CN117460537A - Nucleic acid delivery - Google Patents

Abstract

The present invention provides lipid/dendrimer systems that can deliver therapeutic molecules such as nucleic acids to mammalian cells and into humans and animals. For example, the present invention provides a system capable of delivering nucleic acids efficiently to lymphoid organs, skeletal muscles, brain and adipose tissue, as well as tumor tissue, liver and lung. Delivery of DNA and RNA is provided. In particular, the system of the invention can deliver mRNA.

Description

Nucleic acid delivery

Technical Field

The present invention relates to compositions that can deliver therapeutic molecules (such as nucleic acids) to mammalian cells and into humans and animals. Delivery to certain cell types, such as to lymphoid tissues and leukocytes in the circulation, is shown. Delivery to certain organs and tissues is also shown. For example, the present invention provides compositions capable of delivering nucleic acids efficiently to spleen, lymphoid organs, skeletal muscle, brain and adipose tissue, and lung, tumor tissue, heart, skeletal muscle, adipose tissue, brain, liver and kidney. Delivery of DNA and RNA is provided. In particular, mRNA delivery is shown.

Background

In order for nucleic acid therapies to fully play a potential as the next generation of precision medicine for revolutionary healthcare, the key challenge of drug delivery must be addressed. The limitations of current viral and non-viral vector platforms severely hamper development, and many therapies fail in the clinical transformation stage due to off-target effects, immune system activation, and difficulty in mass production.

In the context of in vivo delivery of nucleic acids, viral derivatizing agents are the most potent vectors, some of which have been greatly advanced clinically (Sheridan et al, 2011). In particular, adeno-associated virus (AAV) is an effective system for in vivo delivery (Wang et al, 2019). However, the use of such systems is limited because they are only capable of transporting DNA of < 5kb, and not RNA or larger. Furthermore, despite advances in viral vectors, problems remain with the use of such nucleic acid delivery vectors, such as potential random insertion and immunotoxicity. For example, AAV delivery can be highly immunogenic, particularly when high doses are required to target tissues other than the liver. AAV delivery systems also have limitations of repeated dosing. Patients often develop immunity to AAV delivery systems, making repeated dosing impractical. Finally, AAV delivery systems are expensive to produce and difficult to mass produce in Good Manufacturing Practice (GMP) scale.

In view of the shortcomings of viral systems, non-viral vectors for delivering nucleic acids to cells and tissues in vivo are currently being investigated. Different delivery techniques have been developed for delivering smaller nucleic acids such as siRNA and antisense oligonucleotides (ASOs). These techniques include bioconjugated oligonucleotide delivery systems in which siRNA or ASO is conjugated to an antibody or ligand (Benizri et al, 2019). However, siRNA and ASO therapies can only achieve gene silencing or exon skipping, and cannot achieve gene expression. For many diseases, it would be advantageous to express functional genes with DNA or mRNA. The use of conjugation systems to efficiently deliver larger gene payloads such as plasmid DNA or mRNA is challenging. Plasmid DNA or mRNA is a large negatively charged molecule that does not readily cross the negatively charged cell membrane. This is useful for effective delivery when the delivery vehicle encapsulates them and neutralizes the charge.

Lipid Nanoparticles (LNPs) have been used for mRNA delivery as encapsulated non-viral delivery vehicles, for example in the COVID-19 vaccine (Qui et al 2021). To teach the immune system against viruses, it is sufficient to locally transfect relatively small numbers of immune cells and muscle cells at the injection site. Thus, an RNA-based COVID-19 vaccine is administered intramuscularly. However, only transfection of many specific types of cells (which may require intravenous injection) is effective in treating a wide variety of diseases. The surface properties of LNPs currently in clinical use make them well suited for targeting the liver when administered intravenously. But LNPs currently in clinical use are not well suited for targeting many other tissues.

Various liposome-based systems have also been studied, for example involving uncharged lipids such as dioleoyl phosphatidylethanolamine (DOPE) and/or cationic lipids such as 1, 2-dioleoyl-3-trimethylammoniopropane chloride (DOTAP) and N- [1- (2, 3-dioleyloxy) propyl ] -N, N, N-trimethylammonium chloride (DOTMA) (Braum, 2019; ren et al, 2000). Similar liposomes have been used clinically for nucleic acid delivery, with results showing modest efficacy.

The addition of peptides to lipid-based carriers has been studied. In peptide/lipid hybrid systems, peptides and lipids associate with nucleic acids to form nanoparticles that can be internalized by cells. The peptide component may be linear (Kwok et al 2016) or branched, such as a peptide dendrimer (Kwok et al 2013). However, this previous work on peptide/lipid hybrid systems was only performed on double-stranded plasmid DNA (pDNA) and short interfering RNA (siRNA) under serum-free conditions in vitro, which is quite different from in vivo conditions. It is not clear whether the system can be used for in vivo nucleic acid delivery. Only short single stranded oligonucleotides were reported for delivery in vivo using peptide dendrimers/lipid nanoparticles (Saher et al, 2018; saher et al, 2019), with only a 20% to 30% low increase in ASO liver delivery observed compared to ASO alone. From this data, it can be concluded that peptide dendrimer systems may not be able to deliver even larger nucleic acids such as mRNA to tissues in vivo. The use of peptide dendrimer/lipid hybrid systems to deliver long nucleic acids such as mRNA in vivo has not previously been demonstrated.

The present invention has been devised in view of the above-mentioned considerations.

Disclosure of Invention

The development of nucleic acid therapies relies on efficient nucleic acid delivery. The present inventors have developed a framework for nucleic acid delivery using peptide dendrimers. These dendrimers are branched peptides that exhibit one, two, three or four amino acid residues between "branching residues" that act as branching units within the dendrimer molecule. The present invention surprisingly allows for the efficient delivery of larger nucleic acids (e.g., larger than antisense oligonucleotides ASOs). Although some smaller nucleic acids such as ASO are easily taken up into cells even without a vector system, the compositions of the invention allow larger nucleic acids such as mRNA to be efficiently delivered. This opens up clinical applications in the fields of CRISPR-mediated gene editing and related technologies, etc., without the need for viral delivery vectors.

The specific development of tissue-specific mRNA delivery systems represents a significant advance because there is currently a lack of clinical mRNA or CRISPR cassette (i.e., sgRNA and Cas9 mRNA) delivery systems that bypass the liver. The present invention can effectively deliver CRISPR cassettes and mRNA in cells to a variety of tissues, particularly lung and immune cell-rich tissues, including spleen, lymph nodes and bone marrow. This allows the development of mRNA and CRISPR therapies for extrahepatic tissue. The ability to deliver mRNA to immune cells, and to a greater extent to myeloid cells, including monocytes, macrophages, neutrophils, and dendritic cells, enables the technology to be developed for the treatment of all cancer types, including solid tumors and blood cancers, such as myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML), autoimmune diseases (e.g., rheumatoid arthritis, crohn's disease, uveitis, inflammatory bowel disease), and other immune cell-related disorders, such as Gaucher's disease.

The inventors have also found that their dendrimer-based systems have a surprising versatility. A range of first, second and third generation dendrimers can be used to deliver DNA and RNA molecules. In one example, a dendrimer-based carrier system such as a G1,2,3-KL dendrimer associated with lipids ("third generation" dendrimer) can increase cell transfection of DNA by 6 to 10 fold compared to some major commercial agents such as Lipofectamine 2000. In another example, a carrier system based on a dendrimer such as a G1,2-RL,3-LR dendrimer associated with a lipid ("third generation" dendrimer) can increase cell transfected nucleic acids such as mRNA by a factor of 10 compared to some major commercial agents such as Lipofectamine 2000. In addition, certain organs and tissues may be targeted. As described in more detail below, the inventors have found that dendrimer-based systems are surprisingly robust and versatile, exhibit high activity in the presence of serum in vitro and unexpected tissue targeting properties in vivo. Effective delivery of DNA and RNA is discussed below.

