least one tracking system comprises at least one fluorescein, including associated with, encoded in, or contained in the reference construct.

In some embodiments, the at least one tracking system comprises at least one fucose comprising, encoded therein, or contained therein, associated with the reference construct.

In some embodiments, the at least one tracking system comprises at least one amine comprising an amino group associated with, encoded in, or contained in a reference construct.

In some embodiments, the at least one tracking system comprises at least one Texas including associating with, encoding therein or contained therein a reference construct

In some embodiments, the at least one tracking system comprises a system comprising at least one reporter sequence or protein associated with or contained in a delivery vehicle. In some embodiments, the at least one tracking system comprises at least one reporter sequence or protein comprising, associated with, encoded in, or contained in a reference construct. Non-limiting examples of reporter sequences or proteins include eGFP; a luciferase; gene editing agents (e.g., cas9 editing, DNA readout); ox-40; beta 6 integrin; CD45; surface markers with HA tag, flag tag with or without TEV protease site; or any combination thereof.

In some embodiments, the at least one tracking system comprises a device comprising at least one functional sequence or protein associated with or contained in a delivery vehicle. In some embodiments, the at least one tracking system comprises at least one functional sequence or protein comprising, being associated with, encoded by, or contained in a reference construct. Non-limiting examples of functional sequences or proteins include fluorescent proteins, surface proteins, cre-recombinases, CRISPR/CAS systems, surface proteins with epitope tags (e.g., HA, FLAG, etc.), or any combination thereof.

In some embodiments, the at least one tracking system comprises at least one functional sequence or protein comprising a protease cleavage site (e.g., TEV) that can be associated with or contained in a delivery vehicle. In some embodiments, the at least one tracking system comprises a nucleic acid comprising at least one functional sequence or protein comprising a protease cleavage site (e.g., TEV) that can be associated with, encoded by, or contained in a reference construct.

In some embodiments, the at least one tracking system comprises at least one functional sequence or protein comprising an affinity tag (e.g., 3xHA, FLAG, his) that can be associated with or contained in a delivery vehicle. In some embodiments, the at least one tracking system comprises at least one functional sequence or protein comprising an affinity tag (e.g., 3xHA, FLAG, his) that can be associated with, encoded in, or contained in a reference construct.

V. pharmaceutical compositions and routes of administration

Pharmaceutical composition and formulation

The initial construct, baseline construct, and targeting system can be formulated using one or more excipients to: (1) increased stability; (2) increasing cell transfection or transduction; (3) allowing sustained or delayed expression of the payload; (4) Altering the biodistribution (e.g., targeting the viral particles to a specific tissue or cell type); (5) increasing translation of the encoded protein; (6) altering the release profile of the encoded protein; and/or (7) achieving adjustable expression of cargo and/or payload.

Formulations can include, but are not limited to, saline, liposomes, lipid nanoparticles, polymers, peptides, proteins, cells transfected with viral vectors (e.g., for transfer or transplantation into a subject), and combinations thereof.

The formulations of the pharmaceutical compositions described herein may be prepared by any method known in the pharmacological arts or developed hereafter. As used herein, the term "pharmaceutical composition" refers to a composition comprising at least one active ingredient and optionally one or more pharmaceutically acceptable excipients.

Generally, such methods of preparation include the step of associating the active ingredient with an excipient and/or one or more other accessory ingredients. As used herein, the phrase "active ingredient" generally refers to an initial construct or reference construct having a payload region or cargo or payload as described herein.

The formulations of the initial constructs, reference constructs, and targeting systems described herein and pharmaceutical compositions may be prepared by any method known in the pharmacological arts or developed hereafter. Generally, such preparation methods comprise the following steps: the active ingredient is associated with excipients and/or one or more other adjunct ingredients, and the product is then divided, shaped and/or packaged as necessary and/or desired into single or multi-dose units.

Pharmaceutical compositions according to the present disclosure may be prepared, packaged and/or sold in bulk, in single unit dosage form and/or in multiple single unit dosage forms. As used herein, "unit dose" refers to discrete amounts of a pharmaceutical composition comprising a predetermined amount of an active ingredient. The amount of active ingredient is typically equal to the dose of active ingredient to be administered to the subject and/or a suitable fraction of such dose, for example one half or one third of such dose.

In some embodiments, the pharmaceutically acceptable excipient may be at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% pure. In some embodiments, the excipient is approved for human and veterinary use. In some embodiments, the excipient may be approved by the U.S. food and drug administration (United States Food and Drug Administration). In some embodiments, the excipient may be pharmaceutical grade. In some embodiments, the excipient may meet the standards of United states pharmacopoeia (United States Pharmacopoeia; USP), european pharmacopoeia (European Pharmacopoeia; EP), british pharmacopoeia (British Pharmacopoeia), and/or International pharmacopoeia (International Pharmacopoeia).

The relative amounts of the active ingredient, pharmaceutically acceptable excipients, and/or any additional ingredients in the pharmaceutical compositions according to the present disclosure may vary depending on the identity, size, and/or condition of the subject being treated and further depending on the route of administration of the composition. For example, the composition may comprise between 0.1% and 99% (w/w) of the active ingredient. For example, the composition may comprise between 0.1% and 100%, such as between 0.5% and 50%, between 1-30%, between 5-80% or at least 80% (w/w) of the active ingredient.

In one aspect, the present disclosure further provides a delivery system for delivering the therapeutic payloads disclosed herein. In some embodiments, a delivery system suitable for delivering the therapeutic payloads disclosed herein comprises a Lipid Nanoparticle (LNP) formulation.

In some embodiments, the LNP of the present disclosure comprises an ionizable lipid, a structural lipid, a pegylated lipid (also referred to as a PEG lipid), and a phospholipid. In alternative embodiments, the LNP comprises ionizable lipids, structural lipids, pegylated lipids (also referred to as PEG lipids), and zwitterionic amino acid lipids. In some embodiments, the LNP further comprises a 5 th lipid in addition to any of the aforementioned lipid components. In some embodiments, the LNP encapsulates one or more elements of the active agents of the present disclosure. In some embodiments, the LNP further comprises a targeting moiety that is covalently or non-covalently bound to the outer surface of the LNP. In some embodiments, the targeting moiety is one that binds to or facilitates uptake by cells of a particular organ system.

In some embodiments, the LNP has a diameter of at least about 20nm, 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, or 90nm. In some embodiments, the LNP has a diameter less than about 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, or 160nm. In some embodiments, the LNP has a diameter of less than about 100nm. In some embodiments, the LNP has a diameter of less than about 90nm. In some embodiments, the LNP has a diameter of less than about 80nm. In some embodiments, the LNP has a diameter of about 60-100nm. In some embodiments, the LNP has a diameter of about 75-80nm.

In some embodiments, the lipid nanoparticle compositions of the present disclosure are described in terms of the corresponding molar ratios of the component lipids in the formulation. As one non-limiting example, the mole% of the ionizable lipid can be from about 10 mole% to about 80 mole%. As one non-limiting example, the mole% of the ionizable lipid can be from about 20 mole% to about 70 mole%. As one non-limiting example, the mole% of the ionizable lipid can be from about 30 mole% to about 60 mole%. As one non-limiting example, the mole% of the ionizable lipid can be from about 35 mole% to about 55 mole%. As one non-limiting example, the mole% of the ionizable lipid can be from about 40 mole% to about 50 mole%.

In some embodiments, the mole% of phospholipids may be from about 1 mole% to about 50 mole%. In some embodiments, the mole% of phospholipids may be about 2 mole% to about 45 mole%. In some embodiments, the mole% of phospholipids may be about 3 mole% to about 40 mole%. In some embodiments, the mole% of phospholipids may be about 4 mole% to about 35 mole%. In some embodiments, the mole% of phospholipids may be about 5 mole% to about 30 mole%. In some embodiments, the mole% of phospholipids may be about 10 mole% to about 20 mole%. In some embodiments, the mole% of phospholipids may be about 5 mole% to about 20 mole%.

In some embodiments, the mole% of the structural lipid may be about 10 mole% to about 80 mole%. In some embodiments, the mole% of the structural lipid may be from about 20 mole% to about 70 mole%. In some embodiments, the mole% of the structural lipid may be about 30 mole% to about 60 mole%. In some embodiments, the mole% of the structural lipid may be about 35 mole% to about 55 mole%. In some embodiments, the mole% of the structural lipid may be about 40 mole% to about 50 mole%.

In some embodiments, the mol% of PEG lipid may be about 0.1mol% to about 10mol%. In some embodiments, the mol% of PEG lipid may be about 0.2mol% to about 5mol%. In some embodiments, the mol% of PEG lipid may be about 0.5mol% to about 3mol%. In some embodiments, the mol% of PEG lipid may be about 1mol% to about 2mol%. In some embodiments, the mol% of PEG lipid may be about 1.5mol%.

In some embodiments, the nanoparticle comprises an ionizable lipid, a phospholipid, a PEG lipid, and a structural lipid. In certain embodiments, the lipid component of the nanoparticle composition comprises from about 30mol% to about 60mol% ionizable lipid, from about 0mol% to about 30mol% phospholipid, from about 18.5mol% to about 48.5mol% structural lipid, and from about 0mol% to about 10mol% peg lipid, provided that the total mol% is no more than 100%. In some embodiments, the lipid component of the nanoparticle composition includes about 35mol% to about 55mol% ionizable lipids, about 5mol% to about 25mol% phospholipids, about 30mol% to about 40mol% structural lipids, and about 0mol% to about 10mol% peg lipids. In a particular embodiment, the lipid component comprises about 50mol% ionizable lipid, about 10mol% phospholipid, about 38.5mol% structural lipid, and about 1.5mol% peg lipid. In another particular embodiment, the lipid component comprises about 40mol% ionizable lipid, about 20mol% phospholipid, about 38.5mol% structural lipid, and about 1.5mol% peg lipid. In another particular embodiment, the lipid component comprises about 48.5mol% ionizable lipid, about 10mol% phospholipid, about 40mol% structural lipid, and about 1.5mol% peg lipid. In another particular embodiment, the lipid component comprises about 48.5mol% ionizable lipid, about 10mol% phospholipid, about 39mol% structural lipid, and about 2.5mol% peg lipid. In some embodiments, the phospholipid may be DOPE or DSPC. In other embodiments, the PEG lipid may be PEG-DMG, and/or the structural lipid may be cholesterol. The amount of active agent in the nanoparticle composition can depend on the size, composition, desired target and/or application or other characteristics of the nanoparticle composition, as well as the characteristics of the active agent. For example, the amount of active agent suitable for use in the nanoparticle composition may depend on the size, sequence, and other characteristics of the active agent. The relative amounts of active agent and other elements (e.g., lipids) in the nanoparticle composition can also vary. In some embodiments, the weight/weight ratio of lipid component to enzyme in the nanoparticle composition can be from about 5:1 to about 60:1, such as 5:1、6:1、7:1、8:1、9:1、10:1、11:1、12:1、13:1、14:1、15:1、16:1、17:1、18:1、19:1、20:1、25:1、30:1、35:1、40:1、45:1、50:1 and 60:1. The amount of enzyme in the nanoparticle composition can be measured, for example, using absorption spectroscopy (e.g., ultraviolet visible spectroscopy).

In some embodiments, nanoparticle compositions comprising the active agents of the present disclosure are formulated to provide a particular E: P ratio. The E: P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in the RNA active agent. In general, lower E to P ratios are preferred. The one or more enzymes, lipids, and amounts thereof may be selected to provide an E: P ratio of about 2:1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In certain embodiments, the E:P ratio may be from about 2:1 to about 8:1. In other embodiments, the E:P ratio is from about 5:1 to about 8:1. For example, the E:P ratio may be about 5.0:1, about 5.5:1, about 5.67:1, about 6.0:1, about 6.5:1, or about 7.0:1.

The characteristics of the nanoparticle composition may depend on its components. For example, nanoparticle compositions that include cholesterol as a structural lipid may have different characteristics than nanoparticle compositions that include a different structural lipid. Similarly, the characteristics of a nanoparticle composition may depend on the absolute or relative amounts of its components. For example, nanoparticle compositions comprising higher mole fractions of phospholipids may have different characteristics than nanoparticle compositions comprising lower mole fractions of phospholipids. The characteristics may also vary depending on the method and conditions under which the nanoparticle composition is prepared. Nanoparticle compositions can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of nanoparticle compositions. Dynamic light scattering or potentiometry (e.g., potentiometric titration) can be used to measure zeta potential. Dynamic light scattering can also be used to determine particle size. Instruments such as Zetasizer Nano ZS (Malvern Instruments Ltd, malvern, worcestershire, UK) can also be used to measure various characteristics of nanoparticle compositions, such as particle size, polydispersity index, and zeta potential.

The average size of the nanoparticle composition may be between ten and one hundred nanometers, as measured by Dynamic Light Scattering (DLS), for example. For example, the average size may be about 40nm to about 150nm, such as about 40nm、45nm、50nm、55nm、60nm、65nm、70nm、75nm、80nm、85nm、90nm、95nm、100nm、105nm、110nm、115nm、120nm、125nm、130nm、135nm、140nm、145nm or 150nm. In some embodiments, the nanoparticle composition can have an average size of about 50nm to about 100nm, about 50nm to about 90nm, about 50nm to about 80nm, about 50nm to about 70nm, about 50nm to about 60nm, about 60nm to about 100nm, about 60nm to about 90nm, about 60nm to about 80nm, about 60nm to about 70nm, about 70nm to about 100nm, about 70nm to about 90nm, about 70nm to about 80nm, about 80nm to about 100nm, about 80nm to about 90nm, or about 90nm to about 100nm. In certain embodiments, the nanoparticle composition can have an average size of about 70nm to about 100nm. In a particular embodiment, the average size may be about 80nm. In other embodiments, the average size may be about 100nm.

The nanoparticle composition can be relatively homogeneous. The polydispersity index may be used to indicate the homogeneity of the nanoparticle composition, such as the particle size distribution of the nanoparticle composition. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. The nanoparticle composition can have a polydispersity index of about 0to about 0.25, such as 0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.10、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.20、0.21、0.22、0.23、0.24 or 0.25.

The zeta potential of the nanoparticle composition can be used to indicate the zeta potential of the composition. For example, the zeta potential may describe the surface charge of the nanoparticle composition. Nanoparticle compositions having relatively low positive or negative charges are generally desirable because more highly charged species may undesirably interact with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the nanoparticle composition may be from about-10 to about +20mV, from about-10 to about +15mV, from about-10 to about +10mV, from about-10 to about +5mV, from about-10 to about 0mV, from about-10 to about-5 mV, from about-5 to about +20mV, from about-5 to about +15mV, from about-5 to about +10mV, from about-5 to about +5mV, from about-5 to about 0mV, from about 0 to about +20mV, from about 0 to about +15mV, from about 0 to about +10mV, from about 0 to about +5mV, from about +5 to about +20mV, from about +5 to about +15mV, or from about +5 to about +10mV.

Encapsulation efficiency of the payload describes the amount of payload that is encapsulated or otherwise combined with the nanoparticle composition after preparation relative to the initial amount provided. Encapsulation efficiency is preferably high (e.g., near 100%). Encapsulation efficiency may be measured, for example, by comparing the amount of payload in a solution containing the nanoparticle composition before and after decomposing the nanoparticle composition with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free payload in solution. For nanoparticle compositions described herein, the encapsulation efficiency of the therapeutic and/or prophylactic agent can be at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In certain embodiments, the encapsulation efficiency may be at least 90%.

Lipids and methods for their preparation are disclosed, for example, in the following: U.S. patent No. 8,569,256, 5,965,542; and U.S. patent publication nos. 2016/0199485, 2016/0009637, 2015/0273068, 2015/0265708, 2015/0203446, 2015/0005363, 2014/0308304, 2014/0200257, 2013/086373, 2013/0338210, 2013/03032369, 2013/024567, 2013/0195920, 2013/012338, 2013/0022649, 2013/0017223, 2012/0295832, 2012/0183581, 2012/0172411, 2012/0027803; 2012/0058188, 2011/0311583, 2011/0311582, 2011/0262527, 2011/0216622, 2011/017125, 2011/0091525, 2011/007635, 2011/0060032, 2010/013088, 2007/0042031, 2006/0243093, 2006/0083780, 2006/0008910, 2005/0175682, 2005/017054, 2005/0110253, 2005/0064595, 2004/0142025, 2007/0042031, 1999/009076; and PCT publications WO 99/39741, WO 2017/117528, WO 2017/004143, WO 2017/075531, WO 2015/199952, WO 2014/008334, WO 2013/086373, WO 2013/086322, WO 2013/016058, WO 2013/086373, WO2011/141705, and WO 2001/07548; and Semple et al Nature Biotechnology,2010,28,172-176, the entire disclosures of which are incorporated herein by reference in their entirety for all purposes.

Nanoparticle compositions can include any material suitable for use in pharmaceutical compositions. For example, nanoparticle compositions may include one or more pharmaceutically acceptable excipients or adjunct ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersing aids, suspending aids, granulating aids, disintegrants, fillers, glidants, liquid vehicles, binders, surfactants, isotonic agents, thickening or emulsifying agents, buffers, lubricants, oils, preservatives and other substances. Excipients, for example waxes, fats, colorants, coating agents, flavoring agents and fragrances may also be included. Pharmaceutically acceptable excipients are well known in the art (see, e.g., remington THE SCIENCE AND PRACTICE of Pharmacy, 21 st edition, A.R. Gennaro: lippincott, williams & Wilkins, baltimore, md., 2006).

Excipients and diluents

As used herein, excipients include, but are not limited to, any and all solvents, dispersion media, diluents or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives and the like suitable for the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing such compositions are known in the art (see Remington: THE SCIENCE AND PRACTICE of Pharmacy, 21 st edition, a.r. gennaro, lippincott, williams & Wilkins, baltimore, MD,2006; incorporated herein by reference in its entirety). Unless any conventional excipient medium is incompatible with the substance or derivative thereof, e.g., to produce any undesirable biological effect or otherwise interact in a deleterious manner with any other component of the pharmaceutical composition, the use of conventional excipient media is contemplated within the scope of the present disclosure.

Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium lactose phosphate, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, sugar powder, and the like, and/or combinations thereof.

Ionizable lipids

In some embodiments, the LNP disclosed herein comprises an ionizable lipid. In some embodiments, the LNP comprises two or more ionizable lipids. In some embodiments, the ionizable lipid is any of the ionizable lipids disclosed herein or any combination thereof.

Structural lipids

In some embodiments, the LNP comprises a structural lipid. The structural lipid may be selected from the group consisting of (but not limited to): cholesterol, stigmasterol, fucosterol, beta sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, lycorine, cholic acid, stigmastanol, lithocholic acid, lycopin, ursolic acid, alpha-tocopherol, vitamin D3, vitamin D2, calcipotriol, botulinum toxin, lupeol, oleanolic acid, beta-sitosterol-acetate and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid is a cholesterol analog disclosed by Patel et al, nat Commun, 11,983 (2020), which is incorporated herein by reference in its entirety. In some embodiments, the structural lipids include cholesterol and corticosteroids (e.g., prednisolone (prednisolone), dexamethasone (dexamethasone), prednisone (prednisone), and hydrocortisone (hydrocortisone)) or any combination thereof. In some embodiments, structural lipids are described in international patent application WO2019152557A1, which is incorporated herein by reference in its entirety.

In some embodiments, the structural lipid is a cholesterol analog. The use of cholesterol analogues may enhance endosomal escape as described in Patel et al ,Naturally-occuring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA,Nature Communications(2020), incorporated herein by reference.

In some embodiments, the structural lipid is a phytosterol. The use of phytosterols may enhance endosomal escape as described in Herrera et al ,Illuminating endosomal escape of polymorphic lipid nanoparticles that boost mRNA delivery,Biomaterials Science(2020), incorporated herein by reference.

In some embodiments, the structural lipid contains a plant sterol mimetic for enhanced endosomal release.

Pegylated lipids

The polyethylene glycol lipid is a polyethylene glycol modified lipid. In some embodiments, the LNP comprises a compound of formula I as described above or a pharmaceutically acceptable salt thereof. In some embodiments, the LNP comprises a compound of formula II as described above or a pharmaceutically acceptable salt thereof. In some embodiments, the LNP comprises a compound set forth in table (I) as described above.

In some embodiments, the LNP comprises additional pegylated lipids or PEG-modified lipids. The pegylated lipids may be selected from the non-limiting group consisting of: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. For example, the PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC or PEG-DSPE lipid.

In some embodiments, the LNP comprises a pegylated lipid ：US 2019/0240354、US 2010/0130588、US 2021/0087135、WO 2021/204179、US 2021/0128488、US 2020/0121809、US 2017/0119904、US 2013/0108685、US 2013/0195920、US 2015/0005363、US 2014/0308304、US 2013/0053572、WO 2019/232095A1、WO 2021/077067、WO 2019/152557、US 2015/0203446、US 2017/0210697、US 2014/0200257 or WO 2019/089828A1 disclosed in one of the following, each of which is incorporated herein by reference in its entirety.

In some embodiments, the LNP comprises a pegylated lipid alternative rather than a pegylated lipid. All embodiments disclosed herein that cover pegylated lipids are understood to be applicable to pegylated lipid alternatives as well. In some embodiments, the LNP comprises a poly-sarcosine-lipid conjugate, such as those disclosed in US 2022/0001025 A1, which is incorporated herein by reference in its entirety.

Phospholipid

In some embodiments, the LNP of the present disclosure comprises a phospholipid. Phospholipids suitable for use in the compositions and methods may be selected from the non-limiting group consisting of: 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DLPC), 1, 2-dimyristoyl-sn-glycero-phosphorylcholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-bisundecanoyl-sn-glycero-phosphorylcholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (POPC), 1, 2-di-O-octadecenyl-sn-glycero-3-phosphorylcholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphorylcholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphorylcholine (C16 Lyso PC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine, 1, 2-di-arachidonoyl-sn-glycero-3-phosphorylcholine, 1, 2-di-docosahexaenoic acid-sn-glycero-3-phosphorylcholine, 1, 2-di-phytyl-glycerol-3-phosphate ethanolamine (ME16.0PE), 1, 2-di-stearoyl-sn-glycerol-3-phosphate ethanolamine, 1, 2-di-oleoyl-sn-glycerol-3-phosphate ethanolamine, 1, 2-di-linolenoyl-sn-glycerol-3-phosphate ethanolamine, 1, 2-di-arachidonoyl-sn-glycerol-3-phosphate ethanolamine, 1, 2-di-docosahexaenoic acid-sn-glycerol-3-phosphate ethanolamine, 1, 2-di-oleoyl-sn-glycerol-3-phosphate-rac- (1-glycerol) sodium salt (DOPG), (S) -2-ammonio-3- ((((R) -2- (oleoyloxy) -3- (stearyloxy) propoxy) phosphoryl) oxy) propanoate) sodium (L- α -phosphatidylserine; Brain PS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoyl phosphatidylglycerol (DMPG), dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal), dioleoyl phosphatidylglycerol (DOPG), 1, 2-dioleoyl-sn-glycerol-3- (phospho-L-serine) (DOPS), cell fusion phospholipid (DPhPE), dipalmitoyl phosphatidylethanolamine (DPPE), dipalmitoyl phosphatidylglycerol (DPPG), Dipalmitoyl phosphatidylserine (DPPS), distearoyl phosphatidylcholine (DSPC), distearoyl-phosphatidyl-ethanolamine (DSPE), distearoyl-ethanolamine-imidazole phosphate (DSPEI), 1, 2-bis-undecanoyl-sn-glycero-choline phosphate (DUPC), phosphatidylcholine (EPC), 1, 2-dioleoyl-sn-glycero-3-phosphate (18:1 pa; DOPA), ammonium bis ((S) -2-hydroxy-3- (oleoyloxy) propyl) phosphate (18:1 DMP; LBPA), 1, 2-dioleoyl-sn-glycerol-3-phosphate- (1' -inositol) (DOPI; 18:1 PI), 1, 2-distearoyl-sn-glycero-3-phospho-L-serine (18:0 PS), 1, 2-dioleoyl-sn-glycero-3-phospho-L-serine (18:2 PS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (16:0-18:1 PS; POPS), 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (18:0-18:1 PS), 1-stearoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine (18:0-18:2 PS), 1-oleoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18:1 lyso PS), 1-stearoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18:0 lyso PS), and sphingomyelin. In some embodiments, the LNP comprises a DSPC. In certain embodiments, the LNP comprises DOPE. In some embodiments, the LNP includes both DSPC and DOPE.

In some embodiments, the phospholipid tail may be modified so as to facilitate endosomal escape, as described in U.S.2021/0121411, incorporated herein by reference.

In some embodiments, the LNP comprises a phospholipid ：US2019/0240354、US 2010/0130588、US 2021/0087135、WO 2021/204179、US 2021/0128488、US 2020/0121809、US 2017/0119904、US 2013/0108685、US 2013/0195920、US 2015/0005363、US 2014/0308304、US 2013/0053572、WO 2019/232095A1、WO 2021/077067、WO 2019/152557、US 2017/0210697 or WO 2019/089828A1 disclosed in one of the following, each of which is incorporated herein by reference in its entirety.

In some embodiments, the phospholipids disclosed in US 2020/011809 have the following structure:

Wherein R ₁ and R ₂ are each independently branched or straight, saturated or unsaturated carbon chains (e.g., alkyl, alkenyl, alkynyl).

Targeting moiety

In some embodiments, the lipid nanoparticle further comprises a targeting moiety. The targeting moiety may be an antibody or fragment thereof. The targeting moiety may be capable of binding to the antigen of interest.

In some embodiments, the pharmaceutical composition comprises a targeting moiety operably linked to the lipid nanoparticle. In some embodiments, the targeting moiety is capable of binding to the antigen of interest. In some embodiments, the antigen of interest is expressed in the organ of interest. In some embodiments, the target antigen is expressed more in the target organ than it is expressed in the liver.

In some embodiments, the targeting moiety is an antibody as described in WO2016189532A1, which is incorporated herein by reference. For example, in some embodiments, the targeted particles are conjugated with a specific anti-CD 38 monoclonal antibody (mAb), which allows for specific delivery of siRNA encapsulated within the particles to B cell lymphocyte malignancies (e.g., MCL) in greater percentages than to other subtypes of leukocytes.

In some embodiments, the lipid nanoparticle may be targeted upon conjugation/attachment/association with a targeting moiety, e.g., an antibody.

Zwitterionic amino lipids

In some embodiments, the LNP comprises a zwitterionic lipid. In some embodiments, the LNP comprising a zwitterionic lipid does not comprise a phospholipid.

Zwitterionic amino lipids have been shown to self-assemble into LNPs without phospholipid loading, stabilize them and release mRNA intracellularly, as described in U.S. patent application 20210121411, incorporated herein by reference in its entirety. Zwitterionic, ionizable, and persistent cationic helper lipids enable tissue-selective delivery of mRNA into spleen, liver, and lung and CRISPR-Cas9 gene editing therein, as described in Liu et al ,Membrane-destablizing ionizable phospholipids for organ-selective mRNA delivery and CRISPR-Cas gene editing,Nat Mater.(2021), which is incorporated herein by reference in its entirety.

Zwitterionic lipids can have a headgroup containing a cationic amine and an anionic carboxylate, as described in Walsh et al ,Synthesis,Characterization and Evaluation of Ionizable Lysine-Based Lipids for siRNA Delivery,Bioconjug Chem.(2013), which is incorporated herein by reference in its entirety. Ionizable lysine-based lipids containing a lysine head group linked to a long chain dialkylamine via an amide linkage at the lysine alpha amine can reduce immunogenicity as described in Walsh et al ,Synthesis,Characterization and Evaluation of Ionizable Lysine-Based Lipids for siRNA Delivery,Bioconjug Chem.(2013).

Additional lipid component

In some embodiments, the LNP compositions of the present disclosure further comprise one or more additional lipid components capable of affecting the directionality of the LNP. In some embodiments, the LNP further comprises at least one lipid selected from the group consisting of: DDAB, EPC, 14PA, 18BMP, DODAP, DOTAP and C12-200 (see Cheng et al, nat nanotechnol.2020, month 4; 15 (4): 313-320.; dillard et al, PNAS2021, volume 118, 52.).

Polynucleotide

In some embodiments, the LNP of the present disclosure contains an active agent. In some embodiments, the active agent is a polynucleotide. In some embodiments, the LNP is capable of delivering the polynucleotide to a target organ. Polynucleotides, in the broadest sense of the term, include any compound and/or substance that is incorporated into or can be incorporated into an oligonucleotide strand. Exemplary polynucleotides for use in accordance with the present disclosure include, but are not limited to, one or more of the following: deoxyribonucleic acid (DNA), ribonucleic acid (RNA), including messenger mRNA (mRNA), hybrids thereof, RNAi-inducing agents, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that induces triple helix formation, aptamers, vectors, and the like. RNAs suitable for use in the compositions and methods described herein may be selected from the group consisting of (but not limited to): short timer (shortimer), antagomir, antisense, ribozyme short interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microrna (miRNA), dicer substrate RNA (dsRNA), short hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In some embodiments, the polynucleotide is mRNA. In some embodiments, the polynucleotide is a circular RNA. In some embodiments, the polynucleotide encodes a protein. The polynucleotide may encode any polypeptide of interest, including any naturally or non-naturally occurring polypeptide or modified polypeptide. The polypeptide may be of any size and may have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA may have a therapeutic effect when expressed in a cell.

In other embodiments, the polynucleotide is an siRNA. The siRNA may be capable of selectively knocking down or down-regulating expression of the gene of interest. For example, the siRNA can be selected to silence a gene associated with a particular disease, disorder, or condition after administration of a nanoparticle composition comprising the siRNA to a subject in need thereof. The siRNA may comprise a sequence complementary to an mRNA sequence encoding a gene or protein of interest. In some embodiments, the siRNA can be an immunomodulatory siRNA.

In some embodiments, the polynucleotide is an shRNA or a vector or plasmid encoding the same. shRNA may be produced inside the target cell after delivery of the appropriate construct to the nucleus. Constructs and mechanisms related to shRNA are well known in the relevant arts.

The polynucleotide may include a first region (e.g., a coding region) encoding a linked nucleoside of a polypeptide of interest, a first flanking region (e.g., 5 '-UTR) located at the 5' end of the first region, a second flanking region (e.g., 3 '-UTR) located at the 3' end of the first region, at least one 5 '-cap region, and a 3' -stabilizing region. In some embodiments, the polynucleotide further comprises a poly a region or Kozak sequence (e.g., in the 5' -UTR). In some cases, a polynucleotide may contain one or more intron nucleotide sequences that are capable of excision from the polynucleotide. In some embodiments, a polynucleotide (e.g., mRNA) can include a 5' cap structure, a chain termination nucleotide, a stem loop, a poly a sequence, and/or a polyadenylation signal. Any region of a nucleic acid may include one or more alternative components (e.g., alternative nucleosides). For example, the 3 '-stabilizing region may contain alternative nucleosides, such as L-nucleosides, reverse thymidine, or 2' -O-methyl nucleosides; and/or the coding region, 5'-UTR, 3' -UTR, or cap region may include alternative nucleosides, such as 5-substituted uridine (e.g., 5-methoxyuridine), 1-substituted pseudouridine (e.g., 1-methyl-pseudouridine or 1-ethyl-pseudouridine), and/or 5-substituted cytidine (e.g., 5-methyl-cytidine). In some embodiments, the polynucleotide contains only naturally occurring nucleosides.

In some cases, the polynucleotide is greater than 30 nucleotides in length. In another embodiment, the polynucleotide molecule is greater than 35 nucleotides in length. In another embodiment, at least 40 nucleotides in length. In another embodiment, at least 45 nucleotides in length. In another embodiment, at least 55 nucleotides in length. In another embodiment, at least 50 nucleotides in length. In another embodiment, at least 60 nucleotides in length. In another embodiment, at least 80 nucleotides in length. In another embodiment, at least 90 nucleotides in length. In another embodiment, at least 100 nucleotides in length. In another embodiment, at least 120 nucleotides in length. In another embodiment, at least 140 nucleotides in length. In another embodiment, at least 160 nucleotides in length. In another embodiment, at least 180 nucleotides in length. In another embodiment, at least 200 nucleotides in length. In another embodiment, at least 250 nucleotides in length. In another embodiment, at least 300 nucleotides in length. In another embodiment, at least 350 nucleotides in length. In another embodiment, at least 400 nucleotides in length. In another embodiment, at least 450 nucleotides in length. In another embodiment, at least 500 nucleotides in length. In another embodiment, at least 600 nucleotides in length. In another embodiment, at least 700 nucleotides in length. In another embodiment, at least 800 nucleotides in length. In another embodiment, at least 900 nucleotides in length. In another embodiment, at least 1000 nucleotides in length. In another embodiment, at least 1100 nucleotides in length. In another embodiment, at least 1200 nucleotides in length. In another embodiment, at least 1300 nucleotides in length. In another embodiment, at least 1400 nucleotides in length. In another embodiment, at least 1500 nucleotides in length. In another embodiment, at least 1600 nucleotides in length. In another embodiment, at least 1800 nucleotides in length. In another embodiment, at least 2000 nucleotides in length. In another embodiment, at least 2500 nucleotides in length. In another embodiment, at least 3000 nucleotides in length. In another embodiment, at least 4000 nucleotides in length. In another embodiment, at least 5000 nucleotides or greater than 5000 nucleotides in length.

In some embodiments, the polynucleotide molecules, formulas, compositions, or methods related thereto comprise one or more polynucleotides ：WO2002/098443、WO2003/051401、WO2008/052770、WO2009/127230、WO2006/122828、WO2008/083949、WO2010/088927、WO2010/037539、WO2004/004743、WO2005/016376、WO2006/024518、WO2007/095,976、WO2008/014979、WO2008/077592、WO2009/030481、WO2009/095226、WO2011/069586、WO2011/026641、WO2011/144358、WO2012/019780、WO2012/013326、WO2012/089338、WO2012/113513、WO2012/116811、WO2012/116810、WO2013/113502、WO2013/113501、WO2013/113736、WO2013/143698、WO2013/143699、WO2013/143700、WO2013/120626、WO2013/120627、WO2013/120628、WO2013/120629、WO2013/174409、WO2014/127917、WO2015/024669、WO2015/024668、WO2015/024667、WO2015/024665、WO2015/024666、WO2015/024664、WO2015/101415、WO2015/101414、WO2015/024667、WO2015/062738、WO2015/101416,, which contain features as described in the following, are all incorporated herein by reference.

Polynucleotides, such as circular RNAs, may contain an Internal Ribosome Entry Site (IRES). IRES may serve as the sole ribosome binding site, or may serve as one of the multiple ribosome binding sites of mRNA. Polynucleotides containing more than one functional ribosome binding site can encode a number of peptides or polypeptides (e.g., polycistronic mRNA) that are independently translated from the ribosome. When the polynucleotide has an IRES, a second translatable region is further optionally provided. Examples of IRES sequences that may be used according to the present disclosure include, but are not limited to, those from picornaviruses (e.g., FMDV), pest viruses (CFFV), polioviruses (PV), encephalomyocarditis viruses (ECMV), foot and Mouth Disease Viruses (FMDV), hepatitis C Viruses (HCV), classical Swine Fever Viruses (CSFV), murine Leukemia Viruses (MLV), simian Immunodeficiency Viruses (SIV), or cricket paralysis viruses (CrPV).

In some embodiments, the polynucleotide comprises one or more microrna binding sites. In some embodiments, the microrna binding site is recognized by micrornas in a non-target organ. In some embodiments, the microrna binding site is recognized by micrornas in the liver. In some embodiments, the microrna binding site is recognized by micrornas in hepatocytes.

Inactive ingredients

In some embodiments, the formulations described herein may comprise at least one inactive ingredient. As used herein, the term "inactive ingredient" refers to one or more agents that have no effect on the activity of the active ingredient of the pharmaceutical composition contained in the formulation. In some embodiments, the inactive ingredients useful in the formulations of the present disclosure may be all, none, or some of which are approved by the U.S. Food and Drug Administration (FDA).

In some embodiments, the formulations disclosed herein can include a cation or an anion. Formulations include metal cations such as, but not limited to, zn ²⁺、Ca²⁺、Cu²⁺、Mn²⁺、Mg²⁺ and combinations thereof. As one non-limiting example, the formulation may include polymers and complexes with metal cations.

The formulations of the present disclosure may also include one or more pharmaceutically acceptable salts. As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting the acid or base moiety present to its salt form (e.g., by reacting the free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali metal or organic salts of acidic residues such as carboxylic acids; etc. Representative acid addition salts include acetates, acetic acid, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorinate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, caproate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, and the like. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Pharmaceutically acceptable salts of the present disclosure include, for example, conventional non-toxic salts of the parent compound formed from non-toxic inorganic or organic acids.

Solvates may be prepared by crystallization, recrystallization or precipitation from solutions comprising organic solvents, water or mixtures thereof. Examples of suitable solvents are ethanol, water (e.g., monohydrate, dihydrate, and trihydrate), N-methylpyrrolidone (NMP), dimethylsulfoxide (DMSO), N '-Dimethylformamide (DMF), N' -Dimethylacetamide (DMAC), 1, 3-dimethyl-2-imidazolidone (DMEU), 1, 3-dimethyl-3, 4,5, 6-tetrahydro-2- (1H) -pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate, and the like. When water is the solvent, the solvate is referred to as a "hydrate".

