Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
  • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
  • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
  • A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
  • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/51—Nanocapsules; Nanoparticles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/5434—IL-12

Definitions

• IL12 protein Due to its ability to activate both NK cells and cytotoxic T cells, IL12 protein has been studied as a promising anti-cancer therapeutic since 1994. See Nastala, C. L. el al, J Immunol 153: 1697-1706 (1994). But despite high expectations, early clinical studies did not yield satisfactory results. Lasek W. et ah, Cancer Immunol Immunother 63: 419-435, 424 (2014). Repeated administration of IL12, in most patients, led to adaptive response and a progressive decline of IL12-induced interferon gamma (IFN-g) levels in blood. Id.
• IFN-g interferon gamma
• IFN-g IL12-induced anti-cancer activity is largely mediated by the secondary secretion of IFNy
• the concomitant induction of IFN-g along with other cytokines (e.g ., TNF-a) or chemokines (IP- 10 or MIG) by IL12 caused severe toxicity. Id.
• the marginal efficacy of the IL12 therapy in clinical settings may be caused by the strong immunosuppressive environment in humans.
• Id. To minimize IFN-g toxicity and improve IL12 efficacy, scientists tried different approaches, such as different dose and time protocols for IL12 therapy. See Sacco, S. et al, Blood 90: 4473-4479 (1997); Leonard, J. P. et al, Blood 90: 2541-2548 (1997); Coughlin, C. M. et al, Cancer Res. 57: 2460-2467 (1997); Asselin-Paturel, C. et al, Cancer 91: 113-122 (2001); and Saudemont, A.
• the present disclosure provides a lipid nanoparticle comprising (i) one or more types of lipid; and (ii) a modified mRNA comprising a sequence that encodes an interleukin (IL)-12 molecule; wherein the lipid nanoparticle is capable of triggering immunogenic cell death.
• the one or more types of lipid comprises a cationic lipid.
• the cationic lipid is a compound of Formula I: salts thereof; wherein each R 1 is independently unsubstituted alkyl; each R 2 is independently unsubstituted alkyl; each R 3 is independently hydrogen or substituted or unsubstituted alkyl; and each m is independently 3, 4, 5, 6, 7, or 8.
• at least one R 1 is C11H23.
• at least one R 3 is hydrogen.
• at least one m is 3.
• the cationic lipid is Nl,N3,N5-tris(3- (didodecylamino)propyl)benzene-l, 3, 5 -tri carboxamide (TT3), having the structure: a nd sa lt s thereof, wherein m is 3.
• the lipid nanoparticle comprises TT3, 1,2- Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, and C14-PEG2000.
• DOPE 1,2- Dioleoyl-sn-glycero-3-phosphoethanolamine
• the modified RNA comprises a modified 5’-cap.
• the modified 5’-cap is selected from the group consisting of m2 7,2 °Gpp s pGRNA, m 7 GpppG, m 7 Gppppm 7 G, m2 (7,3 0) GpppG, m2 (7,2 0) GppspG(Dl), m2 (7,2 0) GppspG(D2), m2 7 ’ 3 °Gppp(mi 2 °)ApG, (m 7 G-3' mppp-G; which may equivalently be designated 3' O- Me-m7G(5')ppp(5')G), N7,2'-0-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m 7 Gm-ppp-G, N7-(4-chlorophenoxyethyl)-G(5')ppp(5')G,
• the modified RNA further comprises a half-life extending moiety.
• the half-life extending moiety comprises an Fc, an albumin or a fragment thereof, an albumin binding moiety, a PAS sequence, a HAP sequence, transferrin or a fragment thereof, an XTEN, or any combinations thereof.
• the IL-12 molecule is selected from the group consisting of IL-12, an IL-12 subunit, or a mutant IL-12 molecule that retains the immunomodulatory function.
• the IL-12 comprises an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 1.
• the IL-12 molecule comprises IL-12a and/or IL-12b subunits.
• the IL-12a subunit comprises an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 2.
• the IL- 12b subunit comprises an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 3.
• the IL-12a subunit and the IL-12P subunit are linked by a linker.
• the linker comprises an amino acid linker of at least about 2, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, or at least about 20 amino acids.
• the linker comprises a (GS) linker.
• the GS linker has a formula of (Gly4Ser)n or S(Gly4Ser)n, wherein n is a positive integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, or 100.
• the (Gly4Ser)n linker is (Gly4Ser)3 or (Gly4Ser)4.
• the IL12 molecule comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 4 or SEQ ID NO: 5.
• the modified mRNA comprises a nucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 6.
• the modified RNA comprises a nucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 7.
• the modified RNA further comprises a regulatory element.
• the regulatory element is selected from the group consisting of at least one translation enhancer element (TEE), a translation initiation sequence, at least one microRNA binding site or seed thereof, a 3’ tailing region of linked nucleosides, an AU rich element (ARE), a post transcription control modulator, and combinations thereof.
• the 3’ tailing region of linked nucleosides comprises a poly-A tail, a polyA- G quartet, or a stem loop sequence.
• the modified RNA comprises at least one modified nucleoside.
• the at least one modified nucleoside is selected from the group consisting of 6-aza-cytidine, 2-thio-cytidine, a-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1 -methyl-pseudouridine, 5,6-dihydrouridine, a-thio-uridine, 4-thio- uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, pseudo-uridine, inosine, a- thio-guanosine, 8-oxo-guanosine, 06-methyl-guanosine, 7-deaza-guanosine, N1 -methyl adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, 6-chloro-
• the modified RNA comprises a nucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 8.
• the modified RNA comprises a nucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 9.
• the lipid nanoparticle has a diameter of about 30-500 nm. In some aspects, the lipid nanoparticle has a diameter of about 50-400 nm. In some aspects, the lipid nanoparticle has a diameter of about 70-300 nm. In some aspects, the lipid nanoparticle has a diameter of about 100-200 nm. In some aspects, the lipid nanoparticle has a diameter of about 100-175 nm. In some aspects, the lipid nanoparticle has a diameter of about 100-120 nm. In some aspects, the lipid nanoparticle and the modified RNA have a mass ratio of about 1 :2 to about 2: 1.