The inventors have found that for DNA delivery, G1,2,3-KL and G1,2,3-RL ("third generation" dendrimers) and to some extent G1,2-KL, G1,2-RL (both "second generation" dendrimers) are capable of transfecting HeLa and Neuro2A cells in the presence of serum. (serum components present challenges for in vivo DNA delivery, especially systemic delivery, as components such as albumin may interfere with cationic formulations.) this unexpected finding prompted the inventors to investigate the mechanism behind cell entry and assess the ability of such carrier systems in vivo. Surprisingly, it was found that G1,2,3-RL based vectors mediate efficient delivery of functional nucleic acids into certain tissues after systemic delivery. Has high targeting to liver and skeletal muscle. Endocytic pathway analysis indicated that the G1,2,3-RL DNA complex delivers DNA by clathrin, cell-mediated endocytosis, and megaloblastic.

The inventors have found that dendritic polymers with 1 or 2 passages (e.g. RHCG1-R, RHCG1,2-R, RHCG1-LR, RHCG1-RL, 2-LR) can effectively transfect cells and mediate similar transfection efficiencies as dendritic polymers with 3 passages for mRNA delivery (see fig. 28A, which shows the efficacy of transfecting HeLa cells under complete growth medium conditions (including serum). This indicates the specific versatility of the hybrid system for RNA delivery.

Dendrimers used in the present invention are first-, second-or third-generation peptide dendrimers, which means that they have up to three "layers" of peptide motifs (which are typically dipeptide motifs) interspersed between "branching" residues such as lysine. The first generation of dendrimers had the following structure, which is shown in the N-terminal to C-terminal orientation and treats Lys as a branching unit:

(N-terminal-Pep 1) ₂ Lys- (core) - (C-terminal)

The second generation dendrimers have the following structure, which is shown in an N-terminal to C-terminal orientation and treats Lys as a branching unit:

(N-terminal-Pep 2) ₄ -Lys ₂ -(Pep1) ₂ Lys- (core) - (C-terminal)

Third generation dendrimers have the following structure, which is shown in an N-terminal to C-terminal orientation and treats Lys as a branching unit:

(N-terminal-Pep 3) ₈ -Lys ₄ -(Pep2) ₄ -Lys ₂ -(Pep1) ₂ Lys- (core) - (C-terminal)

The third generation dendrimer is represented graphically in fig. 31 (N-terminal on the left and C-terminal on the right).

Circles represent core sequences. Each triangle represents a branching residue, such as lysine. Each rectangle represents a peptide motif. The third generation dendrimer has two peptide motifs in the first layer, four peptide motifs in the second layer, and eight peptide motifs in the third layer. The N-and C-termini may be derivatized with additional chemical motifs, as discussed herein. For example, while in the underivatized embodiment the C-terminus is a carboxylic acid, in other embodiments it is As a result of the chemical route for synthesizing the dendrimer, the C-terminus is derivatized, for example to contain a primary amide group CONH ₂ (rather than COOH). Functionally important derivatisation such as targeting moieties (e.g. antibodies, peptide groups, sugar groups and/or lipid chains) are also envisaged, which may be linked to the N-terminal and/or C-terminal, or at other positions along the dendrimer.

As described herein, the dendrimer may be first generation, second generation, or third generation. This may be defined structurally as follows: the first generation dendrimers comprise a core peptide sequence, a first branching residue and two first peptide motifs. The two first peptide motifs independently consist of a single amino acid, dipeptide, tripeptide or tetrapeptide motif. The second generation dendrimer further comprises two second branching residues (e.g., lysine) and four second peptide motifs, wherein one of the second branching residues is covalently bonded to one of the first peptide motifs and the other second branching residue is covalently bonded to the other first peptide motif, and wherein each second branching residue is covalently bonded to two second peptide motifs. The four second peptide motifs independently consist of single amino acid, dipeptide, tripeptide or tetrapeptide motifs. The third generation dendrimer further comprises four third branching residues (e.g., lysine) and eight third peptide motifs, wherein each second peptide motif is covalently bonded to one of the third branching residues, respectively, such that each third branching residue is covalently bonded to one second peptide motif, and wherein each third branching residue is covalently bonded to two third peptide motifs. The eight third peptide motifs independently consist of single amino acid, dipeptide, tripeptide or tetrapeptide motifs. When present, the first, second, and third peptide motifs may each comprise (1) an amino acid having a basic side chain, such as, but not limited to, lysine (K) or arginine (R) or histidine (H), (2) an amino acid having an acidic side chain, such as, but not limited to, aspartic acid (D) and glutamic acid (E), (3) an amino acid having a nonpolar side chain, such as, but not limited to, glycine (G), alanine (a), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), β -alanine (B), tryptophan (W), proline (P), aminocaproic acid (X), and cysteine (C), and (4) an amino acid having an uncharged polar side chain, such as, but not limited to, asparagine (N), glutamine (Q), serine (S), threonine (T), and tyrosine (Y).

Preferred dendrimers are given in table 2 below. Some examples are discussed in particular. For example, in dendrimers where each peptide motif is an Arg-Leu (RL) dipeptide, the structure may be represented as G1-RL, G1,2-RL and G1,2,3-RL. In dendrimers where each peptide motif is a Lys-Leu (KL) dipeptide, the structure is denoted G1-KL, G1,2-KL and G1,2,3-KL. In dendrimers where each peptide motif is a Leu-Arg (LR) dipeptide, the structure is denoted as G1-LR, G1,2-LR and G1,2,3-LR.

The "1 st generation", "2 nd generation" and "3 rd generation" peptide motifs of the first, second and third layers, respectively, ("G1", "G2" and "G3") each amino acid residue may be an L-amino acid or a D-amino acid.

Thus, in a first aspect, the present invention provides a composition for use in medicine, wherein the composition comprises a first, second or third generation peptide dendrimer, a nucleic acid and a lipid. The composition further comprises nucleic acids and lipids. The peptide dendrimer comprises at least: a core peptide sequence, a first branching residue and two first peptide motifs. The branching residue may be lysine, 2, 4-diaminobutyric acid, ornithine or diaminopropionic acid. The nucleic acid comprises a nucleic acid of at least 30, at least 35, at least 40, at least 45, 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 100 nucleotides.

In a second aspect, the present invention provides a composition comprising a peptide dendrimer, a nucleic acid and a lipid, wherein the peptide dendrimer comprises at least: a core peptide sequence, a first branching residue and two first peptide motifs. The branching residue may be lysine, 2, 4-diaminobutyric acid, ornithine or diaminopropionic acid. The nucleic acid comprises a single stranded nucleic acid of at least 30, at least 35, at least 40, at least 45, or 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 100 nucleotides.

In a third aspect, the invention provides a method of delivering a nucleic acid into cells of a subject in need of delivery, the method comprising administering to the subject a pharmaceutically effective amount of the composition. The composition comprises a peptide dendrimer, a nucleic acid and a lipid, wherein the peptide dendrimer comprises at least: a core peptide sequence, a first branching residue and two first peptide motifs. The branching residue may be lysine, 2, 4-diaminobutyric acid, ornithine or diaminopropionic acid. The nucleic acid comprises a nucleic acid of at least 30, at least 35, at least 40, at least 45, 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 100 nucleotides. The nucleic acid may be single stranded.

In some embodiments, the nucleic acid is RNA. For example, the RNA may be selected from mRNA, ssRNA, dsRNA, sgRNA, crRNA, tracrRNA, incRNA, siRNA, saRNA and/or self-amplifying RNA. Preferably, the RNA is mRNA.

In some embodiments, the nucleic acid is DNA. For example, the DNA may include ssDNA, dsDNA, plasmids, and/or cDNA.

To avoid any doubt; the composition may comprise more than one nucleic acid (e.g., more than one type of RNA molecule). Similarly, the composition may comprise more than one lipid. The composition may comprise more than one peptide dendrimer.

In some embodiments, the composition comprises RNA nucleic acids and DNA nucleic acids. The RNA nucleic acid and the DNA nucleic acid may be part of a single nucleic acid molecule.

In some embodiments, the nucleic acid comprises a modified nucleic acid. Exemplary nucleic acid modifications are described herein.