Route of administration

The initial constructs, reference constructs, and targeting systems described herein can be administered by any delivery route that produces a therapeutically effective result. These include, but are not limited to: enteral (into the intestines), gastrointestinal tract, epidural (into the dura), oral (by means of the oral cavity), transdermal, brain (into the brain), brain ventricle (into the brain ventricle), upper epidermis (coated onto the skin), intradermal (into the skin itself), subcutaneous (under the skin), nasal administration (via the nose), intravenous (into the vein), intravenous bolus, intravenous drip, intra-arterial (into the artery), intramuscular (into the muscle), intracardiac (into the heart), intra-osseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraparenchymal (into the brain tissue), intraperitoneal (infusion or injection into the peritoneum), subcutaneous (under the skin), nasal administration (via the nose), intravenous (into the vein), Intravesical infusion, intravitreal (via the eye), intracavitary injection (into the pathological cavity), intracavitary (into the fundus of the penis), intravaginal administration, intrauterine, extraamniotic administration, transdermal (via intact skin diffusion for systemic distribution), transmucosal (via mucosal diffusion), transvaginal, insufflation (sniffing), sublingual, subcchear, enema, eye drops (onto the conjunctiva), ear drops, aural (in or by means of the ear), buccal (directed towards the cheek), conjunctiva, skin, teeth (to one or more teeth), electroosmosis, intracervical, dou Daona, intratracheal, extracorporeal, hemodialysis, invasive, interstitial, intraabdominal, Intra-amniotic, intra-articular, intra-biliary, intra-bronchial, intra-cystic, intra-cartilage (in cartilage), intra-caudal (in the cauda equina), intra-cerebral-cisterna (in the greater cisterna cerebellum), intra-corneal (in the cornea), intra-dental crown, intra-coronary (in the coronary artery), intra-corpora cavernosa (in the inflatable space of the corpora cavernosa), intra-discal (in the intervertebral disc), intra-tubular (in the gland duct), intra-duodenal (in the duodenum), intra-dural (in or under the dura mater), intra-epidermal (to the epidermis), intra-oesophageal (to the oesophagus), intra-gastric (in the stomach), intra-gingival (in the gingiva), intra-ileal (in the distal portion of the small intestine), intra-cardiac (in the distal portion of the small intestine), Intralesional (in a local intralesional or directly introduced to a local intralesional), intraluminal (in a lumen), intralymphatic (in a lymph), intramedullary (in a bone marrow cavity of a bone), meningeal (in a meninge), myocardial (in a myocardium), intraocular (in an eye), ovarian (in an ovary), pericardial (in a pericardium), pleural (in a pleura), prostate (in a prostate gland), pulmonary (in a lung or its bronchi), sinus (in a nasal or periorbital sinus), spinal (in a spinal column), synovial (in a synovial cavity of a joint), tendon (in a tendon), testicular (in a testis), intrathecal (in cerebrospinal fluid at any level of the spinal axis), intrathecal (in a spinal fluid of the brain), Intrathoracic (intrathoracic), intratubular (intratubular in the organ), intratumoral (intratumoral), intrathecal (in the middle ear), intravascular (in one or more blood vessels), intraventricular (in the room), iontophoretic (by means of electric current, wherein ions of soluble salts migrate into tissues of the body), lavage (flushing or irrigating open wounds or body cavities), laryngeal (directly behind the larynx), nasogastric tube (via the nose and into the stomach), occlusive dressing techniques (topical route administration, which is then covered by a dressing that occludes the area), ophthalmic (to the outside of the eye), oropharyngeal (directly to the mouth and pharynx), parenteral, transdermal, periarticular Epidural, peri-nerve, periodontal, transrectal, respiratory tract (by oral or nasal inhalation into the respiratory tract for local or systemic effects), retrobulbar (postcerebral or postocular), soft tissue, subarachnoid, subconjunctival, submucosal, topical, transplacental (through or across the placenta), transtracheal (through the tracheal wall), transtympanic (across or through the tympanic cavity), ureter (to ureter), urethra (to urethra), transvaginal, caudal block, diagnosis, nerve block, biliary tract perfusion, cardiac perfusion, photopheresis, and spinal cord.

In some embodiments, the composition may be administered in a manner that allows it to cross the blood brain barrier, vascular barrier, or other epithelial barrier. The initial construct, reference construct and targeting system can be administered in any suitable form, in the form of a liquid solution or suspension, in the form of a solid suitable for a liquid solution or in the form of a suspension of a liquid solution. The initial construct, baseline construct, and targeting system can be formulated with any suitable and pharmaceutically acceptable excipients.

In some embodiments, the initial construct, the reference construct, and the targeting system can be delivered to the subject via a single route of administration.

In some embodiments, the initial construct, the reference construct, and the targeting system can be delivered to the subject via a multi-site route of administration. The subject may be administered at 2, 3, 4, 5, or more than 5 sites.

In some embodiments, the initial construct, the reference construct, and the targeting system may be administered to the subject using bolus injection.

In some embodiments, the initial construct, the reference construct, and the targeting system may be administered to the subject using a period of sustained delivery for minutes, hours, or days. Infusion rates may vary depending on the subject, the distribution, the formulation, or another delivery parameter.

In some embodiments, the initial construct, the reference construct, and the targeting system can be delivered by an intramuscular delivery route. Non-limiting examples of intramuscular administration include intravenous injection or subcutaneous injection.

In some embodiments, the initial construct, the reference construct, and the targeting system can be delivered by oral administration. Non-limiting examples of oral delivery include digestive tract administration and buccal administration.

In some embodiments, the initial construct, the reference construct, and the targeting system can be delivered by an intraocular delivery route. Non-limiting examples of intraocular delivery include intravitreal injection.

In some embodiments, the initial construct, the reference construct, and the targeting system can be delivered by an intranasal delivery route. Non-limiting examples of intranasal delivery include nasal drops or nasal sprays.

In some embodiments, the initial construct, the reference construct, and the targeting system can be administered to the subject by peripheral injection. Non-limiting examples of peripheral injections include intraperitoneal, intramuscular, intravenous, conjunctival or joint injections.

In some embodiments, the initial construct, the reference construct, and the targeting system may be delivered by injection into the cerebrospinal fluid. Non-limiting examples of delivery to the cerebrospinal fluid include intrathecal and intraventricular administration.

In some embodiments, the initial construct, the reference construct, and the targeting system can be delivered by systemic delivery. As one non-limiting example, systemic delivery may be by intravascular administration.

In some embodiments, the initial construct, the reference construct, and the targeting system can be administered to the subject by intracranial delivery.

In some embodiments, the initial construct, the reference construct, and the targeting system can be administered to the subject by intraparenchymal administration.

In some embodiments, the initial construct, the reference construct, and the targeting system can be administered to the subject by intramuscular administration.

In some embodiments, the initial construct, the reference construct, and the targeting system are administered to the subject and transduce the muscle of the subject. As one non-limiting example, the initial construct, the reference construct, and the targeting system are administered by intramuscular administration.

In some embodiments, the initial construct, the reference construct, and the targeting system can be administered to the subject by intravenous administration.

In some embodiments, the initial construct, the reference construct, and the targeting system can be administered to the subject by subcutaneous administration.

In some embodiments, the initial construct, the reference construct, and the targeting system can be administered to the subject by topical administration.

In some embodiments, the initial construct, the reference construct, and the targeting system can be delivered by more than one route of administration.

The initial constructs, reference constructs, and targeting systems described herein can be co-administered in combination with one or more initial constructs, reference constructs, targeting systems, or therapeutic agents or moieties.

Target region, tissue or cell for delivery

Delivery of nucleic acid sequences, polypeptides or peptides and formulations thereof can be targeted to specific target areas, tissues or cells using the methods and targeted delivery systems described herein.

Tumor(s)

In some embodiments, delivery of the nucleic acid sequence, polypeptide or peptide and formulations thereof can be targeted to a tumor. The tumor may be a benign tumor or a malignant tumor.

In some embodiments, delivery of the nucleic acid sequence, polypeptide or peptide and formulations thereof is localized to connective tissue tumors such as, but not limited to, adult fibrous tissue, embryonic (myxoma) fibrous tissue, adipose tissue, cartilage, bone, and spinal cord. As one non-limiting example, a tumor is a benign tumor called fibroma located in adult fibrous tissue. As a non-limiting example, a tumor is a malignancy in adult fibrotissue known as fibrosarcoma. As a non-limiting example, a tumor is a benign tumor called myxoma located in embryonic fibrous tissue. As a non-limiting example, the tumor is a malignancy located in embryonic fibrous tissue called myxosarcoma. As one non-limiting example, a tumor is a benign tumor in adipose tissue called a lipoma. As one non-limiting example, the tumor is a malignancy in adipose tissue called liposarcoma. As one non-limiting example, a tumor is a benign tumor called a chondrioma located in cartilage. As one non-limiting example, the tumor is a malignancy located in cartilage called chondrosarcoma. As one non-limiting example, a tumor is a benign tumor located in bone known as a osteoma. As a non-limiting example, a tumor is a malignancy in bone known as osteosarcoma. As one non-limiting example, the tumor is a malignancy located in the chordae known as chordoma. As one non-limiting example, a tumor is a benign tumor called a fibrocytoma located in connective tissue. As one non-limiting example, a tumor is a malignancy in connective tissue called malignant fibrous histiocytoma.

In some embodiments, delivery of the nucleic acid sequence, polypeptide or peptide and formulations thereof is localized to endothelial and/or mesothelial tumor tissue, such as, but not limited to, blood vessels, lymphatic vessels, and mesothelium. As one non-limiting example, a tumor is a benign tumor called a hemangioma located in a blood vessel. As one non-limiting example, a tumor is a benign tumor located in a blood vessel known as a vascular epidermoid tumor. As a non-limiting example, a tumor is a malignancy in a blood vessel known as a vascular endothelial tumor. As a non-limiting example, a tumor is a malignant tumor located in a blood vessel known as a angiosarcoma. As a non-limiting example, a tumor is a benign tumor located in lymphatic vessels called lymphangioma. As a non-limiting example, the tumor is a malignancy located in lymphatic vessels called lymphangiosarcoma. As one non-limiting example, the tumor is a malignancy in the mesothelium known as mesothelioma.

In some embodiments, delivery of the nucleic acid sequence, polypeptide or peptide and formulations thereof is localized to blood and lymphocyte tissue, such as, but not limited to, hematopoietic cells and lymphoid tissue. As a non-limiting example, a tumor is a benign tumor in hematopoietic cells known as a pre-leukemia. As one non-limiting example, a tumor is a benign tumor known as a myeloproliferative disorder located in hematopoietic cells. As a non-limiting example, a tumor is a malignancy in hematopoietic cells called leukemia. As one non-limiting example, a tumor is a benign tumor called plasmacytoid located in lymphoid tissue. As one non-limiting example, the tumor is a malignancy called plasmacytoma located in lymphoid tissue. As one non-limiting example, a tumor is a malignancy in lymphoid tissue known as multiple myeloma. As a non-limiting example, the tumor is a malignancy called Hodgkin's lymphoma (Hodgkin's lymphoma) located in lymphoid tissue. As one non-limiting example, the tumor is a malignancy in lymphoid tissue known as non-hodgkin's lymphoma.

In some embodiments, delivery of the nucleic acid sequence, polypeptide or peptide and formulations thereof is localized to muscle tissue, such as, but not limited to, smooth muscle and striated muscle. As one non-limiting example, a tumor is a benign tumor called a smooth myoma that is located in smooth muscle. As one non-limiting example, the tumor is a malignancy in smooth muscle called leiomyosarcoma. As one non-limiting example, the tumor is a benign tumor called rhabdomyoma located in rhabdomyoma. As one non-limiting example, the tumor is a malignancy in the rhabdomyoma called rhabdomyosarcoma.

In some embodiments, delivery of the nucleic acid sequence, polypeptide or peptide and formulations thereof is targeted to epithelial tissue, such as, but not limited to, stratified squamous tissue, glandular epithelial tissue (e.g., liver, kidney, bile duct), transitional epithelial tissue, placenta, and testis. As one non-limiting example, a tumor is a benign tumor called papilloma, located in stratified squamous tissue. As one non-limiting example, a tumor is a benign tumor called seborrheic keratosis located in stratified squamous tissue. As one non-limiting example, a tumor is a malignancy called squamous cell carcinoma that is located in stratified squamous tissue. As one non-limiting example, a tumor is a malignancy called epidermoid carcinoma that is located in stratified squamous tissue. As one non-limiting example, a tumor is a benign tumor called an adenoma, which is located in glandular epithelial tissue. As one non-limiting example, a tumor is a benign tumor called a liver adenoma located in the liver glandular epithelial tissue. As one non-limiting example, the tumor is a benign tumor called a tubular adenoma located in the epithelial tissue of the kidney gland. As one non-limiting example, the tumor is a benign tumor called cholangiocarcinoma located in the cholangiopancreatic tissue. As one non-limiting example, the tumor is a malignancy located in the glandular epithelium, known as adenocarcinoma. As one non-limiting example, the tumor is a malignancy located in the liver glandular epithelial tissue called liver cancer. As one non-limiting example, the tumor is a malignancy located in the liver glandular epithelial tissue called hepatocellular carcinoma. As one non-limiting example, the tumor is a malignancy called renal cell carcinoma located in the epithelial tissue of the kidney gland. As one non-limiting example, the tumor is a malignancy called adrenal tumor located in the epithelial tissue of the kidney gland. As one non-limiting example, the tumor is a malignancy called cholangiocarcinoma located in the biliary gland epithelial tissue. As one non-limiting example, a tumor is a benign tumor called transitional cell papilloma located in transitional epithelial tissue. As one non-limiting example, a tumor is a malignancy called transitional cell carcinoma located in transitional epithelial tissue. As one non-limiting example, the tumor is a benign tumor in the placenta called a water-sac-like block. As one non-limiting example, the tumor is a malignancy in the placenta called choriocarcinoma. As one non-limiting example, the tumor is a malignancy called seminoma located in the testes. As one non-limiting example, the tumor is a malignancy in the testis called an embryonic cell carcinoma tumor.

In some embodiments, delivery of the nucleic acid sequence, polypeptide or peptide and formulations thereof is localized to a neural tissue, such as, but not limited to, glial cells, neural cells, meninges, and schwann. As one non-limiting example, the tumor is a malignancy called glioma (grade I-III) located in glial cells. As one non-limiting example, the tumor is a malignancy called glioblastoma multiforme (grade I-III) located in glial cells. As one non-limiting example, the tumor is a malignancy called glioblastoma multiforme (grade IV) located in glial cells. As one non-limiting example, a tumor is a benign tumor called gangliocytoma located in a nerve cell. As one non-limiting example, the tumor is a malignancy called neuroblastoma located in a nerve cell. As one non-limiting example, the tumor is a malignancy called a neural tube blastoma located in a nerve cell. As one non-limiting example, the tumor is a benign tumor called a meningioma located in meningeal tissue. As one non-limiting example, the tumor is a malignancy in meningeal tissue known as malignant spinal cord tumor. As one non-limiting example, the tumor is a benign tumor called a schwannoma located in the nerve sheath. As one non-limiting example, the tumor is a benign tumor called a schwannoma located in the nerve sheath. As one non-limiting example, the tumor is a benign tumor called neurofibromatosis located in the nerve sheath. As one non-limiting example, the tumor is a malignancy called malignant spinal cord tumor located in the nerve sheath. As one non-limiting example, the tumor is a malignant tumor called malignant schwannoma located in the nerve sheath. As one non-limiting example, the tumor is a malignant tumor called neurofibrosarcoma located in the nerve sheath.

In some embodiments, delivery of the nucleic acid sequence, polypeptide, or peptide and formulations thereof is targeted to an Amine Precursor Uptake and Decarboxylation (APUD) system, such as, but not limited to, pituitary tissue, parathyroid tissue, thyroid tissue, bronchial tissue, adrenal medullary tissue, pancreatic tissue, stomach and intestine, carotid body, and chemoreceptor system tissue. The APUD system is a series of cells that have endocrine functions and secrete a variety of small amine or polypeptide hormones. As a non-limiting example, a tumor is a benign tumor called a basophilic adenoma located in the pituitary tissue. As a non-limiting example, a tumor is a benign tumor called eosinophilic adenoma located in the pituitary tissue. As a non-limiting example, a tumor is a benign tumor called a refractory cellular adenoma located in the pituitary tissue. As one non-limiting example, the tumor is a benign tumor called parathyroid adenoma located in the parathyroid gland. As one non-limiting example, the tumor is a malignancy in the parathyroid gland known as parathyroid carcinoma. As one non-limiting example, a tumor is a benign tumor called C-cell proliferation located in thyroid tissue (C-cells). As one non-limiting example, a tumor is a malignancy in thyroid tissue (C cells) known as medullary thyroid carcinoma. As one non-limiting example, a tumor is a malignancy called bronchogenic carcinoma located in the endobronchial layer (Kultschitzky cells). As one non-limiting example, the tumor is a malignancy called oat cell carcinoma located in the bronchial lining (Kultschitzky cells). As one non-limiting example, the tumor is a benign tumor called pheochromocytoma located in the adrenal medulla. As one non-limiting example, the tumor is a malignancy in the adrenal medulla called malignant pheochromocytoma. As one non-limiting example, the tumor is a benign tumor located in the pancreas called an islet cell adenoma. As one non-limiting example, a tumor is a benign tumor located in the pancreas called an insulinoma. As one non-limiting example, the tumor is a benign tumor called a gastrinoma located in the pancreas. As one non-limiting example, the tumor is a malignancy in the pancreas called islet cell carcinoma. As one non-limiting example, a tumor is a benign tumor known as carcinoid located in the stomach and intestines. As one non-limiting example, a tumor is a malignancy in the stomach and intestines known as a malignant carcinoid. As one non-limiting example, a tumor is a benign tumor called chemoreceptor tumor located in the carotid body and chemoreceptor system. As one non-limiting example, a tumor is a benign tumor called a paraganglioma located in the carotid body and the chemoreceptor system. As one non-limiting example, a tumor is a malignancy that is located in the carotid body and chemoreceptor system, known as malignant carcinoid. As one non-limiting example, a tumor is a malignancy called malignant paraganglioma located in the carotid body and the chemoreceptor system.

In some embodiments, delivery of the nucleic acid sequence, polypeptide or peptide and formulations thereof is localized in cells of neuro-spinal origin, such as, but not limited to, pigment-producing cells (e.g., skin and eye), sheath cells of the peripheral nervous system, and merkel (merkel) cells in squamous epithelium. As one non-limiting example, a tumor is a benign tumor called a mole that is located in pigment-producing cells (e.g., skin and eyes). As one non-limiting example, a tumor is a malignancy called melanoma that is located in pigment-producing cells (e.g., skin and eyes). As one non-limiting example, a tumor is a benign tumor called a schwannoma or schwannoma located in the schwann cells of the peripheral nervous system. As one non-limiting example, a tumor is a malignancy called malignant schwannoma located in the schwann cells of the peripheral nervous system. As one non-limiting example, the tumor is a malignancy called a merkel cell neoplasm in merkel cells located in squamous epithelium.

In some embodiments, delivery of the nucleic acid sequence, polypeptide or peptide and formulations thereof is localized in breast tissue. As one non-limiting example, a tumor is a benign tumor known as fibroadenoma. As a non-limiting example, the tumor is a malignancy called She Zhuangnang sarcoma.

In some embodiments, delivery of the nucleic acid sequence, polypeptide or peptide and formulations thereof is localized in renin (RENAL ANLAGE) tissue. As a non-limiting example, the tumor is a malignancy known as Wilms tumor (Wilms tumor).

In some embodiments, delivery of the nucleic acid sequence, polypeptide or peptide and formulations thereof is localized in ovarian tissue.

In some embodiments, delivery of the nucleic acid sequence, polypeptide or peptide and formulations thereof is localized in testicular tissue.

In some embodiments, delivery of the nucleic acid sequence, polypeptide or peptide and formulations thereof is localized in germ cell tumor tissue. Non-limiting examples of germ cell tumors include seminomas, asexual cytomas, choriocarcinomas, embryonal carcinomas, endodermal sinus tumors, and teratocarcinomas.

In some embodiments, delivery of the nucleic acid sequence, polypeptide or peptide and formulations thereof is localized in connective tissue matrix. Non-limiting examples of such tumors are Sertoli-Leydig cell tumors, ovarian male blastomas, granulosa-follicular membrane cell tumors, portal cell tumors, lipid cell tumors.

Organ

In some embodiments, delivery of the nucleic acid sequence, polypeptide or peptide and formulations thereof can be localized to an organ. Non-limiting examples of organs include anal canal, artery, ascending colon, bladder, bone marrow, brain, bronchi, bronchioles, glomerulonephritis, capillaries, cecum, cerebellum, hemispheres of the brain, cervix, choroid plexus, clitoris, cranial nerves, descending colon, metaencephalon, duodenum, ear, enteric nervous system, epididymis, esophagus, external genitalia, fallopian tube, gall bladder, ganglion, taste, intestinal-related lymphoid tissue, heart, ileum, endophytic organ, interstitium, jejunum, joint, kidney, large intestine, larynx, ligament, liver, lung, lymph node, lymphatic vessel, breast, medulla, mesenteric, midbrain, oral cavity, respiratory muscle, nasal cavity, nerves, olfaction, ovary, pancreas, parotid gland, penis, pharynx, placenta, brain bridge, prostate, rectum, salivary gland, scrotum, seminal vesicle, sigmoid colon, skeleton (eleton), skin, small intestine, spinal nerves, viscera, stomach, subcutaneous tissue, sublingual gland, submaxillary gland, teeth, testis, testicle, tendon, ventricle, spinal canal, brain, ureter, uterine canal, urinary tract, colon, uterine canal, urinary tract, and colon.

Tissue of

In some embodiments, delivery of the nucleic acid sequence, polypeptide or peptide and formulations thereof can be localized to a tissue. Non-limiting examples of tissue include adrenal medulla, adult fibrous tissue, blood vessels, bone, breast, bronchial lining, carotid body, cartilage, connective tissue, embryonic (myxoma) fibrous tissue, epithelial cells, epithelium, fat, glandular epithelium (liver, kidney, bile duct), gonads, hematopoietic cells, lymphatic vessels, lymphoid tissue, meninges, mesothelium, muscle, nerve sheath, neuro, chordae, ovary, pancreas, parathyroid, pituitary, placenta, renin, smooth muscle, stomach and intestine, layered squamous tissue, striated muscle, stroma, testis, thyroid, and transitional epithelium. As one non-limiting example, the tissue is connective tissue.

Cells

In some embodiments, delivery of a nucleic acid sequence, polypeptide or peptide and formulations thereof can be targeted to a particular cell type. Non-limiting examples of cells include adipocytes; adrenergic nerve cells; alpha cells; no axon nerve cells; glazing cells; anterior lens epithelial cells; anterior/intermediate pituitary cells; apocrine sweat gland cells; astrocytes; auditory endohair cells of the organ of coti (corti); auditory outer hair cells of the organ of Kotiva; b cells; pasteur (bartholin's) gland cells; basal cells (stem cells) of cornea, tongue, oral cavity, nasal cavity, distal anal canal, distal urethra and distal vagina; The basal cells of the olfactory epithelium; basket cells; basophils and precursors; beta cells; belleville cells (betz cell); bone marrow reticulocytes; boundary cells of the organ of Kotiva; boundary cells; Bowman's (bowman's) gland cells, brown adipocytes, buchner (brunner's) gland cells, bulbar urinary gland cells, clusterin cells (bushy cell), c cells, cajal-retzius cells, cardiomyocytes (cardiac muscle cell), cardiomyocytes (cardiac muscle cells), wheel cells, glucocorticoid-producing fascicular band cells, mineralocorticoid-producing fascicular band cells, androgen-producing reticulocytes, adrenal cortex cells, cementoblasts, and, Glandular cells, earwax gland cells, chandelier cells, chemoreceptor angioglomus cells of carotid somatic cells, master cells, cholinergic neurons, pheochromocytes, rod cells (club cells), cold-sensitive primary sensory neurons, connective tissue macrophages (all types), corneal fibroblasts (keratocytes), progesterone-secreting ruptured follicular corpus luteum cells, cortical hair stem cells, corticotropin cells, lens fibroblasts containing lens globulin, stratum corneum hair stem cells, cytotoxic t cells, d cells, delta cells, dendritic cells, double-bouquet cells, tubular cells, exocrine sweat gland clear cells, exocrine sweat gland dark cells, efferent vessel cells, elastic cartilage cells, endothelial cells, enteric glial cells, enteric chromiphilic like cells, enteric endocrine cells, eosinophilic leukocyte granules and precursors, ependymal cells, epidermal basal cells, epidermal langerhans cells (LANGERHANS CELL), epididymal basal cells, epididymal primary cells, epithelial reticulocytes, epsilon cells, erythrocytes, fibrocartilage cells, forked neurons, glenoid cells (foveolar cell), g cells, gall bladder epithelial cells, germ cells, Littley cells (LITTRE CELL) glands, molar cells (moll cell) glands in the eyelid, glial cells, golgi cells, gonadal stromal cells, gonadotrophin cells (gonadotrope), granulosa cells, granulosa lutein cells, grid cells, head facing cells and hematopoietic stem cells. In some embodiments, the at least one cell type comprises a cancer cell. In some embodiments, the at least one cell type comprises a non-cancerous cell. In some embodiments, the at least one cell type comprises both cancerous and non-cancerous types. In some embodiments, the cancerous state of the at least one cell type is unknown.

Physiological system

In some embodiments, delivery of the nucleic acid sequence, polypeptide or peptide and formulations thereof can be targeted to a physiological system. Non-limiting examples of physiological systems include the auditory system, cardiovascular system, central nervous system, chemical receptor system, circulatory system, digestive system, endocrine system, excretory system, exocrine system, genital system, epidermal system, lymphatic system, muscular system, musculoskeletal system, nervous system, peripheral nervous system, renal system, reproductive system, respiratory system, urinary system, and visual system.

Detection method and analysis

Detection of a directionality exploration platform that includes a targeting system (e.g., a candidate targeting system and a validated targeting system) can be performed via a variety of techniques that can be selected based on a usage tracking system (i.e., detection techniques or analysis techniques, both of which can be used interchangeably herein).

In some embodiments, the targeting systems described herein are detected using nuclear imaging techniques. As used herein, nuclear imaging techniques are intended to encompass any imaging, detection, counting (couniting) or sorting technique that utilizes radioactive emissions (emitted from a subject or external source). Nuclear imaging techniques may include, without limitation, X-ray, magnetic Resonance Imaging (MRI) (including functional magnetic resonance imaging (fMRI) and magnetic resonance imaging), computed Tomography (CT), positron Emission Tomography (PET), single Photon Emission Computed Tomography (SPECT), absorption imaging, or any combination thereof. The general principles and procedures for these approaches are known in the art, see Perez-Medina et al ,Nuclear imaging approaches facilitating nanomedicine translation.Advanced Drug Delivery Reviews 154-155(2020)123-141,, the contents of which are incorporated herein by reference in their entirety, as it relates to nuclear imaging techniques.

In some embodiments, detection of the targeting system described herein in a subject can be performed using MRI techniques. This route may be performed by any method known or discovered. While not wishing to be bound by theory, MRI makes use of the detection of certain nuclear species rotation characteristics. In some embodiments, MRI in combination with the targeting systems described herein can be used as a non-invasive detection technique that comprises an MRI contrast agent, such as gadolinium-based small molecules, manganese-based small molecules, iron oxide nanoparticles, ¹⁹ F-based compounds, and any combination thereof. As one example, MRI techniques may allow for in vivo detection of targeted systems in specific organs and tissues of a subject, as well as changes in those distributions over time.

In some embodiments, detection of the targeting system described herein in a subject can be performed using CT techniques. This route may be performed by any method known or discovered. While not wishing to be bound by theory, CT uses the interaction of X-ray photons with a substance, CT binding targeting systems can be used as non-invasive detection techniques that include CT contrast agents, such as gold high density lipoprotein nanoparticles (Au-HDL). As one example, CT techniques may allow for in vivo detection of targeted systems in specific organs and tissues of a subject, as well as changes in those distributions over time.

In some embodiments, detection of the targeting system described herein in a subject can be performed using PET techniques. This route may be performed by any method known or discovered. While not wishing to be bound by theory, PET utilizes detection of photon emissions from exogenously administered radiological substances (i.e., radiotracers). Mainly, PET scanners detect two photons emitted in opposite directions after positron-electron annihilation (coincidence events). PET binding targeting systems can be used As invasive or non-invasive detection techniques that include suitable radiolabels such As 111In, 99mTc, 13N, 68Ga, 18F, 64Cu, 86Y, 76Br, 89Zr, 72As, 124I, 74As, fluorine-18, gallium-68, nitrogen-13, copper-64, bromine-76, iodine-125, arsenic-74, carbon-11, iodine-131, 153Sm, 177Lu, 186Re, 188Re, 198Au, and 225Ac. These labels may be conjugated to structural elements, cargo components, or both. PET scanning can be performed to detect in vivo the distribution of the targeting system over the subject (including those changes in distribution over time) or over resected samples of the subject. PET technology can allow detection of a targeted system in a subject from the organ/tissue level down to the cell type level. Some PET techniques may allow detection of the targeting system at the intracellular level.

In some embodiments, detection of the targeting system described herein in a subject can be performed using SPECT techniques. This route may be performed by any method known or discovered. While not wishing to be bound by theory, SPECT utilizes detection of photon emission from exogenously administered radioactive materials (i.e., radiotracers). Mainly, SPECT scanners detect X-rays and gamma photons associated with nuclear state transitions. SPECT binding targeting systems can be used As a invasive or non-invasive detection technique that includes suitable radiolabels such As 111In, 99mTc, 13N, 68Ga, 18F, 64Cu, 86Y, 76Br, 89Zr, 72As, 124I, 74As, fluoro-18, gallium-68, nitrogen-13, copper-64, bromo-76, iodo-125, arsenic-74, carbon-11, iodo-131, 153Sm, 177Lu, 186Re, 188Re, 198Au, and 225Ac. These labels may be conjugated to structural elements, cargo components, or both. SPECT scans can be performed to detect in vivo the distribution of the targeting system over the subject (including those changes in distribution over time) or over resected samples of the subject. SPECT techniques can allow detection of a targeted system in a subject from the organ/tissue level down to the cell type level. Some SPECT techniques may allow detection of the targeting system at the intracellular layer level.

In some implementations, multi-core imaging techniques may be used with a targeting system, including a single tracking system. In some implementations, multi-core imaging techniques may be used with targeting systems, including multi-tracking systems.

In some embodiments, the targeting systems described herein are detected using optical imaging techniques. As used herein, optical imaging techniques are intended to encompass any imaging, detection, counting or sorting technique that utilizes the light emission and specific characteristics of photons (emitted from a subject or external source). Optical imaging techniques may include, without limitation, visible light microscopy, raman spectroscopy (Raman spectroscopy), fluorescence microscopy, bioluminescence imaging (BLI), optical coherence tomography, or any combination thereof. The general principles and procedures of these approaches are known in the art, see Drummen.Fluorescent Probes and Fluorescence(Microscopy)Techniques-Illuminating Biological and Biomedical Research.Molecules 2012,17,14067-14090,Boutorine et al ,Fluorescent Probes for Nucleic Acid Visualization in Fixed and Live Cells.Molecules 2013,18,15357-15397, and Juskowiak,Nucleic acid-based fluorescent probes and their analytical potential.Anal.Bioanal.Chem.(2011)399:3157-3176,, the contents of which are incorporated herein by reference in their entirety, for optical imaging techniques.

In some embodiments, detection of the targeting systems described herein in a subject can be performed using visible fluorescence microscopy. Fluorescence microscopy techniques include a wide range of techniques known in the art including, but not limited to, confocal fluorescence microscopy, fluorescence reflectance imaging, fluorescence molecular tomography, andResonance Energy Transfer (FRET). In general, all fluorescence microscopy techniques use detection of light emitted from an endogenously existing or exogenously applied fluorescent compound (i.e., a compound that absorbs light or other electromagnetic radiation and re-emits it at a longer wavelength). Fluorescence microscopy in combination with targeting systems can be used as a invasive or non-invasive detection technique comprising at least one tracking system comprising a suitable fluorescent compound. Without limitation, such fluorescent compounds may include green fluorescent protein, yellow fluorescent protein, red fluorescent protein 、Sirius、EBFP2、CFP、Cerulean、EGFP、EYFP、mOrange、mCherry、mPlum、NIR、iRFP、EosFP、PamCherry、Dronpa、Dreiklang、asFP595、mMaple、mGeo、mEos2、Dendra2、psCFP2、2,3,5,6- tetracarbazole-4-cyano-pyridine (CPy), fluorescent nanoparticles, or fluorescent lipids, fluorescein, TAMRA, cy dye, texas red, HEX, JOE, oregon green, rhodamine 6G, coumarin, pyrene, diOC6 (3, 3' -dihexyloxycarbocyanine iodide), or any combination thereof. In some embodiments, the targeting system for detection with fluorescence microscopy will comprise at least one fluorophore, which may include, but is not limited to, quantum dots, coumarins, naphthalimides, luciferins, BODIPYs, cyanines, dibenzopyrans, oxazines, oligothiophenes, and phthalocyanine derivatives (PcDer). These fluorescent compounds may be incorporated into the structure of the targeting system as cargo or payload, as product expression of the cargo or payload, or any combination thereof. Fluorescence microscopy can be performed to detect in vivo the distribution of the targeting system over a subject (including those changes in distribution over time) or over an excised sample of a subject. Fluorescence microscopy can allow detection of a targeted system in a subject from the organ/tissue level down to the cell type level. Some fluorescence microscopy techniques may allow detection of the targeting system at the intracellular level. In some embodiments, fluorescence microscopy can be used to sort samples of cells after administration using Fluorescence Activated Cell Sorting (FACS).

In some embodiments, detection of the targeting system in the subject may be performed using bioluminescence imaging (BLI) techniques. This route may be performed by any method known or discovered. While not wishing to be bound by theory, BLI imaging utilizes exogenously supplied compounds that emit light under physiological conditions as a product of a chemical reaction. These emissions can be detected via various techniques of light and fluorescence microscopy. In some embodiments, the BLI technology can be used in conjunction with a targeting system that includes a bioluminescent compound. Such compounds may be incorporated into nanoparticles or as cargo or payload for post-delivery expression. In some embodiments, the bioluminescent compound may include, but is not limited to, a luciferase, including renilla luciferase, metridia luciferase, nanoluc luciferase, firefly luciferase, kowtow luciferase, or any combination thereof. The BLI technique can be performed to detect in vivo the distribution of the tropism exploration platform over a subject (including those changes over time) or over an resected sample of a subject. The BLI may allow detection of a targeted system in a subject from the organ/tissue level down to the cell type level. Some BLI techniques may allow detection of the targeting system at the intracellular level. In some embodiments, the BLI technique may include quantifying luciferase expression from different organs using an In Vivo Imaging System (IVIS).

In some embodiments, detection of the targeting systems described herein can be performed using nucleotide sequencing techniques. Nucleotide sequencing techniques can be used to detect the presence of a known sequence of nucleotides in a sample, such as an identifier (e.g., bar code) sequence. Non-limiting examples of nucleotide sequencing techniques that can be used to detect the targeting system include high throughput sequencing, PCR, deep sequencing, and any combination thereof.

In some embodiments, detection of the targeting systems described herein can be performed by detecting the products of a tracking system comprising a functional polynucleotide (e.g., DNA, mRNA, or oRNA) encoding a known peptide sequence or protein (i.e., a reporter sequence). In some embodiments, the functional polynucleotide may comprise a sequence encoding a unique non-functional polypeptide sequence (i.e., a peptide or protein). In some embodiments, the reporter sequence may comprise a beta-galactosidase (beta-gal) sequence. In some embodiments, the reporter sequence may comprise eGFP; a luciferase; gene editing agents (e.g., cas9 editing, DNA readout); ox-40; beta 6 integrin; CD45; surface markers with a (3 x) -HA tag, (with or without a TEV protease site) a (3 x) -flag tag, or any combination thereof. In some embodiments, the reporter sequence may comprise a luciferase or fluorescent compound sequence. In some embodiments, expression of the functional sequence and the associated targeting system presence can be performed by any of the techniques previously disclosed. In some embodiments, detecting the product of the tracking system comprising the reporter sequence may be performed using any method known or found to detect the expressed product. Such techniques include, but are not limited to, liquid/gas chromatography, mass spectrometry, optical spectroscopy (absorbance), gel electrophoresis, quantitative enzyme-linked immunosorbent assay (ELISA), western blotting (Western blotting), dot blotting, northern blotting, protein immunostaining, protein immunoprecipitation, or any combination thereof.

In some embodiments, detection of the targeting system described herein may be performed by utilizing a detection system selected for use in particular in matching a designed tracking system. As one non-limiting example, the targeting system described herein can be detected by electron microscopy, thermal imaging, ultrasound imaging, photoacoustic imaging, laboratory assays, and any combination thereof.

In some embodiments, detection of the targeting system described herein can be performed by using cell sorting techniques including, but not limited to, the following in combination with tracking system nanoparticles comprising components recognized by the cell sorting method: magnetic beads, flow cytometry, peptide lysis with LC-MS/MS, fluorescence Activated Cell Sorting (FACS), or any combination thereof.

In some embodiments, the detection technique may analyze only one formulation or cargo at a time. In some embodiments, the detection technique may analyze multiple agents or cargo at a time. In some embodiments, the assay technique can analyze about 1 formulation, 2 formulations, 3 formulations, 4 formulations, 5 formulations, 6 formulations, 7 formulations, 8 formulations, 9 formulations, 10 formulations, 11 formulations, 12 formulations, 13 formulations, 14 formulations, 15 formulations, 16 formulations, 17 formulations, 18 formulations, 19 formulations, 20 formulations, 21 formulations, 22 formulations, 23 formulations, 24 formulations, 25 formulations, or more at a time. In some embodiments, the detection technique may analyze between about 1 and 100 agents. As one non-limiting example, the detection technique may analyze about 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, or 1-90 preparations. In some embodiments, the detection technique can analyze more than 100 formulations at a time.

In some embodiments, libraries of targeting systems can be analyzed. As one non-limiting example, the targeting system can have the same formulation and different identifier sequences or portions. As another non-limiting example, the targeting system can have the same formulation and the same identifier sequence or portion. As another non-limiting example, the targeting system can have different agents and the same identifier sequences or portions. As another non-limiting example, the targeting system can have different agents and different identifier sequences or portions.