• the lipid nanoparticle and the modified RNA have a mass ration of 1:2, 1:1.5, 1:1.2, 1:1.1, 1:1, 1.1:1, 1.2:1, 1.5:1, or 2:1. In some aspects, the lipid and the modified RNA have a mass ratio of about 1:1.
• the prevent disclosure also provides a pharmaceutical composition, comprising the lipid nanoparticle disclosed herein and a pharmaceutically acceptable carrier.
• the pharmaceutical composition is formulated for intratumoral, intrathecal, intramuscular, intravenous, subcutaneous, inhalation, intradermal, intralymphatic, intraocular, intraperitoneal, intrapleural, intraspinal, intravascular, nasal, percutaneous, sublingual, submucosal, transdermal, or transmucosal administration.
• the present disclosure also includes a method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of the lipid nanoparticle or the pharmaceutical composition disclosed herein.
• the subject is a human patient having or suspected of having a cancer.
• the human patient has a cancer selected from the group consisting of melanoma, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, gastric cancer, and head and neck cancer.
• the lipid nanoparticle or the pharmaceutical composition is administered to the subject in a single dose.
• the pharmaceutical composition is administered to the subject by intratumoral injection, intramuscular injection, subcutaneous injection, or intravenous injection.
• FIG. 1A shows IL-12 expression level between self-replicating mRNA and modified RNA.
• Y axis shows the expression level in B16.F10 cell line and the X axis shows hours post transfection.
• FIG. IB shows IL-12 expression level between self-replicating mRNA and modified RNA.
• Y axis shows the expression level in 4T1 cell line.
• FIG. 2A shows the efficacy (tumor size) of modified RNA and self replicating RNA in a highly difficult to treat mouse model.
• the top line is for control, and the bottom two lines are for modRNA-IL-12 and repRNA-IL-12.
• FIG. 2B shows the efficacy (tumor size) between modified RNA-IL-12 and repRNA-IL-12.
• FIG. 3 shows IL-12 payload expression in the modified RNA and replicating RNA.
• FIG. 4 shows probability of survival of mice administered modRNA-IL12 or repRNA-IL12 after B16-F10 cells were introduced subcutaneously. The mRNAs were dosed as a single dose at about 350mm 3 tumor size.
• a or “an” entity refers to one or more of that entity; for example, “a polynucleotide,” is understood to represent one or more polynucleotides.
• the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
• the term "at least" prior to a number or series of numbers is understood to include the number adjacent to the term “at least,” and all subsequent numbers or integers that could logically be included, as clear from context.
• the number of nucleotides in a nucleic acid molecule must be an integer.
• "at least 18 nucleotides of a 21- nucleotide nucleic acid molecule” means that 18, 19, 20, or 21 nucleotides have the indicated property.
• At least is present before a series of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range.
• “At least” is also not limited to integers (e.g., "at least 5%” includes 5.0%, 5.1%, 5.18% without consideration of the number of significant figures.
• Polynucleotide or “nucleic acid” as used herein means a sequence of nucleotides connected by phosphodiester linkages. Polynucleotides are presented herein in the direction from the 5' to the 3' direction.
• a polynucleotide of the present disclosure can be a deoxyribonucleic acid (DNA) molecule or ribonucleic acid (RNA) molecule. Nucleotide bases are indicated herein by a single letter code: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I) and uracil (U).
• polypeptide encompasses both peptides and proteins, unless indicated otherwise.
• coding sequence or sequence “encoding” is used herein to mean a DNA or RNA region (the transcribed region) which “encodes” a particular protein, e.g., such as an IL-12.
• a coding sequence is transcribed (DNA) and translated (RNA) into a polypeptide, in vitro or in vivo , when placed under the control of an appropriate regulatory region, such as a promoter.
• the boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
• a coding sequence can include, but is not limited to, cDNA from prokaryotes or eukaryotes, genomic DNA from prokaryotes or eukaryotes, and synthetic DNA sequences.
• a transcription termination sequence can be located 3' to the coding sequence.
• a Kozak consensus sequence is known as a sequence which occurs on eukaryotic mRNA and has the consensus (gcc)gccRccAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another “G.”
• the polynucleotide comprises a nucleic acid sequence having at least 95%, at least 99% sequence identity, or more to the Kozak consensus sequence. In some aspects, the polynucleotide comprises a Kozak consensus sequence.
• RNA is used herein to mean a molecule which comprises at least one ribonucleotide residue.
• “Ribonucleotide” relates to a nucleotide with a hydroxyl group at the 2'-position of a b-D-ribofuranosyl group.
• the term comprises double-stranded RNA, single-stranded RNA, isolated RNA such as partially or completely purified RNA, essentially pure RNA, synthetic RNA, recombinantly generated RNA such as modified RNA which differs from naturally occurring RNA by addition, deletion, substitution and/or alteration of one or more nucleotides.
• mRNA means “messenger-RNA” and relates to a “transcript” which is generated by using a DNA template and encodes a peptide or protein.
• an mRNA comprises a 5'-UTR, a protein coding region and a 3'- UTR.
• mRNA only possesses limited half-life in cells and in vitro.
• mRNA may be generated by in vitro transcription from a DNA template.
• the in vitro transcription methodology is known to the skilled person. For example, there is a variety of in vitro transcription kits commercially available.
• the RNA preferably the mRNA, is modified with a 5 '-cap structure.
• sequence identity is used herein to mean a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In certain aspects, sequence identity is calculated based on the full length of two given SEQ ID NO or on part thereof. Part thereof can mean at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of both SEQ ID NO, or any other specified percentage. The term “identity” can also mean the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences.
• methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs.
• “Substantial homology” or “substantial similarity,” means, when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95 to 99% of the sequence.
• alkyl as used herein is a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetracedcyl, hexadecyl, eicosyl, tetracosyl, and the like.
• the alkyl group can also be substituted or unsubstituted.