Preferably, the nucleic acid encodes a transgene and is capable of expressing the transgene in a target cell. The transgene may be a protein or peptide. Additionally or alternatively, the nucleic acid may modulate expression or activity of an endogenous gene. The modulation may be an increase in the expression of the gene and/or an increase in the exogenous expression of an additional copy of the gene, or the modulation may be a decrease in the expression of the gene.

In some embodiments, the regulated endogenous gene is a gene that expresses a protein or peptide.

In some embodiments, the protein or peptide comprises an antigen, hormone, receptor, chimeric antigen receptor, transcription factor, and/or cytokine.

In some embodiments, the transgene comprises a tumor antigen, a viral protein, a bacterial protein, or a protein of a microorganism parasitic to a mammal.

In some embodiments, the composition may be used as a vaccine.

In some embodiments, the nucleic acid comprises or encodes self-amplifying RNA.

In some embodiments, the use comprises treating a genetic disorder in a subject.

In some embodiments, the nucleic acid expresses a functional form of a gene that is nonfunctional, down-regulated, inactivated or impaired in the subject.

In some embodiments, the nucleic acid encodes and/or comprises one or more components of a system for editing a genome or a system for altering gene expression. For example, the system for editing the genome or the system for altering gene expression can be a CRISPR/Cas system. The nucleic acid may encode a Cas protein or peptide, and/or include sgrnas, crrnas, and/or tracrrnas. The nucleic acid may include mRNA encoding a Cas protein or peptide, and an RNA sequence containing sgRNA. The composition may comprise an mRNA encoding a Cas protein or peptide, and another RNA (as a separate molecule) comprising an sgRNA. In some embodiments, when present, one or more of sgRNA, crRNA, tracrRNA and the nucleic acid encoding the Cas protein is part of a single nucleic acid. In some embodiments, when present, one or more of sgRNA, crRNA, tracrRNA and the nucleic acid encoding the Cas protein is present on two or more nucleic acids.

In some embodiments, the composition targets spleen, lymphoid tissue, skeletal muscle, brain, and adipose tissue, as well as targets lung, tumor tissue, heart, skeletal muscle, adipose tissue, brain, liver, and kidney.

In some embodiments, the composition targets spleen, lymphoid tissue, lung, and/or bone. In these embodiments, the nucleic acid may be RNA, such as mRNA.

In some embodiments, the nucleic acid is delivered to a cell that is a leukocyte, such as a B lymphocyte, T lymphocyte, monocyte, neutrophil, dendritic cell, macrophage, or monocyte; lymph node tissue cells, myeloid cells, fibroblasts, muscle cells, skeletal muscle cells, endothelial cells, liver cells, astrocytes, neurons, astrocytes, spleen cells, lung cells, myocardial cells, kidney cells, adipocytes, stem cells and/or tumor cells.

In some embodiments, the composition is administered to a subject such that the nucleic acid is delivered to immune cells.

In some embodiments, the nucleic acid expresses an immune molecule or transcription factor in a target cell. The immune molecule may be a T cell receptor, chimeric antigen receptor, cytokine, decoy receptor, antibody, co-stimulatory receptor, co-stimulatory ligand, checkpoint inhibitor, immunoconjugate or tumor antigen.

In some embodiments, the cell is a B lymphocyte, T lymphocyte, monocyte, neutrophil, dendritic cell, macrophage, monocyte, myeloid-derived suppressor cell (MDSC), tumor-associated macrophage, or tumor-associated neutrophil.

In some embodiments, the nucleic acid is RNA, e.g., mRNA.

In some embodiments, the composition is used in a method of treating cancer in a subject. The cancer may be a blood cancer, such as leukemia, lymphoma, myeloma, or myelodysplastic syndrome; or lung cancer, cardiac carcinoma, sarcoma or liver cancer. The treatment may also include administration of an anti-cancer agent. The cancer may be non-small cell lung cancer, advanced melanoma, prostate cancer, ovarian cancer, breast cancer, lung cancer, cholangiocarcinoma (cholangiocellular carcinoma), gall bladder cancer, neuroendocrine tumor, hepatocellular carcinoma, colorectal cancer, pancreatic cancer, and solid tumors.

In some embodiments, the composition is used in a method of treating a pulmonary disease. For example, the composition may be used to treat Chronic Obstructive Pulmonary Disease (COPD) or Cystic Fibrosis (CF).

In some embodiments, the composition is used in a method of treating an autoimmune disease in a subject.

In some embodiments, the composition is for treating pompe disease, muscle wasting disease, myopathy, or muscular dystrophy, e.g., duchenne muscular dystrophy, in a subject. In these embodiments, the nucleic acid may be DNA.

In some embodiments, the composition is for treating limb ischemia, such as diabetic limb ischemia, in a subject, and wherein the transgene is Hepatocyte Growth Factor (HGF), vascular Endothelial Growth Factor (VEGF), and/or Fibroblast Growth Factor (FGF).

In some embodiments, the two first peptide motifs independently consist of a single amino acid, dipeptide, tripeptide or tetrapeptide motif.

In some embodiments, the peptide dendrimer further comprises two second branching residues (e.g., lysine) and four second peptide motifs, wherein one of the second branching residues is covalently bonded to one of the first peptide motifs and the other second branching residue is covalently bonded to the other first peptide motif, and wherein each second branching residue is covalently bonded to two second peptide motifs.

In some embodiments, the four second peptide motifs independently consist of a single amino acid, dipeptide, tripeptide or tetrapeptide motif.

In some embodiments, the peptide dendrimer further comprises four third branching residues (e.g., lysine) and eight third peptide motifs, wherein each second peptide motif is covalently bonded to one of the third branching residues, respectively, such that each third branching residue is covalently bonded to one second peptide motif, and wherein each third branching residue is covalently bonded to two third peptide motifs.

In some embodiments, the eight third peptide motifs independently consist of a single amino acid, dipeptide, tripeptide or tetrapeptide motif.

Each peptide motif independently comprises a naturally occurring L-or D-amino acid and/or a non-naturally occurring L-or D-amino acid, such as β -alanine (B) or aminocaproic acid (X).

In some embodiments, the first peptide motif, the second peptide motif, and/or the third peptide motif comprise an amino acid having a basic side chain.

In some embodiments, the core sequence comprises an amino acid residue having an ionizable group, such as histidine.

In some embodiments, the first peptide motif, the second peptide motif, and/or the third peptide motif comprise an amino acid having a nonpolar side chain.

In some embodiments, the first peptide motif, the second peptide motif, and/or the third peptide motif comprise an amino acid having an acidic side chain.

In some embodiments, the first peptide motif, the second peptide motif, and/or the third peptide motif comprise amino acids having uncharged polar side chains.

In some embodiments, the first, second, and third peptide motifs (when present) comprise a) arginine (R) or lysine (K); and/or b) leucine (L), valine (V), histidine (H) or isoleucine (I).

In some embodiments, the first, second, and/or third peptide motifs comprise leucine (L) and/or arginine (R) residues.

In some embodiments, the peptide dendrimer comprises the structures listed in table 2.

In some embodiments, the peptide dendrimer further comprises a tissue and/or cell targeting motif. The tissue or cell targeting motif may include a muscle targeting motif such as GAASSLNIA (SEQ ID NO: 1), an integrin targeting motif such as arginine-glycine-aspartic acid (RGD), or a chemical modification, e.g., comprising mannose glycosylation.

In some embodiments, the peptide dendrimer further comprises a cell penetrating peptide. The cell penetrating peptide may comprise a TAT derivative sequence. The cell penetrating peptide may comprise the peptide sequence XRXRRBRRXRRBRXB (SEQ ID NO: 2), wherein X is 6-aminocaproic acid and B is beta-alanine.

In some embodiments, the peptide dendrimer further comprises alkyl chains, alkenyl chains, antibodies or fragments thereof, sugars and/or fatty acids. The alkyl or alkenyl chain may be conjugated to the core peptide sequence, for example, at the C-terminus of the peptide dendrimer. Alternatively, the alkyl or alkenyl chain may be conjugated to the N-terminus of the peptide dendrimer.