In some embodiments, a library of targeting systems may have one identifier sequence or portion for analysis.

In some embodiments, a library of targeting systems can have at least two identifier sequences or portions for analysis. The library may have 2-10 identifier sequences or portions for analysis. The library may have 2-100 identifier sequences or portions for analysis. The library may have 2-500 identifier sequences or portions for analysis. The library may have 100-500 identifier sequences or portions for analysis. The library may have 2-1000 identifier sequences or portions for analysis. The library may have 2-2500 identifier sequences or portions for analysis. The library may have 1000-2500 identifier sequences or portions for analysis. The library may have 1000-5000 identifier sequences or portions for analysis. The library may have 2500-5000 identifier sequences or portions for analysis. The library may have 4000-5000 identifier sequences or portions for analysis.

In some embodiments, the library of targeting systems can have at least one initial construct or baseline construct formulated in a nanoparticle delivery vehicle. The library may have 1-10000 nanoparticles. The library may have 1-10 nanoparticles. The library may have 1-100 nanoparticles. The library may have 1-500 nanoparticles. The library may have 100-500 nanoparticles. The library may have 1-1000 nanoparticles. The library may have 100-500 nanoparticles. The library may have 1000-5000 nanoparticles. The library may have 2500-5000 nanoparticles. The library may have 1-5000 nanoparticles. The library may have 1-10000 nanoparticles. The library may have 5000-10000 nanoparticles. As one non-limiting example, the nanoparticle may be a lipid nanoparticle.

VIII methods of use

In some embodiments, the isotropic delivery systems described herein may be used as therapeutic agents for diagnosing, preventing, treating, and/or managing diseases, disorders, and conditions, or as diagnostic agents. The therapeutic agents may be used in personalized medicine, immunooncology, cancer, vaccines, gene editing (e.g., CRISPR).

In some embodiments, the isotropic delivery systems described herein may be used for diagnostic purposes or as a diagnostic tool.

In some embodiments, the delivery systems described herein can be used to treat food-borne afflictions, gastroenteritis, infectious diseases, neglected topical diseases, tropical diseases, disease-mediated biological diseases, toxin exposure.

The pharmaceutical composition may be delivered as described in PCT publication WO2012135805, incorporated herein by reference in its entirety.

The present disclosure provides methods comprising administering a pharmaceutical composition to a subject in need thereof. The pharmaceutical composition can be administered to a subject using any amount and any route of administration effective to prevent, treat, diagnose, or image a disease, disorder, and/or condition. The precise amount required will vary from subject to subject depending on factors such as, but not limited to, the species, age and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. The pharmaceutical composition can be administered to an animal such as a mammal (e.g., human, domestic animal, cat, dog, monkey, mouse, rat, etc.). The payload of the pharmaceutical composition is a polynucleotide.

In some embodiments, the pharmaceutical composition, the prophylactic composition, the diagnostic composition, or the imaging composition thereof is administered to a human.

In some embodiments, the active agent is administered by one or more of the following various routes, including but not limited to: topical, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, intradermal, rectal, intravaginal, intraperitoneal, topical (e.g., by powder, ointment, cream, gel, lotion and/or drops), mucosal, nasal, buccal, enteral, vitreous, intratumoral, sublingual; by intratracheal instillation, bronchial instillation and/or inhalation; as an oral spray, nasal spray, and/or aerosol, and/or via a portal vein catheter.

In some embodiments, the active agent is administered by systemic intravenous injection.

In some embodiments, the active agent is administered intravenously and/or orally.

In particular embodiments, the active agent may be administered in a manner that allows the active agent to cross the blood brain barrier, vascular barrier, or other epithelial barrier.

Injectable formulations, such as sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing, wetting and/or suspending agents. The sterile injectable preparation may be a sterile injectable solution, suspension and/or emulsion in a non-toxic parenterally acceptable diluent and/or solvent, for example, as a solution in 1, 3-butanediol. Among the acceptable vehicles and solvents that may be used are water, ringer's solution, U.S. P. and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids such as oleic acid find use in the preparation of injectables.

The injectable formulation may be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which may be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

Dosage forms for topical, and/or transdermal administration of the pharmaceutical composition may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and/or patches. In addition, the present disclosure encompasses the use of transdermal patches, which generally have the added advantage of providing a controllable delivery of a compound to the body. Such dosage forms may be prepared, for example, by dissolving and/or partitioning the compound in an appropriate medium. Alternatively or additionally, the rate may be controlled by providing a rate controlling membrane and/or by dispersing the compound in the polymer matrix and/or gel.

Formulations suitable for topical administration include, but are not limited to, liquid and/or semi-liquid formulations, such as wipes, lotions, oil-in-water and/or water-in-oil emulsions, such as creams, ointments and/or pastes, and/or solutions and/or suspensions. For example, a topically applicable formulation may contain from about 1% to about 10% (w/w) of the active ingredient, but the concentration of the active ingredient may be up to the limit of dissolution of the active ingredient in the solvent. Formulations for topical application may also include one or more of the additional ingredients described herein.

The pharmaceutical compositions may be prepared, packaged and/or sold in a formulation suitable for ocular administration. Such formulations may, for example, be in the form of eye drops, including, for example, 0.1/1.0% (w/w) solutions and/or suspensions of the active ingredient in an aqueous or oily liquid vehicle. Such drops may also include buffers, salts, and/or one or more other ingredients of any of the additional ingredients described herein. Other ophthalmically administrable formulations suitable for use include those comprising an active ingredient in microcrystalline form and/or in the form of a liposomal formulation. Ear drops and/or eye drops are contemplated within the scope of the present disclosure.

In general, most suitable routes of administration will depend on a variety of factors, including the nature of the active agent to be delivered (e.g., its stability in the environment of the gastrointestinal tract, blood flow, etc.), the condition of the patient (e.g., whether the patient is able to tolerate a particular route of administration), and the like. The present disclosure contemplates the delivery of active agents by any suitable route, taking into account possible advances in the drug delivery sciences.

In certain embodiments, the pharmaceutical compositions according to the present disclosure may be administered at a dosage level sufficient to deliver: about 0.0001mg/kg to about 100mg/kg, about 0.01mg/kg to about 50mg/kg, about 0.1mg/kg to about 40mg/kg, about 0.5mg/kg to about 30mg/kg, about 0.01mg/kg to about 10mg/kg, about 0.1mg/kg to about 10mg/kg, or about 1mg/kg to about 25mg/kg of subject body weight per day, one or more times a day, to obtain a desired therapeutic, diagnostic, or prophylactic effect. The desired dose may be delivered three times a day, twice a day, once a day, every other day, every third day, weekly, biweekly, every third week, or every fourth week. In certain embodiments, the desired dose may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, twelve, thirteen, fourteen or more administrations). When multiple administrations are employed, a divided dosing regimen, such as those described herein, may be used.

According to the present disclosure, administration of an active agent in a divided dosage regimen can produce higher levels of protein in a mammalian subject. As used herein, a "split dose" is a single unit dose or total daily dose divided into two or more doses. As used herein, a "single unit dose" is a dose of any therapeutic agent administered in one dose/disposable/in a single route/single point of contact (i.e., a single administration event). As used herein, a "total daily dose" is an amount given or specified in a 24 hour period. Which may be administered in a single unit dosage form. In one embodiment, the active agents of the present disclosure are administered to a subject in divided doses. In some embodiments, the active agent is formulated in a buffer only or in a formulation described herein.

The LNP of the present disclosure can be used or administered in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. "in combination with … …" is not intended to imply that the agents must be administered simultaneously and/or formulated for delivery together, but such delivery methods are within the scope of the present disclosure. The pharmaceutical composition may be administered simultaneously with, before or after one or more other desired therapeutic or medical procedures. Generally, each agent will be administered at a dosage and/or schedule determined for the agent. In some embodiments, the present disclosure encompasses the delivery of a pharmaceutical, prophylactic, diagnostic, or imaging composition in combination with an agent that can improve its bioavailability, reduce and/or modulate its metabolism, inhibit its excretion, and/or modulate its distribution in the body.

It will be further appreciated that the therapeutic, prophylactic, diagnostic or imaging agents used in combination may be administered together in a single pharmaceutical composition or separately in different pharmaceutical compositions. Generally, we expect that the therapeutic agents used in combination are used at levels not exceeding their levels used alone. In some embodiments, the combined use level will be lower than the single use level. In one embodiment, the combination (individually or together) can be administered according to the split dosing regimen described herein.

The particular combination of therapies (therapeutic agents or procedures) used in the combination regimen will take into account the compatibility of the desired therapeutic agent and/or procedure and the desired therapeutic effect to be achieved. It will also be appreciated that the therapy employed may achieve a desired effect for the same disorder (e.g., a pharmaceutical composition suitable for treating cancer according to the present disclosure may be administered concurrently with a chemotherapeutic agent), or it may achieve a different effect (e.g., control of any side effects).

Pharmaceutical compositions containing the LNPs disclosed herein are formulated for administration as follows: intramuscular, intraarterial, intraocular, vaginal, rectal, intraperitoneal, intravenous, intranasal, subcutaneous, endoscopic, transdermal, intramuscular, intraventricular, intradermal, intrathecal, topical (e.g., by powder, ointment, cream, gel, lotion and/or drops), transmucosal, nasal, enteral, intratumoral, by intratracheal instillation, bronchial instillation and/or inhalation; nasal sprays and/or aerosols, and/or via portal vein catheters.

The pharmaceutical composition may also be formulated for delivery directly to an organ or tissue in any of several ways in the art, including but not limited to: direct infusion or bathing, via catheters, through gels, powders, ointments, creams, gels, lotions and/or drops, by use of a substrate such as a fabric or biodegradable material coated or impregnated with a pharmaceutical composition, and the like. In some embodiments, the pharmaceutical composition is formulated for extended release. In particular embodiments, the active agent and/or pharmaceutical, prophylactic, diagnostic or imaging compositions thereof may be administered in a manner that allows the active agent to cross the blood brain barrier, vascular barrier or other epithelial barrier.

In some aspects of the disclosure, the active agents of the disclosure are spatially entrapped within or near the target tissue. Methods of providing a pharmaceutical composition to a target tissue of a mammalian subject are provided by contacting the target tissue (which contains one or more target cells) with the pharmaceutical composition under conditions such that the pharmaceutical composition, particularly an active agent component of the pharmaceutical composition, is substantially retained in the target tissue, meaning that at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or greater than 99.99% of the pharmaceutical composition is retained in the target tissue. Advantageously, retention is by measuring the amount of the component of the active agent present in the pharmaceutical composition that enters the one or more target cells. For example, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or greater than 99.99% of the active agent administered to the subject is present in the cell at a time after administration.

Aspects of the present disclosure relate to methods of providing a pharmaceutical composition to a target tissue or organ of a mammalian subject by contacting the target tissue (containing one or more target cells) or organ (containing one or more target cells) with the pharmaceutical composition under conditions such that the pharmaceutical composition is substantially retained in the target tissue or organ. The pharmaceutical composition contains an effective amount of an active agent.

Pharmaceutical compositions that may be administered intramuscularly and/or subcutaneously may include, but are not limited to, polymers, copolymers and gels. The polymer, copolymer, and/or gel may be further tuned to modify the release kinetics by adjusting factors such as, but not limited to, molecular weight, particle size, payload, and/or ratio of monomers. As one non-limiting example, an intramuscularly and/or subcutaneously administered formulation may include a copolymer, such as poly (lactic-co-glycolic acid).

The localized delivery of the pharmaceutical compositions described herein may be administered by methods such as, but not limited to, topical delivery, ocular delivery, transdermal delivery, and the like. The pharmaceutical composition may also be topically applied to parts of the body that are not normally available for localized delivery, such as, but not limited to, when the subject's body is open to the environment during treatment. The pharmaceutical composition may further be delivered by bathing, soaking and/or surrounding the body part with the pharmaceutical composition.

However, the present disclosure contemplates the possible advances in drug delivery science to deliver the active agents disclosed herein and/or pharmaceutical, prophylactic, diagnostic, or imaging compositions thereof by any suitable route.

Method for producing polypeptide in cell

The present disclosure provides methods of producing a polypeptide of interest in a mammalian cell. Methods of producing a polypeptide involve contacting a cell with a formulation of the present disclosure comprising an LNP comprising mRNA encoding the polypeptide of interest. After contacting the cells with the lipid nanoparticle, the mRNA can be taken up into the cells and translated therein to produce the polypeptide of interest.

In general, the step of contacting the mammalian cell with an LNP comprising mRNA encoding the polypeptide of interest can be performed in vivo, ex vivo, in culture, or in vitro. The amount of lipid nanoparticle contacted with the cell and/or the amount of mRNA therein may depend on: the type of cell or tissue contacted, the means of administration, the lipid nanoparticle and the physiochemical characteristics (e.g., size, charge and chemical composition) of the mRNA therein, and other factors. In general, an effective amount of lipid nanoparticles will allow for efficient production of polypeptides in cells. Measures of efficiency may include polypeptide translation (indicated by polypeptide expression), the level of mRNA degradation, and immune response indicators.

The step of contacting the LNP comprising mRNA with the cell may involve or cause transfection. The phospholipids included in the lipid component of LNP can facilitate transfection and/or increase transfection efficiency, e.g., by interacting with and/or fusing with cells or intracellular membranes. Transfection may allow translation of mRNA within the cell.

In some embodiments, the lipid nanoparticles described herein may be used therapeutically. For example, mRNA contained in the LNP can encode a therapeutic polypeptide (e.g., in a translatable region) and produce the therapeutic polypeptide upon contact with the cell and/or entry (e.g., transfection) into the cell. In other embodiments, the mRNA contained in the LNP can encode a polypeptide that can improve or increase the immunity of the subject. In some embodiments, the mRNA may encode a granulocytic colony stimulating factor or trastuzumab (trastuzumab).

In some embodiments, the mRNA contained in the LNP can encode a recombinant polypeptide that can replace one or more polypeptides that can be substantially absent in cells contacted with the lipid nanoparticle. The one or more substantially absent polypeptides may be absent due to mutations in the gene encoding the gene or its regulatory pathways. Or the recombinant polypeptide produced by translation of the mRNA can antagonize the activity of endogenous proteins present in, on the surface of, or secreted by the cell. Antagonizing recombinant polypeptides may be desirable to combat adverse effects caused by the activity of endogenous proteins, such as altered activity or localization caused by mutations. In another alternative, the recombinant polypeptide produced by translation of the mRNA may indirectly or directly antagonize the activity of a biological moiety present in, on the surface of, or secreted by the cell. Antagonistic biological moieties can include, but are not limited to, lipids (e.g., cholesterol), lipoproteins (e.g., low density lipoproteins), nucleic acids, carbohydrates, and small molecule toxins. Recombinant polypeptides produced by translation of mRNA may be engineered to be located within a cell, for example, a specific compartment such as the nucleus, or may be engineered for secretion from a cell or for translocation to the plasma membrane of a cell.

In some embodiments, contacting the cell with an LNP comprising mRNA can reduce the innate immune response of the cell to the exogenous nucleic acid. The cell may be contacted with a first lipid nanoparticle comprising a first amount of a first exogenous mRNA comprising a translatable region, and the level of innate immune response of the cell to the first exogenous mRNA may be determined. Subsequently, the cell can be contacted with a second composition comprising a second amount of the first exogenous mRNA, the second amount being a smaller amount of the first exogenous mRNA than the first amount. Or the second composition may include a first amount of a second exogenous mRNA that is different from the first exogenous mRNA. The step of contacting the cells with the first composition and the second composition may be repeated one or more times. In addition, the efficiency of producing a polypeptide (e.g., translation) in a cell can optionally be determined, and the cell can be repeatedly contacted with the first composition and/or the second composition until the efficiency of target protein production is achieved.

Methods of delivering therapeutic agents to cells and organs

Provided herein are methods of treating a disease or disorder comprising administering to a subject in need thereof a pharmaceutical composition of the present disclosure, e.g., a pharmaceutical composition comprising an LNP as described herein.

The present disclosure provides methods of delivering an active agent and/or prophylactic agent (e.g., a nucleic acid) to a mammalian cell or organ. Delivering a therapeutic and/or prophylactic agent to a cell involves administering to a subject a formulation of the present disclosure that includes an LNP that includes the therapeutic and/or prophylactic agent (e.g., a nucleic acid), wherein administering a composition involves contacting the cell with the composition. In some embodiments, a protein, cytotoxic agent, radioactive ion, chemotherapeutic agent, or nucleic acid (e.g., RNA, such as mRNA) may be delivered to a cell or organ. Where the therapeutic and/or prophylactic agent is an mRNA, after contacting the cell with the lipid nanoparticle, the translatable mRNA can be translated in the cell to produce the polypeptide of interest. However, substantially nontranslatable mRNA may also be delivered to the cell. Substantially nontranslatable mRNA may be useful as a vaccine, and/or may sequester the translated components of the cell to reduce the expression of other substances in the cell.

In some embodiments, the LNP may target a particular type or class of cells (e.g., cells of a particular organ or system thereof). In some embodiments, LNP including therapeutic and/or prophylactic agents of interest can be specifically delivered to the liver, kidney, spleen, femur, or lung of a mammal. "specifically delivering" to a particular class of cells, organs, or systems or groups thereof implies that, for example, after administration of LNP to a mammal, the proportion of lipid nanoparticles including therapeutic and/or prophylactic agents delivered to a destination of interest (e.g., tissue) is higher relative to other destinations. In some embodiments, specific delivery can result in a greater than 2-fold, 5-fold, 10-fold, 15-fold, or 20-fold increase in the amount of therapeutic and/or prophylactic agent per 1g of tissue of the targeted destination (e.g., tissue of interest, such as liver) compared to another destination (e.g., spleen). In some embodiments, the tissue of interest is selected from the group consisting of: vascular endothelium in the liver, kidneys, lungs, spleen, femur, blood vessels (e.g., intracoronary or intrafemoral) or kidneys, and tumor tissue (e.g., via intratumoral injection).

As another example of targeted or specific delivery, LNP may include mRNA encoding a protein binding partner (e.g., an antibody or functional fragment thereof, a scaffold protein or peptide) or a receptor on the cell surface. mRNA may additionally or alternatively be used to direct synthesis and extracellular localization of lipids, carbohydrates, or other biological moieties. Or other therapeutic and/or prophylactic agents or elements (e.g., lipids or ligands) of the LNP may be selected based on their affinity for a particular receptor (e.g., a low density lipoprotein receptor) so that the LNP can more readily interact with a target cell population that includes the receptor. In some embodiments, the ligand may include, but is not limited to, members of specific binding pairs, antibodies, monoclonal antibodies, fv fragments, single chain Fv (scFv) fragments, fab 'fragments, F (ab') 2 fragments, single domain antibodies, camelbody and fragments thereof, humanized antibodies and fragments thereof, and multivalent forms thereof; multivalent binding reagents, including mono-or bispecific antibodies, such as disulfide stabilized Fv fragments, scFv concatamers, bifunctional antibodies, trifunctional antibodies, or tetrafunctional antibodies; and aptamers, receptors, and fusion proteins.

In some embodiments, the ligand may be a surface-bound antibody, which may allow tuning of cell targeting specificity. This is particularly applicable because highly specific antibodies can be raised against the epitope of interest at the desired targeting site. In some embodiments, multiple antibodies are expressed on the cell surface, and each antibody may have a different specificity for a desired target. Such pathways may increase the avidity and specificity of the targeted interactions.

The ligand may be selected, for example, by one skilled in the art of biology based on the desired localization or function of the cell. In some embodiments, an estrogen receptor ligand, such as tamoxifen (tamoxifen), can target cells to estrogen-dependent breast cancer cells with an increased number of estrogen receptors on the cell surface. Other non-limiting examples of ligand/receptor interactions include CCR1 (e.g., for treatment of inflamed joint tissue or brain in rheumatoid arthritis and/or multiple sclerosis), CCR7, CCR8 (e.g., for targeting lymph node tissue), CCR6, CCR9, CCR10 (e.g., for targeting intestinal tissue), CCR4, CCR10 (e.g., for targeting skin), CXCR4 (e.g., for general enhanced trans-migration), HCELL (e.g., for treatment of inflammation and inflammatory disorders, bone marrow), α4β7 (e.g., for intestinal mucosa targeting), and VLA-4NCAM-1 (e.g., for targeting the inner skin). In general, any receptor involved in targeting (e.g., cancer metastasis) can be used in the methods and compositions described herein.

The cells targeted may include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, nerve cells, heart cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulosa cells, and tumor cells.

In some embodiments, the LNP may target hepatocytes. Apolipoproteins such as apolipoprotein E (apoE) have been shown to associate with lipid nanoparticles containing neutral or near neutral lipids in vivo, and are known to associate with receptors present on the surface of hepatocytes, such as Low Density Lipoprotein Receptors (LDLR). Thus, LNP comprising a lipid component with a neutral or near neutral charge administered to a subject can acquire apoE in the subject and subsequently deliver therapeutic and/or prophylactic agents (e.g., RNA) to hepatocytes, including LDLR, in a targeted manner.

Methods of treating diseases and disorders

The lipid nanoparticle is useful for treating a disease, disorder or condition. In particular, such compositions are useful for treating diseases, disorders or conditions characterized by a loss or abnormal protein or polypeptide activity. In some embodiments, a formulation of the present disclosure comprising LNP comprising mRNA encoding a deleted or aberrant polypeptide can be administered or delivered to a cell. Subsequent translation of the mRNA may produce the polypeptide, thereby reducing or eliminating problems caused by the absence or abnormal activity of the polypeptide. Because translation can occur rapidly, the methods and compositions can be useful for treating acute diseases, disorders, or conditions, such as sepsis, stroke, and myocardial infarction. The therapeutic and/or prophylactic agents contained in the LNP may also be capable of altering the transcription rate of a given substance, thereby affecting gene expression.

Diseases, disorders and/or conditions characterized by dysfunctional or aberrant protein or polypeptide activity of the administrable composition include, but are not limited to: rare diseases, infectious diseases (as both vaccines and therapeutics), cancer and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases. A variety of diseases, disorders and/or conditions may be characterized by a lack (or substantial decrease such that proper protein function cannot occur) of protein activity. Such proteins may be absent or they may be substantially nonfunctional. A specific example of a dysfunctional protein is a missense mutant variant of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which produces a dysfunctional protein variant of the CFTR protein that causes cystic fibrosis. The present disclosure provides a method for treating such diseases, disorders and/or conditions in a subject by administering LNP comprising RNA and a lipid component comprising the pegylated lipid compounds, phospholipids (optionally unsaturated), optionally second pegylated lipids and structural lipids disclosed herein, wherein the RNA can be mRNA encoding a polypeptide that antagonizes or otherwise overcomes the aberrant protein activity present in the cells of the subject.

In some embodiments, the lipid nanoparticles disclosed herein comprise a polynucleotide encoding an antigen protein. In some embodiments, the polynucleotide is an mRNA or circular RNA encoding an antigenic protein. In some embodiments, the polynucleotide encodes a protein selected from SEQ ID NOS.1-54, or a sequence having about 60% sequence identity, about 70% sequence identity, about 80% sequence identity, about 90% sequence identity, or about 95% sequence identity with a protein selected from SEQ ID NOS.1-54.

The application contains a sequence table submitted electronically in XML format. Sequence Listing XML is incorporated herein by reference in its entirety. The XML file was created at 2022, 9, 13, named REG006_SL and is 97,563 bytes in size.

In some embodiments, the lipid nanoparticles disclosed herein are useful in methods of treating a disease or disorder. In some embodiments, the disease or disorder is a food-borne illness or gastroenteritis. In some embodiments, the food-borne affliction is caused by a pathogen selected from the group consisting of: campylobacter jejuni (Campylobacter jejuni bacteria), clostridium difficile (Clostridium difficile bacteria), amoeba histolyticum (Entamoeba histolytica), enterotoxin B (Enterotoxin B), norwalk virus/norovirus (Norovirus), helicobacter pylori (Helicobacter pyroli) and rotavirus (Rotavirus).

In some embodiments, the disease or disorder is an infectious disease. In some embodiments, the infectious agent is the result of an infection with an infectious agent selected from the group consisting of: candida yeasts, coronaviruses (e.g., SARS-CoV-2, MERS-CoV), enteroviruses 71, epstein-barr viruses, gram negative bacteria (e.g., bordetella), gram positive bacteria (e.g., clostridium tetani, franciscensis, streptococcus bacteria, staphylococcus bacteria), hepatitis viruses, human cytomegaloviruses, HIV, HPV, influenza viruses, JCV, mycobacteria, poxviruses, pseudomonas aeruginosa, toxoplasma gondii, varicella zoster viruses, chikungunya viruses, dengue viruses, rabies viruses, trypanosomes, ebola viruses, plasmodium falciparum, marburg viruses, japanese encephalitis viruses, st.Louis encephalitis viruses, west nile viruses, and yellow fever viruses.

Preventive application

In some embodiments, the isotropic delivery systems described herein may be used to prevent disease or stabilize disease progression.

In some embodiments, the isotropic delivery systems described herein may be used as a prophylactic agent to prevent future diseases or conditions.

In some embodiments, the isotropic delivery systems described herein can be used to interrupt further progression of a disease or disorder.

Vaccine

In some embodiments, the isotropic delivery systems described herein may be used as a vaccine and/or in a manner similar to a vaccine. As used herein, a "vaccine" is a biological agent that improves immunity to a particular disease or infectious agent.

In some embodiments, the isotropic delivery systems described herein may be used as vaccines and/or in a vaccine-like manner for the therapeutic arts, such as, but not limited to, cardiovascular, CNS, dermatological, endocrinology, oncology, immunology, respiratory tract, and anti-infection. In some embodiments, the isotropic delivery systems described herein may be used as vaccines to diagnose, prevent, treat, and/or manage food-borne afflictions. In some embodiments, the isotropic delivery systems described herein may be used as vaccines to diagnose, prevent, treat, and/or manage gastroenteritis. In some embodiments, the isotropic delivery systems described herein may be used as vaccines to diagnose, prevent, treat, and/or manage influenza. In some embodiments, the isotropic delivery systems described herein may be used as a prophylactic agent to diagnose, prevent, treat, and/or manage HIV. In some embodiments, the isotropic delivery systems described herein may be used as a prophylactic agent to diagnose, prevent, treat, and/or manage coronaviruses. In some embodiments, the isotropic delivery systems described herein may be used as a prophylactic agent to diagnose, prevent, treat, and/or manage COVID-19. In some embodiments, the isotropic delivery systems described herein may be used as a prophylactic agent to diagnose, prevent, treat, and/or manage polio. In some embodiments, the isotropic delivery systems described herein may be used as a prophylactic agent to diagnose, prevent, treat, and/or manage tetanus. In some embodiments, the isotropic delivery systems described herein may be used as a prophylactic agent to diagnose, prevent, treat, and/or manage hepatitis a. In some embodiments, the isotropic delivery systems described herein may be used as a prophylactic agent to diagnose, prevent, treat, and/or manage hepatitis b. In some embodiments, the isotropic delivery systems described herein may be used as a prophylactic agent to diagnose, prevent, treat, and/or manage hepatitis c. In some embodiments, the isotropic delivery systems described herein may be used as a prophylactic agent to diagnose, prevent, treat, and/or manage rubella. In some embodiments, the isotropic delivery systems described herein may be used as a prophylactic agent to diagnose, prevent, treat, and/or manage Hib (haemophilus influenzae type b). In some embodiments, the isotropic delivery systems described herein may be used as a prophylactic agent to diagnose, prevent, treat, and/or manage measles. In some embodiments, the isotropic delivery systems described herein may be used as a prophylactic agent to diagnose, prevent, treat, and/or manage pertussis (Pertussis/Whooping Cough). In some embodiments, the isotropic delivery systems described herein may be used as prophylactic agents to diagnose, prevent, treat, and/or manage streptococcus pneumoniae disease. In some embodiments, the isotropic delivery systems described herein can be used as a prophylactic agent to diagnose, prevent, treat, and/or manage rotaviruses. In some embodiments, the isotropic delivery systems described herein may be used as a prophylactic agent to diagnose, prevent, treat, and/or manage mumps. In some embodiments, the isotropic delivery systems described herein may be used as a prophylactic agent to diagnose, prevent, treat, and/or manage varicella. In some embodiments, the isotropic delivery systems described herein may be used as a prophylactic agent to diagnose, prevent, treat, and/or manage diphtheria.

Contraceptive

In some embodiments, the isotropic delivery systems described herein may be used as and/or in a manner similar to contraceptives. As used herein, the term "contraceptive agent (contraceptive)" may be defined as any agent or method that may be used to prevent pregnancy.

In some embodiments, the contraceptive may be used for short or long periods of time.

In some embodiments, the contraceptive may be reversible or permanent.

Diagnosis of

In some embodiments, the isotropic delivery systems described herein may be used for diagnostic purposes or as a diagnostic tool for any of the foregoing diseases or conditions.

In some embodiments, the directional delivery systems described herein can be used to detect biomarkers for disease diagnosis.

In some embodiments, the isotropic delivery systems described herein may be used for diagnostic imaging purposes, such as MRI, PET, CT or ultrasound.

Study of

In some embodiments, the isotropic delivery systems described herein may be used for diagnostic purposes or as a research tool for any of the foregoing diseases or conditions.

In some embodiments, the directional delivery systems described herein can be used to detect biomarkers for research.

In some embodiments, the isotropic delivery systems described herein may be used in any research experiment, such as in vivo or in vitro experiments.

In some embodiments, the isotropic delivery systems described herein can be used in cultured cells. The cultured cells may be derived from any source known to those skilled in the art, and as one non-limiting example, may be derived from a stable cell line, an animal model, or a human patient or control subject.

In some embodiments, the isotropic delivery systems described herein can be used in vivo experiments in animal models (i.e., mice, rats, rabbits, dogs, cats, non-human primates, guinea pigs, ferrets, caenorhabditis elegans, drosophila, zebra fish, or any other animal known in the art for research purposes).

In some embodiments, the isotropic delivery systems described herein may be used in human research experiments or human clinical trials.

In some embodiments, the isotropic delivery systems described herein may be used in stem cells and/or cell differentiation.

IX. the illustrated embodiment

In one embodiment of the present disclosure, provided herein is a pharmaceutical composition comprising:

a) A polynucleotide encoding at least one protein of interest, and

B) Delivery vehicle comprising at least one lipid

Wherein the composition elicits an immune response in the subject.

In one embodiment, the polynucleotide is DNA.

In one embodiment, the polynucleotide is RNA.

In one embodiment, the RNA is short interfering RNA (siRNA).

In one embodiment, the siRNA inhibits or suppresses expression of a target of interest in the cell.

In one embodiment, the inhibition or inhibition is about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30％、20-40％、20-50％、20-60％、20-70％、20-80％、20-90％、20-95％、20-100％、30-40％、30-50％、30-60％、30-70％、30-80％、30-90％、30-95％、30-100％、40-50％、40-60％、40-70％、40-80％、40-90％、40-95％、40-100％、50-60％、50-70％、50-80％、50-90％、50-95％、50-100％、60-70％、60-80％、60-90％、60-95％、60-100％、70-80％、70-90％、70-95％、70-100％、80-90％、80-95％、80-100％、90-95％、90-100％ or 95-100%.

In one embodiment, the polynucleotide is substantially circular.

In one embodiment, the polynucleotide comprises an internal ribosome entry site sequence operably linked to a payload sequence region.

In one embodiment, the IRES sequence comprises a sequence derived from picornavirus complement DNA, encephalomyocarditis virus (EMCV) complement DNA, poliovirus complement DNA, or an antennapedia gene from drosophila melanogaster.

In one embodiment, the polynucleotide comprises a termination element, wherein the termination element comprises at least one termination codon.

In one embodiment, the polynucleotide comprises a regulatory element.

In one embodiment, the polynucleotide comprises at least one masking agent.

In one embodiment, in vitro transcription is used to produce a substantially circular polynucleotide.

In one embodiment, the payload sequence region comprises a non-coding nucleic acid sequence.

In one embodiment, the payload sequence region comprises a coding nucleic acid sequence.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest of campylobacter jejuni.

In one embodiment, the encoding nucleic acid sequence encodes a protein of interest of clostridium difficile.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest that is amoeba histolytica.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest of enterotoxin B.

In one embodiment, the coding nucleic acid sequence encodes a norwalk virus or a protein of interest of a norwalk virus.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest of helicobacter pylori.

In one embodiment, the encoding nucleic acid sequence encodes a protein of interest of a rotavirus.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest of candida yeast.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest of a coronavirus.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest of SARS-CoV.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest of SARS-CoV-2.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest of MERS-CoV.

In one embodiment, the encoding nucleic acid sequence encodes a protein of interest of enterovirus 71.

In one embodiment, the encoding nucleic acid sequence encodes a protein of interest of epstein-barr virus.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest of a gram-negative bacterium.

In one embodiment, the gram negative bacterium is bordetella.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest of a gram positive bacterium.

In one embodiment, the gram positive bacterium is clostridium tetani.

In one embodiment, the gram positive bacterium is Francisella tularensis.

In one embodiment, the gram positive bacterium is a streptococcus bacterium.

In one embodiment, the gram positive bacterium is a staphylococcus bacterium.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest for hepatitis.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest of human cytomegalovirus.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest of a human immunodeficiency virus.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest of human papillomavirus.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest of an influenza virus.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest of JC virus.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest of a mycobacterium.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest of a poxvirus.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest of Pseudomonas aeruginosa.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest of respiratory syncytial virus.

In one embodiment, the encoding nucleic acid sequence encodes a protein of interest of a rubella virus.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest of varicella zoster virus.

In one embodiment, the encoding nucleic acid sequence encodes a protein of interest of a chikungunya virus.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest of a dengue virus.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest of rabies virus.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest of trypanosoma cruzi and/or chagas disease.

In one embodiment, the encoding nucleic acid sequence encodes a protein of interest of ebola virus.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest of plasmodium falciparum.

In one embodiment, the encoding nucleic acid sequence encodes a protein of interest of a marburg virus.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest of Japanese encephalitis virus.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest of the St.Louis encephalitis virus.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest of west nile virus.

In one embodiment, the encoding nucleic acid sequence encodes a protein of interest of yellow fever virus.

In one embodiment, the encoding nucleic acid sequence encodes a protein of interest of bacillus anthracis.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest of a botulinum toxin.

In one embodiment, the coding nucleic acid sequence encodes a protein of interest of ricin.

In one embodiment, the coding nucleic acid sequence encodes a shiga toxin and/or a protein of interest of the shiga toxin.

In one embodiment, the polynucleotide comprises at least one modification.

In one embodiment, at least one modification is pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl uridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 5-methyl-uridine, 1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetyl-cytidine, 5-formyl-cytidine, N4-methyl-cytidine, 5-hydroxymethyl-cytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, zebralin, 5-aza-zebralin, 5-methyl-zebralin, 5-aza-2-thio-zebralin, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1-methyladenosine, N6-isopentenyl adenosine, N6- (cis-hydroxyisopentenyl) adenosine, 2-methylsulfanyl-N6- (cis-hydroxyisopentenyl) adenosine, N6-glycylcarbamoyl adenosine, N6-threonyl carbamoyl adenosine, 2-methylsulfanyl-N6-threonyl carbamoyl adenosine, N6-dimethyl adenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine, inosine, 1-methyl-inosine, hurusoside, hurusin, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl guanosine, N2-dimethyl guanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, n2-methyl-6-thio-guanosine or N2, N2-dimethyl-6-thio-guanosine.

In one embodiment, the pharmaceutical composition comprises at least one cationic lipid selected from the group consisting of: any lipid in table (I), any lipid having the structure of formula (CY-II), any lipid having the structure of formula (CY-III), any lipid having the structure of formula (CY-IV), and combinations thereof.

In one embodiment, the cationic lipid is any lipid having the structure of formula (CY-I).

In one embodiment, the cationic lipid is selected from the group consisting of compounds CY1, CY2, CY3, CY9, CY10, CY11, CY12, CY22, CY23, CY24, CY30, CY31, CY32, CY33, CY43, CY44, CY45, CY50, CY51, CY52, and CY 53.

In one embodiment, the cationic lipid is any lipid having the structure of formula (CY-II).

In one embodiment, the cationic lipid is selected from the group consisting of compounds CY4, CY5, CY16, CY17, CY18, CY25, CY26, CY37, CY38, CY39, CY46, CY56, and CY 57.

In one embodiment, the cationic lipid is any lipid having the structure of formula (CY-III).

In one embodiment, the cationic lipid is selected from the group consisting of compounds CY6, CY14, CY27, CY35, CY47, and CY 55.

In one embodiment, the cationic lipid is any lipid having the structure of formula (CY-IV).

In one embodiment, the cationic lipid is selected from the group consisting of compounds CY7, CY8, CY19, CY20, CY21, CY28, CY29, CY40, CY41, CY42, CY48, CY49, CY58, CY59, and CY 60.

In one embodiment, the pharmaceutical composition comprises an additional cationic lipid.

In one embodiment, the pharmaceutical composition comprises a neutral lipid.

In one embodiment, the pharmaceutical composition comprises an anionic lipid.

In one embodiment, the pharmaceutical composition comprises a helper lipid.

In one embodiment, the pharmaceutical composition comprises stealth lipids.

In one embodiment, the weight ratio of lipid to polynucleotide is between 100:1 and 1:1.

In one embodiment, the pharmaceutical compositions disclosed herein preferentially target immune cells, such as T cells, e.g., T regulatory cells. For example, the disclosed pharmaceutical compositions may preferentially target immune cells over hepatocytes. Exemplary non-limiting immune cells include cd8+, cd4+ or cd8+cd4+ cells. In some embodiments, the disclosed pharmaceutical compositions deliver cargo or payload to immune cells without the need for peptide, protein, or aptamer-based targeting ligands. See, for example, WO 2021/021634.