• the alkyl group can be substituted with one or more groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxyl, ketone, nitro, silyl, sulfoxo, sulfonyl, sulfoxide, or thiol.
• alkyl halogenated alkyl, alkoxy, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxyl, ketone, nitro, silyl, sulfoxo, sulfonyl, sulfoxide, or thiol.
• the terms "effective amount,” “therapeutically effective amount,” and a “sufficient amount” of, e.g., a gene therapy composition comprising a polynucleotide disclosed herein, refer to a quantity sufficient to, when administered to the subject, including a human, effect beneficial or desired results, including clinical results, and, as such, an "effective amount” or synonym thereto depends on the context in which it is being applied.
• the amount of a given therapeutic agent or composition will correspond to such an amount will vary depending upon various factors, such as the given agent, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject (e.g., age, sex, and/or weight) or host being treated, and the like.
• half-life relates to the period of time which is needed to eliminate half of the activity, amount, or number of molecules.
• the half-life of an RNA is indicative for the stability of said RNA.
• the present disclosure relates to the delivery of biologically active molecules to cells using lipid nanoparticles.
• the disclosure relates to an IL-12- expressing modified RNA or circular RNA encapsulated by lipid nanoparticles, the composition thereof, and use of the composition thereof to treat a subject having cancer or suspected of having cancer.
• a lipid nanoparticle refers to a vesicle, such as a spherical vesicle, having a contiguous lipid bilayer. Lipid nanoparticles can be used in methods by which pharmaceutical therapies are delivered to targeted locations. Non-limiting examples of LNPs include liposomes, bolaamphihiles, solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), and monolayer membrane structures (e.g., archaeosomes and micelles).
• the lipid nanoparticle comprises one or more types of lipids.
• a lipid refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and in some aspects are characterized by being insoluble in water, but soluble in many organic solvents. They are usually divided into at least three classes: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids,” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.
• Non-limiting examples of lipids include triglycerides (e.g. tristearin), diglycerides (e.g.
• the one or more types of lipids in the LNP comprises a cationic lipid.
• a cationic lipid refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as a physiological pH.
• lipids include, but are not limited to Nl,N3,N5-tris(3-(didodecylamino)propyl)benzene-l, 3, 5 -tri carboxamide (TT3), N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); lipofectamine; l,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-
• Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA); dioctadecyldimethylammonium (DODMA), Distearyldimethylammonium (DSDMA), N,N-dioleyl-N,N,-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)- N,N,N-trimethylammonium chloride (DOTMA); N-N-distearyl-N,N-dimethylammonium bromide (DDAB); 3-(N — (N , ,N’-dimethylaminoethane)-carbamoyl)cholesterol (DC- Chol) and N-(l,2-dimyristyloxprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE).
• DODMA dioctadecyl
• the cationic lipid is represented by Formula I: (Formula I) and salts thereof; wherein each R 1 is independently unsubstituted alkyl; each R 2 is independently unsubstituted alkyl; each R 3 is independently hydrogen or substituted or unsubstituted alkyl; and each m is independently 3, 4, 5, 6, 7, or 8.
• each R 1 is independently unsubstituted alkyl; each R 2 is independently unsubstituted alkyl; R 3 is hydrogen; and each m is 3.
• at least one R 1 is unsubstituted Ci-24 alkyl.
• at least one R 1 is unsubstituted C1-18 alkyl.
• At least one R 2 is unsubstituted Ci-24 alkyl. In some aspects, at least one R 2 is unsubstituted C1-18 alkyl. In some aspects, at least one R 2 is unsubstituted Ci-12 alkyl. In some aspects, at least one R 2 is unsubstituted C6-18 alkyl. In some aspects, at least one R 2 is unsubstituted C6-12 alkyl. In some aspects, at least one R 2 is unsubstituted C8-12 alkyl. In some aspects, at least one R 2 is unsubstituted Cio-12 alkyl. In some aspects, at least one R 2 is unsubstituted C11 alkyl.
• At least two R 1 are unsubstituted Ci-24 alkyl. In some aspects, at least two R 1 are unsubstituted C1-18 alkyl. In some aspects, at least two R 1 are unsubstituted Ci-12 alkyl. In some aspects, at least two R 1 are unsubstituted C6-18 alkyl. In some aspects, at least two R 1 are unsubstituted C6-12 alkyl. In some aspects, at least two R 1 are unsubstituted C8-12 alkyl. In some aspects, at least two R 1 are unsubstituted C 10-12 alkyl. In some aspects, at least two R 1 are unsubstituted C11 alkyl.
• At least two R 2 are unsubstituted Ci-24 alkyl. In some aspects, at least two R 2 are unsubstituted C1-18 alkyl. In some aspects, at least two R 2 are unsubstituted Ci-12 alkyl. In some aspects, at least two R 2 are unsubstituted C6-18 alkyl. In some aspects, at least two R 2 are unsubstituted C6-12 alkyl. In some aspects, at least two R 2 are unsubstituted C8-12 alkyl. In some aspects, at least two R 2 are unsubstituted C 10-12 alkyl. In some aspects, at least two R 2 are unsubstituted C11 alkyl.
• all instances of R 1 are unsubstituted Ci-24 alkyl. In some aspects, all instances of R 1 are unsubstituted C1-18 alkyl. In some aspects, all instances of R 1 are unsubstituted C 1-12 alkyl. In some aspects, all instances of R 1 are unsubstituted C6-18 alkyl. In some aspects, all instances of R 1 are unsubstituted C6-12 alkyl. In some aspects, all instances of R 1 are unsubstituted C8-12 alkyl. In some aspects, all instances of R 1 are unsubstituted C 10-12 alkyl. In some aspects, all instances of R 1 are unsubstituted C11 alkyl.
• all instances of R 2 are unsubstituted Ci-24 alkyl. In some aspects, all instances of R 2 are unsubstituted Ci-is alkyl. In some aspects, all instances of R 2 are unsubstituted Ci-12 alkyl. In some aspects, all instances of R 2 are unsubstituted C6-18 alkyl. In some aspects, all instances of R 2 are unsubstituted Ce-u alkyl. In some aspects, all instances of R 2 are unsubstituted C8-12 alkyl. In some aspects, all instances of R 2 are unsubstituted C 10-12 alkyl. In some aspects, all instances of R 2 are unsubstituted C11 alkyl.