In some embodiments, the alkyl or alkenyl chain comprises from about 5 carbons to about 50 carbons, preferably from about 12 to about 30 carbons.

In some embodiments, the peptide dendrimer comprises fatty acids conjugated to the C-terminus of the peptide dendrimer. In other embodiments, the peptide dendrimer comprises fatty acids conjugated to the N-terminus of the peptide dendrimer.

In some embodiments, the lipid of the composition comprises a cationic lipid, a neutral lipid, an anionic lipid, and/or an ionizable lipid.

In some embodiments, the lipid of the composition comprises a saturated fatty acid. Additionally or alternatively, the lipid of the composition may comprise an unsaturated fatty acid.

In some embodiments, the lipid comprises 1, 2, 3, 4, 5, or 6 fatty acid chains. Preferably, the lipid comprises 2, 3, 4 or 6 fatty acid chains.

In some embodiments, the lipid comprises dioleoyl phosphatidylethanolamine (DOPE) and/or N- [1- (2, 3-dioleyloxy) propyl ] -N, N-trimethylammonium chloride (DOTMA). In some embodiments, the lipid comprises dioleoyl phosphatidylethanolamine (DOPE) and dioleoyl phosphatidylglycerol (DOPG).

In some embodiments, the N/P ratio is between about 0.01:1 and 100:1. For example, the N/P ratio may be between about 0.05:1 and 50:1, or between about 0.1:1 and 30:1. Narrower ranges are also contemplated, such as between 0.2:1 and 25:1 and between 0.5:1 and 20:1.

In some embodiments, the N/P ratio is between 1:1 and 50:1. In some embodiments, a higher proportion of the composition is observed in the spleen and/or lymph nodes than in the liver following administration to a subject.

In some embodiments, the N/P ratio is between 0.01:1 and 1:1. In some embodiments, a higher proportion of the composition is observed in the lung, spleen and/or lymph nodes than in the liver following administration to a subject.

In some embodiments, the peptide dendrimer, nucleic acid, and lipid form positively charged particles.

In other embodiments, the peptide dendrimer, nucleic acid and lipid form negatively charged particles or neutral charged particles.

In some embodiments, the delivery of the nucleic acid to the target tissue or target cell is increased by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 75%, 85%, 90%, 95%, 100% as compared to delivering the same nucleic acid to the same tissue or cell type using a lipid-based nucleic acid delivery system. The target cell or target tissue is a cell type or organ/tissue as defined herein. For example, the target tissue may be spleen, lymphoid organs, skeletal muscle, brain and adipose tissue, as well as lung, tumor tissue, heart, skeletal muscle, adipose tissue, brain, liver and kidney. The lipid-based nucleic acid delivery system may be DOTMA/DOPE.

The composition may be administered to the subject intravenously, intramuscularly, intratumorally, subcutaneously, intradermally, or intraperitoneally.

In some embodiments, the composition is contained within a liquid. In other embodiments, the composition is provided as a dry composition (e.g., a dry powder). The dried composition may be prepared using lyophilization and/or freeze-drying techniques.

In particular embodiments; the third generation peptide dendrimer comprises: a first lysine residue and two first peptide motifs; two second lysine residues and four second peptide motifs; four third lysine residues and eight third peptide motifs; and a core peptide sequence covalently bonded to the first lysine residue,

(i) Wherein the first lysine residue is covalently bonded to two first peptide motifs, which are each covalently bonded to the two second lysine residues;

(ii) Wherein each second lysine residue is covalently bonded to two second peptide motifs, wherein each second peptide motif is covalently bonded to one of the third lysine residues, respectively; and

(iii) Wherein each third lysine residue is covalently bonded to two of the third peptide motifs, wherein the first peptide motif, the second peptide motif and the third peptide motif are independently mono-, di-, tri-, or tetrapeptide motifs. Each of the first peptide motif, the second peptide motif, and the third peptide motif may comprise arginine (R) or lysine (K); and/or b) leucine (L), valine (V), histidine (H) or isoleucine (I). ( The term "covalent bonding" refers to direct covalent bonding between the recited moieties without any intervening atoms. Thus, only a single, dipeptide, tripeptide or tetrapeptide motif is present between the first lysine residue and each second lysine residue; and only a mono-, di-, tri-, or tetrapeptide motif is present between each second and each third lysine residue. )

In some embodiments, when present, the first, second, and third peptide motifs may each comprise (1) an amino acid having a basic side chain, such as, but not limited to, lysine (K) or arginine (R) or histidine (H), (2) an amino acid having an acidic side chain, such as, but not limited to, aspartic acid (D) and glutamic acid (E), (3) an amino acid having a nonpolar side chain, such as, but not limited to, glycine (G), alanine (a), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), β -alanine (B), tryptophan (W), proline (P), aminocaproic acid (X), and cysteine (C), and (4) an amino acid having an uncharged polar side chain, such as, but not limited to, asparagine (N), glutamine (Q), serine (S), threonine (T), and tyrosine (Y).

Preferably, at least one of the first peptide motif, the second peptide motif and the third peptide motif comprises arginine (R). At least two of the first peptide motif, the second peptide motif, and the third peptide motif may comprise arginine (R). In some embodiments, the first peptide motif, the second peptide motif, and the third peptide motif all comprise arginine (R).

Preferably, at least one of the first peptide motif, the second peptide motif and the third peptide motif comprises leucine (L). At least two of the first peptide motif, the second peptide motif, and the third peptide motif can comprise leucine (L). In some embodiments, the first peptide motif, the second peptide motif, and the third peptide motif all comprise leucine (L).

In some embodiments, each of the first peptide motif, the second peptide motif, and the third peptide motif is a dipeptide motif. In some embodiments, each of the first peptide motif, the second peptide motif, and the third peptide motif is a tripeptide motif. In some embodiments, each of the first peptide motif, the second peptide motif, and the third peptide motif is a tetrapeptide motif. In some embodiments, the first peptide motif, the second peptide motif, and the third peptide motif are each independently a mono-, di-, tri-, or tetrapeptide motif. Preferably, at least one of the first, second and third peptide motifs is a dipeptide motif comprising both leucine (L) and arginine (R). In some embodiments, at least two of the first, second, and third peptide motifs are dipeptide motifs comprising both leucine (L) and arginine (R). In a particularly preferred embodiment, each peptide motif is a dipeptide motif comprising both leucine (L) and arginine (R). Each amino acid residue is independently selected from the L-isoform or the D-isoform.

In some embodiments, the peptide dendrimer is selected from any one of the peptide dendrimers listed in table 2.

In some embodiments, the peptide dendrimer is a polymer comprising (RL) ₈ (KRL) ₄ (KRL) ₂ G1,2,3-RL of the K-core.

In some embodiments, the peptide dendrimersThe article is a composition comprising (RL) ₈ (KRL) ₄ (KLR) ₂ G1-LR, G2,3-RL of the K-core.

In some embodiments, the peptide dendrimer is a polymer comprising (rl) ₈ (krl) ₄ (krl) ₂ G1,2,3-rl of the k-core (where "r" is D-arginine, T is D-leucine and "k" is D-lysine).

In some embodiments, the peptide dendrimer is a polypeptide comprising (LR) ₈ (KRL) ₄ (KRL) ₂ K-core G1,2-RL, G3-LR.

In some embodiments, the peptide dendrimer is a polymer comprising (RL) _s (KLR) ₄ (KLR) ₂ K-core G1,2-LR, G3-RL.

In some embodiments, the peptide dendrimer is a polypeptide comprising (LR) ₈ (KLR) ₄ (KLR) ₂ K-core G1,2,3-LR.

In some embodiments, the peptide dendrimer is RHCG1-R comprising (R) 2 KRCC-NH 2.

In some embodiments, the peptide dendrimer is RHCG1,2-R comprising (R) 4 (KR) 2 KRCC-NH 2.

In some embodiments, the peptide dendrimer is RHCG1-LR comprising (LR) 2 KRCC-NH 2.

In some embodiments, the peptide dendrimer is RHCG1-RL,2-LR comprising (LR) 4 (KRL) 2 KRCC-NH 2.