In one embodiment, the pharmaceutical compositions disclosed herein are delivered to mammalian liver immune cells, spleen T cells, or lung endothelial cells in the absence of a targeting ligand. Specific delivery to a particular class or class of cells indicates that the proportion of the pharmaceutical composition delivered to the class or class of cells of interest is high. For example, specific delivery may cause an increase in the amount of cargo or payload per 1g of tissue targeted to the destination of greater than 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, or 20-fold.

In one embodiment, the vaccine formulation comprises a pharmaceutical composition.

In one embodiment, the vaccine is prepared having any one of formulas (I) - (VI).

In one embodiment, provided herein is a method of vaccinating a subject against an infectious agent, the method comprising contacting the subject with a vaccine formulation or formulation and eliciting an immune response.

In one embodiment, the infectious agent is campylobacter jejuni, clostridium difficile, endo-amoeba histolyticum, enterotoxin B, norwalk or norovirus, helicobacter pylori, rotavirus, candida yeast, coronavirus (including SARS-CoV, SARS-CoV-2 and MERS-CoV), enterovirus 71, epstein-barr virus, gram negative bacteria (including bordetella), gram positive bacteria (including clostridium tetani, franciscensis, streptococci bacteria and staphylococci bacteria), and hepatitis, human cytomegalovirus, human immunodeficiency virus, human papillomavirus, influenza virus, JC virus, mycobacteria, poxvirus, pseudomonas aeruginosa, respiratory syncytial virus, rubella virus, varicella zoster virus, chikungunya virus, dengue virus, rabies virus, trypanosoma and/or calixas virus, ebola virus, malignancies, japanese encephalitis virus, st-lewis toxin, shiga virus, or shiga-roll virus.

The present disclosure includes the following illustrative embodiments

1. A pharmaceutical composition comprising:

a) A polynucleotide encoding at least one protein of interest, and

B) Delivery vehicle comprising at least one lipid

Wherein the composition elicits an immune response in the subject.

2. The pharmaceutical composition of embodiment 1, wherein the polynucleotide is DNA.

3. The pharmaceutical composition of embodiment 1, wherein the polynucleotide is RNA.

4. The pharmaceutical composition of embodiment 3, wherein the RNA is short interfering RNA (siRNA).

5. The pharmaceutical composition of embodiment 4, wherein the siRNA inhibits or suppresses expression of a target of interest in a cell.

6. The pharmaceutical composition of embodiment 5, wherein the inhibition or inhibition is about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30％、20-40％、20-50％、20-60％、20-70％、20-80％、20-90％、20-95％、20-100％、30-40％、30-50％、30-60％、30-70％、30-80％、30-90％、30-95％、30-100％、40-50％、40-60％、40-70％、40-80％、40-90％、40-95％、40-100％、50-60％、50-70％、50-80％、50-90％、50-95％、50-100％、60-70％、60-80％、60-90％、60-95％、60-100％、70-80％、70-90％、70-95％、70-100％、80-90％、80-95％、80-100％、90-95％、90-100％ or 95-100%.

7. The pharmaceutical composition of embodiment 2 or 3, wherein the polynucleotide is substantially circular.

8. The pharmaceutical composition of embodiment 7, wherein the polynucleotide comprises an Internal Ribosome Entry Site (IRES) sequence operably linked to a payload sequence region.

9. The pharmaceutical composition of embodiment 8, wherein the IRES sequence comprises a sequence derived from picornavirus complement DNA, encephalomyocarditis virus (EMCV) complement DNA, poliovirus complement DNA, or an antennapedia gene from drosophila melanogaster.

10. The pharmaceutical composition of embodiment 7, wherein the polynucleotide comprises a termination element, wherein the termination element comprises at least one termination codon.

11. The pharmaceutical composition of embodiment 7, wherein the polynucleotide comprises a regulatory element.

12. The pharmaceutical composition of any one of embodiments 7-11, wherein the polynucleotide comprises at least one masking agent.

13. The pharmaceutical composition of any one of embodiments 7-12, wherein the substantially circular polynucleotide is produced using in vitro transcription.

14. The pharmaceutical composition of any one of embodiments 7-14, wherein the payload sequence region comprises a non-coding nucleic acid sequence.

15. The pharmaceutical composition of any one of embodiments 7-13, wherein the payload sequence region comprises a coding nucleic acid sequence.

16. The pharmaceutical composition of embodiment 15, wherein the coding nucleic acid sequence encodes a protein of interest of campylobacter jejuni.

17. The pharmaceutical composition of embodiment 15, wherein the encoding nucleic acid sequence encodes a protein of interest of clostridium difficile.

18. The pharmaceutical composition of embodiment 15, wherein the coding nucleic acid sequence encodes a protein of interest of amoeba histolytica.

19. The pharmaceutical composition of embodiment 15, wherein the encoding nucleic acid sequence encodes a protein of interest of enterotoxin B.

20. The pharmaceutical composition of embodiment 15, wherein the coding nucleic acid sequence encodes a norwalk virus or a norovirus protein of interest.

21. The pharmaceutical composition of embodiment 15, wherein the coding nucleic acid sequence encodes a protein of interest of helicobacter pylori.

22. The pharmaceutical composition of embodiment 15, wherein the encoding nucleic acid sequence encodes a protein of interest of a rotavirus.

23. The pharmaceutical composition of embodiment 15, wherein the coding nucleic acid sequence encodes a protein of interest of candida yeast.

24. The pharmaceutical composition of embodiment 15, wherein the encoding nucleic acid sequence encodes a protein of interest of a coronavirus.

25. The pharmaceutical composition of embodiment 15, wherein the encoding nucleic acid sequence encodes a protein of interest of SARS-CoV.

26. The pharmaceutical composition of embodiment 15, wherein the encoding nucleic acid sequence encodes a protein of interest of SARS-CoV-2.

27. The pharmaceutical composition of embodiment 15, wherein the coding nucleic acid sequence encodes a MERS-CoV protein of interest.

28. The pharmaceutical composition of embodiment 15, wherein the encoding nucleic acid sequence encodes a protein of interest of enterovirus 71.

29. The pharmaceutical composition of embodiment 15, wherein the encoding nucleic acid sequence encodes a protein of interest of epstein barr virus.

30. The pharmaceutical composition of embodiment 15, wherein the coding nucleic acid sequence encodes a protein of interest of a gram-negative bacterium.

31. The pharmaceutical composition of embodiment 30, wherein the gram-negative bacterium is bordetella.

32. The pharmaceutical composition of embodiment 15, wherein the coding nucleic acid sequence encodes a protein of interest of a gram-positive bacterium.

33. The pharmaceutical composition of embodiment 32, wherein the gram positive bacterium is clostridium tetani.

34. The pharmaceutical composition of embodiment 32, wherein the gram positive bacterium is franciscensis terrestris.

35. The pharmaceutical composition of embodiment 32, wherein the gram positive bacterium is a streptococcus bacterium.

36. The pharmaceutical composition of embodiment 32, wherein the gram positive bacterium is a staphylococcus bacterium.

37. The pharmaceutical composition of embodiment 15, wherein the encoding nucleic acid sequence encodes a protein of interest for hepatitis.

38. The pharmaceutical composition of embodiment 15, wherein the coding nucleic acid sequence encodes a protein of interest of human cytomegalovirus.

39. The pharmaceutical composition of embodiment 15, wherein the encoding nucleic acid sequence encodes a protein of interest of a human immunodeficiency virus.

40. The pharmaceutical composition of embodiment 15, wherein the coding nucleic acid sequence encodes a protein of interest of human papillomavirus.

41. The pharmaceutical composition of embodiment 15, wherein the encoding nucleic acid sequence encodes a protein of interest of an influenza virus.

42. The pharmaceutical composition of embodiment 15, wherein the coding nucleic acid sequence encodes a protein of interest of JC virus.

43. The pharmaceutical composition of embodiment 15, wherein the coding nucleic acid sequence encodes a protein of interest of a mycobacterium.

44. The pharmaceutical composition of embodiment 15, wherein the coding nucleic acid sequence encodes a protein of interest of a poxvirus.

45. The pharmaceutical composition of embodiment 15, wherein the coding nucleic acid sequence encodes a protein of interest of pseudomonas aeruginosa.

46. The pharmaceutical composition of embodiment 15, wherein the coding nucleic acid sequence encodes a protein of interest of respiratory syncytial virus.

47. The pharmaceutical composition of embodiment 15, wherein the encoding nucleic acid sequence encodes a protein of interest of a rubella virus.

48. The pharmaceutical composition of embodiment 15, wherein the coding nucleic acid sequence encodes a protein of interest of varicella zoster virus.

49. The pharmaceutical composition of embodiment 15, wherein the encoding nucleic acid sequence encodes a protein of interest of a chikungunya virus.

50. The pharmaceutical composition of embodiment 15, wherein the encoding nucleic acid sequence encodes a protein of interest of dengue virus.

51. The pharmaceutical composition of embodiment 15, wherein the encoding nucleic acid sequence encodes a protein of interest of rabies virus.

52. The pharmaceutical composition of embodiment 15, wherein the coding nucleic acid sequence encodes a protein of interest of trypanosoma cruzi and/or chagas disease.

53. The pharmaceutical composition of embodiment 15, wherein the encoding nucleic acid sequence encodes a protein of interest of ebola virus.

54. The pharmaceutical composition of embodiment 15, wherein the coding nucleic acid sequence encodes a protein of interest of plasmodium falciparum.

55. The pharmaceutical composition of embodiment 15, wherein the encoding nucleic acid sequence encodes a protein of interest of a marburg virus.

56. The pharmaceutical composition of embodiment 15, wherein the coding nucleic acid sequence encodes a protein of interest of japanese encephalitis virus.

57. The pharmaceutical composition of embodiment 15, wherein the encoding nucleic acid sequence encodes a protein of interest of the st.

58. The pharmaceutical composition of embodiment 15, wherein the coding nucleic acid sequence encodes a protein of interest of west nile virus.

59. The pharmaceutical composition of embodiment 15, wherein the encoding nucleic acid sequence encodes a protein of interest of yellow fever virus.

60. The pharmaceutical composition of embodiment 15, wherein the encoding nucleic acid sequence encodes a protein of interest of bacillus anthracis.

61. The pharmaceutical composition of embodiment 15, wherein the encoding nucleic acid sequence encodes a protein of interest of a botulinum toxin.

62. The pharmaceutical composition of embodiment 15, wherein the encoding nucleic acid sequence encodes a protein of interest of ricin.

63. The pharmaceutical composition of embodiment 15, wherein the coding nucleic acid sequence encodes a shiga toxin and/or a protein of interest of a shiga toxin.

64. The pharmaceutical composition of any one of embodiments 3-63, wherein the polynucleotide comprises at least one modification.

65. The pharmaceutical composition of embodiment 64, wherein at least 20% of the bases are modified.

66. The pharmaceutical composition of embodiment 64, wherein at least 30% of the bases are modified.

67. The pharmaceutical composition of embodiment 64, wherein at least 40% of the bases are modified.

68. The pharmaceutical composition of embodiment 64, wherein at least 50% of the bases are modified.

69. The pharmaceutical composition of embodiment 64, wherein at least 60% of the bases are modified.

70. The pharmaceutical composition of embodiment 64, wherein at least 70% of the bases are modified.

71. The pharmaceutical composition of embodiment 64, wherein at least 80% of the bases are modified.

72. The pharmaceutical composition of embodiment 64, wherein at least 90% of the bases are modified.

73. The pharmaceutical composition of embodiment 64, wherein at least 100% of the bases are modified.

74. The pharmaceutical composition of embodiment 64, wherein a particular base comprises at least one modification.

75. The pharmaceutical composition of embodiment 74, wherein the base is adenine.

76. The pharmaceutical composition of embodiment 75, wherein at least 20% of the adenine bases are modified.

77. The pharmaceutical composition of embodiment 75, wherein at least 30% of the adenine bases are modified.

78. The pharmaceutical composition of embodiment 75, wherein at least 40% of the adenine bases are modified.

79. The pharmaceutical composition of embodiment 75, wherein at least 50% of the adenine bases are modified.

80. The pharmaceutical composition of embodiment 75, wherein at least 60% of the adenine bases are modified.

81. The pharmaceutical composition of embodiment 75, wherein at least 70% of the adenine bases are modified.

82. The pharmaceutical composition of embodiment 75, wherein at least 80% of the adenine bases are modified.

83. The pharmaceutical composition of embodiment 75, wherein at least 90% of the adenine bases are modified.

84. The pharmaceutical composition of embodiment 75, wherein at least 100% of the adenine bases are modified.

85. The pharmaceutical composition of embodiment 74, wherein the base is guanine.

86. The pharmaceutical composition of embodiment 85, wherein at least 20% of the guanine bases are modified.

87. The pharmaceutical composition of embodiment 85, wherein at least 30% of the guanine bases are modified.

88. The pharmaceutical composition of embodiment 85, wherein at least 40% of the guanine bases are modified.

89. The pharmaceutical composition of embodiment 85, wherein at least 50% of the guanine bases are modified.

90. The pharmaceutical composition of embodiment 85, wherein at least 60% of the guanine bases are modified.

91. The pharmaceutical composition of embodiment 85, wherein at least 70% of the guanine bases are modified.

92. The pharmaceutical composition of embodiment 85, wherein at least 80% of the guanine bases are modified.

93. The pharmaceutical composition of embodiment 85, wherein at least 90% of the guanine bases are modified.

94. The pharmaceutical composition of embodiment 85, wherein at least 100% of the guanine bases are modified.

95. The pharmaceutical composition of embodiment 74, wherein the base is cytosine.

96. The pharmaceutical composition of embodiment 95, wherein at least 20% of the cytosine bases are modified.

97. The pharmaceutical composition of embodiment 95, wherein at least 30% of the cytosine bases are modified.

98. The pharmaceutical composition of embodiment 95, wherein at least 40% of the cytosine bases are modified.

99. The pharmaceutical composition of embodiment 95, wherein at least 50% of the cytosine bases are modified.

100. The pharmaceutical composition of embodiment 95, wherein at least 60% of the cytosine bases are modified.

101. The pharmaceutical composition of embodiment 95, wherein at least 70% of the cytosine bases are modified.

102. The pharmaceutical composition of embodiment 95, wherein at least 80% of the cytosine bases are modified.

103. The pharmaceutical composition of embodiment 95, wherein at least 90% of the cytosine bases are modified.

104. The pharmaceutical composition of embodiment 95, wherein at least 100% of the cytosine bases are modified.

105. The pharmaceutical composition of embodiment 74, wherein the base is uracil.

106. The pharmaceutical composition of embodiment 105, wherein at least 20% of uracil bases are modified.

107. The pharmaceutical composition of embodiment 105, wherein at least 30% of uracil bases are modified.

108. The pharmaceutical composition of embodiment 105, wherein at least 40% of uracil bases are modified.

109. The pharmaceutical composition of embodiment 105, wherein at least 50% of uracil bases are modified.

110. The pharmaceutical composition of embodiment 105, wherein at least 60% of uracil bases are modified.

111. The pharmaceutical composition of embodiment 105, wherein at least 70% of uracil bases are modified.

112. The pharmaceutical composition of embodiment 105, wherein at least 80% of uracil bases are modified.

113. The pharmaceutical composition of embodiment 105, wherein at least 90% of uracil bases are modified.

114. The pharmaceutical composition of embodiment 105, wherein at least 100% of uracil bases are modified.

115. The pharmaceutical composition of any of embodiments 64-114, wherein the at least one modification is pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl uridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetyl cytidine, 5-formyl cytidine, N4-methylcytidine, 5-hydroxymethyl cytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, zebralin, 5-aza-zebralin, 5-methyl-zebralin, 5-aza-2-thio-zebralin, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1-methyladenosine, N6-isopentenyl adenosine, N6- (cis-hydroxyisopentenyl) adenosine, 2-methylthio-N6- (cis-hydroxyisopentenyl) adenosine, N6-glycylcarbamoyl adenosine, N6-threonyl carbamoyl adenosine, 2-methylthio-N6-threonyl carbamoyl adenosine, N6, N6-dimethyl adenosine, 7-methyl adenine, 2-methylsulfanyl-adenine, and 2-methoxy-adenine, inosine, 1-methyl-inosine, hudroside, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl guanosine, N2-dimethyl guanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine or N2, N2-dimethyl-6-thio-guanosine.

116. The pharmaceutical composition of embodiment 1, further comprising at least one cationic lipid selected from the group consisting of: any lipid in table (I), any lipid having the structure of formula (CY-II), any lipid having the structure of formula (CY-III), any lipid having the structure of formula (CY-IV), and combinations thereof.

117. The pharmaceutical composition of embodiment 116, wherein the cationic lipid is any lipid having the structure of formula (CY-I).

118. The pharmaceutical composition of embodiment 117, wherein the cationic lipid is selected from the group consisting of compounds CY1, CY2, CY3, CY9, CY10, CY11, CY12, CY22, CY23, CY24, CY30, CY31, CY32, CY33, CY43, CY44, CY45, CY50, CY51, CY52, and CY 53.

119. The pharmaceutical composition of embodiment 116, wherein the cationic lipid is any lipid having the structure of formula (CY-II).

120. The pharmaceutical composition of embodiment 119, wherein the cationic lipid is selected from the group consisting of compounds CY4, CY5, CY16, CY17, CY18, CY25, CY26, CY37, CY38, CY39, CY46, CY56, and CY 57.

121. The pharmaceutical composition of embodiment 116, wherein the cationic lipid is any lipid having the structure of formula (CY-III).

122. The pharmaceutical composition of embodiment 121, wherein the cationic lipid is selected from the group consisting of compounds CY6, CY14, CY27, CY35, CY47, and CY 55.

123. The pharmaceutical composition of embodiment 116, wherein the cationic lipid is any lipid having the structure of formula (CY-IV).

124. The pharmaceutical composition of embodiment 123, wherein the cationic lipid is selected from the group consisting of compounds CY7, CY8, CY19, CY20, CY21, CY28, CY29, CY40, CY41, CY42, CY48, CY49, CY58, CY59, and CY 60.

125. The pharmaceutical composition of any one of embodiments 116-124, further comprising an additional cationic lipid.

126. The pharmaceutical composition of any one of embodiments 116-125, further comprising a neutral lipid.

127. The pharmaceutical composition of any one of embodiments 116-126, further comprising an anionic lipid.

128. The pharmaceutical composition of any one of embodiments 116-127, further comprising a helper lipid.

129. The pharmaceutical composition of any one of embodiments 116-128, further comprising a stealth lipid.

130. The pharmaceutical composition of any one of embodiments 116-129, wherein the weight ratio of the lipid to the polynucleotide is about 100:1 to about 1:1.

131. A vaccine formulation comprising the pharmaceutical composition of any one of embodiments 1-130.

132. A vaccine formulation comprising the pharmaceutical composition of any one of embodiments 116-130.

133. A method of vaccinating a subject against an infectious agent, the method comprising:

a) Contacting a subject with a vaccine formulation as described in embodiment 131 or a vaccine formulation as described in embodiment 132, and

B) An immune response is elicited.

134. The method of embodiment 133, wherein the infectious agent is jejunum, clostridium difficile, endo-amoeba histolyticum, enterotoxin B, norwalk or norovirus, helicobacter pylori, rotavirus, candida yeast, coronavirus (including SARS-CoV, SARS-CoV-2 and MERS-CoV), enterovirus 71, epstein-barr virus, gram negative bacteria (including bordetella), gram positive bacteria (including clostridium tetani, francis, streptococcus and staphylococcus), and hepatitis, human cytomegalovirus, human immunodeficiency virus, human papilloma virus, influenza virus, JC virus, mycobacteria, poxvirus, pseudomonas aeruginosa, respiratory syncytial virus, rubella virus, varicella zoster virus, kukken's virus, rabies virus, trypanosoma and/or chagas virus, ebola virus, malignant river virus, marburg virus, encephalitis virus, lewis toxin, shigella virus, shigella, or shiga virus.

135. The method of embodiment 133, wherein the contacting is enteral (into the intestines), gastrointestinal tract, epidural (into the dura mater), oral (by way of the oral cavity), transdermal, intracerebral (into the brain), intraventricular (into the ventricle), epicutaneous (coated onto the skin), intradermal (into the skin itself), subcutaneous (under the skin), nasal administration (via the nose), intravenous (into the vein), intravenous bolus, intravenous instillation, intra-arterial (into the artery), intramuscular (into the muscle), intracardiac (into the heart), intra-osseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraparenchymal (into the brain tissue), intracorporal (into the brain tissue), Intraperitoneal (infusion or injection into the peritoneum), intravesical infusion, intravitreal (via eye), intracavernosal injection (into the pathological cavity), intracavitary (into the fundus of the penis), intravaginal administration, intrauterine, extraamniotic administration, transdermal (via intact skin diffusion for systemic distribution), transmucosal (via mucosal diffusion), transvaginal, insufflation (sniffing), sublingual, subccheilial, enema, eye drops (onto the conjunctiva), ear drops, ear (in the ear or with the aid of the ear), cheek (directed towards the cheek), conjunctiva, skin, teeth (to one or more teeth), electroosmosis, intracervical, dou Daona, intratracheal, extracorporeal, external, Hemodialysis, infiltration, interstitial, intraabdominal, intra-amniotic, intra-articular, intrabiliary, intrabronchial, intracapsular, intracartilaginous (in cartilage), caudal (in the caudal horse), intracisternal (in the greater cisterna spinosa), intracorneal (in the cornea), intracoronary (in the coronary artery), intracavernosal (in the inflatable space of the corpora cavernosa), intradiscal (in the intervertebral disc), intraductal (in the gland duct), intraduodenal (in the duodenum), intradural (in or under the dura mater), intracutaneous (to the epidermis), intraesophageal (to the esophagus), intragastric (in the stomach), intragingival (in the gingiva), intraduodenal (in the gingiva), the ileum (in the distal part of the small intestine), intralesional (in the local focus or directly introduced to the local focus), intraluminal (in the lumen), intralymphatic (in the lymph), intramedullary (in the bone marrow cavity of the bone), meningeal (in the meninges), intramyocardial (in the myocardium), intraocular (in the eye), ovarian (in the ovary), pericardial (in the pericardium), intrapleural (in the pleura), prostatic (in the prostate gland), pulmonary (in the lung or bronchi thereof), sinus (in the nasal or periorbital sinuses), spinal (in the spinal column), synovial (in the synovial cavity of the joint), tendinous (in the tendon), and the like, Intrathecal (within the testis), intrathecal (within the cerebrospinal fluid at any level of the cerebrospinal shaft), intrathoracic (within the chest), intratubular (within the tubules of the organ), intratumoral (within the tumor), intrathecal (within the middle ear), intravascular (within one or more blood vessels), intraventricular (within the chamber), iontophoretic (by means of an electric current, wherein ions of soluble salts migrate into the tissues of the body), lavage (flushing or rinsing open wounds or body cavities), laryngeal (directly behind the larynx), nasogastric (via the nose and into the stomach), occlusive dressing techniques (topical route application which is then covered by a dressing closing the area), ophthalmic (to the outside of the eye), parenteral (to the outside of the eye), Oropharynx (directly to the mouth and pharynx), parenteral, transdermal, periarticular, epidural, peri-nerve, periodontal, transrectal, respiratory (by oral or nasal inhalation into the respiratory tract for local or systemic effects), retrobulbar (postcerebral or postocular), soft tissue, subarachnoid, subconjunctival, submucosal, superficial, transplacental (through or across the placenta), transtracheal (through the tracheal wall), transtympanic (across or through the tympanic cavity), ureter (to the ureter), urethra (to the urethra), transvaginal, tail block, diagnosis, nerve block, biliary tract perfusion, heart perfusion, photopheresis, or spinal column.

136. A method of delivering a polynucleotide encoding at least one protein of interest into an immune cell of a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of any one of embodiments 1-130.

137. The method of embodiment 136, wherein the immune cell is a T cell.

138. The method of embodiment 137, wherein said T cell is a cd8+ T cell.

139. The method of embodiment 137, wherein said T cell is a T regulatory cell.

140. The method of embodiment 137, wherein said T cell is a cd4+ T cell.

141. The method of embodiment 136, wherein the immune cell is a macrophage, dendritic cell, or liver immune cell.

Definition of X

As used herein, the term "compound" or "structure" is intended to include all stereoisomers, geometric isomers, tautomers and isotopes of the depicted structures.

The compounds or structures described herein may be asymmetric (e.g., have one or more stereocenters). Unless indicated otherwise, all stereoisomers, such as enantiomers and diastereomers, are contemplated. Compounds of the present disclosure containing asymmetrically substituted carbon atoms may be isolated in optically active or racemic forms. Methods of how to prepare optically active forms from optically active starting materials are known in the art, for example by resolution of racemic mixtures or stereoselective synthesis. Many geometric isomers of olefins, c=n double bonds, etc., may also be present in the compounds described herein, and all such stable isomers are encompassed by the present disclosure. The cis and trans geometric isomers of the compounds of the present disclosure are described and may be separated as mixtures of isomers or as separate isomeric forms.

The compounds or structures of the present disclosure also include tautomeric forms. Tautomeric forms result from the exchange of single bonds with adjacent double bonds and concomitant proton migration. Tautomeric forms include proton transfer tautomers in the isomerised protonated state of the same experimental formula and total charge. Examples of proton transfer tautomers include cyclic forms in which one keto-enol pair, amide-imide pair, lactam-lactam pair, amide-imide pair, enamine-imide pair, and one proton can occupy two or more positions of the heterocyclic system, such as 1H-and 3H-imidazole, 1H-, 2H-and 4H-1,2, 4-triazole, 1H-and 2H-isoindole, and 1H-and 2H-pyrazole. Tautomeric forms may be in equilibrium or sterically locked into one form by appropriate substitution.

The compounds or structures of the present disclosure also include all isotopes of atoms present in the intermediates or final compounds. "isotope" refers to atoms having the same atomic number but different mass numbers due to the different numbers of neutrons in the nuclei. Isotopes of hydrogen include, for example, tritium and deuterium.

The compounds or structures and salts of the present disclosure can be prepared by conventional methods in combination with solvents or water molecules to form solvates and hydrates.

The term "alkyl" refers to groups of saturated aliphatic groups, including straight chain alkyl, branched alkyl, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl, and cycloalkyl substituted alkyl groups.

In some embodiments, the linear or branched alkyl group has 30 or fewer carbon atoms in its backbone (e.g., for linear, C ₁-C₃₀; for branched, C ₃-C₃₀), 20 or fewer, 12 or fewer, or 7 or fewer. Also, in some embodiments, cycloalkyl groups have 3 to 10 carbon atoms in their ring structure, for example 5, 6 or 7 carbons in the ring structure. The term "alkyl" (or "lower alkyl") as used throughout the specification, examples and claims is intended to include both "unsubstituted alkyl" and "substituted alkyl", the latter referring to an alkyl moiety in which hydrogen on one or more carbons of the hydrocarbon backbone is replaced by one or more substituents. Such substituents include, but are not limited to, halogen, hydroxy, carbonyl (e.g., carboxy, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (e.g., thioester, thioacetate, or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate (hosphinate), amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties.

As used herein, unless the number of carbons is otherwise specified, "lower alkyl" means an alkyl group as defined above but having one to ten carbons or one to six carbon atoms in the backbone structure. Likewise, "lower alkenyl" and "lower alkynyl" have similar chain lengths. In some embodiments, the alkyl is lower alkyl. In some embodiments, the substituent designated herein as alkyl is lower alkyl.

As used herein, the term "alkylene" refers to a divalent group that is a straight or branched chain alkyl group. In one embodiment, the alkylene is a divalent form of C _1-12 alkyl, i.e., C ₁-C₁₂ alkylene. In one embodiment, the alkylene is a divalent form of C _2-6 alkyl, i.e., C ₁-C₁₀ alkylene. In one embodiment, the alkylene is a divalent form of C _2-14 alkyl, i.e., C ₁-C₈ alkylene. In one embodiment, the alkylene is a divalent form of unsubstituted C _1-6 alkyl, i.e., C ₁-C₆ alkylene. In another embodiment, the alkylene is a divalent form of unsubstituted C _1-4 alkyl, i.e., C ₁-C₄ alkylene. Non-limiting exemplary alkylene groups include -CH₂-、-CH₂CH₂-、-CH₂CH₂CH₂-、-CH₂CH(CH₃)CH₂-、-CH₂(CH₂)₂CH₂-、-CH(CH₂)₃CH₂- and-CH ₂(CH₂)₄CH₂ -.

As used herein, the term "cycloalkylene" refers to a divalent radical of a cycloalkyl group. In one embodiment, the cycloalkylene is a divalent form of C _3-8 cycloalkyl, i.e., C ₃-C₈ cycloalkylene. Non-limiting exemplary cycloalkylene groups include:

It will be appreciated by those skilled in the art that the substituted moiety on the hydrocarbon chain may itself be substituted as appropriate. For example, substituents of substituted alkyl groups can include halogen, hydroxy, nitro, thiol, amino, azido, imino, amide, phosphoryl (including phosphonates and phosphonites), sulfonyl (including sulfates, sulfonamides, sulfamoyl and sulfonates), and silyl groups, as well as ethers, alkylthio, carbonyl (including ketones, aldehydes, carboxylates, and esters), -CF ₃, -CN, and the like. Cycloalkyl groups may be substituted in the same manner.

As used herein, the term "heteroalkyl" refers to a straight or branched or cyclic carbon-containing group containing at least one heteroatom, or a combination thereof. Suitable heteroatoms include, but are not limited to O, N, si, P, se, B and S, wherein the phosphorus and sulfur atoms are optionally oxidized and the nitrogen heteroatom is optionally quaternized. Heteroalkyl may be substituted as defined above with respect to alkyl.

The term "alkylthio" refers to an alkyl group as defined above attached to a thio group. In some embodiments, the "alkylthio" moiety is represented by one of-S-alkyl, -S-alkenyl, and-S-alkynyl. Representative alkylthio groups include methylthio and ethylthio. The term "alkylthio" also encompasses cycloalkyl, alkene and cycloalkenyl groups, and alkyne groups. "arylthio" refers to aryl or heteroaryl. Alkylthio groups may be substituted as defined above for alkyl groups.

The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups similar in length and possible substitution to the alkyl groups described above, but containing at least one double or triple bond, respectively. In one embodiment, the alkenyl group contains one double bond. In another embodiment, the alkenyl group contains two double bonds. In another embodiment, the alkenyl group contains three double bonds.

As used herein, the term "alkenylene" refers to a divalent group of an alkenyl group. In one embodiment, the alkenylene group is a divalent form of C _2-12 alkenyl, i.e., C ₂-C₁₂ alkenylene. In one embodiment, the alkenylene group is a divalent form of C _2-6 alkenyl, i.e., C ₂-C₁₀ alkenylene. In one embodiment, the alkenylene group is a divalent form of C _2-14 alkenyl, i.e., C ₂-C₈ alkenylene. In one embodiment, the alkylene is a divalent form of unsubstituted C _2-6 alkenyl, i.e., C ₂-C₆ alkenylene. In another embodiment, the alkylene is a divalent form of unsubstituted C _2-4 alkyl, i.e., C ₂-C₄ alkenylene. Non-limiting exemplary alkenylene groups include-ch=ch-, -CH ₂CH＝CH-、-CH₂CH₂CH＝CHCH₂ -, and-CH ₂CH＝CHCH₂CH＝CHCH₂CH₂ -.

As used herein, the term "alkoxy" refers to an alkyl group as defined above attached to an oxy group. Representative alkoxy groups include methoxy, ethoxy, propoxy, and t-butoxy. An "ether" is two hydrocarbons covalently linked by oxygen. Thus, the substituent of the alkyl group that makes the alkyl group an ether is or is analogous to an alkoxy group, such as may be represented by one of-O-alkyl, -O-alkenyl, and-O-alkynyl. Aryloxy may be represented by-O-aryl or O-heteroaryl, wherein aryl and heteroaryl are defined below. Alkoxy and aryloxy groups may be substituted as described above for alkyl groups.

The terms "amine" and "amino" are art-recognized and refer to unsubstituted and substituted amines, such as moieties that may be represented by the general formula:

wherein R ₉、R₁₀ and R' ₁₀ each independently represent hydrogen, alkyl, alkenyl, - (CH ₂)_m-R₈), or R ₉ and R ₁₀ together with the N atom to which they are attached complete a heterocyclic ring having 4 to 8 atoms in the ring structure, R ₈ represents aryl, cycloalkyl, cycloalkenyl, heterocyclic or polycyclic, and m is zero or an integer in the range of 1 to 8.

The term "amide" is well known in the art as carbonyl substituted with an amino group and includes moieties that may be represented by the general formula:

Wherein R ₉ and R ₁₀ are as defined above.

As used herein, "aryl" refers to a C ₅-C₁₀ membered aromatic, heterocyclic, fused aromatic, fused heterocyclic, biaromatic, or bi-heterocyclic ring system. As used herein, "aryl" is broadly defined to include 5, 6,7, 8, 9, and 10 membered monocyclic aromatic groups, which may include from zero to four heteroatoms, such as benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles" or "heteroaromatics". The aromatic ring may be substituted at one or more ring positions with one or more substituents including, but not limited to: halogen, azido, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, alkoxy, amino (or quaternized amino), nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moiety, -CF ₃, -CN; and combinations thereof.

The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are shared by two adjoining rings (i.e., a "fused ring"), in which at least one ring is an aromatic ring, examples of heterocycles include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothienyl, benzoxazolyl, benzoxazolinyl, benzothiazolyl, benzotriazole, benzothiazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromen, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro [2,3-b ] tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl indolizinyl, indolyl, 3H-indolyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2, 3-oxadiazolyl, 1,2, 4-oxadiazolyl, 1,2, 5-oxadiazolyl, 1,3, 4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, phenanthridinyl, piperazinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridoxazol, pyridoimidazole, pyridothiazole, pyridinyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2, 5-thiadiazinyl, 1,2, 3-thiadiazolyl, 1,2, 4-thiadiazolyl, 1,2, 5-thiadiazolyl, 1,3, 4-thiadiazolyl, thienyl, thiazolyl, thienyl thiazolyl, thienothiazolyl, thienyl and oxaanthracenyl. One or more of the rings may be substituted as defined above with respect to "aryl".

As used herein, the term "aralkyl" refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).

As used herein, the term "carbocycle" refers to an aromatic or non-aromatic ring in which each atom of the ring is carbon.

As used herein, "heterocycle (Heterocycle/heterocyclics)" refers to a cyclic group attached via a monocyclic or bicyclic ring carbon or nitrogen, containing 3-10 ring atoms, e.g., 5-6 ring atoms, consisting of carbon and one to four heteroatoms each selected from the group consisting of: non-peroxy, thio and N (Y), wherein Y is absent or H, O, (C ₁-C₁₀) alkyl, phenyl or benzyl, and optionally contains 1 to 3 double bonds and is optionally substituted with one or more substituents. Examples of heterocycles include, but are not limited to, benzimidazolyl, benzofuranyl, benzothienyl, benzoxazolyl, benzoxazolinyl, benzothiazolyl, benzotriazole, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4 aH-carbazolyl, carbolinyl, chromanyl, chromene, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro [2,3-b ] tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolinyl, 3H-indolyl, isatoiyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolyl, oxadiazolyl, 1,2, 3-oxadiazolyl, 1,2, 4-oxadiazolyl, 1,2, 5-oxadiazolyl, 1,3, 4-oxadiazolyl, oxazolidinyl, oxazolyl, oxacycloheptyl, oxetanyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, oxazolyl, and the like, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridoxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolyl, tetrahydropyranyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2, 5-thiadiazinyl, 1,2, 3-thiadiazinyl, 1,2, 4-thiadiazinyl, 1,2, 5-thiadiazinyl, 1,3, 4-thiadiazinyl, Thienyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thienyl and oxaanthracyl. The heterocyclyl may be as defined above with respect to alkyl and aryl groups, optionally substituted at one or more positions with one or more substituents, such as halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF3 and-CN.

The term "carbonyl" is art recognized and includes moieties that may be represented, for example, by the general formula:

Wherein X is a bond or represents oxygen or sulfur, and R ₁₁ represents hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, or alkynyl, R' ₁₁ represents hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, or alkynyl. In the case where X is oxygen and R ₁₁ or R' ₁₁ is not hydrogen, the formula represents an "ester". Where X is oxygen and R ₁₁ is as defined above, the moiety is referred to herein as a carboxyl group, and in particular when R ₁₁ is hydrogen, the formula represents a "carboxylic acid". In the case where X is oxygen and R' ₁₁ is hydrogen, the formula represents a "formate". In general, where an oxygen atom of the above formula is replaced with sulfur, the formula represents "thiocarbonyl". In the case where X is sulfur and R ₁₁ or R' ₁₁ is not hydrogen, the formula represents a "thioester". In the case where X is sulfur and R ₁₁ is hydrogen, the formula represents "thiocarboxylic acid". In the case where X is sulfur and R' ₁₁ is hydrogen, the formula represents "thioformate". On the other hand, in the case where X is a bond and R ₁₁ is not hydrogen, the above formula represents a "ketone" group. In the case where X is a bond and R ₁₁ is hydrogen, the above formula represents an "aldehyde" group.

As used herein, the term "heteroatom" means an atom of any element other than carbon or hydrogen. Examples of heteroatoms are boron, nitrogen, oxygen, phosphorus, sulfur and selenium. Other suitable heteroatoms include silicon and arsenic.

As used herein, the term "nitro" means-NO ₂; the term "halogen" refers to-F, -Cl, -Br or-I; the term "sulfhydryl" means-SH; the term "hydroxy" means-OH; and the term "sulfonyl" means-SO ₂ -.

As used herein, the term "substituted" refers to all permissible substituents of compounds described herein. In the broadest sense, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, but are not limited to, halogen, hydroxy, or any other organic group containing any number of carbon atoms (e.g., 1-14 carbon atoms), and optionally include one or more heteroatoms, such as oxygen, sulfur, or nitrogen groupings, in a linear, branched, or cyclic structural form. Representative substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxy, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aryloxy, substituted aryloxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl, C ₃-C₂₀ cyclic groups, substituted C ₃-C₂₀ cyclic groups, heterocyclic, substituted heterocyclic, amino acids, peptides and polypeptide groups.