• At least two R 3 are hydrogen. In some aspects, at least two R 3 are substituted or unsubstituted alkyl. In some aspects, at least two R 3 are substituted or unsubstituted C1-18 alkyl. In some aspects, at least two R 3 are substituted or unsubstituted Ci-12 alkyl. In some aspects, at least two R 3 are substituted or unsubstituted Ci- 6 alkyl. In some aspects, at least two R 3 are substituted or unsubstituted Ci-4 alkyl. In some aspects, at least two R 3 are substituted or unsubstituted C2-4 alkyl. In some aspects, at least two R 3 are substituted or unsubstituted methyl.
• At least two R3 are substituted alkyl, wherein the substituted alkyl is substituted with a halogen. In some aspects, at least two R3 are substituted alkyl, wherein the substituted alkyl is substituted with fluorine. In some aspects, at least two R3 are substituted alkyl, wherein the substituted alkyl is substituted with halogenated alkyl.
• all instances of R 3 are substituted alkyl, wherein the substituted alkyl is substituted with a halogen. In some aspects, all instances of R 3 are substituted alkyl, wherein the substituted alkyl is substituted with fluorine. In some aspects, all instances of R 3 are substituted alkyl, wherein the substituted alkyl is substituted with halogenated alkyl.
• all instances of m are 3. In some aspects, all instances of m are 4.
• all instances of m are 5. In some aspects, all instances of m are 6. In some aspects, all instances of m are 7. In some aspects, all instances of m are 8.
• TT3 is capable of forming lipid nanoparticles for delivery of various biologic active agents into the cells.
• the present disclosure also demonstrates that an unloaded TT3-LNP can induce immunogenic cell death (ICD) in cancer cells in vivo and in vitro.
• ICD immunogenic cell death
• Immunogenic cell death refers to a form of cell death that can induce an effective immune response through activation of dendritic cells (DCs) and consequent activation of specific T cell response.
• the cells that undergo immunogenic cell death are tumor cells. Immunogenic tumor cell death can trigger an effective anti-tumor immune response.
• the lipid nanoparticle comprises TT3-LNP encapsulating a modified RNA (modRNA) encoding only a reporter gene (TT3-LNP-modRNA).
• modified RNA can work synergistically with the TT3-LNP to induce higher level of ICD in tumor cells compared to TT3-LNP alone.
• the lipid nanoparticle comprises a TT3-LNP encapsulating a modRNA encoding an IL-12 molecule.
• IL-12 which is an immunoregulatory cytokine, elicits a potent immune response against the local tumor.
• TT3-LNP modRNA
• IL-12 IL-12
• the cationic lipid is lipofectamine.
• Lipfectamine as used herein, is a common transfection reagent, produced and sold by Invitrogen, used in molecular and cellular biology. It is used to increase the transfection efficiency of RNA (including mRNA and siRNA) or plasmid DNA into in vitro cell cultures by lipofection.
• RNA including mRNA and siRNA
• Lipofectamine contains lipid subunits that can form liposomes or lipid nanoparticles in an aqueous environment, which entrap the transfection payload, e.g. modRNA.
• Particle size of lipid nanoparticles can affect drug release rate, bio-distribution, mucoadhesion, cellular uptake of water and buffer exchange to the interior of the nanoparticles, and protein diffusion.
• the diameter of the LNPs ranges from 30 to 500 nm. In some aspects of the disclosure, the diameter of the LNPs ranges from about 30 to about 500 nm, about 50 to about 400 nm, about 70 to about 300 nm, about 100 to about 200 nm, about 100 to about 175 nm, or about 100 to about 120 nm. In some aspects of the disclosure, the diameter of the LNPs ranges from 100-120 nm.
• the diameter of the LNPs can be 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 101 nm, 102 nm, 103 nm, 104 nm, 105 nm, 106 nm, 107 nm, 108 nm, 109 nm, 110 nm, 111 nm, 112 nm, 113 nm, 114 nm, 115 nm, 116 nm, 117 nm, 118 nm, 119 nm, or 120 nm.
• the zeta potential of the LNPs can be 3 mv, 3.1 mv, 3.2 mv, 3.3 mv, 3.4 mv, 3.5 mv, 3.6 mv, 3.7 mv, 3.8 mv, 3.9 mv, 4 mv, 4.1 mv, 4.2 mv, 4.3 mv, 4.4 mv, 4.5 mv, 4.6 mv, 4.7 mv, 4.8 mv, 4.9 mv, 5 mv, 5.1 mv, 5.2 mv, 5.3 mv, 5.4 mv, 5.5 mv, 5.6 mv, 5.7 mv, 5.8 mv, 5.9 mv, or 6 mv.
• RNA messenger RNA
• mRNA refers to any polynucleotide which encodes a polypeptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ or ex vivo.
• the modified RNA is synthetic.
• the modification can be located on the sugar moiety of the nucleotide. In some aspects, the modification can be located on the phosphate backbone of the nucleotide.
• the modified RNA comprises a degradation domain, which is capable of being acted on in a directed manner within a cell.
• the modified RNA comprises at least one of a modified 5’-cap, a half-life extending moiety, or a regulatory element.
• the modified RNA is cyclized (e.g., circular mRNA), or concatemerized, to generate a translation competent molecule to assist interactions between poly-A binding proteins and 5 '-end binding proteins.
• the mechanism of cyclization or concatemerization may occur through at least 3 different routes: 1) chemical, 2) enzymatic, and 3) ribozyme catalyzed.
• the newly formed 5 '-/3 '-linkage may be intramolecular or intermolecular.
• multiple distinct nucleic acids, modified RNA or primary constructs may be linked together through the 3 '-end using nucleotides which are modified at the 3 '-terminus.
• Chemical conjugation can be used to control the stoichiometry of delivery into cells.