In some embodiments, the peptide dendrimer is a polypeptide comprising (LR) ₈ (KRL) ₄ (KRL) ₂ KGSC-NH ₂ G1,2-RL,3-LR.

In some embodiments, the peptide dendrimer is RHCG1-RLR comprising (RLR) 2 KRCC-NH 2.

In some embodiments, the peptide dendrimer is RHCG1,2-RLR comprising (RLR) 4 (KRRR) 2 KRRC-NH 2

In some embodiments, the peptide dendrimer is a G1-LRLR comprising (LRLR) 2KGSC-NH 2.

In some embodiments, the peptide dendrimer is RHCG1,2-RL,3-LR comprising (LR) 8 (KRL) 4 (KRL) 2 KRCC-NH 2.

In some embodiments, the peptide dendrimer is a polymer comprising (RL) s (KRL) 4 (KRL) ₂ KGSC-NH ₂ G1,2,3-RL.

In some embodiments, the peptide dendrimer is NTX1 comprising (LR) 8 (KRL) 4 (KRL) 2 kgscgaasslniaxrrrrrrrxrrbrxb-NH 2 (where x=6-aminocaproic acid and b=β -alanine).

( "G1" refers to the "1 st generation" peptide motif of the first layer. "G2" refers to the "2 nd generation" peptide motif of the second layer. "G3" refers to the "3 rd generation" peptide motif of the third layer. )

Accordingly, the present invention provides a composition for use in medicine, the composition comprising: nucleic acids, lipids and dendrimers described herein. The dendritic polymer may be a first generation dendritic polymer or a second generation dendritic polymer or a third generation dendritic polymer. Preferably, the N/P ratio (i.e., the amount of peptide (measured by the number N of 1+ charged nitrogen atoms on the peptide) to the amount of nucleic acid (measured by the number P of 1-charged phosphate groups in the backbone)) is greater than 0.05:1, for example greater than 0.1:1. (N/P ratio terms may be denoted as "N/P", "N: P", or "NP") in some embodiments, the N/P ratio is 0.15:1 or about 0.15:1 or at least 0.15:1. In some embodiments, the N/P ratio is 0.16:1 or about 0.16:1 or at least 0.16:1. In some embodiments, the N/P ratio is at least or greater than 1:1, such as about 2:1 or greater, about 2.5:1 or greater, about 3:1 or greater, about 4:1 or greater, about 5:1 or greater, about 10:1 or up to 20:1. In some embodiments, the N/P ratio is about 5:1, about 8:1, about 10:1, or about 20:1. In some embodiments, the N/P ratio is in the range of about 2:1 to about 20:1 or about 2.5:1 to about 10:1.

The lipid component of the composition may include DOTMA, DOPE, DOPC and/or DOPG.

The amount of lipid component can be expressed as weight relative to the amount of nucleic acid in the composition: the weight ratio ("w/w" or "w: w") may be in the range of 1:50 to 50:1. More preferably, the amount of lipid (by weight) relative to the amount of nucleic acid (by weight) is from 1:1 to 50:1, or from 2:1 to 20:1. The ratio of lipid nucleic acids may be at least 2:1. Most preferably, the lipid: the weight to weight ratio of nucleic acid was about 10:1. These ratios relate to the weight of total lipid. As described herein, the composition may comprise lipids containing more than one lipid component, such as a mixture of two, three, or four lipids. The weight of the lipid components is the total (combined) weight of these lipid components. Preferably, each lipid component is mixed in approximately equal proportions.

In a related aspect, the invention provides a pharmaceutical composition comprising a nucleic acid, a lipid, and a first, second or third generation dendrimer as described herein, and a pharmaceutically acceptable excipient.

The nucleic acid forming part of the composition of the invention may be an antisense oligonucleotide (ASO). Preferably, the ASO is at least 20 nucleotides long, at least 25 nucleotides long, at least 30 nucleotides long, at least 35 nucleotides long, or has a length as specified in connection with a nucleic acid as disclosed elsewhere herein, e.g., at least 40 nucleotides long.

The nucleic acid forming part of the composition of the invention may be an mRNA molecule. The nucleic acid forming part of the composition of the invention may be an IncRNA molecule. The nucleic acid forming part of the composition of the invention may comprise a CRISPR sequence. The nucleic acid forming part of the composition of the invention may comprise a double stranded region. The nucleic acid may be an siRNA molecule. The nucleic acid may be a small activating RNA (saRNA) molecule. The nucleic acid may be a self-amplifying RNA molecule. The nucleic acid may be a DNA plasmid that can express an siRNA or saRNA molecule in a target cell. The nucleic acid may be a DNA plasmid (linear or circular), such as a plasmid that can express a transgene in a target cell.

The transgene may be a viral protein, a bacterial protein or a protein of a microorganism parasitic to the mammal. Compositions expressing viral proteins, bacterial proteins or parasitic microbial proteins may be used as vaccines. For example, an effective amount of the composition can be delivered systemically (e.g., intravenously) to a subject to effect expression of a viral protein, bacterial protein, or parasitic microbial protein in skeletal muscle of the subject, thereby eliciting an immune response to the viral, bacterial, or parasitic protein. Accordingly, the present invention provides methods of vaccinating a subject, as well as compositions for vaccinating a subject. In such examples, the transgene may be expressed in an immune cell described herein, e.g., a leukocyte, such as a B lymphocyte, T lymphocyte, monocyte, neutrophil, dendritic cell, macrophage or monocyte; lymph node tissue cells.

The transgene may express a therapeutic protein for gene therapy. Gene therapy may be used to treat genetic disorders in a patient. The genetic disorder may be a monogenic genetic disorder, such as muscular dystrophy in a patient. In embodiments where the single gene genetic disorder is muscular dystrophy, the transgene may be a muscular dystrophy protein. In embodiments where the disorder is ischemia, the transgene may be Hepatocyte Growth Factor (HGF), vascular Endothelial Growth Factor (VEGF), and Fibroblast Growth Factor (FGF). In embodiments where the disorder is muscle atrophy, the transgene may be follistatin. In embodiments where the disorder is a neuromuscular disease, the transgene may be acid alpha-Glucosidase (GAA). Although the transgene may be expressed in one or more of the tissues disclosed herein, the expressed protein may be secreted from the tissue into the circulation.

It is contemplated that the invention may be used to deliver nucleic acid therapies to treat myopathies. It is also contemplated that the invention may be used to deliver nucleic acid therapies for the treatment of muscular dystrophy, such as duchenne muscular dystrophy, myotonic dystrophy, facial shoulder brachial muscular dystrophy, becker muscular dystrophy, limb girdle muscular dystrophy, oculopharynx muscular dystrophy, emery-Dreifuss muscular dystrophy, hereditary muscular dystrophy, congenital muscular dystrophy, and distal muscular dystrophy.