As described herein, the compounds of the present disclosure may contain an "optionally substituted" moiety. In general, the term "substituted", whether preceded by the term "optionally" or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have suitable substituents at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituents at each position may be the same or different. The combinations of substituents contemplated by the present disclosure are preferably those that result in the formation of stable or chemically feasible compounds. As used herein, the term "stable" means that a compound does not substantially change when subjected to conditions that allow it to be produced, detected, and (in certain embodiments) recovered, purified, and used for one or more of the purposes disclosed herein.

Suitable monovalent substituents on the substitutable carbon atom of an "optionally substituted" group are independently: halogen ;-(CH₂)_0-4R^○;-(CH₂)_0-4OR^○;-O(CH₂)_0-4R^○,-O-(CH₂)_0-4C(O)OR^○;-(CH₂)_0-4CH(OR^○)₂;-(CH₂)_0-4SR^○;-(CH₂)_0-4Ph, which may be substituted by R ^○; - (CH ₂)_0-4O(CH₂)_0-1 Ph, which may be substituted by R ^○, -CH=CHPh, which may be substituted by R ^○, -CH ₂)_0-4O(CH₂)_0-1 -pyridyl, which may be substituted by R ^○ for ;-NO₂;-CN;-N₃;-(CH₂)_0-4N(R^○)₂;-(CH₂)_0-4N(R^○)C(O)R^○;-N(R^○)C(S)R^○;-(CH₂)_0-4N(R^○)C(O)NR^○ ₂;-N(R^○)C(S)NR^○ ₂;-(CH₂)_0-4N(R^○)C(O)OR^○;-N(R^○)N(R^○)C(O)R^○;-N(R^○)N(R^○)C(O)NR^○ ₂;-N(R^○)N(R^○)C(O)OR^○;-(CH₂)_0-4C(O)R^○;-C(S)R^○;-(CH₂)_0-4C(O)OR^○;-(CH₂)_0-4C(O)SR^○;-(CH₂)_0-4C(O)OSiR^○ ₃;-(CH₂)_0-4OC(O)R^○;-OC(O)(CH₂)_0-4SR^○,SC(S)SR^○;-(CH₂)_0-4SC(O)R^○;-(CH₂)_0-4C(O)NR^○ ₂;-C(S)NR^○ ₂;-C(S)SR^○;-SC(S)SR^○,-(CH₂)_0-4OC(O)NR^○ ₂;-C(O)N(OR^○)R^○;-C(O)C(O)R^○;-C(O)CH₂C(O)R^○;-C(NOR^○)R^○;-(CH₂)_0-4SSR^○;-(CH₂)_0-4S(O)₂R^○;-(CH₂)_0-4S(O)₂OR^○;-(CH₂)_0-4OS(O)₂R^○;-S(O)₂NR^○ ₂;-(CH₂)_0-4S(O)R^○;-N(R^○)S(O)₂NR^○ ₂;-N(R^○)S(O)₂R^○;-N(OR^○)R^○;-C(NH)NR^○ ₂;-P(O)₂R^○;-P(O)R^○ ₂;-OP(O)R^○ ₂;-OP(O)(OR^○)₂;SiR^○ ₃;-(C_1-4 straight or branched chain alkylene) O-N (R ^○)₂, or- (C _1-4 straight or branched chain alkylene) C (O) O-N (R ^○)₂, wherein each R ^○ may be substituted as defined below and is independently hydrogen, a C _1-6 aliphatic, -CH ₂Ph、-O(CH₂)_0-1Ph、-CH₂ - (5-6 membered heteroaromatic ring) or a 5-6 membered saturated, partially unsaturated or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, or, irrespective of the above definition, two independently occurring R ^○ together with their intermediate atoms form a 3-12 membered saturated, partially unsaturated or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, which may be substituted as defined below.

Suitable monovalent substituents on R ^○ (or the ring formed by joining two independently occurring R ^○ together with their intervening atoms) are independently: halogen, - (CH ₂)_0-2R^●, - (halo R^●)、-(CH₂)_0-2OH、-(CH₂)_0-2OR^●、-(CH₂)_0-2CH(OR^●)₂;-O( halo R^●)、-CN、-N₃、-(CH₂)_0-2C(O)R^●、-(CH₂)_0-2C(O)OH、-(CH₂)_0-2C(O)OR^●、-(CH₂)_0-2SR^●、-(CH₂)_0-2SH、-(CH₂)_0-2NH₂、-(CH₂)_0-2NHR^●、-(CH₂)_0-2NR^● ₂、-NO₂、-SiR^● ₃、-OSiR^● ₃、-C(O)SR^●、-(C_1-4 linear OR branched alkylene) C (O) OR ^●, OR-SSR ^●, wherein each R ^● is unsubstituted, OR is substituted with only one OR more halogens when previously "halo", and is independently selected from C _1-4 aliphatic, -CH ₂Ph、-O(CH₂)_0-1 Ph, OR a 5-to 6-membered saturated, partially unsaturated ring, OR aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur suitable divalent substituents on saturated carbon atoms of R ^○ include =o and =s.

Suitable divalent substituents on saturated carbon atoms of an "optionally substituted" group include ：＝O、＝S、＝NNR*₂、＝NNHC(O)R*、＝NNHC(O)OR*、＝NNHS(O)₂R*、＝NR*、＝NOR*、-O(C(R*₂))_2-3O- or-S (C (R x ₂))_2-3 S-, wherein each independently occurring R is selected from hydrogen; a substituted C _1-6 aliphatic group, which may be defined below; or an unsubstituted 5-6 membered saturated, partially unsaturated ring or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur suitable divalent substituents bonded to adjacent substitutable carbon of an "optionally substituted" group include-O (CR ^* ₂)_2-3 O-, wherein each independently occurring R ^* is selected from hydrogen; a substituted C _1-6 aliphatic group, which may be defined below; or an unsubstituted 5-6 membered saturated, partially unsaturated ring or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur).

Suitable substituents on the aliphatic groups of R include halogen, -R ^●, - (halo R ^●)、-OH、-OR^●, -O (halo R ^●)、-CN、-C(O)OH、-C(O)OR^●、-NH₂、-NHR^●、-NR^● ₂ or-NO ₂, wherein each R ^● is unsubstituted or when preceded by "halo" is substituted with one or more halogens only and is independently a C _1-4 aliphatic, -CH ₂Ph、-O(CH₂)_0-1 Ph, or a 5-6 membered saturated, partially unsaturated or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

Suitable substituents on the substitutable nitrogen of an "optionally substituted" group include OrWherein eachIndependently hydrogen; a C _1-6 aliphatic group which may be substituted as defined below; unsubstituted-OPh; or an unsubstituted 5-6 membered saturated, partially unsaturated or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, or two independently occurring/>, regardless of the definition aboveTogether with the intervening atoms thereof form an unsubstituted 3-12 membered saturated, partially unsaturated ring or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

Suitable substituents on the aliphatic groups of (a) include halogen, -R ^●, - (halo R ^●)、-OH、-OR^●, -O (halo R ^●)、-CN、-C(O)OH、-C(O)OR^●、-NH₂、-NHR^●、-NR^● ₂ or-NO ₂) wherein each R ^● is unsubstituted or, when preceded by a "halo", is substituted with one or more halogens only and is independently a C _1-4 aliphatic, -CH ₂Ph、-O(CH₂)_0-1 Ph, or a 5-to 6-membered saturated, partially unsaturated or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

Heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. It is to be understood that "substitution" or "substituted" includes implicit restrictions, such substitution being in accordance with the permissible valence of the substituted atom and substituent, and that the substitution results in a stable compound, i.e., the compound does not spontaneously undergo conversion, such as by rearrangement, cyclization, or elimination.

In one broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, substituents described herein. For suitable organic compounds, the permissible substituents can be one or more and the same or different. Heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms.

In various embodiments, the substituents are selected from the group consisting of alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, aralkyl, carbamate, carboxyl, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxy, ketone, nitro, phosphate, thioether, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thione, each of which is optionally substituted with one or more suitable substituents. In some embodiments, the substituents are selected from the group consisting of alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, aralkyl, carbamate, carboxyl, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl, heterocyclyl, ketone, phosphate, thioether, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone, wherein each of the alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, aralkyl, carbamate, carboxyl, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl, heterocyclyl, ketone, phosphate, thioether, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone may be further substituted with one or more suitable substituents.

Examples of substituents include, but are not limited to, halogen, azido, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, alkoxy, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, thione, ester, heterocyclyl, -CN, aryl, aryloxy, perhaloalkoxy, aralkoxy, heteroaryl, heteroaryloxy, heteroarylalkyl, heteroarylalkoxy, azido, alkylthio, oxo, acylalkyl, carboxyl ester, carboxamide, acyloxy, aminoalkyl, alkylamino aryl, alkylaryl, alkylamino alkyl, alkoxyaryl, arylamino, aralkylamino, alkylsulfonyl, formaminoalkylaryl, formaminoaryl, hydroxyalkyl, haloalkyl, alkylaminoalkylcarboxy, carbamoylalkyl, cyano, alkoxyalkyl, perhaloalkyl, aralkoxyalkyl, and the like. In some embodiments, the substituents are selected from cyano, halogen, hydroxy, and nitro.

Antibody: as used herein, the term "antibody" refers in its broadest sense and specifically covers various embodiments, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies formed from at least two intact antibodies), and antibody fragments (e.g., bifunctional antibodies), so long as they exhibit the desired biological activity (e.g., "functionality"). Antibodies are predominantly amino acid-based molecules, but may also comprise one or more modifications (including, but not limited to, addition of sugar moieties, fluorescent moieties, chemical tags, etc.) non-limiting examples of antibodies or fragments thereof include VH and VL domains, scFv, fab, fab ', F (ab') 2, fv fragments, bifunctional antibodies, linear antibodies, single chain antibody molecules, multispecific antibodies, bispecific antibodies, intracellular antibodies, monoclonal antibodies, polyclonal antibodies, humanized antibodies, codon-optimized antibodies, tandem scFv antibodies, bispecific T cell engaging molecules, mAb2 antibodies, chimeric Antigen Receptors (CARs), tetravalent bispecific antibodies, biosynthetic antibodies, protogenic antibodies, miniaturized antibodies, monoclonal antibodies, maxiantibodies, antibodies against senescent cells, antibodies against conformational isomers, antibodies against disease-specific epitopes, or antibodies against congenital defenses molecules.

Association (Associated): as used herein, the terms "associated with" … …, "conjugated," linked, "and" tethered, "when used with respect to two or more moieties, mean that the moieties are physically associated or linked to one another, either directly or via one or more additional moieties that act as linkers, to form a structure that is stable enough that the moieties remain physically associated under the conditions (e.g., physiological conditions) in which the structure is used. The "association" need not be strictly via direct covalent chemical bonding. It may also indicate that ionic or hydrogen bonding or hybridization-based connectivity is sufficiently stable that "associated" entities remain physically associated.

And (3) cargo: as used herein, the term "cargo (cargo)" or "payload" may refer to one or more molecules or structures encompassed in a delivery vehicle for delivery to or into a cell or tissue. Non-limiting examples of cargo may include nucleic acids, polypeptides, peptides, proteins, liposomes, tags, labels, small chemical molecules, large biological molecules, and any combinations thereof.

Chimeric Antigen Receptor (CAR): as used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial chimeric protein comprising at least one antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular signaling domain, wherein the antigen-specific targeting region comprises a full-length antibody or fragment thereof. Any molecule capable of binding with high affinity to the antigen of interest can be used in the ASTR of the CAR. The CAR may optionally have an extracellular spacer domain and/or a co-stimulatory domain. CARs may also be used to generate CAR-bearing cytotoxic cells.

Circular RNA: as used herein, the term "circular RNA" or "circRNA" refers to RNA that forms a circular structure via covalent or non-covalent bonds.

Co-administration: as used herein, the term "co-administration (co-ADMINISTERED/co-ADMINISTERING)" means that the initial construct, the reference construct, or the targeting system is administered with one or more additional initial constructs, reference constructs, targeting systems, or other therapeutic agents or moieties within a sufficiently close time so that the effect of the initial construct, reference construct, targeting system, or other therapeutic agent or moiety is enhanced.

Complementary and substantially complementary: as used herein, the term "complementary" refers to the ability of polynucleotides to form base pairs with each other. Base pairs are typically formed by hydrogen bonding between nucleotide units in antiparallel polynucleotide strands. The complementary polynucleotide strands may form base pairs in Watson-Crick fashion (e.g., A and T, A and U, C and G), or in any other manner that allows duplex formation. As known to those skilled in the art, uracil rather than thymine is a base that is considered complementary to adenosine when RNA is used instead of DNA. However, unless otherwise stated, when expressed as U in the context of the present disclosure, it is implied that T can be replaced. Perfect complementarity or 100% complementarity refers to the situation where each nucleotide unit of one polynucleotide strand may form a hydrogen bond with a nucleotide unit of a second polynucleotide strand. Sub-perfect complementarity refers to the situation where some, but not all, of the nucleotide units of two strands may form hydrogen bonds with each other. For example, for two 20 mers, a polynucleotide chain exhibits 10% complementarity if only two base pairs on each strand can form hydrogen bonds with each other. In the same example, a polynucleotide chain exhibits 90% complementarity if 18 base pairs on each strand can form hydrogen bonds with each other. As used herein, the term "substantially complementary" means that the sequence of the siRNA (e.g., in the antisense strand) is sufficient to bind to a desired target mRNA and trigger RNA silencing of said target mRNA.

Delivery: as used herein, "delivery" refers to the act or manner of delivering a compound, substance, entity, portion, cargo, or payload.

DNA and RNA: as used herein, the term "RNA" or "RNA molecule" or "ribonucleic acid molecule" refers to a ribonucleotide polymer; the term "DNA" or "DNA molecule" or "deoxyribonucleic acid molecule" refers to a polymer of deoxyribonucleotides. DNA and RNA can be naturally synthesized, for example, by DNA replication and DNA transcription, respectively; or chemical synthesis. DNA and RNA can be single-stranded (i.e., ssRNA or ssDNA, respectively) or multi-stranded (e.g., double-stranded, i.e., dsRNA and dsDNA, respectively). As used herein, the term "mRNA" or "messenger RNA" refers to a single stranded RNA encoding the amino acid sequence of one or more polypeptide chains.

Encapsulation: as used herein, the term "encapsulate" means to enclose, or encase.

Encoding: as used herein, the term "encode" broadly refers to any process whereby information in polymeric macromolecules is used to guide the production of a second molecule that is different from the first molecule. The second molecule may have a chemical structure that differs from the chemical nature of the first molecule.

Enhancing expression of genes: as used herein, the phrase "add-back" or "enhance expression of a gene" means to cause an increase in the amount of the expression product of the gene. The expression product may be RNA transcribed from a gene (e.g., mRNA) or a polypeptide translated from mRNA transcribed from a gene. In general, increased levels of mRNA cause increased levels of polypeptides translated therefrom. Expression levels may be determined using standard techniques for measuring mRNA or protein.

Exosomes: as used herein, an "exosome" is a vesicle secreted by a mammalian cell or complex involved in RNA degradation.

Preparation: as used herein, "formulation" includes at least one compound, substance, entity, moiety, cargo or payload and delivery agent.

Fragments: as used herein, "fragment" refers to a portion. For example, a fragment of a protein may comprise a polypeptide obtained by digestion of a full-length protein isolated from cultured cells.

Homology: as used herein, the term "homology" refers to the overall relatedness between polymeric molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymer molecules are considered "homologous" to each other if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar. The term "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). According to the present disclosure, two polynucleotide sequences are considered homologous if, for at least one extension having at least about 20 amino acids, the polypeptides encoded by them are at least about 50%, 60%, 70%, 80%, 90%, 95% or even 99% identical. In some embodiments, the homologous polynucleotide sequence is characterized by the ability to encode an extension of at least 4-5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode an extension of at least 4-5 uniquely specified amino acids. According to the present disclosure, two protein sequences are considered homologous if the two proteins are at least about 50%, 60%, 70%, 80% or 90% identical for at least one extension having at least about 20 amino acids.

Inactive ingredients: as used herein, the term "inactive ingredient" refers to one or more agents that have no effect on the activity of the active ingredient of the pharmaceutical composition contained in the formulation. In some embodiments, the inactive ingredients useful in the formulations of the present disclosure may be all, none, or some of which are approved by the U.S. Food and Drug Administration (FDA).

IRES: as used herein, the term "internal ribosome entry site" or "IRES" refers to an RNA sequence or structural element ranging in size from 10 nucleotides to 1,000 nucleotides or more that is capable of initiating translation of a polypeptide in the absence of a normal RNA cap structure.

Identity: as used herein, the term "identity" refers to the overall relatedness between polymer molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. For example, the calculation of the percent identity of two polynucleotide sequences may be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first nucleic acid sequence and the second nucleic acid sequence for optimal alignment and the different sequences may be ignored for comparison purposes). In certain embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of the length of the reference sequence. The nucleotides at the corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, which gaps need to be introduced in order to optimally align the two sequences, taking into account the number of gaps and the length of each gap. A mathematical algorithm may be used to achieve sequence comparison and percent identity determination between two sequences. For example, the percent identity between two nucleotide sequences can be determined using, for example, the methods described in: computational Molecular Biology, lesk, a.m. plaited, oxford University Press, new York,1988; Biocomputing: informatics and Genome Projects, smith, D.W. code ,Academic Press,New York,1993;Sequence Analysis in Molecular Biology,von Heinje,G.,Academic Press,1987;Computer Analysis of Sequence Data, section I, griffin, A.M. and Griffin, H.G. code, humana Press, new Jersey,1994; and Sequence ANALYSIS PRIMER, gribskov, m. and Devereux, j. editions, M stock Press, new York,1991; each of which is incorporated herein by reference. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17) which has been incorporated into the ALIGN program (version 2.0) using the PAM120 weight residue table, gap length penalty 12, and gap penalty 4. Alternatively, the percent identity between two nucleotide sequences may be determined using the GAP program in the GCG software package using the nwsgapdna. Methods commonly used to determine the percent identity between sequences include, but are not limited to, those disclosed in Carillo, h. And Lipman, d., SIAM J Applied mate, 48:1073 (1988), incorporated herein by reference. Techniques for determining identity are encoded in publicly available computer programs. Exemplary computer software for determining homology between two sequences includes, but is not limited to, the GCG program package (Devereux, J. Et al, nucleic ACIDS RESEARCH,12 (1), 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S.F. et al, J.molecular.biol., 215,403 (1990)).

Inhibition of gene expression: as used herein, the phrase "knock-down" or "inhibit the expression of a gene (inhibit expression of a gene)" means that the amount of the expression product of the gene is reduced. The expression product may be RNA transcribed from a gene (e.g., mRNA) or a polypeptide translated from mRNA transcribed from a gene. In general, a decrease in mRNA levels results in a decrease in the level of polypeptide translated therefrom. Expression levels may be determined using standard techniques for measuring mRNA or protein.

Ionizable lipids: as used herein, "ionizable lipid" refers to any of a variety of lipid materials that carry a net positive charge at a selected pH.

Lipid nanoparticles: as used herein, "lipid nanoparticle" or "LNP" refers to a delivery vehicle comprising one or more lipids (e.g., cationic lipids, non-cationic lipids, PEG-modified lipids).

Liposome: as used herein, "liposome" generally refers to a vesicle composed of lipids (e.g., amphiphilic lipids) arranged in one or more spherical bilayers.

Modified: as used herein, "modified" refers to a change in state or structure of a molecule. The molecule can be modified in a variety of ways, including chemically, structurally, and functionally modifying the molecule.

Non-cationic lipids: as used herein, "non-cationic lipid" refers to any neutral, zwitterionic, or anionic lipid.

Peptide: as used herein, a "peptide" is less than or equal to 50 amino acids in length, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

Pharmaceutical composition: as used herein, the term "pharmaceutical composition" refers to a composition comprising at least one active ingredient and optionally one or more pharmaceutically acceptable excipients.

PEG: as used herein, "PEG" means any polyethylene glycol or other polyalkylene ether polymer.

Spacer: as used herein, the term "spacer" refers to a region of a polynucleotide or polypeptide in a sequence ranging from 1 residue to hundreds or thousands of residues separating two other elements. The sequence of the spacer may be defined or random. The spacer sequence is typically a non-coding sequence, but may be a coding sequence.

Sterols: as used herein, "sterols (sterol)" are a subset of steroids consisting of steroids.

Structural lipids: as used herein, "structural lipids" refers to sterols and lipids containing sterol moieties.

Transcription: as used herein, the term "transcription" refers to the formation or synthesis of an RNA molecule by an RNA polymerase using a DNA molecule as a template.

Translation: as used herein, the term "translation" refers to the formation of a polypeptide molecule by ribosomes, based on an RNA template.

Treatment and prevention: as used herein, the term "treat" or "prevent" and the words derived therefrom do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. Furthermore, "preventing" may encompass delaying the onset of a disease, symptom or condition thereof.

Unmodified: as used herein, "unmodified" refers to any substance, compound, or molecule that has been previously altered in any way. Unmodified may refer to, but not always refers to, the wild-type or native form of the biomolecule. The molecules may undergo a series of modifications whereby each modified molecule may act as an "unmodified" starting molecule for subsequent modification.

And (3) a carrier: as used herein, a "vector" is any molecule or portion that transports, transduces, or otherwise acts as a carrier for a heterologous molecule. Vectors of the present disclosure may be recombinantly produced and may be based on and/or may comprise viral parent or reference sequences. Such parent or reference viral sequences may serve as the initial, second, third or subsequent sequences for engineering the vector. In non-limiting examples, such parent or reference viral sequences may comprise any one or more of the following sequences: a polynucleotide sequence encoding a polypeptide or polypeptides, which may be wild-type or modified compared to wild-type, and which may encode the full length or partial sequence of a protein, protein domain or one or more subunits of a protein; a polynucleotide comprising a regulatory or regulatory nucleic acid, which sequence may be wild-type or modified compared to wild-type; and transgenes that may or may not be modified compared to the wild-type sequence. These viral sequences may act as "donor" sequences of one or more codons (at the nucleic acid level) or amino acids (at the polypeptide level) or as "acceptor" sequences of one or more codons (at the nucleic acid level) or amino acids (at the polypeptide level). The details of one or more embodiments of the disclosure are set forth in the accompanying description below. Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred materials and methods are now described. Other features, objects, and advantages of the present disclosure will be apparent from the description. In the specification, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present specification will control.

XI particular embodiments

Embodiment 1. A compound having the structure of formula (CY-I):

Or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is-OH,

Wherein Z ¹ is optionally substituted C ₁-C₆ alkyl;

X ¹ is optionally substituted C ₂-C₆ alkylene;

X ² and X ³ are independently a bond, -CH ₂ -or-CH ₂CH₂ -;

y ¹ and Y ² are independently

And

R ² and R ³ are independently optionally substituted C ₄-C₂₀ alkyl.

Embodiment 2. A compound having the structure of formula (CY-II):

Or a pharmaceutically acceptable salt thereof, wherein:

r1 is-OH,

Wherein Z ¹ is optionally substituted C1-C6 alkyl;

X ¹ is optionally substituted C ₂-C₆ alkylene;

X ² and X ³ are independently a bond, -CH ₂ -or-CH ₂CH₂ -;

y ¹ and Y ² are independently

And

R ² and R ³ are independently optionally substituted C ₄-C₂₀ alkyl.

Embodiment 3. A compound having the structure of formula (CY-III):

Or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is-OH,

Wherein Z ¹ is optionally substituted C ₁-C₆ alkyl;

X ¹ is optionally substituted C ₂-C₆ alkylene;

X ² and X ³ are independently a bond, -CH ₂ -or-CH ₂CH₂ -;

y ¹ and Y ² are independently

And

R ² and R ³ are independently optionally substituted C ₄-C₂₀ alkyl.

Embodiment 4. A compound having the structure of formula (CY-IV):

Or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is-OH,

Wherein Z ¹ is optionally substituted C ₁-C₆ alkyl;

X ¹ is optionally substituted C ₂-C₆ alkylene;

X ² and X ³ are independently a bond, -CH ₂ -or-CH ₂CH₂ -;

y ¹ and Y ² are independently

And

R ² and R ³ are independently optionally substituted C ₄-C₂₀ alkyl.

Embodiment 5. A compound having the structure of formula (CY-I):

Or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is-OH, R ^1a,

Wherein Z ¹ is optionally substituted C ₁-C₆ alkyl;

X ¹ is optionally substituted C ₂-C₆ alkylene;

X ² and X ³ are independently a bond, -CH ₂ -or-CH ₂CH₂ -;

y ¹ and Y ² are independently

R ² and R ³ are independently optionally substituted C ₄-C₂₀ alkyl;

R ^1a is:

R ^2a、R^2b and R ^2c are independently hydrogen and C ₁-C₆ alkyl;

r ^3a、R^3b and R ^3c are independently hydrogen and C ₁-C₆ alkyl;

R ^4a、R^4b and R ^4c are independently hydrogen and C ₁-C₆ alkyl; and

R ^5a、R^5b and R ^5c are independently hydrogen and C ₁-C₆ alkyl.

Embodiment 6. The compound of embodiment 5 wherein R ¹ is-OH,

Embodiment 7. A compound having the structure of formula (CY-II):

Or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is-OH, R ^1a,

Wherein Z ¹ is optionally substituted C ₁-C₆ alkyl;

X ¹ is optionally substituted C ₂-C₆ alkylene;

X ² and X ³ are independently a bond, -CH ₂ -or-CH ₂CH₂ -;

y ¹ and Y ² are independently

R ² and R ³ are independently optionally substituted C ₄-C₂₀ alkyl;

R ^1a is:

R ^2a、R^2b and R ^2c are independently hydrogen and C ₁-C₆ alkyl;

r ^3a、R^3b and R ^3c are independently hydrogen and C ₁-C₆ alkyl;

R ^4a、R^4b and R ^4c are independently hydrogen and C ₁-C₆ alkyl; and

R ^5a、R^5b and R ^5c are independently hydrogen and C ₁-C₆ alkyl.

Embodiment 8. The compound of embodiment 7 wherein R ¹ is-OH,

Embodiment 9. A compound having the structure of formula (CY-III):

Or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is-OH, R ^1a,

Wherein Z ¹ is optionally substituted C ₁-C₆ alkyl;

X ¹ is optionally substituted C ₂-C₆ alkylene;

X ² and X ³ are independently a bond, -CH ₂ -or-CH ₂CH₂ -;

y ¹ and Y ² are independently

R ² and R ³ are independently optionally substituted C ₄-C₂₀ alkyl;

R ^1a is:

R ^2a、R^2b and R ^2c are independently hydrogen and C ₁-C₆ alkyl;

r ^3a、R^3b and R ^3c are independently hydrogen and C ₁-C₆ alkyl;

R ^4a、R^4b and R ^4c are hydrogen and C ₁-C₆ alkyl; and

R ^5a、R^5b and R ^5c are hydrogen and C ₁-C₆ alkyl.

Embodiment 10. The compound of embodiment 9 wherein R ¹ is-OH,

Embodiment 11. A compound having the structure of formula (CY-IV):

Or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is-OH, R ^1a,

Wherein Z ¹ is optionally substituted C ₁-C₆ alkyl;

X ¹ is optionally substituted C ₂-C₆ alkylene;

X ² and X ³ are independently a bond, -CH ₂ -or-CH ₂CH₂ -;

y ¹ and Y ² are independently

R ² and R ³ are independently optionally substituted C ₄-C₂₀ alkyl;

R ^1a is:

R ^2a、R^2b and R ^2c are independently hydrogen and C ₁-C₆ alkyl;

r ^3a、R^3b and R ^3c are independently hydrogen and C ₁-C₆ alkyl;

R ^4a、R^4b and R ^4c are independently hydrogen and C ₁-C₆ alkyl; and

R ^5a、R^5b and R ^5c are hydrogen and C ₁-C₆ alkyl.

Embodiment 12. The compound of embodiment 11 wherein R ¹ is-OH,

Embodiment 13. A compound having the structure of formula (CY-V):

Or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is-OH, R ^1a,

Wherein Z ¹ is optionally substituted C ₁-C₆ alkyl;

X ¹ is optionally substituted C ₂-C₆ alkylene;

y ¹ and Y ² are independently

X ⁶ and X ⁷ are independently-CH ₂ -or-CH ₂CH₂ -;

R ² and R ³ are independently optionally substituted C ₄-C₂₀ alkyl;

R ^1a is:

R ^2a、R^2b and R ^2c are independently hydrogen and C ₁-C₆ alkyl;

r ^3a、R^3b and R ^3c are independently hydrogen and C ₁-C₆ alkyl;

R ^4a、R^4b and R ^4c are hydrogen and C ₁-C₆ alkyl; and

R ^5a、R^5b and R ^5c are hydrogen and C ₁-C₆ alkyl.

Embodiment 14. The compound of embodiment 13 wherein R ¹ is-OH,

Embodiment 15. A compound having the structure of formula (CY-I):

Or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is-OH, R ^1a,

Z ¹ is optionally substituted C ₁-C₆ alkyl;

X ¹ is optionally substituted C ₂-C₆ alkylene;

X ² and X ³ are independently a bond, -CH ₂ -or-CH ₂CH₂ -;

y ¹ and Y ² are independently

Wherein the bond labeled with "+" is attached to X ⁴ or X ⁵;

Each Z ² is independently H or optionally substituted C ₁-C₈ alkyl;

Each Z ³ is independently optionally substituted C ₁-C₆ alkylene;

R ² is optionally substituted C ₄-C₂₀ alkyl, optionally substituted C ₂-C₁₄ alkenylene OR-CH (OR ⁶)(OR⁷);

R ³ is optionally substituted C ₄-C₂₀ alkyl, optionally substituted C ₂-C₁₄ alkenylene OR-CH (OR ⁸)(OR⁹);

R ^1a is:

R ^2a、R^2b and R ^2c are independently hydrogen and C ₁-C₆ alkyl;

r ^3a、R^3b and R ^3c are independently hydrogen and C ₁-C₆ alkyl;

R ^4a、R^4b and R ^4c are independently hydrogen and C ₁-C₆ alkyl;

R ^5a、R^5b and R ^5c are independently hydrogen and C ₁-C₆ alkyl;

A is C ₃-C₈ cycloalkylene;

Each m is independently 0,1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; and

Each n is independently 0, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, or 12.

Embodiment 16. The compound of embodiment 15 wherein:

R ¹ is-OH, R ^1a,

Wherein Z ¹ is optionally substituted C ₁-C₆ alkyl;

X ¹ is optionally substituted C ₂-C₆ alkylene;

X ² and X ³ are independently a bond, -CH ₂ -or-CH ₂CH₂ -;

y ¹ and Y ² are independently

R ² and R ³ are independently optionally substituted C ₄-C₂₀ alkyl;

R ^1a is:

R ^2a、R^2b and R ^2c are independently hydrogen and C ₁-C₆ alkyl;

r ^3a、R^3b and R ^3c are independently hydrogen and C ₁-C₆ alkyl;

R ^4a、R^4b and R ^4c are independently hydrogen and C ₁-C₆ alkyl; and

R ^5a、R^5b and R ^5c are independently hydrogen and C ₁-C₆ alkyl.

Embodiment 17. The compound of embodiment 16 wherein:

R ¹ is-OH, R ^1a,

Wherein Z ¹ is optionally substituted C ₁-C₆ alkyl;

X ¹ is optionally substituted C ₂-C₆ alkylene;

X ² and X ³ are independently a bond, -CH ₂ -or-CH ₂CH₂ -;

y ¹ and Y ² are independently

R ² and R ³ are independently optionally substituted C ₄-C₂₀ alkyl;

R ^1a is:

R ^2a、R^2b and R ^2c are independently hydrogen and C ₁-C₆ alkyl;

r ^3a、R^3b and R ^3c are independently hydrogen and C ₁-C₆ alkyl;

R ^4a、R^4b and R ^4c are independently hydrogen and C ₁-C₆ alkyl; and

R ^5a、R^5b and R ^5c are independently hydrogen and C ₁-C₆ alkyl.

Embodiment 18. The compound of embodiment 16 or 17 wherein R ¹ is-OH,

Embodiment 19. The compound of embodiment 16 or 17 wherein Y ¹ and Y ² are independently:

Embodiment 20. The compound of any one of embodiments 15, 18 OR 19, wherein R ² is-CH (OR ⁶)(OR⁷).

Embodiment 21. The compound of any one of embodiments 15 OR 18-20, wherein R ³ is-CH (OR ⁸)(OR⁹).

Embodiment 22. A compound having the structure of formula (CY-II):

Or a pharmaceutically acceptable salt thereof, wherein R ¹、R²、R³、X¹、X²、X³、X⁴、X⁵、Y¹ and Y ² are as defined for formula (CY-I) of embodiment 15.

Embodiment 23. The compound of embodiment 22 wherein:

R ¹ is-OH, R ^1a,

Wherein Z ¹ is optionally substituted C ₁-C₆ alkyl;

X ¹ is optionally substituted C ₂-C₆ alkylene;

X ² and X ³ are independently a bond, -CH ₂ -or-CH ₂CH₂ -;

y ¹ and Y ² are independently

R ² and R ³ are independently optionally substituted C ₄-C₂₀ alkyl;

R ^1a is:

R ^2a、R^2b and R ^2c are independently hydrogen and C ₁-C₆ alkyl;

r ^3a、R^3b and R ^3c are independently hydrogen and C ₁-C₆ alkyl;

R ^4a、R^4b and R ^4c are independently hydrogen and C ₁-C₆ alkyl; and

R ^5a、R^5b and R ^5c are independently hydrogen and C ₁-C₆ alkyl.

Embodiment 24. The compound of embodiment 22 or 23 wherein R ¹ is-OH,

Embodiment 25. The compound of embodiment 22 or 23 wherein Y ¹ and Y ² are independently:

Embodiment 26. The compound of any one of embodiments 22, 24 OR 25, wherein R ² is-CH (OR ⁶)(OR⁷).

Embodiment 27. The compound of any one of embodiments 22 OR 24-26 wherein R ³ is-CH (OR ⁸)(OR⁹).

Embodiment 28. A compound having the structure of formula (CY-III):

Or a pharmaceutically acceptable salt thereof, wherein R ¹、R²、R³、X¹、X²、X³、X⁴、X⁵、Y¹ and Y ² are as defined for formula (CY-I) in embodiment 15.

Embodiment 29. The compound of embodiment 28 wherein:

R ¹ is-OH, R ^1a,

Wherein Z ¹ is optionally substituted C ₁-C₆ alkyl;

X ¹ is optionally substituted C ₂-C₆ alkylene;

X ² and X ³ are independently a bond, -CH ₂ -or-CH ₂CH₂ -;

y ¹ and Y ² are independently

R ² and R ³ are independently optionally substituted C ₄-C₂₀ alkyl;

R ^1a is:

R ^2a、R^2b and R ^2c are independently hydrogen and C ₁-C₆ alkyl;

r ^3a、R^3b and R ^3c are independently hydrogen and C ₁-C₆ alkyl;

R ^4a、R^4b and R ^4c are independently hydrogen and C ₁-C₆ alkyl; and

R ^5a、R^5b and R ^5c are independently hydrogen and C ₁-C₆ alkyl.

Embodiment 30 the compound of embodiment 28 or 29 wherein R ¹ is-OH,

Embodiment 31 the compound of embodiment 28 or 30 wherein Y ¹ and Y ² are independently:

Embodiment 32. The compound of any one of embodiments 28, 30 OR 31, wherein R ² is-CH (OR ⁶)(OR⁷).

Embodiment 33 the compound of any one of embodiments 28 OR 30-32, wherein R ³ is-CH (OR ⁸)(OR⁹).

Embodiment 34. A compound having the structure of formula (CY-IV):

Embodiment 35. The compound of embodiment 34 wherein:

R ¹ is-OH, R ^1a,

Wherein Z ¹ is optionally substituted C ₁-C₆ alkyl;

X ¹ is optionally substituted C ₂-C₆ alkylene;

X ² and X ³ are independently a bond, -CH ₂ -or-CH ₂CH₂ -;

y ¹ and Y ² are independently

R ² and R ³ are independently optionally substituted C ₄-C₂₀ alkyl;

R ^1a is:

R ^2a、R^2b and R ^2c are independently hydrogen and C ₁-C₆ alkyl;

r ^3a、R^3b and R ^3c are independently hydrogen and C ₁-C₆ alkyl;

R ^4a、R^4b and R ^4c are independently hydrogen and C ₁-C₆ alkyl; and

R ^5a、R^5b and R ^5c are independently hydrogen and C ₁-C₆ alkyl

Embodiment 36 the compound of embodiment 34 or 35 wherein R ¹ is-OH,

Embodiment 37. The compound of embodiment 34 or 36 wherein Y ¹ and Y ² are independently:

Embodiment 38. The compound of any one of embodiments 34, 36 OR 37, wherein R ² is-CH (OR ⁶)(OR⁷).

Embodiment 39. The compound of any one of embodiments 34 OR 36-38, wherein R ³ is-CH (OR ⁸)(OR⁹).

Embodiment 40. A compound having the structure of formula (CY-V):

Or a pharmaceutically acceptable salt thereof, wherein X ⁶ and X ⁷ are independently-CH ₂ -or-CH ₂CH₂ -; and R ¹、R²、R³、X¹、X⁴、X⁵、Y¹ and Y ² are as defined for formula (CY-I) of embodiment 15.