• the glyoxylate cycle enzymes, isocitrate lyase and malate synthase can be supplied into HepG2 cells at a 1:1 ratio to alter cellular fatty acid metabolism.
• the two nucleic acids or modified RNA species may be combined in an aqueous solution, in the presence or absence of copper, to form a new covalent linkage via a click chemistry mechanism as described in the literature.
• more than two polynucleotides may be linked together using a functionalized linker molecule.
• a functionalized saccharide molecule may be chemically modified to contain multiple chemical reactive groups (SH — , NH2 — , N3, etc. . . .) to react with the cognate moiety on a 3 '-functionalized mRNA molecule (i.e., a 3'- maleimide ester, 3' — NHS-ester, alkynyl).
• the number of reactive groups on the modified saccharide can be controlled in a stoichiometric fashion to directly control the stoichiometric ratio of conjugated nucleic acid or mRNA.
• nucleic acids modified
• RNA, polynucleotides or primary constructs of the present disclosure can be designed to be conjugated to other polynucleotides, dyes, intercalating agents (e.g. acridines), cross linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g.
• intercalating agents e.g. acridines
• cross linkers e.g. psoralene, mitomycin C
• porphyrins TPPC4, texaphyrin, Sapphyrin
• polycyclic aromatic hydrocarbons e.g., phenazine, dihydrophenazine
• artificial endonucleases e.g.
• biotin e.g., aspirin, vitamin E, folic acid
• transport/absorption facilitators e.g., aspirin, vitamin E, folic acid
• synthetic ribonucleases proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell, hormones and hormone receptors, non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, or a drug.
• a specified cell type such as a cancer cell, endothelial cell, or bone cell
• hormones and hormone receptors non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, or a drug.
• Conjugation may result in increased stability and/or half-life and may be particularly useful in targeting the nucleic acids, modified RNA, polynucleotides or primary constructs to specific sites in the cell, tissue or organism.
• the primary construct is designed to encode one or more polypeptides of interest or fragments thereof.
• a polypeptide of interest may include, but is not limited to, whole polypeptides, a plurality of polypeptides or fragments of polypeptides, which independently may be encoded by one or more nucleic acids, a plurality of nucleic acids, fragments of nucleic acids or variants of any of the aforementioned.
• the term “polypeptides of interest” refers to any polypeptide which is selected to be encoded in the primary construct of the present invention.
• polypeptide means a polymer of amino acid residues (natural or unnatural) linked together most often by peptide bonds.
• polypeptides refers to proteins, polypeptides, and peptides of any size, structure, or function.
• polypeptide encoded is smaller than about 50 amino acids and the polypeptide is then termed a peptide. If the polypeptide is a peptide, it will be at least about 2, 3, 4, or at least 5 amino acid residues long.
• polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing.
• a polypeptide may be a single molecule or may be a multi-molecular complex such as a dimer, trimer or tetramer.
• polypeptides may also comprise single chain or multichain polypeptides such as antibodies or insulin and may be associated or linked. Most commonly disulfide linkages are found in multichain polypeptides.
• polypeptide may also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.
• polypeptide variant refers to molecules which differ in their amino acid sequence from a native or reference sequence.
• the amino acid sequence variants may possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence, as compared to a native or reference sequence.
• variants will possess at least about 50% identity (homology) to a native or reference sequence, and preferably, they will be at least about 80%, more preferably at least about 90% identical (homologous) to a native or reference sequence.
• sequence tags or amino acids such as one or more lysines
• Sequence tags can be used for peptide purification or localization.
• Lysines can be used to increase peptide solubility or to allow for biotinylation.
• amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein can optionally be deleted providing for truncated sequences.
• Certain amino acids e.g., C-terminal or N- terminal residues
• protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of polypeptides of interest of this invention.
• any protein fragment meaning an polypeptide sequence at least one amino acid residue shorter than a reference polypeptide sequence but otherwise identical
• a polypeptide to be utilized in accordance with the invention includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequences provided or referenced herein.
• the modified 5’ -cap is selected from the group consisting of im 7,2 °Gpp s pGRNA, m 7 GpppG, m 7 Gppppm 7 G, im (7,3 0) GpppG, m2 (7,2 0) GppspG(Dl), m 2 (7 ’ 2' - 0) Gpp S pG(D2), m2 7 ’ 3 °Gppp(mi 2 °)ApG, (m 7 G-3' mppp-G; which may equivalently be designated 3' 0-Me-m7G(5')ppp(5')G), N7,2'-0-dimethyl-guanosine-5'- triphosphate-5'-guanosine, m 7 Gm-ppp-G, N7-(4-chlorophenoxyethyl)-G(5')ppp(5')G, N7- (4-chlorophenoxyethy
• the disclosure also includes a polynucleotide that comprises both a 5' Cap and a modified RNA of the disclosure (e.g., a polynucleotide comprising a nucleotide sequence encoding an IL12B polypeptide, an IL12A polypeptide, and/or IL12B and IL12A fusion polypeptides).
• a polynucleotide comprising a nucleotide sequence encoding an IL12B polypeptide, an IL12A polypeptide, and/or IL12B and IL12A fusion polypeptides.
• the 5' cap structure of a natural mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species.
• CBP mRNA Cap Binding Protein
• the cap further assists the removal of 5' proximal introns during mRNA splicing.
• Endogenous mRNA molecules can be 5 '-end capped generating a 5'-ppp-5'- triphosphate linkage between a terminal guanosine cap residue and the 5 '-terminal transcribed sense nucleotide of the mRNA molecule.
• This 5'-guanylate cap can then be methylated to generate an N7-methyl-guanylate residue.
• the ribose sugars of the terminal and/or anteterminal transcribed nucleotides of the 5' end of the mRNA can optionally also be 2'-0-methylated.
• 5'-decapping through hydrolysis and cleavage of the guanylate cap structure can target a nucleic acid molecule, such as an mRNA molecule, for degradation.
• 5' terminal caps can include endogenous caps or cap analogs.
• a 5' terminal cap can comprise a guanine analog.
• Useful 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.