Nucleic acid therapy may be used to treat muscle wasting disorders such as cachexia. Nucleic acid therapy may be used to treat other muscle disorders, such as genetic muscle disorders, e.g., congenital myotonia or familial periodic paralysis. Nucleic acid therapy may be used to treat motor neuron diseases such as ALS (amyotrophic lateral sclerosis), spinal Bulbar Muscular Atrophy (SBMA), or Spinal Muscular Atrophy (SMA). Nucleic acid therapies may be used to treat mitochondrial disorders such as friedel-crafts ataxia (FA), or mitochondrial myopathies such as kanns-ser syndrome (KSS), leydig syndrome (subacute necrotizing encephalopathy), mitochondrial DNA depletion syndrome, mitochondrial encephalomyopathy, lactic acidosis and stroke-like attacks (MELAS), mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), myoclonus seizures with broken red fibers (MERRF), neuropathies, ataxia and retinal pigment degeneration (NARP), pearson syndrome, or progressive extraocular muscle Paralysis (PEO). Nucleic acid therapies may be used to treat congenital myopathies such as cap myopathies, central nuclear myopathies, congenital fiber type imbalance myopathies, axial null myopathies, central axial null myopathies, myoglobin-depositional myopathies, myotubular myopathies, or rhabdomyopathies. Nucleic acid therapies may be used to treat distant myopathies such as GNE myopathies/Nonaka myopathies/Hereditary Inclusion Body Myopathies (HIBM), lashing distant myopathies, markesber-Griggs delayed distant myopathies, miyoshi myopathies, udd myopathies/tibial muscular dystrophies, VCP myopathies/IBMPFD, vocal cord and pharyngeal distant myopathies, or Welander distant myopathies. Nucleic acid therapy may be used to treat endocrinopathy, such as hyperthyroidism or sarcopenia. Nucleic acid therapies may be used to treat inflammatory myopathies, such as dermatomyositis, inclusion body myositis, or polymyositis. Nucleic acid therapy may be used to treat metabolic myopathies such as acid maltase deficiency (AMD, pompe disease), carnitine deficiency, carnitine palmitoyl transferase deficiency, debranching enzyme deficiency (coriolis disease, fobs disease), lactate dehydrogenase deficiency, inosine deaminase deficiency, phosphofructokinase deficiency (vertical well disease), phosphoglycerate kinase deficiency, phosphoglycerate mutase deficiency, or phosphorylase deficiency (mecalder disease). Nucleic acid therapy may be used to treat myofibrillar myopathy or shoulder fibular myopathy. Nucleic acid therapies may be used to treat neuromuscular junction diseases such as congenital muscle weakness syndrome (CMS), lambert-eaton muscle weakness syndrome (LEMS), or Myasthenia Gravis (MG). Nucleic acid therapy may be used to treat peripheral nerve diseases such as fibular muscular dystrophy (CMT) or Giant Axonal Neuropathy (GAN). Nucleic acid therapies may be used to treat cardiovascular diseases such as thromboangiitis obliterans/berg disease, diabetic peripheral neuropathy (also tested in ALS, critical limb ischemia and foot ulcers), peripheral arterial disease, limb ischemia, critical limb ischemia (also known as chronic limb threatening ischemia and diabetic limb ischemia), critical Peripheral Arterial Occlusive Disease (PAOD), or intermittent claudication/arteriosclerosis. Nucleic acid therapies may be used to treat infectious diseases such as covd-19, HIV, HBV, HCV, ebola virus and marburg virus, west nile fever, SARS, avian influenza, HPV, cytomegalovirus or malaria. Nucleic acid therapy may be used to treat cancer, such as sarcoma, melanoma, breast cancer, lung cancer, pancreatic cancer, prostate cancer, liver cancer, acute myelogenous leukemia, or B-cell lymphoma. Nucleic acid therapy may be used to treat allergies, such as peanut allergies. Nucleic acid therapy may be used to treat Multiple Sclerosis (MS). Nucleic acid therapies may be used to treat myelodysplastic syndrome (MDS).

Pompe disease is caused by a deficiency in human acid alpha-Glucosidase (GAA), a lysosomal enzyme that cleaves the ends alpha 1-4 and alpha 1-6 glucose from glycogen. The composition of the invention may be used for the treatment of pompe disease. The compositions of the invention comprising a GAA-encoding nucleic acid may be administered to a subject suffering from pompe disease to deliver the nucleic acid to a target tissue of the subject to express GAA in the target tissue described herein (particularly liver and skeletal muscle). The enzyme may be secreted from the tissue into the circulation.

Follistatin is an inhibitor of TGF- β superfamily ligands that repress skeletal muscle growth and promote muscle atrophy. A composition of the invention comprising a nucleic acid encoding follistatin may be administered to a subject suffering from a muscular dystrophy to deliver the nucleic acid to a target tissue of the subject to express follistatin in the target tissue described herein (particularly liver and skeletal muscle). The protein may be secreted from the tissue into the circulation.

Accordingly, the present invention provides methods for treating such disorders and compositions for use in such treatment.

The core peptide motif of a dendrimer is a single amino acid residue or a short peptide motif, such as a dipeptide or tripeptide motif. The core sequence may comprise any amino acid (L-and/or D-isomer), such as glycine (G), serine(s), cysteine (C), alanine (A), lysine (K), leucine (L), valine (V), isoleucine (I), phenylalanine (F), methionine (M), tyrosine (Y), tryptophan (W), proline (P), threonine (T), asparagine (N), glutamine (Q), aspartic acid (D), glutamic acid (E), arginine (R) and/or histidine (H). The core sequence may also comprise non-naturally occurring amino acids (L-and/or D-isomers), such as β -alanine (B) and/or aminocaproic acid (X). Where the core is a tripeptide motif, it may comprise glycine (G), serine (S), and cysteine (C) or alanine (a). Preferably, the core comprises ionizable residues such as histidine (H). The core sequence may comprise arginine (R), histidine (H) and cysteine (C). The core sequence may comprise arginine (R) or glycine (G), histidine (H) or serine (S), and cysteine (C) or alanine (a). For example, the core sequence may be GSC or RHC. Tripeptide motifs may include alanine (a), lysine (K), and leucine (L). For example, the core sequence may be KLA. The core peptide may be covalently bound to another moiety, such as a cell-specific targeting peptide, or may be derivatized with a lipid molecule. One, some or all of the amino acids (of the core peptide motif, for example) of the dendrimer may be covalently bonded to another moiety, such as an antibody, a cell-specific targeting peptide, a glycoligand such as glucose, mannose, galactose and GalNAc (or glycans containing them), and/or a lipid substituent. The skilled artisan can readily select additional moieties that do not adversely affect solubility or nucleic acid binding characteristics.

The lipid component of the composition may comprise a mixture of lipids, including cationic lipids. For example, the lipid component may include dioleoyl phosphatidylethanolamine (DOPE) and N- [1- (2, 3-dioleyloxy) propyl ] -N, N, N-trimethylammonium chloride (DOTMA). The DOPE to DOTMA ratio can be readily determined to achieve the best characteristics for a given application, but is typically in the range of 1:5 to 5:1.

Preferably, the range is 3:1 to 1:3, or 2:1 to 1:2. Most preferably, the DOPE to DOTMA ratio is 1:1. In other embodiments, the lipid comprises 1, 2-dioleoyl-3-trimethylammoniopropane chloride (DOTAP), e.g., as the sole lipid or in combination with DOPE.

In other embodiments, the lipid component of the composition may include other lipids in addition to (or in lieu of) DOPE and DOTMA. Exemplary lipid components are set forth below:

cationic lipid:

n- [1- (2, 3-dioleyloxy) propyl ] -N, N, N-trimethylammonium chloride (DOTMA)

1, 2-dioleoyl-3-trimethylammoniopropane chloride (DOTAP)

2, 3-dioleyloxy-N- (2 [ spermine-carboxamide ] ethyl) -N, N-dimethyl-1-propylamine trifluoroacetate (DOSPA)

3p- [ N- (N ', N' -dimethylaminoethyl) -carbamoyl ] cholesterol hydrochloride (DC-chol)

Neutral lipids:

1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)

1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC)

Cholesterol

Anionic lipid:

1, 2-dioleoyl-sn-glycerol-3-phosphoryl- (1' -rac-glycerol) (DOPG)

Ionizable lipids:

DLin-DMA

DLin-KC2-DMA

DLin-MC3-DMA

other potential lipids include 4- (2-aminoethyl) -morpholino-cholesterol-hemisuccinate (MoChol), cholesterol Hemisuccinate (CHEMS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), cholesterol- (3-imidazol-1-yl propyl) Carbamate (CHIM), dioctadecyl Dimethyl Ammonium Bromide (DDAB), dioleoyl phosphatidyl silkAmino acids (DOPS), dioleoyl phosphatidyl glycerol (DOPG), cholesterol sulfate (chol-S0) ₄ )。

It is contemplated that any of the foregoing lipids may be used alone or in combination with one another in the compositions of the present invention. In addition, lipids can be derivatized by attachment to a PEG group such as PEG 2000.