Embodiment 41. The compound of embodiment 40 wherein:

R ¹ is-OH, R ^1a,

Wherein Z ¹ is optionally substituted C ₁-C₆ alkyl;

X ¹ is optionally substituted C ₂-C₆ alkylene;

X ² and X ³ are independently a bond, -CH ₂ -or-CH ₂CH₂ -;

y ¹ and Y ² are independently

R ² and R ³ are independently optionally substituted C ₄-C₂₀ alkyl;

R ^1a is:

R ^2a、R^2b and R ^2c are independently hydrogen and C ₁-C₆ alkyl;

r ^3a、R^3b and R ^3c are independently hydrogen and C ₁-C₆ alkyl;

R ^4a、R^4b and R ^4c are independently hydrogen and C ₁-C₆ alkyl; and

R ^5a、R^5b and R ^5c are independently hydrogen and C ₁-C₆ alkyl

Embodiment 42 the compound of embodiment 40 or 41 wherein R ¹ is-OH,

Embodiment 43 the compound of embodiment 40 or 41 wherein Y ¹ and Y ² are independently:

Embodiment 44 the compound of any one of embodiments 40, 42 OR 43, wherein R ² is-CH (OR ⁶)(OR⁷).

Embodiment 45 the compound of any one of embodiments 40 OR 42-44, wherein R ³ is-CH (OR ⁸)(OR⁹).

Embodiment 46. A compound having the structure of formula (CY-VI):

or a pharmaceutically acceptable salt thereof, wherein R¹、R⁶、R⁷、R⁸、R⁹、X¹、X²、X⁴、X⁵、Y¹ and Y ² are as defined for formula (CY-I) of embodiment 15.

Embodiment 47. The compound of embodiment 46, or a pharmaceutically acceptable salt thereof, wherein R ¹ is-OH.

Embodiment 48. The compound of embodiment 46 or 47, or a pharmaceutically acceptable salt thereof, wherein X ¹ is C ₂-C₆ alkylene.

Embodiment 49 the compound of any one of embodiments 46-48, or a pharmaceutically acceptable salt thereof, wherein X ² is-CH ₂CH₂ -.

Embodiment 50. The compound of any one of embodiments 46-49, or a pharmaceutically acceptable salt thereof, wherein X ⁴ is C ₂-C₆ alkylene.

Embodiment 51 the compound of any one of embodiments 46-50, or a pharmaceutically acceptable salt thereof, wherein X ⁵ is C ₂-C₆ alkylene.

Embodiment 52 the compound of any one of embodiments 46-41, or a pharmaceutically acceptable salt thereof, wherein Y ¹ is:

Embodiment 53 the compound of any one of embodiments 46-52, or a pharmaceutically acceptable salt thereof, wherein Y ² is:

Embodiment 54 the compound of any one of embodiments 46-53, or a pharmaceutically acceptable salt thereof, wherein each Z ³ is independently optionally substituted C ₁-C₆ alkylene.

Embodiment 55 the compound of any one of embodiments 46-54, or a pharmaceutically acceptable salt thereof, wherein each Z ³ is-CH ₂CH₂ -.

Embodiment 56 the compound of any one of embodiments 46-55, or a pharmaceutically acceptable salt thereof, wherein R ⁶ is C ₅-C₁₄ alkyl.

Embodiment 57 the compound of any one of embodiments 46-56, or a pharmaceutically acceptable salt thereof, wherein R ⁷ is C ₆-C₁₄ alkyl.

Embodiment 58 the compound of any one of embodiments 46-55 or 57, or a pharmaceutically acceptable salt thereof, wherein R ⁶ is C ₅-C₁₄ alkenyl.

Embodiment 59 the compound of any one of embodiments 46-56 or 58, or a pharmaceutically acceptable salt thereof, wherein R ⁷ is C ₆-C₁₄ alkenyl.

Embodiment 60. The compound of any one of embodiments 46-59, or a pharmaceutically acceptable salt thereof, wherein R ⁸ is C ₅-C₁₆ alkyl.

Embodiment 61 the compound of any one of embodiments 46-60, or a pharmaceutically acceptable salt thereof, wherein R ⁹ is C ₆-C₁₄ alkyl.

Embodiment 62 the compound of any one of embodiments 46-59 or 61, or a pharmaceutically acceptable salt thereof, wherein R ⁸ is C ₅-C₁₆ alkenyl.

Embodiment 63 the compound of any one of embodiments 46-60 or 62, or a pharmaceutically acceptable salt thereof, wherein R ⁹ is C ₆-C₁₄ alkyl.

The disclosure is further illustrated by the following non-limiting examples.

XII. Examples

Method for preparing lipid

The lipids of the present disclosure may be prepared using any suitable method. In one reasonable approach, lipids are constructed from their individual components. As is known in the art, components may be covalently bound to each other via functional groups, where such functional groups may be present on the component or introduced onto the component using one or more steps (e.g., oxidation reaction, reduction reaction, cleavage reaction, etc.). Functional groups useful for covalently bonding components together to produce lipids: hydroxyl, sulfhydryl, amino, and the like. If necessary and/or desired, certain portions of the components may be protected with end capping groups as known in the art, see, e.g., green & Wuts, protective Groups in Organic Synthesis (John Wiley & Sons) (1991).

Alternatively, the lipids can be generated using known combinatorial methods to generate a larger library of potential lipids, which can then be screened to identify lipids with the desired functionality.

Method of preparing a delivery vehicle

Delivery vehicles, such as LNPs of the present disclosure, can be prepared using any suitable method. In one non-limiting example, the LNP is formed by mixing an equal volume of lipid dissolved in alcohol with an oligonucleotide payload dissolved in citrate buffer using an impact spray method.

Lipid solutions contain the cationic lipid compounds, helper lipids, neutral lipids, and pegylated lipids of the present disclosure. The payload to total lipid ratio was approximately 1:20 (wt/wt). LNP is formed by mixing an equal volume of a lipid ethanol solution with an oligonucleotide payload dissolved in citrate buffer via a mixing device using an impact spray method. The mixed LNP solution was kept at room temperature for 0-24 hours prior to the dilution step.

The solution is then concentrated and diafiltered with a suitable buffer by ultrafiltration or dialysis methods using a membrane. The final product was sterile filtered and stored at 4 ℃.

Evaluation of candidate LNP targeting systems

A library of candidate targeting systems is prepared, wherein the candidate targeting systems comprise at least one identifier sequence or portion in a formulation and at least one identifier sequence and/or payload in a nucleic acid construct.

Candidate targeting system generation

A population of Lipid Nanoparticle (LNP) formulations is produced, wherein the cationic lipid component is labeled with at least one identifier sequence or moiety. The LNP formulation produced may include LNP as follows: wherein (a) the components of all formulations are the same and the molar ratio of each component of all LNP formulations is the same, (b) the components of all formulations are the same but the molar ratio of each component of all LNP formulations is different, or (c) the components of the LNP formulations are different. Each of the different LNP formulations can include a different identifier sequence or portion in order to track the targeting system after administration. Nucleic acid constructs comprising at least one identifier sequence or payload (e.g., a reporter gene) are generated and formulated in the LNP population in order to generate candidate targeting systems to be administered to a subject.

Screening and validation of candidate targeting systems

The candidate targeting system is then administered to the subject at a predetermined dose and dosing interval. After administration, the whole subject or a region of the subject is screened to determine LNP formation and/or the location of the payload of the reference construct. The subject may be scanned by various methods known in the art, including Positron Emission Tomography (PET) and Computed Tomography (CT) using ⁶⁴ Cu radiolabels. The LNP formation and/or the positioning of the payload will be determined by visual inspection of the area of the PET image with the greatest ⁶⁴ Cu concentration and the anatomical location of the PET results will be confirmed using the results of the CT scan. Scanning may be repeated to determine if the position fix changes over time.

At the desired time point, samples will be obtained from the area of the subject that showed localization of LNP and/or payload in the whole animal localization screen performed above for higher resolution screening of the distribution.

The samples can then be prepared for Fluorescence Activated Cell Sorting (FACS) via instructions provided by the cell sorter. These cell populations are then prepared for deep sequencing to determine the presence and identity of the payload and/or identifier sequences.

These results from screening LNP libraries will provide directionality of the LNP formulation and nucleic acid construct administered to the subject.

Synthesis of exemplary ionizable lipids

Synthesis of selected intermediates

Synthesis of 3-pentyloxy-2-enoic acid ethyl ester (L1-2)

To a solution of triethyl phosphonoacetate (26.3 g,118 mmol) in anhydrous THF (33 mL) under nitrogen at-10 to-15℃was added dropwise THF (118 mL,118 mmol) containing 1M NaHMDS. After the addition was completed, the mixture was stirred at-10 to-15 ℃ for 30min and then at 0 ℃ for 1h. To this mixture was added dropwise 6-undecanone (10.0 g,59 mmol) at 0℃and the reaction mixture was allowed to warm to room temperature and stirred overnight. The mixture was then warmed to 45 ℃ and stirred for 24h. Saturated aqueous NH ₄ Cl (8 mL) was added and THF was evaporated. The residue was mixed with Et ₂ O (80 mL) and H ₂ O (100 mL) and the resulting phases were separated. The aqueous phase was extracted with Et ₂ O (80 mL). The combined organic phases were washed with H ₂ O (100 ml×2) and dried over anhydrous Na ₂SO₄. Filtration and concentration gave a crude material which was purified by flash column chromatography (SiO ₂: 0 to 4% ethyl acetate/hexanes gradient) to give ethyl 3-pentycine-2-enoate as a colorless oil L1-2(11.6g,82％).¹H-NMR(300MHz,CDCl₃)δ5.60(s,1H),4.13(q,J＝7.1Hz,2H),2.57(t,J＝7.6Hz,2H),2.12(t,J＝7.4Hz,2H),1.52-1.19(m,15H),0.89(t,J＝7.2Hz,6H);CIMS m/z 241[M+H]⁺.

Synthesis of ethyl 3-pentyctanoate (L1-3)

To a solution of L1-2 (11.0 g,2.1 mmol) in EtOAc (90 mL) was added 10% Pd/C (0.5 g). The resulting mixture was stirred under a hydrogen balloon for one day. The mixture was then filtered through celite. The celite was rinsed with EtOAc (25 ml×3). The combined filtrates were evaporated to give ethyl 3-amyl octanoate as a pale yellow oil L1-3(9.0g,83％).¹H-NMR(300MHz,CDCl₃)δ4.10(q,J＝7.1Hz,2H),2.20(d,J＝6.8Hz,2H),1.82(s,1H),1.40-1.12(m,19H),0.88(t,J＝7.0Hz,6H).

Synthesis of 3-pentylsin-1-ol (L1-4)

To a solution of lithium aluminum hydride in 2.0M THF (28 mL,56 mmol) at 0deg.C under nitrogen was slowly added a solution of L1-3 (7.0 g,29 mmol) in THF (33 mL). The resulting mixture was stirred at 0 ℃ for 1h and then at room temperature overnight. The reaction was quenched by addition of a saturated aqueous solution of Na ₂SO₄ under ice water bath cooling to give a milky white solution. The organic phase was separated and the aqueous phase was extracted with Et ₂ O (50 ml x 2). The combined organic phases were dried over Na ₂SO₄. Filtration and concentration gave a crude material which was purified by flash column chromatography (SiO ₂: 0 to 15% ethyl acetate/hexanes gradient) to give 3-pentylsin-1-ol as a yellowish oil L1-4(4.0g,70％).¹H-NMR(300MHz,CDCl₃)3.65(t,J＝4.4Hz,2H),1.51(dd,J＝13.7Hz,6.8Hz,2H),1.46-1.12(m,17H),0.88(t,J＝7.1Hz,6H).

Synthesis of 4, 4-bis (3, 7-dimethyloctyl) oxy) butyronitrile (L4L-2) [ procedure A ]

To a 100mL round bottom flask was added 4, 4-dimethoxybutyronitrile (3.0 g,23.2 mmol), ethanol (11.0 g,69.7 mmol) and pyridinium p-toluenesulfonate (0.29 g,1.2 mmol). The resulting mixture was stirred at 120 ℃ for 4h and cooled to room temperature. EtOAc (50 mL) and H ₂ O (20 mL) were added and the resulting phases separated. The aqueous phase was extracted with EtOAc (50 mL). The combined organic extracts were washed with H ₂ O (20 mL) and dried over anhydrous MgSO ₄. Filtration and concentration gave a crude material which was purified by flash column chromatography (SiO ₂: 0 to 10% ethyl acetate/hexanes gradient) to give a colorless oil L4L-2(6.6g,74％);¹HNMR(CDCl₃)δ4.50-4.53(t,1H),3.58-3.60(m,2H),3.41-3.49(m,2H),2.39-2.44(t,2H),1.92-1.94(q,2H),1.50-1.55(m,6H),1.38-1.42(m,2H),1.11-1.14(m,14H)0.88-0.84(t,18H);CIMS m/z[M+H]⁺381.

Synthesis of 4, 4-bis ((3, 7-dimethyloctyl) oxy) butanoic acid (L4L-3) [ procedure B ]

To a 100mL round bottom flask containing a solution of L4L-2 (8.2 g,21 mmol) in ethanol (50 mL) was added a solution of KOH (3.6 g,64 mmol) in water (50 mL). After the addition was completed, the mixture was stirred at 120 ℃ for 20h. Volatiles were removed and the reactant pH was adjusted to 5. EtOAc (150 mL) and H ₂ O (60 mL) were added and the resulting phases separated. The aqueous phase was extracted with EtOAc (50 mL). The combined organic extracts were washed with H ₂ O (60 ml x 2) and dried over anhydrous MgSO ₄. Filtration and concentration gave L4L-3 (6.4 g, 74%) which was used in the next step without further purification .¹HNMR(CDCl₃)δ4.54(t,1H),3.60-3.65(m,2H),3.45-3.49(m,2H),2.39-2.44(t,2H),1.92-1.94(m,2H),1.50-1.95(m,6H),1.26-1.55(m,8H),1.11-1.14(m,6H),0.84-0.88(d,18H);CIMS m/z[M-H]^-399.

Synthesis of selected Compounds

EXAMPLE 1 Synthesis of bis (2-hexyldecanoic acid) (1- (4-hydroxybutyl) pyrrolidine-3, 4-diyl) bis (butane-4, 1-diyl) ester (Compound CY 43)

Synthesis of 2, 3a,4,7 a-hexahydro-1H-isoindole (L19-2)

To a stirred solution of 3a,4,7 a-tetrahydro-1H-isoindole-1, 3 (2H) -dione L19-1 (10 g,66.1 mmol) in THF (200 mL) cooled to 0deg.C was added dropwise THF (82.5 mL,165 mmol) containing 2M lithium aluminum hydride. The reaction mixture was allowed to warm to room temperature and stirred for 12h. After starting material depletion was observed by TLC, the reaction mixture was cooled to 0 ℃ over 2h and quenched with THF/water (40 mL, v/v 9:1), 15% aqueous NaOH (40 mL) and water (100 mL) in sequence. The resulting mixture was stirred at room temperature for 1h and filtered through celite, followed by washing with DCM (3×100 mL). The collected filtrate was concentrated under reduced pressure to give L19-2 (5.8 g, 71%) as a brown liquid, which was used in the next step without further purification. CIMS m/z 124.2[ M+H ] ⁺.

Synthesis of tert-butyl 1, 3a,4,7 a-hexahydro-2H-isoindole-2-carboxylate (L19-3)

A solution of crude L19-2 (5.8 g,47.1 mmol) in THF (100 mL) was cooled to 0deg.C under nitrogen. Triethylamine (9.8 mL,70.6 mmol) and di-tert-butyl dicarbonate (11.4 g,52.2 mmol) were added and the reaction mixture was stirred at room temperature for 12h. Water and DCM were added and the aqueous phase was extracted with DCM. The organic extract was washed with saturated aqueous sodium bicarbonate and dried over Na ₂SO₄. Filtration and concentration gave a colorless oil L19-3(7.5g,71％).¹H-NMR(300MHz,CDCl₃)δ5.62(s,2H),3.42-3.33(m,2H),3.17-3.03(m,2H),2.30-2.16(m,4H),1.91-1.85(m,2H),1.44(s,9H).

Synthesis of tert-butyl 3, 4-bis (2-oxoethyl) pyrrolidine-1-carboxylate (L19-4)

L19-3 (3.0 g,13.4mmol,1 eq) was dissolved in DCM (200 mL) and the solution was cooled to-78 ℃. Ozone is bubbled until the color of the solution turns blue. The reaction was then quenched with dimethyl sulfide and stirred under nitrogen for 30min. The solvent was removed under reduced pressure to give the crude material which was used in the next step without further purification (2.31 g, 67%).

Synthesis of 4,4'- (1- (tert-butoxycarbonyl) pyrrolidine-3, 4-diyl) (2E, 2' E) -bis (but-2-enoic acid) diethyl ester (L19-5)

To a solution of triethyl phosphonoacetate (11.2 g,50.1 mmol) in THF (60 mL) cooled to-15℃under nitrogen was added dropwise 1M NaHMDS (10.1 mL,50.1 mmol). After the addition was completed, the mixture was stirred at the same temperature for 30min, and then at 0 ℃ for 60min. The resulting mixture was slowly added to crude L19-4 (3.2 g,12.5 mmol) at 0deg.C. The reaction mixture was allowed to reach room temperature and stirred overnight. The reaction was quenched with aqueous ammonium chloride and extracted with ethyl acetate and dried over anhydrous Na ₂SO₄. Filtration and concentration gave a crude material which was purified by flash column chromatography (SiO ₂: 0 to 35% ethyl acetate/hexanes gradient) to give a colorless oil L19-5(1.01g,20％).¹H-NMR(300MHz,CDCl₃)δ6.91-6.82(m,2H),5.88-5.83(m,2H),4.22-4.14(m,4H),3.43-3.37(m,2H),3.18-3.09(m,2H),2.36-2.11(m,6H),1.44(s,9H),1.28(t,6H);CIMS m/z 296.1[M-Boc+H]⁺.

Synthesis of diethyl 4,4' - (1- (tert-butoxycarbonyl) pyrrolidine-3, 4-diyl) dibutyrate (L19-6)

To a solution of compound L19-5 (0.58 g,1.46 mmol) in ethyl acetate (20 mL) was added 10% P/C (0.2 g). The mixture was stirred at room temperature under a hydrogen balloon for 12h and filtered through a celite pad. After washing with ethyl acetate, the filtrate was concentrated and the crude was used in the next step without further purification (0.57g,97％).¹H-NMR(300MHz,CDCl₃)δ4.15-4.08(m,4H),3.40-3.30(m,2H),3.15-3.01(m,4H),2.29(t,4H),2.09-2.03(m,2H),1.65-1.52(m,6H),1.44(s,9H),1.24(t,6H);CIMS m/z 300.2[M-Boc+H]⁺.

Synthesis of diethyl 4,4' - (pyrrolidine-3, 4-diyl) dibutyrate TFA salt (L19-7)

To a solution of compound L19-6 (0.57 g,1.42 mmol) in DCM (5 mL) was added TFA (5 mL) and the mixture was stirred at room temperature for 12h. Volatile components were removed under reduced pressure and the crude product was used in the next step without further purification (0.57 g, tfa salt) ).¹H-NMR(300MHz,CDCl₃)δ4.15-4.08(m,4H),3.37-3.10(m,4H),2.35-2.30(m,7H),1.61-1.43(m,7H),1.24(t,6H);CIMS m/z 300.2[M-Boc+H]⁺.

Synthesis of diethyl 4,4' - (1- (4- (benzyloxy) butyl) pyrrolidine-3, 4-diyl) dibutyrate (L19-8)

To a solution of compound L19-7 (460 mg,1.5 mmol) and benzyl 4-bromobutyl ether (411 mg,1.69 mmol) in CPME (5 mL) and ACN (5 mL) under nitrogen was added K ₂CO₃ (850 mg,6.1 mmol) and KI (255 mg,1.53 mmol). The reaction mixture was heated at 60℃for 18h. After cooling to room temperature, the reaction mixture was filtered through celite, washed with ethyl acetate, and the solvent was removed in vacuo to give the crude product, which was purified by flash chromatography (40 g SiO ₂: 0 to 10% methanol/dichloromethane gradient) to give the compound as a colorless oil L19-8(0.41g,57％).¹H-NMR(300MHz,CDCl₃)δ7.30-7.25(m,5H),4.43(s,2H),4.09-4.04(m,4H),3.60-3.46(m,4H),3.13-3.06(m,4H),2.29(t,4H),1.75-1.33(m,14H),1.22(t,6H);CIMS m/z 462.2[M+H]⁺.

Synthesis of 4,4' - (1- (4- (benzyloxy) butyl) pyrrolidine-3, 4-diyl) bis (butan-1-ol) (L19-9)

To a solution of compound L19-8 (0.4 g,0.88 mmol) in THF (10 mL) cooled to 0deg.C was added dropwise THF (1.1 mL,1.1 mmol) containing 1M lithium aluminum hydride. The reaction mixture was allowed to reach room temperature and stirred for 12h. After starting material depletion as observed by TLC, the reaction mixture was cooled to 0 ℃ and diluted with THF and quenched with 15% NaOH solution. The resulting mixture was stirred at room temperature for 1h and filtered through celite, followed by washing with ethyl acetate. The filtrate was concentrated to give the crude product (0.21 g, 62%) which was used in the next step without further purification. CIMS m/z 378.3[ M+H ] ⁺.

Synthesis of bis (2-hexyldecanoic acid) (1- (4- (benzyloxy) butyl) pyrrolidine-3, 4-diyl) bis (butane-4, 1-diyl) ester (L19-10)

To a solution of compound L19-9 (200 mg,0.53 mmol) in dichloromethane (6 mL) were added DMAP (65 mg,0.53 mmol) and EDC (0.319 g,3.18 mmol), followed by acid L12-1 (0.135 g,0.53 mmol). The reaction mixture was stirred at room temperature for 24h and evaporated under vacuum. The residue was dissolved in dichloromethane (100 mL) and washed with brine (80 mL x 3). After drying over anhydrous Na ₂SO₄, the solvent was evaporated and the crude was purified by column chromatography (40 g SiO ₂: 0 to 10% methanol/dichloromethane gradient) to give the compound as a colorless oil L19-10.(0.41g,57％).¹H-NMR(300MHz,CDCl₃)δ7.33-7.26(m,5H),4.46(s,2H),4.04(t,4H),3.48(t,2H),2.91(s,2H),2.33-2.24(m,4H),1.88-1.23(m,68H),0.85(t,12H);CIMS m/z 854.7[M+H]⁺.

Synthesis of bis (2-hexyldecanoic acid) (1- (4-hydroxybutyl) pyrrolidine-3, 4-diyl) bis (butane-4, 1-diyl) ester (Compound CY 43)

To a solution of compound L19-10 (125 mg,0.14 mmol) in ethyl acetate (3 mL) was added 10% P (OH) ₂/C (50 mg). The reaction mixture was stirred under a hydrogen balloon at room temperature for 6h. The mixture was filtered through a pad of celite, the filtrate was concentrated, and the crude was purified by column chromatography (12 g SiO ₂:0 to 10% methanol/dichloromethane gradient) to give compound CY43(43mg,38％).¹H-NMR(300MHz,CDCl₃)δ4.05(t,4H),3.64(t,2H),3.30(s,1H),2.91(s,2H),2.33-2.24(m,4H),1.87-1.23(m,68H),0.85(t,12H);CIMS m/z 864.7[M+H]⁺. as a colorless oil as an analytical HPLC column: agilent Zorbax SB-C18,5 μm, 4.6X105 mm, mobile phase A: acetonitrile containing 0.1% trifluoroacetic acid, mobile phase B: water containing 0.1% trifluoroacetic acid using a gradient: a/B5% to 95% in 15min, flow rate: 1mL/min, column temperature: 20±2 ℃, detector: ELSD, t _R =11.7 min, purity: 97.66%; UPLC column: thermo SCIENTIFIC HYPERSIL GOLD C4, mobile phase a: acetonitrile containing 0.1% trifluoroacetic acid, mobile phase B: water containing 0.1% trifluoroacetic acid using a gradient: a/B60% to 100% within 15min, flow rate: 0.5mL/min, column temperature: 20±2 ℃, detector: CAD, t _R =14.0 min, purity: 88.54%.

EXAMPLE 2 Synthesis of bis (2-hexyldecanoic acid) (1- (4-hydroxybutyl) piperidine-4, 4-diyl) bis (ethane-2, 1-diyl) ester (Compound CY 61)

Synthesis of 9-benzyl-2, 4-dioxo-3, 9-diazaspiro [5.5] undecane-1, 5-dinitrile (L20-2')

To ice-cooled 7M ammonia/methanol (120 mL) was added 1-benzyl-4-piperidone (40 g,212 mmol) followed by ethyl cyanoacetate (45 mL,2 mmol). The resulting mixture was allowed to stand in a refrigerator at-2℃for five days. The precipitate was filtered and washed with cold methanol. Oven drying overnight to give L20-2' (23 g, 30%) as an off-white solid; CIMS M/z [ M+H ] ⁺ 323,323.

Synthesis of diethyl 2,2' - (1-benzylpiperidine-4, 4-diyl) diacetate (L20-2)

A mixture of L20-2' (5.0 g,1.6 mmol), water (5.1 mL) and concentrated sulfuric acid (6 mL) was heated at 100deg.C for 48 hours. After cooling to room temperature, ethanol (60 mL) was added to the mixture and concentrated. The procedure was repeated four times. Ethanol (40 mL) was then added to the crude product and the solution was heated at reflux for 3 days. After ice cooling, na ₂CO₃ (6 g) and water were added and the mixture was concentrated. Ethyl acetate was added and the solution was washed with water and brine and dried over anhydrous Na ₂SO₄. Filtration and concentration gave a crude material which was purified by flash chromatography (SiO ₂: ethyl acetate/hexane 0-100%, 1% triethylamine in eluent) to give a pale yellow oil L20-2(1.85g,82％).¹H-NMR(300MHz,CDCl₃)δ7.32-7.21(m,5H),4.11(q,J＝6.5Hz,4H),3.50(s,2H),2.56(s,4H),2.43(t,J＝5.5Hz,4H),1.68(t,J＝6.8Hz,4H),1.24(t,J＝7.1Hz,6H);CIMS m/z[M+H]⁺348.

Synthesis of 2,2' - (1-benzylpiperidine-4, 4-diyl) bis (ethan-1-ol) (L22-1)

To a 2.0M ice-cooled solution of lithium aluminum hydride in THF (5.0 mL,10 mmol) was slowly added a solution of L20-2 (1.85 g,5.3 mmol) in anhydrous THF (25 mL) under nitrogen. The resulting mixture was stirred at room temperature overnight. Water (0.38 mL), 15% aqueous sodium hydroxide (0.38 mL) and water (1.15 mL) were added sequentially using an ice-water bath for cooling. Filtration through celite and concentration gave L22-1 as an oil which slowly solidified to an off-white solid (1.32g,94％).¹H-NMR(300MHz,CDCl₃)δ7.39-7.18(m,5H),3.74(t,J＝6.5Hz,4H),3.49(s,2H),2.40(t,J＝5.2Hz,4H),1.67(t,J＝6.8Hz,4H),1.50(t,J＝7.1Hz,6H);CIMS m/z[MH⁺]264.

Synthesis of bis (2-hexyldecanoic acid) (1-benzylpiperidine-4, 4-diyl) bis (ethane-2, 1-diyl) ester (L22-2)

To a solution of L22-1 (1.32 g,5 mmol) in DCM (50 mL) was added L12-1 (3.4 g,13 mmol) followed by DMAP (0.61 g,5 mmol) and EDC (3.7 g,20 mmol). The resulting mixture was stirred at room temperature under nitrogen atmosphere for 48h. The reaction mixture was diluted with DCM (50 mL) and washed with saturated aqueous NaHCO ₃ (50 mL), water (25 mL) and brine (25 mL). The organic phase was dried over anhydrous Na ₂SO₄. Filtration and concentration gave a crude material which was purified by flash column chromatography (SiO ₂: ethyl acetate/hexane 0-100%, 1% triethylamine in eluent) to give L22-2 as an oil which slowly solidified into a white solid (2.6g,70％).¹H-NMR(300MHz,CDCl₃)δ7.31-7.19(m,5H),4.12(q,J＝7.1Hz,4H),3.49(s,2H),2.49-2.22(m,6H),1.73-1.12(m,56H),0.87(t,J＝6.3Hz,12H);CIMS m/z[M+H]⁺740.

Synthesis of bis (2-hexyldecanoic acid) piperidine-4, 4-diylbis (ethane-2, 1-diyl) ester (L22-3)

To a solution of L22-2 (2.6 g,3.5 mmol) in 2-propanol (60 mL) was added 10% Pd/C (1.5 g) and EtOAc (10 mL) containing 1M HCl. The resulting mixture was stirred under a hydrogen balloon and heated in an oil bath at 80 ℃ for 20h. The reaction mixture was filtered through celite. The celite was rinsed with 2-propanol, dichloromethane and EtOAc. The combined filtrates were evaporated to give a pale yellow oil L22-3(2.2g,95％);4.20-4.00(m,4H),3.49-2.90(m,4H),2.35-2.15(m,2H),1.95-0.90(m,56H),0.87(t,J＝6.6Hz,12H);CIMS m/z[M+H]⁺650.

Synthesis of bis (2-hexyldecanoic acid) (1- (4- (benzyloxy) butyl) piperidine-4, 4-diyl) bis (ethane-2, 1-diyl) ester (L22-4)

To a solution of L22-3 (1.5 g,2.3 mmol) and 4-benzyloxybutyraldehyde (0.8 g,4.6 mmol) in dichloroethane (60 mL) was added sodium triacetoxyborohydride (1.5 g,6.9 mmol) followed by acetic acid (0.16 mL,2.3 mmol). The resulting mixture was stirred at room temperature under nitrogen atmosphere for two days. The reaction mixture was diluted with DCM (40 mL) and washed with saturated aqueous NaHCO ₃ (50 mL), water (25 mL) and brine (25 mL). The organic phase was dried over anhydrous Na ₂SO₄. Filtration and concentration gave a crude material which was purified by flash column chromatography (SiO ₂: 0 to 100% ethyl acetate/hexanes gradient) to give a pale yellow oil L22-4(0.9g,48％).¹H-NMR(300MHz,CDCl₃)δ7.35-7.21(m,5H),4.49(s,2H),4.13(q,J＝7.1Hz,4H),3.47(t,J＝5.7Hz,2H),2.49-2.20(m,8H),1.75-1.12(m,60H),0.87(t,J＝6.0Hz,12H);CIMS m/z[M+H]⁺812.

Synthesis of bis (2-hexyldecanoic acid) (1- (4-hydroxybutyl) piperidine-4, 4-diyl) bis (ethane-2, 1-diyl) ester (Compound CY 61)

To a solution of L22-4 (0.9 g,2.1 mmol) in EtOAc (40 mL) was added 10% Pd/C (0.5 g) and EtOAc (8 mL) with 1M HCl. The resulting mixture was stirred under a hydrogen balloon overnight. It was then filtered through celite. The celite was rinsed with EtOAc (25 ml x 3). Concentration gave a crude material which was purified by flash column chromatography (SiO ₂: ethyl acetate/hexane 0-100%, 1% triethylamine in eluent) to afford compound CY61(130mg,16％).¹H-NMR(300MHz,CDCl₃)δ4.12(q,J＝7.1Hz,4H),3.55(m,2H),2.55-2.20(m,8H),1.75-1.12(m,60H),0.87(t,J＝6.3Hz,12H);MS(CI):m/z[M+H]⁺722.6; as a pale yellow oil as an analytical HPLC column: agilent Zorbax SB-C18,5 μm, 4.6X105 mm, mobile phase A: acetonitrile containing 0.1% trifluoroacetic acid, Mobile phase B: water containing 0.1% trifluoroacetic acid using a gradient: a/B5% to 95% in 15min, flow rate: 1mL/min, column temperature: 20±2 ℃, detector: ELSD, t _R =11.2 min, purity: >99%; UPLC column: thermo SCIENTIFIC HYPERSIL GOLD C4, mobile phase a: acetonitrile containing 0.1% trifluoroacetic acid, mobile phase B: water containing 0.1% trifluoroacetic acid using a gradient: a/B60% to 100% within 15min, flow rate: 0.5mL/min, column temperature: 20±2 ℃, detector: CAD, t _R =12.1 min, purity: 99.21%. The acetylated product CY62(550mg).¹H NMR(300MHz,CDCl₃):δppm 4.13(q,J＝7.1Hz,4H),4.06(q,J＝6.3Hz,2H),2.46-2.21(m,8H),2.03(s,3H),1.72-1.15(m,60H),0.87(t,J＝6.3Hz,12H);MS(CI):m/z[M+H]⁺764.6; was also isolated as a pale yellow oil on an analytical HPLC column: agilent Zorbax SB-C18,5 μm, 4.6X105 mm, mobile phase A: acetonitrile containing 0.1% trifluoroacetic acid, mobile phase B: water containing 0.1% trifluoroacetic acid using a gradient: a/B5% to 95% in 15min, flow rate: 1mL/min, column temperature: 20±2 ℃, detector: ELSD, t _R =11.3 min, Purity: 99.83%; UPLC column: thermo SCIENTIFIC HYPERSIL GOLD C4, mobile phase a: acetonitrile containing 0.1% trifluoroacetic acid, mobile phase B: water containing 0.1% trifluoroacetic acid, using a gradient of 60% to 100% a/B over 15min, flow rate: 0.5mL/min, column temperature: 20±2 ℃, detector: CAD, t _R =13.7 min, purity: 97.57%.

EXAMPLE 3 Synthesis of bis (2-hexyldecanoic acid) (1- (4-hydroxybutyl) piperidine-4, 4-diyl) bis (ethane-2, 1-diyl) ester (Compound CY 57)

Synthesis of tert-butyl 3, 5-bis (4- (benzyloxy) but-1-en-1-yl) piperidine-1-carboxylate (L21-3)

To a cooled solution of L21-1 (500 mg,1.6 mmol) in dry ice-acetone bath in anhydrous toluene (8 mL) was added toluene (3.4 mL,3.4 mmol) containing 1.0M diisobutylaluminum hydride under nitrogen atmosphere. The resulting mixture was stirred at-72℃for 2h. About half of the precooled (-72 ℃) benzyloxypropylene triphenylphosphine solution ("Wittig reagent", obtained by adding potassium tert-butoxide (1.1 g,9.3 mmol) to a solution of (3-benzyloxypropyl) triphenylphosphine bromide L21-2 (4.86 g,9.6 mmol) in anhydrous toluene (8 mL) at 0deg.C) was stirred at room temperature for 2h. The reaction mixture was warmed to room temperature and stirred for 16h. The remaining Wittig reagent solution was added and the reaction was stirred at room temperature for an additional 16h. The reaction was then quenched by the addition of water (15 mL) and extracted with ethyl acetate (25 mL x 3). The combined organic extracts were washed with water (25 ml x 3) and dried over anhydrous Na ₂SO₄. Filtration and concentration gave a crude material which was purified by flash column chromatography (SiO ₂: ethyl acetate/hexane 0-100) to give a colorless oil L21-3(250mg,30％).¹H-NMR(300MHz,CDCl₃)δ7.39-7.18(m,10H),5.55-5.40(m,2H),5.17(t,J＝9.1Hz,2H),4.51(s,4H),3.99(s,br,4H),3.49(t,J＝6.9Hz,4H),2.58-2.23(m,6H),1.77-1.68(m,1H),1.45(s,9H),1.09-0.96(m,1H);CIMS m/z[M-Boc+H]⁺405.7.

Synthesis of tert-butyl 3, 5-bis (4-hydroxybutyl) piperidine-1-carboxylate (L21-4)

A mixture of L21-3 (470 mg,0.9 mmol) and 10% Pd/C (100 mg) in methanol (12 mL) was stirred under a hydrogen balloon at room temperature for 20h. The reaction mixture was filtered through celite. The celite was washed with methanol. The combined filtrates were evaporated to give a pale yellow oil L21-4(300mg,98％);4.20-3.95(m,4H),3.63(t,J＝6.3Hz,4H),2.25-2.05(m,2H),1.93-1.82(m,1H),1.70-1.05(m,23H),0.69-0.53(m,1H);CIMS m/z[M-Boc+H]⁺230.

Synthesis of bis (2-hexyldecanoic acid) (1- (t-butoxycarbonyl) piperidine-3, 5-diyl) bis (butane-4, 1-diyl) ester (L21-5)

To a solution of L21-4 (300 mg,0.9 mmol) in DCM (10 mL) was added L12-1 (580 mg,2.3 mmol) followed by DMAP (110 mg,0.9 mmol) and EDC (700 mg,3.6 mmol). The resulting mixture was stirred at room temperature under nitrogen atmosphere for 20h. The reaction mixture was diluted with DCM (15 mL) and washed with brine (10 mL). The organic phase was dried over anhydrous Na ₂SO₄. Filtration and concentration gave a crude material which was purified by flash column chromatography (SiO ₂: ethyl acetate/hexane 0-100%) to give a colorless oil L21-5(600mg,82％).¹H-NMR(300MHz,CDCl₃)δ4.20-3.95(m,4H),4.05(t,J＝6.6Hz,4H),2.39-2.22(m,2H),2.21-2.05(m,2H),1.91-1.80(m,1H),1.68-1.11(m,69H),0.86(t,J＝6.3Hz,12H)0.69-0.53(m,1H);CIMS m/z[M-Boc+H]⁺706.7.

Synthesis of bis (2-hexyldecanoic acid) piperidine-3, 5-diylbis (butane-4, 1-diyl) ester (L21-6)

To a solution of L21-5 (450 mg,0.56 mmol) in dichloromethane (3 mL) was added TFA (3 mL) at 0deg.C and the reaction mixture was stirred at room temperature for 4h. Volatile components were removed under reduced pressure and crude L21-6 (450 mg) was used in the next step without further purification .¹H-NMR(300MHz,CDCl₃)δ4.05(t,J＝6.3Hz,4H),3.49–2.80(m,4H),2.51–2.22(m,4H),2.02–1.01(m,61H),0.69-0.53(m,13H);CIMS m/z[M+H]⁺706.7.