• the 5' terminal cap structure is a CapO, Capl, ARC A, inosine, Nl- methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino- guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5' methylG cap, or an analog thereof.
• the regulatory element is selected from the group consisting of at least one translation enhancer element (TEE), a translation initiation sequence, at least one microRNA binding site or seed thereof, a 3’ tailing region of linked nucleosides, an AU rich element (ARE), a post transcription control modulator, and combinations thereof.
• TEE translation enhancer element
• ARE AU rich element
• the 3' end of the transcript can be cleaved to free a
• poly-A polymerase adds a chain of adenine nucleotides to the RNA.
• the process called polyadenylation, adds a poly-A tail that can be between, for example, approximately 80 to approximately 250 residues long, including approximately 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 residues long.
• UTRs Untranslated Regions
• the regulatory features of a UTR can be incorporated into the modified RNA of the present disclosure to enhance the stability of the molecule.
• the specific features can also be incorporated to ensure controlled down-regulation of the transcript in case they are misdirected to undesired organs sites.
• 5'UTR secondary structures involved in elongation factor binding can interact with other RNA binding molecules in the 5'UTR or 3 TR to regulate gene expression.
• the elongation factor EIF4A2 binding to a secondarily structured element in the 5'UTR is necessary for microRNA mediated repression (Meijer H A et ah, Science, 2013, 340, 82-85, herein incorporated by reference in its entirety).
• the different secondary structures in the 5'UTR can be incorporated into the flanking region to either stabilize or selectively destabilize mRNAs in specific tissues or cells.
• non-UTR sequences may be incorporated into the 5' (or 3' UTR) UTRs.
• introns or portions of introns sequences may be incorporated into the flanking regions of the nucleic acids or mRNA of the invention. Incorporation of intronic sequences may increase protein production as well as mRNA levels.
• Nucleotides can be mutated, replaced and/or removed from the 5' (or 3') UTRs.
• one or more nucleotides upstream of the start codon can be replaced with another nucleotide.
• the nucleotide or nucleotides to be replaced can 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, 30, 35, 40, 45, 50, 55, 60 or more than 60 nucleotides upstream of the start codon.
• one or more nucleotides upstream of the start codon can be removed from the UTR.
• the 5'UTR of the modified RNA comprises at least one translational enhancer polynucleotide, translation enhancer element, translational enhancer elements (collectively referred to as “TEE”s).
• TEE translational enhancer polynucleotide, translation enhancer element, translational enhancer elements
• the TEE is located between the transcription promoter and the start codon.
• the modified RNA with at least one TEE in the 5'UTR comprises a cap at the 5'UTR.
• the at least one TEE may be located in the 5'UTR of modified RNA undergoing cap-dependent or cap- independent translation.
• TEE refers to sequences that increase the amount of polypeptide or protein produced from an mRNA.
• TEEs are conserved elements in the UTR which can promote translational activity of a nucleic acid such as, but not limited to, cap-dependent or cap- independent translation.
• a nucleic acid such as, but not limited to, cap-dependent or cap- independent translation.
• the modified RNA has at least one TEE that has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity with the disclosed in U.S. Application Number 2014/0147454, which is hereby incorporated by reference in its entirety.
• the modified RNA includes at least one TEE that has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity with the TEEs described in US Patent Publication Nos.
• the 5'UTR of the modified RNA may include at least 1, 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 16, at least 17, at least 18 at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55 or more than 60 TEE sequences.
• the TEE sequences in the 5'UTR of the modified RNA are the same or different TEE sequences.
• the TEE sequences are in a pattern such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than three times.
• each letter, A, B, or C represent a different TEE sequence at the nucleotide level.
• the TEE used in the 5'UTR of the modified RNAof the present invention is an IRES sequence such as, but not limited to, those described in U.S. Pat. No. 7,468,275 and International Patent Publication No. W02001055369, each of which is herein incorporated by reference in its entirety.
• the spacer separating two TEE sequences includes other sequences known in the art which regulate the translation of the modified RNA, such as, but not limited to, miR sequences described herein (e.g., miR binding sites and miR seeds).
• miR sequences described herein e.g., miR binding sites and miR seeds.
• each spacer used to separate two TEE sequences includes a different miR sequence or component of a miR sequence (e.g., miR seed sequence).
• MicroRNAs are 19-25 nucleotide long noncoding RNAs that bind to the 3'UTR of nucleic acid molecules and down-regulate gene expression either by reducing nucleic acid molecule stability or by inhibiting translation.
• the modified RNA comprises one or more microRNA target sequences, microRNA sequences, or microRNA seeds.
• Such sequences may correspond to any known microRNA such as those taught in US Publication US2005/0261218 and US Publication US2005/0059005, the contents of which are incorporated herein by reference in their entirety.
• known microRNAs, their sequences and seed sequences in human genome are described in U.S. Application No. 2014/0147454, which is herein incorporated by reference in its entirety.
• tissues where microRNA are known to regulate mRNA, and thereby 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), myeloid cells (miR-142-3p, miR- 142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart (miR-ld, miR-149), kidney (miR-192, miR-194, miR-204), and lung epithelial cells (let-7, miR-133, miR-126).
• liver miR-122
• muscle miR-133, miR- 206, miR-208
• endothelial cells miR-17-92, miR-126
• myeloid cells miR-142-3p, miR- 142-5p, miR-16, miR-21, mi
• a modified messenger RNA comprises microRNA binding region sites that either have 100% identity to known seed sequences or have less than 100% identity to seed sequences.
• the seed sequence can be partially mutated to decrease microRNA binding affinity and as such result in reduced downmodulation of that mRNA transcript.
• the degree of match or mis-match between the target mRNA and the microRNA seed can act as a rheostat to more finely tune the ability of the microRNA to modulate protein expression.
• mutation in the non-seed region of a microRNA binding site may also impact the ability of a microRNA to modulate protein expression.
• cells can be transfected with different ARE-engineering molecules and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hr, 12 hr, 24 hr, 48 hr, and 7 days post-transfection.