The invention provides a method of delivering a nucleic acid into a target cell, the method comprising contacting the target cell with a composition of the invention. The target cell may be a muscle cell, liver cell, astrocyte, neuron, astrocyte, spleen cell, lung cell, cardiomyocyte, kidney cell, adipocyte, myeloid-derived suppressor cell (MDSC), tumor-associated macrophage or tumor-associated neutrophil, stem cell or tumor cell. The method may be performed in vitro. The cells may be obtained from the patient and may be administered to the patient after the method is performed, i.e. the method may be performed ex vivo. Alternatively, the method may be performed in vivo as part of a medical use or treatment. The method involves entering the cell by clathrin-mediated endocytosis, litter-mediated endocytosis and/or microcellular potion. Such in vivo treatments may involve administering a composition to a subject to deliver nucleic acids to a specific target tissue or tissues; such as muscle and/or liver, delivered to a subject. The composition may be administered as a single dose or as two or more doses.

The present invention also provides novel dendritic polymers such as those shown in table 2. In some embodiments, the dendrimer is a third generation dendrimer, such as a G1,2,3-LR dendrimer, G1,2,3-RL dendrimer, G1-LR, G2,3-RL dendrimer, G1,2-RL, G3-LR dendrimer, and G1,2-LR, G3-RL dendrimer, which may be collectively defined as a peptide dendrimer comprising: a first lysine residue and two first dipeptide motifs; two second lysine residues and four second dipeptide motifs; four third lysine residues and eight third dipeptide motifs; and a core peptide sequence covalently bonded to a first lysine residue, (i) wherein the first lysine residue is covalently bonded to two first dipeptide motifs, each of which is covalently bonded to two second lysine residues; (ii) Wherein each second lysine residue is covalently bonded to two second dipeptide motifs, wherein each second peptide motif is covalently bonded to one of the third lysine residues, respectively; and (iii) wherein each third lysine residue is covalently bonded to both of the third dipeptide motifs, wherein the first dipeptide motif, the second dipeptide motif, and the third dipeptide motif each comprise leucine (L) and arginine (R), and wherein at least one of the first dipeptide motif, the second dipeptide motif, and the third dipeptide motif consists of a leucine-arginine (LR) dipeptide motif (wherein the "LR" motif is recited according to standard N-terminal to C-terminal convention). Each amino acid residue is independently selected from the L-isoform or the D-isoform. These novel dendrimers may form part of the compositions of the present invention and may be used in other aspects of the invention disclosed herein.

The invention includes combinations of aspects and preferred features described unless such combinations are clearly not permitted or explicitly avoided.

Drawings

Embodiments and experiments illustrating the principles of the present invention will now be discussed with reference to the accompanying drawings, in which:

FIG. 1 comparison of transfection efficiencies of dendrimers for DNA delivery with different lysine and leucine arrangements. Changing the position of lysine and leucine in the generation did not significantly alter transfection efficiency in either (a) HeLa or (B) Neuro2A cells. Modification of the dendrimer G1,2,3-KL structure to its variants such as G1,2,3-K (G1, 2,3-KL version with all leucine removed) and substitution of all L-amino acids to D-form did not significantly alter transfection efficiency in (C) HeLa or (D) Neuro2A cells. G0,1,2,3-KL (G1, 2,3-KL version with zero generation of KL motif) was not significantly different in (E) HeLa or (F) Neuro2A cells. Conditions are as follows: transfection with 0.25. Mu.g of pCI-Luc 1X 10 ⁴ Individual cells. Error bars refer to mean ± SEM of experiments performed in triplicate. D/D is DOTMA/DOPE.

FIG. 2. Having different generations (G1, G2 and G3) and cationic residues (e.gKL vs RL) transfection efficiency of dendrimers for DNA delivery. (A) Transfection efficiency in HeLa cells, (B) transfection efficiency in Neuro2A cells. Cells were transfected with luciferase-expressing plasmid (pCI-Luc) in the presence of peptide dendrimers and D/D (24 hours post-transfection). Luminescence values were normalized by dividing the luminescence values by similar values for cells treated with the D/D DNA complex (w/w 1:1, 0.25 pg) to yield% transfection. Conditions are as follows: transfection with 0.25. Mu.g of pCI-Luc 1X 10 ⁴ Individual cells. Error bars refer to mean ± SEM of experiments performed in triplicate. * Representing p < 0.05.D/D is DOTMA/DOPE.

FIG. 3 comparison of transfection efficiencies of G1,2,3-RL with other commercial transfection reagents for DNA delivery, including DOTMA/DOPE, polyethylenimine and Lipofectamine 2000. (a) transfection in HeLa cells in the absence of serum, (B) transfection in HeLa cells in the presence of serum, (C) transfection in Neuro2A cells in the absence of serum, and (D) transfection in Neuro2A cells in the presence of serum. Cells were transfected with luciferase-expressing plasmid (pCI-Luc) in the presence of G1,2,3-RL and D/D (24 hours post-transfection). Luminescence values were normalized by dividing the luminescence values by similar values for cells treated with D/D DNA complexes to yield% transfection. Conditions are as follows: transfection of 1X 10 with 0.25. Mu.g of pCI-Luc in the absence of serum or in the presence of 10% serum ⁴ Cells were grown for 4 hours. Error bars refer to mean ± SEM of experiments performed in triplicate. In HeLa and Neuro2A cells, G1,2,3-RL mediates transfection significantly more strongly than other agents. * P < 0.05, p < 0.001, and p < 0.0001.D/D is DOTMA/DOPE.

FIG. 4 comparison of transfection efficiencies of dendrimers for DNA delivery with different generations (G1, G2 and G3) and cationic residues (e.g. KL versus RL) under serum conditions. (A) Transfection efficiency in HeLa cells, (B) transfection efficiency in Neuro2A cells. Cells were transfected with luciferase-expressing plasmid (pCI-Luc) in the presence of peptide dendrimers and D/D (24 hours post-transfection). By dividing the luminescence by the similarity of cells treated with the D/D DNA complex (w/w 1: 1,0.25. Mu.g)The values were normalized to luminescence values to yield% transfection. Conditions are as follows: 1X 10 transfection with 0.25. Mu.g of pCI-Luc in the presence of 10% serum ⁴ Cells were grown for 4 hours. Error bars refer to mean ± SEM of experiments performed in triplicate. In HeLa and Neuro2A cells, G1,2,3-RL (N/P between 5 and 20) mediated transfection significantly more efficiently than the other groups. * Represents p < 0.05; g1,2,3-RL (N/P is 10, 20) mediates transfection significantly more strongly than the other groups, indicating P < 0.001.D/D is DOTMA/DOPE.

FIG. 5 cell uptake pathway of G1,2,3-RL-D/D-DNA nanoparticles. Chlorpromazine inhibits clathrin-mediated endocytosis; genistein and coarse bran Chai Gusu inhibit pit-mediated endocytosis and microporosity, respectively.

FIG. 6 expression of follistatin in skeletal muscle following intravenous administration of a composition comprising G1,2,3-RL with D/D and follistatin-expressing plasmid DNA. Mice were injected with G1,2,3-RL complex twice and tissues were harvested to determine follistatin mRNA expression levels by qPCR.

Figure 7 body weight of mice after intravenous administration of the disclosed compositions.

FIG. 8 luciferase expression in mouse tissues following intravenous administration of compositions comprising G1,2,3-RL or G1,2,3-LR with DOTMA/DOPE (w/w=1:1 relative to DNA) and microring DNA expressing luciferase. These compositions were injected at a NP ratio of 20:1. Mice were injected with these compositions and after 24 hours tissues were harvested to measure luciferase signals in heart, kidney, liver, lung, spleen and skeletal muscle.

FIG. 9 luciferase expression in mouse skeletal muscle following intramuscular administration of a composition comprising G1,2-RL,3-LR with DOTMA/DOPE (w/w=1:1 relative to DNA) and microring DNA expressing luciferase. The composition was injected at a NP ratio of 20:1. Mice were injected with these compositions and skeletal muscle tissue was harvested after 48 hours to measure luciferase signals.