Synthesis of bis (2-hexyldecanoic acid) (1- (4- (benzyloxy) butyl) piperidine-4, 4-diyl) bis (ethane-2, 1-diyl) ester (L21-7)

To a solution of L21-6 (450 mg,0.55 mmol) and 4-benzyloxybutyraldehyde (198 mg,1.1 mmol) in 1, 2-dichloroethane (15 mL) was added sodium triacetoxyborohydride (354 mg,1.6 mmol), followed by acetic acid (36. Mu.L, 0.55 mmol). The resulting mixture was stirred at room temperature under nitrogen atmosphere for 20h. The reaction mixture was diluted with DCM (20 mL) and a saturated aqueous solution of sodium bicarbonate was slowly added until no bubbles were generated. The two phases were separated and the aqueous phase was extracted with DCM (20×2 ml). The combined organic extracts were dried over anhydrous Na ₂SO₄. Filtration and concentration gave a crude material which was purified by flash column chromatography (SiO ₂: ethyl acetate/hexane 0-100%, 1% triethylamine in eluent) to give a pale yellow oil L21-7(340mg,71％).¹H-NMR(300MHz,CDCl₃)δ7.35-7.21(m,5H),4.49(s,2H),4.05(t,J＝6.3Hz,4H),3.47(m,2H),2.95-2.92(m,2H),2.50-2.10(m,6H),1.81-1.70(m,1H),1.65-1.15(m,66H),0.86(t,J＝6.9Hz,12H),0.55-0.42(m,1H);CIMS m/z[M+H]⁺868.

Synthesis of bis (2-hexyldecanoic acid) (1- (4-hydroxybutyl) piperidine-4, 4-diyl) bis (ethane-2, 1-diyl) ester (Compound CY 57)

A mixture of L21-7 (340 mg,0.4 mmol) and 10% Pd (OH) ₂/C (120 mg) in EtOAc (12 mL) was stirred under a hydrogen balloon for 70h. It was then filtered through celite. The celite was rinsed with EtOAc (10 ml x 3). Concentration gave a crude material which was purified by flash column chromatography (SiO ₂: ethyl acetate/hexane 0-100%, 1% triethylamine in eluent) to afford compound CY57(171mg,56％).¹H-NMR(300MHz,CDCl₃)δ4.04(t,J＝6.3Hz,4H),3.54(m,2H),2.95-2.92(m,2H),2.61-2.22(m,6H),1.85-1.75(m,1H),1.76-1.12(m,66H),0.87(t,J＝6.9Hz,12H),0.55-0.42(m,1H);MS(CI):m/z[M+H]⁺778.7; as a pale yellow oil as an analytical HPLC column: agilent Zorbax SB-C18,5 μm, 4.6X105 mm, mobile phase A: acetonitrile containing 0.1% trifluoroacetic acid, mobile phase B: water containing 0.1% trifluoroacetic acid using a gradient: a/B5% to 95% in 15min, flow rate: 1mL/min, column temperature: 20±2 ℃, detector: ELSD, t _R =11.6 min, purity: >99%; UPLC column: thermo SCIENTIFIC HYPERSIL GOLD C4, mobile phase a: acetonitrile containing 0.1% trifluoroacetic acid, mobile phase B: water containing 0.1% trifluoroacetic acid using a gradient: a/B60% to 100% within 15min, flow rate: 0.5mL/min, column temperature: 20±2 ℃, detector: CAD, t _R =13.3 min, purity: 97.05%.

EXAMPLE 4 Synthesis of bis (4, 4-bis (octyloxy) butanoic acid) (1- (4-hydroxybutyl) piperidine-4, 4-diyl) bis (ethane-2, 1-diyl) ester (CY 63)

Synthesis of diethyl 2,2' - (piperidine-4, 4-diyl) diacetate (L27-1)

A solution of L20-2 (5.4 g,15.4 mmol) in ethanol (107 ml) was treated with 10% Pd/C (1.1 g) at room temperature under a nitrogen atmosphere. The reaction mixture was evacuated and purged with H ₂ gas (3×) and then vigorously stirred at room temperature under an atmosphere of H ₂ (1 atm, H ₂ balloon). After 24h, the reaction mixture was filtered through celite and the filtrate was concentrated in vacuo to give the crude product L27-1 (4 g) which was used in the next step without further purification. APCI MS M/z [ M+H ] ⁺ 257.16.

Synthesis of diethyl 2,2' - (1- (4- (benzyloxy) butyl) piperidine-4, 4-diyl) diacetate (L27-2)

To a mixture of L27-1 (4 g,15.5 mmol) and 4- (benzyloxy) butanal (5.5 g,31.1 mmol) in 1, 2-dichloroethane (180 mL) was added Na (OAc) ₃ BH (9.9 g,46.6 mmol) and acetic acid (1 mL). The reaction mixture was subjected to vacuum/N ₂ cycle (3X) and stirred at room temperature for 18h. The reaction was quenched by slow addition of saturated NaHCO ₃ (100 mL) at 0deg.C. The aqueous phase was extracted with ethyl acetate (100 ml,3 x) and the combined organic phases were dried over anhydrous Na ₂SO₄. Filtration followed by concentration gave the crude material, which was dissolved in DCM. Silica gel (40 g) and triethylamine (40 mL) were added to the crude material and shaken for 10-15min, and the solvent was removed under vacuum. The residue was loaded onto an empty flash drum, which was then connected to a flash purification system loaded with 80g of silica gel column and purified by flash chromatography (SiO ₂: 0 to 10% ethyl acetate/hexane (10% triethylamine)) to give ethyl as a pale yellow oil L27-2(3.7g,57％).¹H-NMR(300MHz,CDCl₃)δ7.31-7.30(m,5H),4.46(s,2H),4.09-4.04(m,4H),3.47-3.43(m,2H),2.52-2.31(m,10H),1.68-1.57(m,8H),1.22(t,6H);APCI MS m/z[M+H]⁺420.3.

Synthesis of 2,2' - (1- (4- (benzyloxy) butyl) piperidine-4, 4-diyl) bis (ethan-1-ol) (L27-3)

A solution of L27-2 (0.75 g,1.78 mmol) in THF (14 mL) was cooled in an ice bath (0deg.C) and THF (3.56 mL,7.14 mmol) containing 2M LiAlH ₄ was added dropwise thereto. The ice bath was removed and the reaction mixture was stirred at room temperature for 18h. The mixture was diluted with Et ₂ O (50 mL), cooled in an ice bath, and carefully quenched with water (10 mL), 20% NaOH (10 mL), and water (30 mL). After stirring for 30min, the aqueous phase was extracted with 20mL DCM (3×), and the combined organic phases were dried (Na ₂SO₄), filtered and concentrated to give L27-3 as a white solid (0.54 g,91% yield). APCI MS M/z [ M+H ] ⁺ 336.3.3.

Synthesis of 4, 4-bis (nonyloxy) butanoic acid (L4-3 (T9))

Prepared following procedure B described in the synthesis of compound L4L. Compound L4-3 (T9) was isolated as a pale yellow oil in yield 11.8g(98％).¹HNMR(CDCl₃)δ:4.53-4.56(t,1H),3.57-3.60(m,2H),3.40-3.43(m,2H),2.39-2.41(t,2H),1.90-1.95(m,2H),1.54-1.56(M,4H),1.26(bs,28H),0.85-0.87(t,6H);CIMS m/z[M-H]^-371.

Synthesis of bis (4, 4-bis (octyloxy) butanoic acid) (1- (4-hydroxybutyl) piperidine-4, 4-diyl) bis (ethane-2, 1-diyl) ester (L27-4) [ procedure E ]

To a 250mL round bottom flask containing L4-3 (T9) (1 g,2.9mmol,2.5 eq), EDC (1.01 g,5.28mmol,4 eq), DMAP (161 mg,1.32mmol,1 eq) and L27-3 (440 mg,1.32mmol,1 eq) was added anhydrous dichloromethane (20 mL) and the reaction mixture was stirred at room temperature overnight. After the reaction was completed, about 30g of flash silica was added and the contents were thoroughly stirred to give a homogeneous mixture. The solvent was removed from this mixture under vacuum. The residue was loaded onto an empty flash cartridge, which was then connected to a flash purification system loaded with a flash silica column and purified by flash chromatography (SiO ₂: hexane (10% triethylamine)/ethyl acetate 0-20%) to give the compound as a pale yellow oil L27-4(0.94g,73％).¹H-NMR(300MHz,CDCl₃)δ7.33-7.31(m,5H),4.48-4.47(m,4H),4.10-4.08(m,4H),3.56-3.37(m,10H),2.37-2.32(m,10H),1.90-1.80(m,20H),1.31-1.10(m,40H),0.84(t,12H);APCI MS m/z[M+H]⁺988.8.

Synthesis of bis (4, 4-bis (octyloxy) butanoic acid) (1- (4-hydroxybutyl) piperidine-4, 4-diyl) bis (ethane-2, 1-diyl) ester (CY 63) [ procedure F ]

To a 250mL round bottom flask containing L27-4 (560 mg,0.56 mmol) and 10% Pd/C (186 mg) was added ethyl acetate (20 mL) and then the reaction mixture was subjected to vacuum/N ₂ cycle (3X) followed by another vacuum/H ₂ cycle (3X). The reaction mixture was placed under 1atm H ₂ (hydrogen balloon) and allowed to stir overnight. The reaction mixture was diluted with ethyl acetate (100 mL) and then filtered through celite, washed with ethyl acetate, and then the solvent was removed under vacuum to dryness to give the crude product CY63(132mg,26％).¹H-NMR(300MHz,CDCl₃)δ4.48(t,4H),4.12-4.07(m,4H),3.55-3.39(m,10H),2.46-2.31(m,10H),1.90-1.88(m,4H),1.66-1.51(m,20H),1.30-1.00(m,40H),0.86(t,12H);APCI MS m/z[M+H]⁺898.8; as a light brown oil analytical HPLC column: agilent Zorbax SB-C18,5 μm, 4.6X105 mm, mobile phase A: acetonitrile containing 0.1% trifluoroacetic acid, mobile phase B: water containing 0.1% trifluoroacetic acid using a gradient: a/B5% to 95% in 15min, flow rate: 1mL/min, column temperature: 20±2 ℃, detector: ELSD, t _R =11.6 min, purity: >99%; UPLC column: waters Aquity ACSHTM, C18,1.7 μm, 3.0X105 mm, (part number 186005302), mobile phase A: acetonitrile containing 0.1% trifluoroacetic acid, mobile phase B: water containing 0.1% trifluoroacetic acid using a gradient: a/B5% to 95% in 15min, flow rate: 1mL/min, column temperature: 20±2 ℃, detector: CAD, t _R =13.8 min, purity: >99%. /(I)

EXAMPLE 5 Synthesis of heptadec-9-yl 2- (1- (4-hydroxybutyl) -4- (2-oxo-2- ((3-pentyloxy) oxy) ethyl) piperidin-4-yl) acetate (Compound CY 69)

Synthesis of 2,2' - (1- (4- (benzyloxy) butyl) piperidine-4, 4-diyl) diacetic acid (L28A-1)

To a solution of diester L27-2 (0.9 g,2.1 mmol) in THF (15 mL) and methanol (2.5 mL) was added a solution of LiOH (0.36 g,6.4 mmol) in water (5 mL). The mixture was stirred at room temperature for 20h. While cooling in an ice-water bath, the reaction mixture was adjusted to pH 4. Volatile components were removed under reduced pressure and the residue was lyophilized to give an off-white solid which was purified by reverse phase column chromatography (acetonitrile/water 0-100) to give an off-white foam solid L28A-1(0.62g,80％).¹H-NMR(300MHz,CDCl₃)δ7.39-7.18(m,5H),4.47(s,2H),3.48(t,J＝6.3Hz,2H),2.90-2.56(m,6H),2.43(m,4H),2.05-1.56(m,8H);CIMS m/z[M-H]^-361.5.

Synthesis of 9- (4- (benzyloxy) butyl) -3-oxa-9-azaspiro [5.5] undecane-2, 4-dione (L28A-2)

To a solution of L28A-1 (0.5 g,1.4 mmol) in anhydrous DCM (15 mL) and pyridine (2 mL) was added anhydrous DMF (1 drop) and oxalyl chloride (0.15 mL,4.2 mmol) under nitrogen at 0deg.C. After the addition was completed, the mixture was stirred at room temperature for 18h. More oxalyl chloride (0.15 ml,4.2 mmol) was added and the mixture was stirred at room temperature for 20h. The reaction mixture was concentrated and co-evaporated with anhydrous toluene to give L28A-2 (0.48 g, 99%) as a pale yellow oil; CIMS M/z [ M+H ] ⁺ 346.2.

Synthesis of 2- (1- (4- (benzyloxy) butyl) -4- (2-oxo-2- ((3-pentyloxy) oxy) ethyl) piperidin-4-yl) acetic acid (L28A-3)

To a solution of L28A-2 (480 mg,1.4 mmol) in DCM (15 mL) and pyridine (2 mL) at 0deg.C was added L1-4 (800 mg,4.0 mmol). The resulting mixture was stirred at room temperature under nitrogen atmosphere for 18h. More L1-4 (160 mg,1.4 mmol) was added and the mixture was stirred at 50℃for 20h. The reaction mixture was concentrated and the crude material was purified by flash column chromatography (SiO ₂: methanol/DCM 0-30%, 5% triethylamine) to give a pale yellow solid L28A-3(330mg,44％).¹H-NMR(300MHz,CDCl₃)δ7.41-7.15(m,5H),4.46(s,2H),4.06(t,J＝6.5Hz,2H),2.89-2.36(m,10H),1.97-1.15(m,29H),0.87(t,J＝6.8Hz,6H);CIMS m/z[M+H]⁺546.4.

Synthesis of heptadec-9-yl 2- (1- (4- (benzyloxy) butyl) -4- (2-oxo-2- ((3-pentyloxy) oxy) ethyl) piperidin-4-yl) acetate (L28A-4)

To a solution of L28A-3 (320 mg,0.58 mmol) in DCM (10 mL) was added heptadecan-9-ol (L2-1) (225 mg,0.88 mmol), followed by DMAP (38 mg,0.3 mmol) and EDC (225 mg,1.2 mmol). The resulting mixture was stirred at room temperature under nitrogen atmosphere for 18h. The reaction mixture was diluted with DCM (15 mL) and washed with brine (10 mL). The organic phase was dried over anhydrous Na ₂SO₄. Filtration and concentration gave a crude material which was purified by flash column chromatography (SiO ₂: ethyl acetate/hexane 0-100%) to give a colorless oil L28A-4(305mg,66％).¹H-NMR(300MHz,CDCl₃)δ7.38-7.21(m,5H),4.84(m,1H),4.49(s,2H),4.05(t,J＝7.1Hz,2H),3.47(t,J＝5.9Hz,2H),2.60-2.28(m,10H),1.76-1.15(m,55H),0.87(t,J＝6.0Hz,12H);CIMS m/z[M+H]⁺784.8.

Synthesis of heptadec-9-yl 2- (1- (4-hydroxybutyl) -4- (2-oxo-2- ((3-pentyloxy) oxy) ethyl) piperidin-4-yl) acetate (Compound CY 69)

A mixture of L28A-4 (300 mg,0.38 mmol) and 10% Pd (OH) ₂/C (150 mg) in EtOAc (15 mL) was stirred under a hydrogen balloon for 80h. The mixture was then filtered through celite. The celite was rinsed with EtOAc (10 ml x 3). The filtrate was concentrated to give a crude material which was purified by flash column chromatography (SiO ₂: ethyl acetate/hexane 0-100%, 1% triethylamine in eluent) to give compound CY69(241mg,91％).¹H-NMR(300MHz,CDCl₃)δ4.84(m,1H),4.05(t,J＝7.4Hz,2H),3.56(m,2H),2.71-2.35(m,10H),1.82-1.43(m,15H),1.36-1.15(m,40H),0.87(t,J＝5.2Hz,12H);MS(CI):m/z[M+H]⁺694.6; as a pale yellow oil as an analytical HPLC column: agilent Zorbax SB-C18,5 μm, 4.6X105 mm, mobile phase A: acetonitrile containing 0.1% trifluoroacetic acid, mobile phase B: water containing 0.1% trifluoroacetic acid using a gradient: a/B5% to 95% in 15min, flow rate: 1mL/min, column temperature: 20±2 ℃, detector: ELSD, t _R =10.6 min, purity: >99%; UPLC column: waters Aquity ACSHTM, C18,1.7 μm, 3.0X105 mm, (part number 186005302), mobile phase A: acetonitrile containing 0.1% trifluoroacetic acid, mobile phase B: water containing 0.1% trifluoroacetic acid using a gradient: a/B60% to 100% within 15min, flow rate: 0.5mL/min, column temperature: 20±2 ℃, detector: CAD, t _R =13.4 min, purity: >99%.

EXAMPLE 6 Synthesis of bis (2-nonylundecanoic acid) (1- (4-hydroxybutyl) piperidine-4, 4-diyl) bis (ethane-2, 1-diyl) ester (CY 65)

Synthesis of bis (2-nonylundecanoic acid) (1- (4- (benzyloxy) butyl) piperidine-4, 4-diyl) bis (ethane-2, 1-diyl) ester (L49-4)

Prepared following procedure E described in the synthesis of compound L27. The compound was isolated as a colorless oil L49-1(580mg,43％).¹H-NMR(300MHz,CDCl₃)δ7.33-7.26(m,5H),4.48(s,2H),4.12(t,4H),3.46(t,2H),2.45-2.24(m,7H),1.70-1.37(m,19H),1.29-1.15(m,58H),0.86(t,12H);CIMS m/z[M+H]⁺925.56.

Synthesis of bis (2-nonylundecanoic acid) (1- (4-hydroxybutyl) piperidine-4, 4-diyl) bis (ethane-2, 1-diyl) ester (CY 65)

Prepared following procedure F described in the synthesis of compound CY 63. Compound CY65(0.52g,97％).¹H-NMR(300MHz,CDCl₃)δ4.11(m,4H),3.56(t,2H),2.55-241(m,4H),2.27-2.21(m,2H),1.68-1.57(m,16H),1.28-1.15(m,54H),0.86(t,12H);CIMS m/z[M+H]⁺834.1. was isolated as a colorless oil on an analytical HPLC column: agilent Zorbax SB-C18,5 μm, 4.6X105 mm, mobile phase A: acetonitrile containing 0.1% trifluoroacetic acid, mobile phase B: water containing 0.1% trifluoroacetic acid using a gradient: a/B5% to 95% in 15min, flow rate: 1mL/min, column temperature: 20±2 ℃, detector: ELSD, t _R =12.1 min, purity: >99%; UPLC column: waters Aquity ACSHTM, C18,1.7 μm, 3.0X105 mm, (part number 186005302), mobile phase A: acetonitrile containing 0.1% trifluoroacetic acid, mobile phase B: water containing 0.1% trifluoroacetic acid using a gradient: a/B5% to 95% in 15min, flow rate: 1mL/min, column temperature: 20±2 ℃, detector: CAD, t _R =15.4 min, purity: >98.1%.

EXAMPLE 7 Synthesis of bis (4, 4-bis (nonyloxy) butanoic acid) (1- (4-hydroxybutyl) piperidine-3, 5-diyl) bis (ethane-2, 1-diyl) ester (CY 66)

Synthesis of bis (4, 4-bis (nonyloxy) butanoic acid) (1- (4- (benzyloxy) butyl) piperidine-3, 5-diyl) bis (ethane-2, 1-diyl) ester (L50-1)

Prepared following procedure E described in the synthesis of compound CY 63. The compound was isolated as a colorless oil L50-1(0.55g,44％).¹H-NMR(300MHz,CDCl₃)δ7.33-7.32(m,5H),4.49-4.47(m,4H),4.12-4.10(m,4H),3.56-3.37(m,10H),2.38-2.33(m,10H),2.04-1.89(m,4H),1.66-1.50(m,20H),1.40-0.99(m,48H),0.87(t,12H);APCI-MS:m/z[M+H]⁺1045.0.

Synthesis of bis (4, 4-bis (nonyloxy) butanoic acid) (1- (4-hydroxybutyl) piperidine-3, 5-diyl) bis (ethane-2, 1-diyl) ester (CY 66)

Prepared following procedure F described in the synthesis of compound CY 63. Compound CY66(0.2g,44％).¹H-NMR(300MHz,CDCl₃)δ4.47(t,4H),4.13-4.10(m,4H),3.56-3.40(m,10H),2.57-2.39(m,10H),1.91-1.89(m,4H),1.67-1.52(m,26H),1.37-1.00(m,48H),0.87(t,12H);APCI-MS:m/z[M+H]⁺954.7; was isolated as a colorless oil on an analytical HPLC column: agilent Zorbax SB-C18,5 μm, 4.6X105 mm, mobile phase A: acetonitrile containing 0.1% trifluoroacetic acid, mobile phase B: water containing 0.1% trifluoroacetic acid using a gradient: a/B5% to 95% in 15min, flow rate: 1mL/min, column temperature: 20±2 ℃, detector: ELSD, t _R =11.6 min, purity: >99%; UPLC column: waters Aquity ACSHTM, C18,1.7 μm, 3.0X105 mm, (part number 186005302), mobile phase A: acetonitrile containing 0.1% trifluoroacetic acid, mobile phase B: water containing 0.1% trifluoroacetic acid using a gradient: a/B5% to 95% in 15min, flow rate: 1mL/min, column temperature: 20±2 ℃, detector: CAD, t _R =13.8 min, purity: >99%.

EXAMPLE 8 Synthesis of bis (4, 4-bis (decyloxy) butanoic acid) (1- (4-hydroxybutyl) piperidine-4, 4-diyl) bis (ethane-2, 1-diyl) ester (CY 67)

Synthesis of bis (4, 4-bis (decyloxy) butanoic acid) (1- (4- (benzyloxy) butyl) piperidine-4, 4-diyl) bis (ethane-2, 1-diyl) ester (L51-1)

Prepared following procedure E described in the synthesis of compound CY 63. L51-1 (1.16 g, 88%) as a colorless oil ,¹H-NMR(300MHz,CDCl₃)δ7.33-7.25(m,5H),4.51-4.46(m,4H),4.11(t,J＝7.5Hz,4H),3.62-3.33(m,10H),2.45-2.26(m,10H),1.97-1.85(m,4H),1.73-1.41(m,18H),1.40-1.15(m,58H),0.87(t,J＝6.3Hz,12H);MS(CI):m/z[M+H]⁺1100.8.

Synthesis of bis (4, 4-bis (decyloxy) butanoic acid) (1- (4-hydroxybutyl) piperidine-4, 4-diyl) bis (ethane-2, 1-diyl) ester (CY 67)

Prepared following procedure F described in the synthesis of compound CY 63. Compound CY67 (616 mg, 58%) as colorless oil ,¹H-NMR(300MHz,CDCl₃)δ4.48(t,J＝5.6Hz,2H),4.11(t,J＝7.4Hz,4H),3.61-3.32(m,10H),2.65-2.30(m,10H),1.98-1.85(m,4H),1.76-1.15(m,76H),0.91-0.80(m,12H);MS(CI):m/z[M+H]⁺1010.8; analytical HPLC column: agilent Zorbax SB-C18,5 μm, 4.6X105 mm, mobile phase A: acetonitrile containing 0.1% trifluoroacetic acid, mobile phase B: water containing 0.1% trifluoroacetic acid using a gradient: a/B5% to 95% in 15min, flow rate: 1mL/min, column temperature: 20±2 ℃, detector: ELSD, t _R =12.4 min, purity: >99%; UPLC column: waters Aquity ACSHTM, C18,1.7 μm, 3.0X105 mm, (part number 186005302), mobile phase A: acetonitrile containing 0.1% trifluoroacetic acid, mobile phase B: water containing 0.1% trifluoroacetic acid using a gradient: a/B60% to 100% within 15min, flow rate: 0.5mL/min, column temperature: 20±2 ℃, detector: CAD, t _R =16.0 min, purity: 98%.

EXAMPLE 9 Synthesis of bis (4, 4-bis (nonyloxy) butanoic acid) (1- (4- (1H-imidazol-1-yl) butyl) piperidine-4, 4-diyl) bis (ethane-2, 1-diyl) ester (CY 71)

Synthesis of 4-bromobutanal (L57-2)

To a solution of pyridinium chlorochromate (PCC) (12.14 g,56.55 mmol) in DCM (75 mL) was added DCM (25 mL) containing 4-bromobutan-1-ol (5.77 g,37.7 mmol) over 10min (intermittent cooling was required to prevent solvent reflux). The reaction mixture was stirred at room temperature for 2h and then diluted with diethyl ether. The upper ether phase was poured from the flask and filtered through celite and the filter cake was washed with diethyl ether. The combined ether phases were evaporated under reduced pressure to give crude 4-bromobutyraldehyde L57-2, which was used in the next step without further purification (4.5 g, crude); ¹H-NMR(300MHz,CDCl₃ ) δ9.81 (s, 1H), 3.74 (m, 2H), 2.18 (m, 2H), 1.84 (s, 2H).

Synthesis of 4-bromo-1, 1-dimethoxybutane (L57-3)

4-Bromobutyraldehyde L57-2 (4.5 g, crude) was dissolved in methanol (10 mL) and then diethyl ether (10 mL) containing 2N HCl was added. The reaction mixture was stirred at room temperature overnight. The volatile components were evaporated under reduced pressure to give 4-bromo-1, 1-dimethoxybutane L57-3 (3.9 g, crude) as a pale yellow oil; ¹H-NMR(300MHz,CDCl₃ ) δ4.38 (m, 1H), 3.4 (m, 2H), 3.32 (s, 6H), 1.91 (m, 2H), 1.75 (m, 2H).

Synthesis of 1- (4, 4-dimethoxybutyl) -1H-imidazole (L57-4)

To a solution of imidazole (1.48 g,21.76 mmol) in anhydrous THF (40 mL) at 5-10deg.C with stirring was added NaH (948 mg,23.74mmol,60% in mineral oil) in portions. The resulting mixture was then stirred at room temperature for 2h. To the suspension was added dropwise THF (10 mL) containing 4-bromo-1, 1-dimethoxybutane L57-3 (3.9 g,19.79 mmol) over a period of 15min and the reaction was stirred at room temperature for a further 3h to achieve a homogeneous mixture. The reaction mixture was heated at 60 ℃ overnight, cooled to room temperature and filtered. THF was removed under reduced pressure and the residue was purified by flash chromatography (SiO 2:0-5% MeOH/DCM gradient) to give 1- (4, 4-dimethoxybutyl) -1H-imidazole L57-4 (850 mg, over 3 steps) 12％).¹H-NMR(300MHz,CDCl₃)δ7.45(s,1H),7.04(s,1H),6.9(s,1H),4.32(t,J＝5.49Hz,1H),3.95(t,J＝7.14Hz,2H),3.29(s,6H),1.84(m,2H),1.58(m,2H);CIMS m/z[M+H]⁺185.

Synthesis of 1- (4-oxobutyl) -1H-imidazol-1-ium chloride (L57-5)

To a solution of 1- (4, 4-dimethoxybutyl) -1H-imidazole L57-4 (1.05 g,5.7 mmol) in THF (5.0 mL) was added 1.5N HCl (5.0 mL). The reaction mixture was stirred at room temperature overnight. THF was evaporated and the aqueous layer was washed with DCM (10 mL) and EtOAc (10 mL) to remove impurities. The aqueous layer was evaporated under reduced pressure, then co-evaporated with acetonitrile (2 x 10 ml) and toluene (2 x 10 ml) and dried under high vacuum for 24H to give 1- (4-oxobutyl) -1H-imidazol-1-ium chloride L57-5 as a pale yellow gummy solid which was used in the next step (1.0 g, crude) without further purification ).¹H-NMR(300MHz,DMSO-D6)δ9.63(s,1H),9.21(s,1H),7.81(s,1H),7.7(s,1H),4.19(m,2H),3.34-3.62(m,2H)2.05(m,2H);CIMS m/z[M+H]⁺139.

Synthesis of diethyl 2,2' - (1- (4- (1H-imidazol-1-yl) butyl) piperidine-4, 4-diyl) diacetate (L57-6)

To a solution of diethyl 2,2' - (piperidine-4, 4-diyl) diacetate (850 mg,3.5 mmol) in a mixture of DMF (5 mL) and DCE (5 mL) was added DMF (5 mL) containing 1- (4-oxobutyl) -1H-imidazol-1-ium chloride L57-5 (1.0 g,5.74 mmol), followed by Na (OAc) ₃ BH (2.22 g,10.5 mmol) and AcOH (240. Mu.L, 4.2 mmol). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. LC-MS confirmed the reaction was complete. The reaction mixture was diluted with DCM and washed with saturated NaHCO ₃. The aqueous layer was extracted with DCM (3X 50 mL). The combined organic layers were dried over Na ₂SO₄ and concentrated under reduced pressure to give the crude product, which was purified by flash chromatography (SiO 2:0-6% MeOH/DCM gradient) to give diethyl 2,2' - (1- (4- (1H-imidazol-1-yl) butyl) piperidine-4, 4-diyl) diacetate L57-6(600mg,45％).¹H-NMR(300MHz,CDCl₃)δ7.45(s,1H),7.04(s,1H),6.89(s,1H),4.08(q,J＝7.14Hz,4H),3.93(t,J＝7.14Hz,2H),2.53(s,4H),2.36(m,6H),1.78(m,2H),1.67(m,4H),1.5(m,2H),1.23(t,J＝7.14Hz,6H);CIMS m/z[M+H]⁺380.

Synthesis of 2,2' - (1- (4- (1H-imidazol-1-yl) butyl) piperidine-4, 4-diyl) bis (ethan-1-ol) (L57-7)

To a solution of 2,2' - (1- (4- (1H-imidazol-1-yl) butyl) piperidine-4, 4-diyl) diacetate L57-6 (600 mg,1.58 mmol) in anhydrous THF (10 mL) was added dropwise a solution of LiAlH ₄ in anhydrous THF (2.0M, 1.6mL,3.16 mmol) at 0deg.C under nitrogen. The resulting reaction mixture was stirred at room temperature overnight. The reaction mixture was cooled to 0 ℃ and Na ₂SO₄.10H₂ O was slowly added until all gas evolution ceased. After filtration through celite, the filter cake was washed with THF. The combined filtrates were concentrated under reduced pressure to give 2,2' - (1- (4- (1H-imidazol-1-yl) butyl) piperidin-4, 4-diyl) bis (ethan-1-ol) L57-7 as a colorless viscous liquid, which was used in the next step without further purification (440 mg, crude) ).¹H-NMR(300MHz,CDCl₃)δ7.45(s,1H),7.02(s,1H),6.89(s,1H),3.93(t,J＝7.14Hz,2H),3.7(t,J＝6.6Hz,4H),2.33(m,6H),1.77(m,2H),1.65(t,J＝6.75Hz,4H),1.55(m,2H),1.47(m,4H);CIMS m/z[M+H]⁺296.

Synthesis of bis (4, 4-bis (nonyloxy) butanoic acid) (1- (4- (1H-imidazol-1-yl) butyl) piperidine-4, 4-diyl) bis (ethane-2, 1-diyl) ester (CY 71)

To a solution of 4, 4-bis (nonyloxy) butanoic acid (1.21 g,3.27 mmol) in DCM (15 mL) was added DMAP (803 mg,2.98 mmol) and EDC (1.25 g,6.55 mmol). The reaction mixture was stirred at room temperature for 15min, and DCM (5 mL) containing 2,2' - (1- (4- (1H-imidazol-1-yl) butyl) piperidine-4, 4-diyl) bis (ethan-1-ol) L57-7 (440 mg,1.49 mmol) was added to the reaction mixture. The reaction mixture was stirred at room temperature overnight. Product formation was confirmed by LCMS. The reaction mixture was diluted with DCM and then washed with water and brine. The DCM layer was dried over Na ₂SO₄ and concentrated under reduced pressure to give the crude product. The crude product was purified by flash chromatography (SiO ₂: 0-5% MeOH/DCM and 1% NH ₄ OH gradient) to give compound CY71 (404 mg, two step, 26％).¹H-NMR(300MHz,CDCl₃)δ:7.45(s,1H),7.04(s,1H),6.89(s,1H),4.47(t,J＝5.3Hz,2H),4.1(t,J＝7.4Hz,4H),3.93(t,J＝6.75Hz,2H),3.53(m,4H),3.4(m,4H),2.35(m,10H),1.89(m,4H),1.75(m,2H),1.66(m,2H),1.54(m,12H),1.25(m,48H),0.87(m,12H);CIMS m/z[M+H]⁺:1004.1. analytical HPLC column: agilent Zorbax SB-C18,5 μm, 4.6X106 mm, mobile phase A: acetonitrile containing 0.1% trifluoroacetic acid, mobile phase B: water containing 0.1% trifluoroacetic acid, using gradient: A/B5% to 95% within 15min, flow rate: 1mL/min, column temperature: 20.+ -. 2 ℃, detector: ELSD, t _R =8.5 min, purity: >99%; UPLC column: waters Aquity)CSHTM, C18,1.7 μm, 3.0X105 mm, (part number 186005302), mobile phase A: acetonitrile containing 0.1% trifluoroacetic acid, mobile phase B: water containing 0.1% trifluoroacetic acid using a gradient: a/B5% to 95% in 15min, flow rate: 1mL/min, column temperature: 20±2 ℃, detector: CAD, t _R =13.7 min, purity: >99%.

EXAMPLE 10 Synthesis of bis (4, 4-bis (nonyloxy) butanoic acid) (1- (3- (1H-imidazol-1-yl) propyl) piperidine-4, 4-diyl) bis (ethane-2, 1-diyl) ester (CY 70)

Synthesis of 1- (3, 3-dimethoxypropyl) -1H-imidazole (L64-2)

To a solution of imidazole (2.0 g,30 mmol) in anhydrous THF (60 mL) was added NaH (1.31 g,32.78mmol,60% in mineral oil) in portions with stirring. The resulting mixture was stirred at room temperature for 2h. To the resulting suspension was added dropwise THF (15 mL) containing 3-bromo-1, 1-dimethoxypropane (5.0 g,27.32 mmol) over a period of 10min and stirred for a further 3h to give a homogeneous mixture. The reaction mixture was heated at 60 ℃ overnight and then cooled to room temperature and filtered. THF was removed under reduced pressure. DCM was added followed by activated carbon and anhydrous Na ₂SO₄, stirred for 2h and filtered through celite. DCM was removed under reduced pressure to give the crude product as a pale yellow liquid which was purified by flash chromatography (SiO ₂:0-5% MeOH/DCM gradient) to give 1- (3, 3-dimethoxypropyl) -1H-imidazole L64-2(3.64g,78％).¹H-NMR(300MHz,CDCl₃)δ:7.45(s,1H),7.04(s,1H),6.9(s,1H),4.24(t,J＝5.49Hz,2H),4.01(t,J＝7.14Hz,4H),2.05(q,J＝6.84Hz,4H);CIMS m/z[M+H]⁺171.1.

Synthesis of 3- (1H-imidazol-1-yl) propanal hydrochloride (L64- -3)

To a solution of compound 1- (3, 3-dimethoxypropyl) -1H-imidazole L64-2 (3.0 g17.64 mmol) in THF (15.0 mL) was added 1.5N HCl (15.0 mL). The reaction mixture was stirred at room temperature overnight. THF was evaporated and the aqueous layer was washed with DCM and EtOAc to remove impurities. The aqueous layer was evaporated under reduced pressure, then co-evaporated with acetonitrile (2 x 10 ml) and toluene (2 x 10 ml) and dried under high vacuum for 24H to give 3- (1H-imidazol-1-yl) propanal hydrochloride L64-3 (2.7 g) as a pale yellow gummy solid which was used in the next step without further purification .¹H-NMR(300MHz,DMSO-D6)δ:9.67(s,1H),9.14(s,1H),7.75(s,1H),7.66(s,1H),4.42(t,J＝6.45Hz,2H),3.18(t,J＝6.6Hz,2H);CIMS m/z[M+H]⁺125.2.

Synthesis of diethyl 2,2' - (1- (3- (1H-imidazol-1-yl) propyl) piperidine-4, 4-diyl) diacetate (L64-4)

To a solution of diethyl 2,2' - (piperidine-4, 4-diyl) diacetate (555 mg,2.28 mmol) in DCE (10 mL) was added 3- (1H-imidazol-1-yl) propanal hydrochloride L64-3 (730 mg,4.56 mmol), followed by Na (OAc) ₃ BH (1.45 g,6.84 mmol) and AcOH (156 mL,2.73 mmol). The reaction mixture was stirred at room temperature under nitrogen for 18 hours and then heated and stirred at 50 ℃ for 2 hours. Reaction completion was confirmed by LCMS. The reaction mixture was diluted with DCM and washed with saturated NaHCO ₃. The aqueous layer was extracted with DCM. The combined organic layers were dried over Na ₂SO₄ and concentrated under reduced pressure to give the crude product. The crude product was purified by flash chromatography (SiO ₂:0-6% MeOH/DCM gradient) to give diethyl 2,2' - (1- (3- (1H-imidazol-1-yl) propyl) piperidine-4, 4-diyl) diacetate L64-4(440mg,52％).¹H-NMR(300MHz,CDCl₃)δ:7.48(s,1H),7.04(s,1H),6.89(s,1H),4.09(q,J＝7.1Hz,2H),3.99(t,J＝6.84Hz,4H),2.54(s,4H)2.4(m,4H),2.28(t,J＝7.14Hz,2H),1.92(m,2H),1.69(m,4H),1.26(t,J＝7.14Hz,6H);CIMS m/z[M+H]⁺366.2.