• ELISA kit to the relevant protein and assaying protein produced at 6 hr, 12 hr, 24 hr, 48 hr, and 7 days post-transfection.
• the modified RNA comprises a triple helix on the 3' end of the modified nucleic acid, enhanced modified RNA or ribonucleic acid.
• the 3' end of the modified RNA include a triple helix alone or in combination with a Poly- A tail.
• the modified RNA, which comprises the histone stem loop may be stabilized by the addition of at least one chain terminating nucleoside.
• the addition of at least one chain terminating nucleoside may slow the degradation of a nucleic acid and thus can increase the half-life of the nucleic acid.
• Modifications to the RNA of the present disclosure may generate a non- hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5'-ppp-5' phosphorodiester linkages, modified nucleotides may be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, Mass.) may be used with a-thio- guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5'-ppp-5' cap. Additional modified guanosine nucleotides may be used such as a-methyl-phosphonate and seleno-phosphate nucleotides.
• a cap analog of the present invention is a 4-chloro/bromophenoxyethyl analog.
• cap analogs allow for the concomitant capping of a nucleic acid molecule in an in vitro transcription reaction, up to 20% of transcripts remain uncapped. This, as well as the structural differences of a cap analog from an endogenous 5 '-cap structures of nucleic acids produced by the endogenous, cellular transcription machinery, may lead to reduced translational competency and reduced cellular stability.
• providing an RNA with a 5 '-cap or 5 '-cap analog is achieved by in vitro transcription of a DNA template in the presence of said 5 '-cap or 5 '-cap analog, wherein said 5 '-cap is co-transcriptionally incorporated into the generated RNA strand,
• RNA may be generated, for example, by in vitro transcription, and the 5 '-cap may be attached to the RNA post-transcriptionally using capping enzymes, for example, capping enzymes of vaccinia virus.
• capping enzymes for example, capping enzymes of vaccinia virus.
• the modified RNA is capped post-transcriptionally, using enzymes, in order to generate more authentic 5 '-cap structures.
• the phrase “more authentic” refers to a feature that closely mirrors or mimics, either structurally or functionally, an endogenous or wild type feature.
• recombinant Vaccinia Virus Capping Enzyme and recombinant 2'-0- methyltransferase enzyme can create a canonical 5 '-5 '-triphosphate linkage between the 5'- terminal nucleotide of an mRNA and a guanine cap nucleotide wherein the cap guanine contains an N7 methylation and the 5 '-terminal nucleotide of the mRNA contains a 2'-0- methyl.
• This cap results in a higher translational-competency and cellular stability and a reduced activation of cellular pro-inflammatory cytokines, as compared, e.g., to other 5' cap analog structures known in the art.
• Cap structures include 7mG(5')ppp(5')N,pN2p, 7mG(5')ppp(5')NlmpNp, 7mG(5')-ppp(5')NlmpN2 mp and m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up.
• 5' terminal caps include endogenous caps or cap analogs.
• a 5' terminal cap comprises a guanine analog.
• the 5’ cap comprises a 5’ to 5’ triphosphate linkage. In some aspects, the 5’ cap comprises a 5’ to 5’ triphosphate linkage including thiophosphate modification. In some aspects, the 5’ cap comprises a 2 -0 or 3'-0-ribose-methylated nucleotide. In some aspects, the 5’ cap comprises a modified guanosine nucleotide or modified adenosine nucleotide. In some aspects, the 5’ cap comprises 7- methylguanylate.
• Exemplary cap structures include m7G(5’)ppp(5’)G, m7,2'0-mG(5 , )ppSp(5’)G, m7G(5 , )ppp(5’)2'0-mG, and m7,3'0-mG(5 , )ppp(5’)2'0-mA.
• the length is at least 1800 nucleotides. In some aspects, the length is at least 1900 nucleotides. In some aspects, the length is at least 2000 nucleotides. In some aspects, the length is at least 2500 nucleotides. In some aspects, the length is at least 3000 nucleotides.
• the modified RNA comprises a polyA tail andis stabilized by the addition of a chain terminating nucleoside.
• the modified RNA with a polyA tail further comprise a 5' cap structure.
• the modified RNA which comprise a polyA tail or a polyA-G
• one or more uridine in the modified RNA is replaced by a modified nucleoside.
• he modified nucleoside replacing uridine is pseudouridine (y), N1 -methyl-pseudouridine (m 1 y) or 5-methyl-uridine (m5U).
• the modified RNA comprises a modified RNA as described in U. S.
• cytotoxic nucleosides can be incorporated into polynucleotides such as bifunctional modified RNAs or mRNAs.
• Cytotoxic nucleoside anti-cancer agents include, but are not limited to, adenosine arabinoside, cytarabine, cytosine arabinoside, 5- fluorouracil, fludarabine, floxuridine, FTORAFUR® (a combination of tegafur and uracil), tegafur ((RS)-5-fluoro-l-(tetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione), and 6- mercaptopurine.
• a number of prodrugs of cytotoxic nucleoside analogues are also reported in the art. Examples include, but are not limited to, N4-behenoyl-l-beta-D- arabinofuranosylcytosine, N4-octadecyl- 1 -beta-D-arabinofuranosylcytosine, N4- palmitoyl-l-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl) cytosine, and P-4055 (cytarabine 5'-elaidic acid ester).
• the modified RNA comprises one or more gene(s) of experimental or therapeutic interest.
• the gene(s) of experimental or therapeutic interest encode cytokines, chemokines, or growth factors other than IL-12.
• Cytokines are known in the art, and the term itself refers to a generalized grouping of small proteins that are secreted by certain cells within the immune system and have an effect on other cells. Cytokines are known to enhance the cellular immune response and, as used herein, can include, but are not limited to, TNFa, IFN-g, IFN-a, TGFp, IL-1, IL-2, 11-4, IL-10, IL-13, IL-17, IL-18, and chemokines.
• the two components can be separated by an enteric layer that serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release.
• enteric layers or coatings such materials include a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.
• Suitable emulsions may be prepared using commercially available fat emulsions, such as INTRALIPIDTM, LIPOSYNTM, INFONUTROLTM, LIPOFUNDINTM, and LIPIPHYSANTM.