Synthesis of 2,2' - (1- (3- (1H-imidazol-1-yl) propyl) piperidine-4, 4-diyl) bis (ethan-1-ol) (L64-5)

To a solution of diethyl 2,2' - (1- (3- (1H-imidazol-1-yl) propyl) piperidine-4, 4-diyl) diacetate L64-4 (430 mg,1.17 mmol) in anhydrous THF (10 mL) at 0deg.C was added dropwise a solution of 2.0M LiAlH ₄ in THF (1.2 mL,2.35 mmol). The resulting mixture was stirred at room temperature overnight. The reaction mixture was cooled to 0 ℃ and Na ₂SO₄.10H₂ O was slowly added until all gas evolution ceased. After filtration through celite, the filter cake was washed with THF. All the filtrates were concentrated under reduced pressure to give 2,2' - (1- (3- (1H-imidazol-1-yl) propyl) piperidine-4, 4-diyl) bis (ethan-1-ol) L64-5 (375 mg, crude) as a colorless viscous liquid, which was used in the next step without further purification .¹H-NMR(300MHz,CDCl₃)δ7.64(s,1H),7.12(s,1H),6.91(s,1H),4.05(t,J＝6.6Hz,2H),3.61(t,J＝7.6Hz,4H),2.45(m,4H),2.32(t,J＝7.14Hz,2H),1.9(m,2H),1.56(m,8H);CIMS m/z[M+H]⁺282.2.

Synthesis of bis (4, 4-bis (nonyloxy) butanoic acid) (1- (3- (1H-imidazol-1-yl) propyl) piperidine-4, 4-diyl) bis (ethane-2, 1-diyl) ester (CY 70)

To a solution of 4, 4-bis (nonyloxy) butanoic acid (L4-3 (T9)) (375 mg,1.31 mmol) in DCM (15 mL) was added DMAP (320 mg,2.62 mmol) and EDC (1.2 g,6.29 mmol). The reaction mixture was stirred at room temperature for 15min, and DCM (5 mL) containing 2,2' - (1- (3- (1H-imidazol-1-yl) propyl) piperidine-4, 4-diyl) bis (ethan-1-ol) L64-5 was added. The reaction mixture was stirred at room temperature overnight and then diluted with DCM, washed with water and brine. The DCM layer was dried over Na ₂SO₄ and concentrated under reduced pressure. The crude product was purified by flash chromatography (SiO 2:0-5% MeOH/DCM and 1% NH ₄ OH gradient) to give compound CY70 (445 mg, two-step 25％).¹H-NMR(300MHz,CDCl₃)δ7.45(s,1H),7.04(s,1H),6.89(s,1H),4.48(t,J＝5.4Hz,2H),4.11(t,J＝7.4Hz,4H),3.99(t,J＝7.17Hz,2H),3.54(m,4H),3.4(m,4H),2.36(m,8H),2.25(m,2H),1.92(m,6H),1.67(m,2H),1.52(m,10H),1.25(m,50H),0.87(m,12H);CIMS m/z[M+H]⁺990.1. analytical HPLC column: agilent Zorbax SB-C18,5 μm, 4.6X106 mm, mobile phase A: acetonitrile containing 0.1% trifluoroacetic acid, mobile phase B: water containing 0.1% trifluoroacetic acid, using gradient: 15min A/B5% to 95%, flow rate: 1mL/min, column temperature: 20.+ -. 2 ℃, detector: ELSD, t _R =8.3 min, purity >99%; UPLC column: waters Aquity) as colorless oilCSHTM, C18,1.7 μm, 3.0X105 mm, (part number 186005302), mobile phase A: acetonitrile containing 0.1% trifluoroacetic acid, mobile phase B: water containing 0.1% trifluoroacetic acid using a gradient: a/B5% to 95% in 15min, flow rate: 1mL/min, column temperature: 20±2 ℃, detector: CAD, t _R =13.4 min, purity: >99%.

Preparation of lipid nanoparticles-general procedure

Representative lipids of the present disclosure, distearoyl phosphatidylcholine (DSPC), cholesterol, and 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000) were dissolved in pure ethanol at a molar ratio of 48.5:10:40:1.5 (IM administration) or 48.5:10:39:2.5 (IV administration), with a total lipid concentration of 10.8mM. See, e.g., qia et al, PNAS118:e2020401118 (2021). The lipid solution was then mixed with an acidic sodium acetate buffer (pH 4.0) or sodium citrate buffer (pH 4.0) containing mRNA (0.10 mg/mL) using a NanoAssemblr microfluidic system. The mRNA solution and lipid solution were each injected into NanoAssemblr microfluidic devices at a total flow rate of 12mL/min, at a 3:1 ratio of mRNA solution to lipid solution, and the devices allowed for rapid mixing of the two components and thus self-assembly of the LNP. The formulations were further dialyzed overnight against PBS (pH 7.4) or 20mM Tris (pH 7.4) with 8% sucrose solution at 4℃in a dialysis cartridge. Particle size of the formulations was measured by Dynamic Light Scattering (DLS) using a Zetasizer Ultra (MALVERN PANALYTICAL). RNA encapsulation efficiency was characterized by Ribogreen assay.

EXAMPLE 11 LNP formulation A

The ionizable lipid, DSPC, cholesterol, and PEG2K-DMG were dissolved in pure ethanol at a ratio of 48.5:10:39:2.5mol%, with a total lipid concentration of 10.8mM. An acidic buffer (pH 4.0-5.0) containing mRNA encoding human erythropoietin (hEPO) and firefly luciferase (fLuc) (1:2 ratio) was used to prepare a 0.10mg/mL mRNA solution. The nucleotide and lipid solutions were mixed at a 3:1 volume ratio using NanoAssemblr microfluidic system at a total flow rate of 12mL/min, resulting in rapid mixing and self-assembly of LNP. The formulation was further dialyzed overnight against PBS (pH 7.4) at 4 ℃, concentrated using centrifugal filtration and filtered (0.2 μm pore size). The particle size and polydispersity index (PDI) of the formulations were measured by Dynamic Light Scattering (DLS) using a Zetasizer Ultra (MALVERN PANALYTICAL). RNA encapsulation efficiency (EE%) was determined by Ribogreen assay.

Table 2A: LNP formulation

Formulations	Ionizable lipids	Acidic buffer	Size (nm)	PDI	EE％
						F-1	CY57	B	81.2	0.17	86.0
F-2	CY63	B	81.5	0.11	91.0
						F-3	CY65	B	67.8	0.16	94.5
F-4	CY66	B	82.0	0.19	92.5
						F-5	CY67	B	72.8	0.13	92.3
F-6	CY69	B	72.4	0.12	94.9
						F-7	CY70	B	94.9	0.08	90.9

Buffer a:25mM sodium acetate, pH 5.0; buffer B:50mM citrate, pH 4.0

EXAMPLE 12 LNP formulation B

The ionizable lipid, DSPC, cholesterol and PEG2K-DSPE were dissolved in pure ethanol at a ratio of 48.5:10:40:1.5mol%, with a total lipid concentration of 10.8mM. A0.10 mg/mL mRNA solution was prepared using an acidic buffer (pH 4.0-5.0) containing mRNA encoding firefly luciferase (fLuc). The nucleotide and lipid solutions were mixed at a 3:1 volume ratio using NanoAssemblr microfluidic system at a total flow rate of 12mL/min, resulting in rapid mixing and self-assembly of LNP. The formulation was further dialyzed overnight against PBS (pH 7.4) at 4 ℃, concentrated using centrifugal filtration and filtered (0.2 μm pore size). The particle size and polydispersity index (PDI) of the formulations were measured by Dynamic Light Scattering (DLS) using a Zetasizer Ultra (MALVERN PANALYTICAL). RNA encapsulation efficiency (EE%) was determined by Ribogreen assay.

Table 2B: LNP formulation

Formulations	Ionizable lipids	Acidic buffer	Size (nm)	PDI	EE％
						F-8	CY61	B	75.7	0.071	91.39
F-9	CY63	B	87	0.078	95.1
						F-10	CY69	B	73.1	0.05	96.03
F-11	CY66	B	97.8	0.091	95.04
						F-12	CY67	B	104.9	0.066	96.5
F-13	CY65	B	73.7	0.032	96.75
						F-14	CY70	B	126.6	0.083	95.28
F-15	CY71	B	105.3	0.081	95.38

Buffer a:25mM sodium acetate, pH 5.0; buffer B:50mM citrate, pH 4.0

Example 13 LNP formulation C

The ionizable lipid, DSPC, cholesterol, and PEG2K-DMG were dissolved in pure ethanol at a 48.5:10:40:1.5mol% ratio (formulation comprising the ionizable lipid of the present disclosure) or a 50:10:38.5:1.5mol% ratio (SM 102 formulation), wherein the total lipid concentration was 10.8mM. A0.10 mg/mL RNA solution was prepared using an acidic buffer (pH 4.0-5.0) containing circular RNA (oRNA) encoding COVID spike protein or linear mRNA, as indicated in Table XC. The nucleotide and lipid solutions were mixed at a 3:1 volume ratio using NanoAssemblr microfluidic system at a total flow rate of 12mL/min, resulting in rapid mixing and self-assembly of LNP. The formulation was further dialyzed overnight against cold buffer at 4 ℃, concentrated using centrifugal filtration and filtered (0.2 μm pore size). The formulation was then stored at-80 ℃ until use. The particle size and polydispersity index (PDI) of the formulations were measured by Dynamic Light Scattering (DLS) using a Zetasizer Ultra (MALVERN PANALYTICAL). RNA encapsulation efficiency (EE%) was determined by Ribogreen assay.

Table 2C: LNP formulation

Buffer a:25mM sodium acetate, pH 5.0; buffer B:50mM citrate, pH 4.0

Example 14 hEPO and fLUC in vivo reporter assays

LNP (formulated with 0.1mg/kg EPO and 0.2mg/kg Luc, see example 11) was administered to Balb/cAnNCrl (female, 6-8 weeks) by intravenous injection. Plasma samples were collected 5, 23 and 47 hours post-dose for hEPO analysis. After injection of D-fluorescein solution (150 mg/kg, intraperitoneal Injection (IP)), mice were bioluminescent imaged (BLI) for measurement at 6, 24 and 48 hours post-dose using IVIS Lumina III LT system (PerkinElmer). hEPO concentrations were measured using ELISA kit (DEP 00, R & D Systems). Using non-compartmental analysis (non-compartment analysis; NCA) procedureThe maximum concentration or BLI signal (Cmax) and the area under the concentration versus time curve (AUC) of individual mouse plasma hEPO or whole body BLI data were calculated at version 8.3.4 [ PHARSIGHT CORP (Mountain View, CA, USA) ].

Table Y reports the hEPO concentration at 5 hours post-dose and AUC over a period of 48 hours for each formulation tested, overall and relative to the internal standard throughout the experiment. Table Y also reports luciferase bioluminescence imaging measured 6 hours post-dosing and AUC over a period of 48 hours for each formulation tested.

Data legend:

hEPO C5hr(IU/μL)：+＝<10IU/μL；10IU/μL≤++<100IU/μL；100≤+++<1,000IU/μL

hEPO AUC(hr*IU/μL)：+＝<10hr*IU/μL;10hr*IU/μL≤++<100hr*IU/μL;100hr*IU/μL≤+++<1,000hr*IU/μL;1,000hr*IU/μL≤++++<10,000hr*IU/μL

AUC ratio relative to standard: * = <0.1;0.1 <0.5;0.5 <1.0;1.0 <1.5;1.5 <2.0; * ?

Luciferase blic 6hr (photons/second): # = <1 hundred million p/s;1 hundred million p/s is less than or equal to # 10 hundred million p/s;10 hundred million p/s is less than or equal to # # <100 hundred million p/s;100 hundred million p/s is less than or equal to # # # less than 1000 hundred million p/s;1000 hundred million p/s is less than or equal to # # is less than 1 trillion p/s

Luciferase BLI AUC48hr (hr photon/sec): p/s $ <100 hundred million hr; 100 hundred million hr p/s is less than or equal to $ 1000 hundred million hr p/s;1000 hundred million hr p/s is less than or equal to $ 1 trillion hr p/s;1 trillion hr p/s is less than or equal to $ 10 trillion hr p/s

Table 3: in vivo assay data

EXAMPLE 15 in vivo organ tropism assay

Balb/cAnNCrl (female, 6-8 weeks) was given LNP formulation (formulated with 0.2mg/kg Luc mRNA, see example 12) by IV injection. Mice were injected with a D-fluorescein solution (150 mg/kg, intraperitoneal (IP)) 6 hours after LNP dosing. Mice were sacrificed 10 minutes after D-fluorescein administration and organs (liver, spleen, lung, heart, kidney) were collected. Organs from each dosing group were simultaneously bioluminescent imaged using IVIS Lumina III LT system (PerkinElmer).

The sum of bioluminescence from all organs of each individual mouse was summed as total flux (photons/sec). The percent bioluminescence of each individual organ was calculated to determine the organ tropism of the LNP formulation.

Luciferase blic 6hr (photons/second): # = <1 hundred million p/s;1 hundred million p/s is less than or equal to # 10 hundred million p/s

TABLE 4 Total flux of organs and percent flux in each organ

Example 16 in vivo T cell response to RNA encoding spike protein-murine

Dosing regimen

LNP formulations were prepared as described in example 13. On days 0 and 21, each formulation was injected intramuscularly in5 BALB/c mice at 0.02mg/mL of oRNA or linear mRNA encoding COVID spike protein, in a total volume of 0.5 mL. Prior to dosing, BALB/c mice were placed in chambers pre-filled with isoflurane at a flow rate of 0.4-0.8 liters/min until sedated so that no exercise was performed during injection. After both administrations, the stimulation at the injection site was monitored. On day 35, all mice were humanly euthanized by CO ₂ inhalation and spleens were collected and stored on wet ice until treatment. All in vivo experiments in this study were performed under approved animal care guidelines.

Analysis

Spleens were collected and manually isolated as single cell suspensions by filtration using a 70 μm filter (Miltenyi 130-098-462) and washed with 1xPBS (Fisher 10010049) containing 2mM EDTA (thermo Fisher 15575-020) and 0.5% BSA (Miltenyi 130-091-376). Erythrocytes were lysed using ACK lysis buffer (thermo fisher a 1049201) and washed twice with 1x PBS+2mM EDTA+0.5%BSA. After the last wash, the cells were resuspended in 1x PBS and counted (ViCell XR, beckman Coulter 731196). Cells were resuspended in CTL Test Plus medium (C.T.L.CTLTP-005) containing 1x Glutamax (ThermoFisher TP-050122) and 1x Pen/Strep (ThermoFisher 15-140-122) at appropriate concentrations and plated for downstream function assays.

ELISPot assays were performed using the mouse IFN-. Gamma. ELISpotPLUS kit (Mabtech 3321-4 HST-10) according to the manufacturer's protocol. Briefly, plates were washed with 1 XPBS and blocked with RPMI (ThermoFisher 72400-047) containing 10% FBS (ThermoFisher A38400-01) for 1h at 37 ℃. After blocking, cells were plated at 200,000 cells/well (for DMSO and peptide stimulated wells) or 25,000 cells/well (for PMA/Ionomycin (Ionomycin) treatment). Cells were incubated with 1% DMSO (ThermoFisher D12345), 7.5 μg/mL of the S1 or S2 peptide pool (JPT PM-WCPV-S-1) of spike protein spanning SARS-CoV-2, or 1 XPMA/ionomycin (ThermoFisher 00-4970-93) in triplicate. Plates were incubated overnight at 37℃with 5% CO ₂. After incubation, plates were washed and 1 μg/mL detection antibody was added for 2h at room temperature. The wash was repeated and 1x streptavidin-HRP was added and incubated for 1 hour at room temperature. Finally, the plates were washed and TMB substrate was added, incubated in the dark for producing color spots, and then washed off using running water. The plates were dried and counted by ELISpot analyzer (ZellNet Consulting).

For intracellular staining (ICS), 5,000,000 cells/Kong Tupu were stimulated in a 96-well round bottom plate (Costar 3799) and using the same ELISpot conditions as described above, and incubated at 37 ℃ with 5% CO ₂ for a total of 5.5h. Golgi plug (BD 555029) was added to all wells for the last 4.5h stimulation. After incubation, cells were stained for flow cytometry using surface or intracellular antibodies listed in the following table. Briefly, cells were washed with 1XPBS and stained with Live/dead fixable water (Live/Dead Fixable Aqua) (Invitrogen L34966) for 20min at room temperature. Then, the cells were washed twice with a cell staining buffer (BioLegend 420201) and incubated with an Fc blocking agent (BioLegend 156604) at 4 ℃ for 5min followed by surface antibody staining at 4 ℃ for 30min. Thereafter, the cells were washed twice with a cell staining buffer, fixed in an IC fixation buffer (ThermoFisher 88-8824-00) at 4℃for 30min, and infiltrated in a1 Xinfiltration buffer (ThermoFisher 88-8824-00), and intracellular staining was performed at 4℃overnight. Thereafter, cells were washed twice with 1x penetration buffer, resuspended in 1xPBS, and harvested on a cell counter (ThermoFisher Attune NXT with a blue (3)/red (3)/violet (4)/yellow (4) laser configuration) equipped with a high throughput autosampler (ThermoFisher CytKick). Compensation was performed using UltraComp eBeads (ThermoFisher 01-3333-41) and the ArC amine reactive compensation bead kit (ThermoFisher A10346).

Results

Mice with LNP formulation F-16 were shown to respond to the Moderna SM LNP formulations F-16 and F-17 with a spike-specific multifunctional CD 4T cell.

Example 17 in vivo T cell response to spike protein-encoding RNA-non-human primate

Dosing regimen

LNP formulations were prepared as described in example 13. On day 1 (once) and day 22 (once), each formulation was injected via intramuscular injection into 3 non-untreated cynomolgus monkeys at a dose level of 100 μg. All NHPs were temporarily restricted for dosing and did not sedate. Prior to administration, the administration site is shaved and marked as needed for clinical observation. Each dose was administered in the demarcation region using a syringe/needle. Samples were collected throughout the study for clinical pathology parameters, pharmacokinetic analysis, and immunogenicity analysis.

Analysis

Cryopreserved PBMC were thawed in a 37℃water bath and cells were transferred to conical tubes containing complete RPMI (RPMI [ ThermoFisher 72400-047 ]) containing 10% FBS [ ThermoFisher A38400-01] and 1XPen/Strep [ ThermoFisher 15-140-122]. Cells were centrifuged, resuspended in complete RPMI containing 50U/mL Benzonase (EMD 70664-10 KUN) and incubated at 37℃for 15min. Cells were centrifuged, resuspended in complete RPMI and allowed to stand for 3 hours. Cells were centrifuged and resuspended in CTL Test Plus medium (C.T.L.CTLTP-005) containing 1X Glutamax (thermo Fisher TP-050122) and 1xPen/Strep, and counted (Vicell XR, beckman Coulter 731196). The concentration was adjusted and cells plated for downstream functional assays.

ELISPot assays were performed using the monkey IFN-. Gamma. ELISpotPLUS kit (Mabtech 3421M-4 HST-10) according to the manufacturer's protocol. Briefly, plates were washed with 1 XPBS and blocked with RPMI (ThermoFisher 72400-047) containing 10% FBS (ThermoFisher A38400-01) for 1h at 37 ℃. After blocking, cells were plated in triplicate and stimulated under the following conditions: no peptide (1% DMSO (ThermoFisher D12345)), 0.5 μg/mL pool of S1+S2 peptides spanning spike protein of SARS-CoV-2 (JPT PM-WCPV-S-1) and 1 XPMA/ionomycin (ThermoFisher 00-4970-93). 200,000 cells/well (for DMSO and peptide pool stimulation) were plated, and 10,000 cells/well (for PMA/ionomycin stimulation) of pooled samples from each group were plated.

Plates were incubated overnight at 37℃with 5% CO ₂. After incubation, plates were washed and 1 μg/mL detection antibody was added for 2h at room temperature. The wash was repeated and 1x streptavidin-HRP was added and incubated for 1h at room temperature. Finally, the plates were washed and TMB substrate was added, incubated in the dark for producing color spots, and then washed off using running water. The plates were dried and counted using an ELISpot analyzer (ZellNet Consulting).

For intracellular staining (ICS), approximately 2,000,000 cells/Kong Tupu from each animal were in a 96-well round bottom plate (Costar 3799) and stimulated using the same ELISpot conditions described above. After one hour stimulation, golgi plugs (BD 555029) were added to all wells and the plates were incubated overnight at 37 ℃ with 5% CO ₂. Thereafter, the cells were washed and stained for flow cytometry. Briefly, cells were washed with 1x PBS and stained with live/dead fixable water (Invitrogen L34966) for 20min at room temperature. Then, the cells were washed twice with a cell staining buffer (BioLegend 420201) and incubated with an Fc blocking agent (BioLegend 156604) at 4 ℃ for 5min, followed by surface antibody staining at 4 ℃ for 30min. After surface staining, the cells were then washed twice with cell staining buffer, and fixed in IC fixation buffer for 30min at 4℃and permeabilized in 1 Xpermeabilization buffer (ThermoFisher 88-8824-00). Intracellular staining was performed at 4 ℃ for 1 hour. Thereafter, cells were washed twice with 1x penetration buffer, resuspended in 1x PBS, and acquired on a cell counter (ThermoFisher Attune NXT with a blue (3)/red (3)/violet (4)/yellow (4) laser configuration) equipped with a high throughput autosampler (ThermoFisher CytKick). Compensation was performed using UltraComp eBeads (ThermoFisher 01-3333-41) and the ArC amine reactive compensation bead kit (ThermoFisher A10346).

Results

The NHP given to LNP formulation F-16 exhibited a greater or comparable spike protein specific multifunctional CD 4T cell response than Moderna SM LNP formulation 102, as determined by ICS assay. On day 36, formulation F-16 exhibited about a 10-fold higher spike protein specific T cell response compared to control formulation F-17, and a similar response to control formulation F-18. On day 36, T cells from NHPs giving F-16 also exhibited increased spike protein specific IFNγ secreting T cells, 3-fold over F-17 induced T cells and comparable to F-18 preparations, as determined via the ELISPot assay.

XIII equivalent and scope

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. The scope of the present disclosure is not intended to be limited by the foregoing description, but rather is set forth in the following claims.

In the claims, articles such as "a/an" and "the" may mean one or more than one unless indicated to the contrary or otherwise apparent from the context. Unless indicated to the contrary or otherwise apparent from the context, if one, more than one, or all group members are present, used in, or otherwise related to an intended product or method, then the claims or descriptions that include an "or" between one or more members of a group are considered to be satisfied. The present disclosure includes embodiments in which exactly one member of the group is present in, used in, or otherwise related to the intended product or process. The present disclosure includes embodiments in which more than one or all of the group members are present, used in, or otherwise associated with a given product or process.

It should also be noted that the term "comprising" is intended to be open and allows for, but does not require, the inclusion of additional elements or steps. When the term "comprising" is used herein, the term "consisting of … …" is also hereby encompassed and disclosed.

As used herein, the term "about" means within 10% of a given value or range. Thus, "about 10" means 9 to 11.

When ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values expressed as ranges may employ any particular value or subrange within the stated range in different embodiments of the disclosure, reaching one tenth of the unit of the lower limit of the range unless the context clearly dictates otherwise.

In addition, it should be understood that any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are believed to be known to those of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. For any reason, whether or not related to the presence of the prior art, any particular embodiment of the compositions of the present disclosure (e.g., any antibiotic, therapeutic or active ingredient, any method of manufacture, any method of use, etc.) may be excluded from any one or more of the claims.

It is to be understood that the words which have been used are words of description rather than limitation, and that changes may be made within the scope of the appended claims without departing from the true scope and spirit of the disclosure in its broader aspects.

Although the present disclosure has been described with respect to several embodiments described, at a certain length and with some particularity, it is not intended that the present disclosure should be limited to any such details or embodiments or any particular embodiment, but rather should be construed with reference to the appended claims in order to provide the broadest possible interpretation of such claims in view of the prior art and, therefore, to effectively encompass the intended scope of the present disclosure.

Claims

1. A compound of formula (CY)

Or a pharmaceutically acceptable salt thereof,

Wherein:

R ¹ is selected from the group consisting of: -OH, -OAc, R ^1a,

Z ¹ is optionally substituted C ₁-C₆ alkyl;

X ¹ is optionally substituted C ₂-C₆ alkylene;

y ¹ and Y ² are independently selected from the group consisting of:

Wherein the bond labeled with "+" is attached to X ⁴ or X ⁵;

Each Z ² is independently H or optionally substituted C ₁-C₈ alkyl;

Each Z ³ is independently optionally substituted C ₁-C₆ alkylene;

R ^1a is:

R ^2a、R^2b and R ^2c are independently hydrogen and C ₁-C₆ alkyl;

r ^3a、R^3b and R ^3c are independently hydrogen and C ₁-C₆ alkyl;

R ^4a、R^4b and R ^4c are independently hydrogen and C ₁-C₆ alkyl;

R ^5a、R^5b and R ^5c are independently hydrogen and C ₁-C₆ alkyl;

Each a is independently C ₃-C₈ cycloalkylene;

Each m is independently 0, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, or 12;

Each n is independently 0, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, or 12;

p is selected from the group consisting of 0,1,2,3,4,5,6 and 7; and

Q is selected from the group consisting of 0,1, 2, 3, 4, 5, 6 and 7.

2. The compound of claim 1, wherein the compound has the formula (CY-I'), (CY-II), (CY-III), (CY-IV), or (CY-V):

Or a pharmaceutically acceptable salt thereof,

Wherein the method comprises the steps of

X ² and X ³ are independently a bond, -CH ₂ -or-CH ₂CH₂ -; and

X ⁶ and X ⁷ are independently-CH ₂ -or-CH ₂CH₂ -.

3. The compound of claim 1 or 2, wherein R ¹ is selected from the group consisting of: -OH,

4. The compound of claim 1 or 2, wherein R ¹ is-OH.

5. The compound of any one of claims 1-4, wherein Y ¹ and Y ² are independently:

6. The compound of any one of claims 1-5, wherein the compound has formula (CY-VI) or (CY-VII):

Or a pharmaceutically acceptable salt thereof.

7. The compound of any one of claims 1-5, wherein the compound has formula (CY-VIII) or (CY-IX):

Or a pharmaceutically acceptable salt thereof.

8. The compound of any one of claims 1-5, wherein the compound is of formula (CY-IV-a), (CY-IV-b), or (CY-IV-c)

Or a pharmaceutically acceptable salt thereof.

9. The compound of any one of claims 1-5, wherein the compound is of formula (CY-IV-d), (CY-IV-e), or (CY-IV-f)

Or a pharmaceutically acceptable salt thereof.

10. The compound of any one of claims 1-9, wherein X ⁴ is optionally substituted C ₂-C₁₀ alkylene and X ⁵ is optionally substituted C ₂-C₁₀ alkylene.

11. The compound of any one of claims 1-9, wherein X ⁴ is optionally substituted C ₂-C₆ alkylene and X ⁵ is optionally substituted C ₂-C₆ alkylene.

12. The compound of claim 11, wherein X ⁴ is optionally substituted C ₂ alkylene and X ⁵ is optionally substituted C ₂ alkylene.

13. The compound of claim 11, wherein X ⁴ is optionally substituted C ₂ alkylene and X ⁵ is optionally substituted C ₂ alkylene.

14. The compound of any one of claims 1-13, wherein X ¹ is optionally substituted C ₂-C₄ alkylene.

15. The compound of any one of claims 1-13, wherein X ¹ is optionally substituted C ₂ alkylene.

16. The compound of any one of claims 1-15, wherein R ² is- (CH ₂)_pCH(OR⁶)(OR⁷).

17. The compound of claim 16, wherein p is selected from the group consisting of 0, 1 and 2.

18. The compound of claim 16, wherein p is 0.

19. The compound of any one of claims 1-18, wherein R ³ is- (CH ₂)_qCH(OR⁸)(OR⁹).

20. The compound of claim 19, wherein q is selected from the group consisting of 0, 1 and 2.

21. The compound of claim 19, wherein q is 0.

22. The compound of any one of claims 1-15 or 19-21, wherein R ² is optionally substituted C ₄-C₂₀ alkyl.

23. The compound of claim 22, wherein R ² is optionally substituted C ₁₀-C₂₀ alkyl.

24. The compound of claim 22, wherein R ² is optionally substituted C ₁₀-C₁₅ alkyl.

25. The compound of claim 22, wherein R ² is optionally substituted C ₁₅-C₂₀ alkyl.

26. The compound of any one of claims 1-18, wherein R ³ is optionally substituted C ₄-C₂₀ alkyl.

27. The compound of claim 26, wherein R ³ is optionally substituted C ₁₀-C₂₀ alkyl.

28. The compound of claim 26, wherein R ³ is optionally substituted C ₁₀-C₁₅ alkyl.

29. The compound of claim 26, wherein R ³ is optionally substituted C ₁₅-C₂₀ alkyl.

30. The compound of any one of claims 1-21, wherein

R ⁶ is optionally substituted C ₁-C₁₄ alkyl;

R ⁷ is optionally substituted C ₁-C₁₄ alkyl;

R ⁸ is optionally substituted C ₁-C₁₄ alkyl; and

R ⁹ is optionally substituted C ₁-C₁₄ alkyl.

31. The compound of any one of claims 30, wherein

R ⁶ is optionally substituted C ₃-C₁₀ alkyl;

R ⁷ is optionally substituted C ₃-C₁₀ alkyl;

R ⁸ is optionally substituted C ₃-C₁₀ alkyl; and

R ⁹ is optionally substituted C ₃-C₁₀ alkyl.

32. The compound of any one of claims 30, wherein

R ⁶ is optionally substituted C ₆-C₁₀ alkyl;

R ⁷ is optionally substituted C ₆-C₁₀ alkyl;

R ⁸ is optionally substituted C ₆-C₁₀ alkyl; and

R ⁹ is optionally substituted C ₆-C₁₀ alkyl.

33. The compound of any one of claims 1-15, wherein R ² is selected from the group consisting of:

34. The compound of any one of claims 1-15 or 33, wherein R ³ is selected from the group consisting of:

35. A compound selected from the group consisting of:

Or a pharmaceutically acceptable salt thereof.

36. A pharmaceutical composition comprising:

a) A polynucleotide, and

B) A delivery vehicle comprising a compound of any one of claims 1-35.

37. The pharmaceutical composition of claim 36, wherein the polynucleotide is DNA.

38. The pharmaceutical composition of claim 37, wherein the polynucleotide is RNA.

39. The pharmaceutical composition of claim 38, wherein the RNA is short interfering RNA (siRNA).

40. The pharmaceutical composition of claim 39, wherein the siRNA inhibits or inhibits expression of a target of interest in a cell.

41. The pharmaceutical composition of any one of claims 36-39, wherein the polynucleotide comprises at least one modification.

42. The pharmaceutical composition of any one of claims 36-41, further comprising an additional cationic lipid.

43. The pharmaceutical composition of any one of claims 36-42, further comprising a neutral lipid.

44. The pharmaceutical composition of any one of claims 36-43, further comprising an anionic lipid.

45. The pharmaceutical composition of any one of claims 36-44, further comprising a helper lipid.

46. The pharmaceutical composition of any one of claims 36-45, further comprising stealth lipids.

47. The pharmaceutical composition of any one of claims 36-46, wherein the weight ratio of the lipid to the polynucleotide is about 100:1 to about 1:1.

48. A vaccine formulation comprising the pharmaceutical composition of any one of claims 36-47.

49. A vaccine formulation comprising the pharmaceutical composition of any one of claims 36-47.

50. A method of vaccinating a subject against an infectious agent, the method comprising:

c) Contacting a subject with the vaccine formulation of claim 48 or the vaccine formulation of claim 49, and

D) An immune response is elicited.

51. A method of delivering a polynucleotide encoding at least one protein of interest into immune cells of a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of any one of claims 36-47.

52. The method of claim 51, wherein the immune cells are T cells.

53. The method of claim 52, wherein the T cells are CD8+ T cells.

54. The method of claim 52, wherein the T cells are T regulatory cells.

55. The method of claim 52, wherein the T cells are CD4+ T cells.

56. The method of claim 51, wherein the immune cells are macrophages, dendritic cells, or liver immune cells.

57. A Lipid Nanoparticle (LNP) comprising the compound of any one of claims 1-35, or a pharmaceutically acceptable salt thereof.

58. The LNP of claim 57, said LNP further comprising:

(a) PEG-lipid;

(b) Structural lipids; and

(C) Non-ionizable lipids and/or zwitterionic lipids.

59. The LNP of claim 58, wherein the ionizable lipid comprises an ionizable amino lipid.

60. The LNP of any one of claims 58-59, wherein said PEG-lipid is selected from the group consisting of: PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC and PEG-DSPE.

61. The LNP of any one of claims 58-60, wherein said structural lipid is selected from the group consisting of: cholesterol, stigmasterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, lycorine, ursolic acid, alpha-tocopherol.

62. The LNP of any one of claims 58-61, wherein said non-ionizable lipid is a phospholipid selected from the group consisting of: 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DLPC), 1, 2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DPPC), 1, 2-Diundecanoyl-sn-glycero-phosphorylcholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (POPC), 1, 2-di-O-octadecenyl-sn-glycero-3-phosphorylcholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphorylcholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphorylcholine (C16 Lyso PC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine, 1, 2-di-arachidonoyl-sn-glycero-3-phosphorylcholine, 1, 2-Didocosahexaenoic acid-sn-glycerol-3-phosphorylcholine, 1, 2-dioctanoyl-sn-glycerol-3-phosphoethanolamine (ME 16.0 PE), 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-dioctanoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-didodecylhexaenoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-didodecyloyl-sn-glycerol-3-phosphoethanolamine, 1, 2-dioctanoyl-sn-glycerol-3-phospho-rac- (1-glycerol) sodium salt (DOPG), Sodium (S) -2-ammonio-3- ((((R) -2- (oleoyloxy) -3- (stearyloxy) propoxy) phosphoryl) oxy) propionate (L- α -phosphatidylserine; Brain PS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoyl phosphatidylglycerol (DMPG), dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal), dioleoyl phosphatidylglycerol (DOPG), 1, 2-dioleoyl-sn-glycerol-3- (phospho-L-serine) (DOPS), cell fusion phospholipid (DPhPE), dipalmitoyl phosphatidylethanolamine (DPPE), dipalmitoyl phosphatidylglycerol (DPPG), Dipalmitoyl phosphatidylserine (DPPS), distearoyl phosphatidylcholine (DSPC), distearoyl-phosphatidyl-ethanolamine (DSPE), distearoyl-ethanolamine-imidazole phosphate (DSPEI), 1, 2-bis-undecanoyl-sn-glycero-choline phosphate (DUPC), phosphatidylcholine (EPC), 1, 2-dioleoyl-sn-glycero-3-phosphate (18:1 pa; DOPA), ammonium bis ((S) -2-hydroxy-3- (oleoyloxy) propyl) phosphate (18:1 DMP; LBPA), 1, 2-dioleoyl-sn-glycerol-3-phosphate- (1' -inositol) (DOPI; 18:1 PI), 1, 2-distearoyl-sn-glycero-3-phospho-L-serine (18:0 PS), 1, 2-dioleoyl-sn-glycero-3-phospho-L-serine (18:2 PS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (16:0-18:1 PS; POPS), 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (18:0-18:1 PS), 1-stearoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine (18:0-18:2 PS), 1-oleoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18:1 lyso PS), 1-stearoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18:0 lyso PS), and sphingomyelin.

63. An LNP according to any one of claims 58-62, further comprising a targeting moiety.

64. The LNP of claim 63, wherein the targeting moiety is an antibody or fragment thereof.

65. An LNP according to any one of claims 58-64, further comprising an active agent.

66. The LNP of claim 65, wherein the active agent is a nucleic acid.

67. The LNP of claim 66, wherein the nucleic acid is ribonucleic acid.

68. The LNP of claim 67, wherein the ribonucleic acid is at least one ribonucleic acid selected from the group consisting of: small interfering RNAs (siRNA), asymmetric interfering RNAs (aiRNA), micrornas (miRNA), dicer-substrate RNAs (dsRNA), small hairpin RNAs (shRNA), messenger RNAs (mRNA), and long non-coding RNAs (lncRNA).

69. The LNP of claim 66, wherein the nucleic acid is a messenger RNA (mRNA) or a circular RNA.

70. The LNP of claim 69, wherein the mRNA comprises an open reading frame encoding a cancer antigen.

71. The LNP of claim 69, wherein the mRNA comprises an open reading frame encoding an immune checkpoint modulator.

72. The LNP of any one of claims 69-71, wherein said mRNA comprises at least one motif selected from the group consisting of: stem loops, chain terminating nucleosides, poly a sequences, polyadenylation signals and 5' cap structures.

73. The LNP of claim 66, wherein the nucleic acid is a polynucleotide encoding a protein selected from the group consisting of SEQ ID NOs 1-54.

74. The LNP of claim 66, wherein the nucleic acid is suitable for genome editing techniques.

75. The LNP of claim 74, wherein the genome editing technique is Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) or transcription activator-like effector nucleases (TALENs).

76. The LNP of claim 66, wherein said nucleic acid is at least one nucleic acid suitable for genome editing technology selected from the group consisting of: CRISPR RNA (crRNA), transactivation crRNA (tracrRNA), single guide RNA (sgRNA) and DNA repair templates.

77. The LNP of claim 69, wherein the mRNA is at least 30 nucleotides in length.

78. The LNP of claim 69, wherein the mRNA is at least 300 nucleotides in length.

79. A pharmaceutical composition comprising the LNP of any one of claims 57-78 and a pharmaceutically acceptable carrier.

80. The pharmaceutical composition of claim 79, formulated for intravenous or intramuscular administration.

81. The pharmaceutical composition of claim 80, formulated for intravenous administration.

82. A method for delivering a nucleic acid to a cell, the method comprising contacting the cell with the LNP of any one of claims 57-78 or the pharmaceutical composition of any one of claims 79-82.

83. A method for treating a disease characterized by a deficiency in a functional protein, the method comprising administering to a subject suffering from the disease an LNP formulation comprising the LNP of any one of claims 82, wherein mRNA encodes the functional protein or a protein having the same biological activity as the functional protein.

84. A method for treating a disease characterized by overexpression of a polypeptide, the method comprising administering to a subject suffering from the disease an LNP formulation comprising the LNP of any one of claims 57-78 and an siRNA, wherein the siRNA targets expression of the overexpressed polypeptide.