• Administration of one or more lipid nanoparticles or pharmaceutical compositions described herein can be continuous or intermittent, depending, for example, upon the recipient’s physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners.
• the administration of the lipid nanoparticle or pharmaceutical composition described herein may be essentially continuous over a preselected period of time or may be in a series of spaced doses, e.g., either before, during, or after developing a target disease or disorder.
• any combination of the lipid nanoparticle or pharmaceutical composition described herein and another anti-cancer agent may be used in any sequence for treating a cancer.
• the combinations described herein may be selected on the basis of a number of factors, which include but are not limited to, the effectiveness or reducing tumor formation or tumor growth, reducing cancer cells, increasing immune activity, and/or alleviating at least one symptom associated with the cancer, or the effectiveness for mitigating the side effects of another agent of the combination.
• a combined therapy described herein may reduce any of the side effects associated with each individual members of the combination, for example, a side effect associated with the anti-cancer agent.
• the other anti-cancer therapeutic agent is a chemotherapy, a radiation therapy, a surgical therapy, an immunotherapy, or combinations thereof.
• the chemotherapeutic agent is carboplatin, cisplatin, docetaxel, gemcitabine, nab- paclitexal, pemetrexed, vinorelbine, or combinations thereof.
• the radiation therapy is ionizing radiation, gamma-radiation, neutron beam radiotherapy, electron beam radiotherapy, proton therapy, brachytherapy, systemic radioactive isotopes, radiosensitizers, or combinations thereof.
• the surgical therapy is a curative surgery (e.g., tumor removal surgery), a preventative surgery, a laparoscopic surgery, a laser surgery, or combinations thereof.
• the immunotherapy is adoptive cell transfer, therapeutic cancer vaccines, or combinations thereof.
• the other anti-cancer therapeutic is a bacterial treatment.
• Angiogenesis inhibitors prevent the extensive growth of blood vessels (angiogenesis) that tumors require to survive.
• the angiogenesis promoted by tumor cells to meet their increasing nutrient and oxygen demands for example can be blocked by targeting different molecules.
• angiogenesis-mediating molecules or angiogenesis inhibitors which may be combined with the present invention are soluble VEGF (VEGF isoforms VEGF121 and VEGF165, receptors VEGFR1, VEGFR2 and co-receptors Neuropilin-1 and Neuropilin-2) 1 and RP-1, angiopoietin 2, TSP-1 and TSP-2, angiostatin and related molecules, endostatin, vasostatin, calreticulin, platelet factor-4, TIMP and CDAI, Meth-1 and Meth-2, IFN-a, -b and -g, CXCL10, IL-4, -12 and -18, prothrombin (kringle domain-2), antithrombin III fragment, prolactin, VEGI, SPARC,
• the instructions comprise dosage information, dosing schedule, and route of administration.
• the containers are unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses.
• the instructions are written instructions on a label or package insert (e.g., a paper sheet included in the kit).
• the instructions are machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk).
• the label or package insert indicates that the lipid nanoparticle or pharmaceutical composition disclosed herein is used for treating, delaying the onset, and/or alleviating a disease or disorder associated with cancer, such as those described herein. Instructions may be provided for practicing any of the methods described herein.
• kits further comprise additional components such as buffers and interpretive information.
• the kit comprises a container and a label or package insert(s) on or associated with the container.
• the disclosure provides articles of manufacture comprising the contents of the kits described herein.
• RNA For replicon RNA, a VEE replicon vector containing the payload was prepared.
• RNA (modRNA) template the DNA vector was generated using a replicon plasmid with forward primer containing T7 promoter (TAA TAC GAC TCA CTA TA ATG GAC TAC GAC ATA GT; SEQ ID NO: XX) and SGP and a reverse primer in the 3'-UTR (GAA ATA TTA AAA AC A AAA TCC GAT TCG GAA AAG AA; SEQ ID NO: XX).
• the T m for the forward and reverse primers were 68°C and 64°C, respectively. Tables 4 and 5 (below) provide additional information relating to the PCR reaction.
• modified RNA (modRNA) synthesis the UTP component of the kit was replaced with Nl-methylpseudouridine-5' - triphosphate. To begin the in vitro transcription process, the kit components were thawed on ice, mixed, and pulse-spinned in a microfuge. The sample was placed on ice until further use.
• modified RNA modified RNA
• the reaction was diluted to 50 pL with nuclease free water. Then, 5 pL of the enzyme (2 U/pL) was added to 20 pL IVT reaction. Then, the mixture was incubated for 30 minutes at 37 °C. Afterwards, the RNA was purified using Monarch RNA cleanup kit. A 1 pL aliquot was taken for quality control purposes
• Poly(A ) tail synthesis To add the poly(A) tails to the modified RNAs, the components provided in Table 9 (below) were added to a reaction tube. Then, the reaction was incubated for 30 minutes at 37 °C. Then, the reaction was stopped by directly purifying the RNA with the small Monarch cleanup kit. As a quality control, 200 ng of the RNA was run on a 1.2% RNA gel to confirm the size of the RNA. To do so, RNA was denatured with 50% formaldehyde sample buffer for 5 minutes at 65 °C, and then, immediately placed on ice for at least one minute. Then, the denatured RNA was loaded onto a gel and visualized using a Transilluminator.
• Example 2 In Vitro Analysis of Expression Kinetics [0252] To assess the expression efficiency of the RNA constructs disclosed herein, both a self-replicating mRNA (repRNA) and a modified mRNA (modRNA) encoding IL-12 protein was constructed using methods described herein (see, e.g ., Example 1). Then, the mRNA constructs were transfected into two different cells lines (i.e., B16.F10 and 4T1) using messengerMAX, and the expression level of the encoded protein was assessed at 24, 48, and 72 hours post-transfection.
• repRNA self-replicating mRNA
• modified mRNA modified mRNA
• the modified mRNA constructs disclosed herein e.g, encoding IL-12
• the self- replicating mRNAs e.g, encoding IL-12

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