FIG. 10 luciferase expression in mouse tissue following intravenous administration of compositions comprising G1,2,3-RL (upper panel) or G1,2-RL,3-LR (lower panel) with DOTMA/DOPE (w/w=10:1 relative to mRNA) and luciferase-expressing mRNA. Each dendritic polymer composition was injected at NP ratios of 8:1 and 0.15:1. Mice were injected with these compositions and after 6 hours tissues were harvested to measure luciferase signals in muscle (gastrocnemius), liver, lung, heart, spleen, kidney, adipose tissue, brain, cervical lymph nodes and inguinal lymph nodes. When injected at an NP ratio of 0.15:1, the composition comprising G1,2,3-RL and G1,2-RL,3-LR is capable of delivering mRNA to immune cell-rich tissues, including lung, spleen and lymph nodes. G1,2-RL,3-LR injected at NP 8:1 was able to target mRNA delivery to the spleen.

FIG. 11 luciferase expression in mouse tissue following intravenous administration of compositions comprising G1,2,3-RL (upper panel) or G1,2-RL,3-LR (lower panel) with DOTMA/DOPE (w/w=10:1 relative to mRNA) and luciferase-expressing mRNA. Each dendritic polymer composition was injected at NP ratios of 8:1 and 0.15:1. Mice were injected with these compositions and after 6 hours tissues were harvested to measure luciferase signals in muscle (gastrocnemius), liver, heart, kidneys, adipose tissue and brain.

FIG. 12 comparison of luciferase expression in mouse tissues after intravenous administration of compositions comprising G1,2,3-RL or G1,2-RL,3-LR and DOTMA/DOPE (w/w=10:1 relative to mRNA) and luciferase-expressing mRNA at an NP ratio of 8:1. Mice were injected with these compositions and after 6 hours tissues were harvested to measure luciferase signals in muscle (gastrocnemius), liver, lung, heart, spleen, kidney, adipose tissue, brain, cervical lymph nodes and inguinal lymph nodes (fig. 12A, upper panel). The same data is provided to allow comparison between the lung, cervical lymph nodes and inguinal lymph nodes (fig. 12A, lower panel) and muscle (gastrocnemius), liver, heart, kidney, adipose tissue and brain (fig. 12B). Compositions comprising G1,2-RL,3-LR injected at an NP ratio of 8:1 are capable of delivering mRNA to immune cell-rich tissues with greater efficiency than G1,2,3-RL injected at an NP ratio of 8:1.

FIG. 13 luciferase expression in mouse tissues following intravenous administration of two doses of a composition comprising G1,2,3-RL with DOTMA/DOPE (w/w=10:1 relative to mRNA) and luciferase-expressing mRNA. The dendrimer composition was injected at NP ratio = 0.15:1. Mice were injected with these compositions and after 6 hours tissues were harvested to measure luciferase signals in muscle (gastrocnemius), liver, lung, heart, spleen, kidney, adipose tissue, brain, cervical lymph nodes and inguinal lymph nodes.

FIG. 14 luciferase expression in mouse tissues following intravenous administration of compositions comprising G1,2,3-RL, G1,2-RL,3-LR or NTX1 with DOTMA/DOPE (w/w=10:1 relative to mRNA) and luciferase-expressing mRNA. Each dendrimer composition was injected at an NP ratio of 0.15:1. Mice were injected with these compositions and after 6 hours tissues were harvested to measure luciferase signals in muscle (gastrocnemius), liver, lung, heart, spleen, kidney, adipose tissue, brain, cervical lymph nodes and inguinal lymph nodes. Compositions comprising NTX1 (which comprises a skeletal muscle targeting domain and a cell penetrating peptide domain) are capable of delivering mRNA to immune cell-enriched tissue with greater efficiency than compositions comprising G1,2,3-RL and G1,2-RL, 3-LR.

FIG. 15 luciferase expression in mouse tissues following intravenous administration of compositions comprising G1,2,3-RL, G1,2-RL,3-LR or NTX1 with DOTMA/DOPE (w/w=10:1 relative to mRNA) and luciferase-expressing mRNA and comparison to mice injected with compositions comprising DOTMA/DOPE (w/w=10:1 relative to mRNA) and luciferase-expressing mRNA but no dendrimer. Each dendrimer composition was injected at an NP ratio of 0.15:1. Mice were injected with these compositions and after 6 hours tissues were harvested to measure luciferase signals in muscle (gastrocnemius), liver, lung, heart, spleen, kidney, adipose tissue, brain, cervical lymph nodes and inguinal lymph nodes.

FIG. 16 shows luciferase expression in mouse tissue following intravenous administration of a composition comprising G1,2-RL,3-LR and DOTMA/DOPE (w/w=10:1 relative to mRNA) and luciferase-expressing mRNA and comparison to injection of a composition comprising DOTMA/DOPE (w/w=10:1 relative to mRNA) and luciferase-expressing mRNA but without dendrimer to mice. The dendrimer composition was injected at an NP ratio of 8:1. Mice were injected with these compositions and after 6 hours tissues were harvested to measure luciferase signals in muscle (gastrocnemius), liver, lung, heart, spleen, kidney, adipose tissue, brain, cervical lymph nodes and inguinal lymph nodes. Compositions comprising NTX1, G1,2,3-RL and G1,2-RL,3LR each showed increased mRNA delivery to all tissues relative to compositions comprising mRNA alone or compositions comprising DOTMA/DOPE and mRNA.

FIG. 17 luciferase and eGFP expression in HeLa or C2C12 cells after transfection with dendrimer compositions or commercial transfection reagents. HeLa cells were transfected with the following materials: 1) Luciferase mRNA alone, compositions comprising G1,2-RL,3-LR (NP=0.16:1), DOTMA/DOPE (w/w=10:1 relative to mRNA) and luciferase mRNA or compositions comprising Lipofectamine2000 ^TM And luciferase mRNA compositions (upper left panel); or 2) eGFP mRNA alone, a composition comprising G1,2-RL,3-LR (NP=0.16:1), DOTMA/DOPE (w/w=10:1 relative to mRNA) and eGFP mRNA or a composition comprising DLin-MC 3-DMA: cholesterol: DSPC: DMG-PEG lipid nanoparticles and eGFP mRNA (upper right panel). C2C12 cells were transfected with the following: luciferase mRNA alone, composition comprising G1,2-RL,3-LR (NP=8:1), DOTMA/DOPE (w/w=10:1 relative to mRNA) and luciferase mRNA, lipofectamine2000 ^TM And luciferase mRNA or a combination of polyethyleneimine and luciferase mRNA (lower panel). Luciferase and eGFP expression were measured 24 hours after transfection. The peptide dendrimers of the present invention are more effective in transfecting HeLa cells in vitro than commercially available transfection reagents such as Lipofectamine2000 and LNP.

FIG. 18 RNA transfection efficiency in HeLa cells after transfection with fluorescently labeled sgRNA and Cas9 mRNA or with G1,2-RL,3-LR with fluorescently labeled sgRNA and Cas9 mRNA at NP ratios of 0.15:1 or 8:1. The sgrnas were labeled with TM-rhodamine, and Cas9 mRNA was labeled with Cy5 fluorophore. Cells were transfected for 2 hours and assayed by flow cytometry. 100% HeLa cells exposed to compositions comprising G1,2-RL,3LR were double positive for sgRNA and Cas9 mRNA, demonstrating extremely high transfection efficiency.

FIG. 19 RNA transfection efficiency in HeLa cells after transfection with tracrRNA (trRNA), crRNA and Cas9 mRNA or with G1,2-RL,3-LR and tracrRNA, crRNA and Cas9 mRNA at NP ratios of 0.15:1 or 8:1. The tracrRNA is labeled with ATTO 55 fluorophore, crRNA is labeled with fluorescein, and Cas9 mRNA is labeled with Cy5 fluorophore. Cells were transfected for 2 hours and assayed by flow cytometry.

FIG. 20 RNA transfection efficiency in HeLa cells after transfection with tracrRNA (trRNA), crRNA or Cas9 mRNA. Cells were transfected with or without G1,2-RL, 3-LR. The tracrRNA is labeled with ATTO 55 fluorophore, crRNA is labeled with fluorescein, and Cas9 mRNA is labeled with Cy5 fluorophore. Cells were transfected for 2 hours and assayed by flow cytometry.

Fig. 21.Mrna delivery to immune cells in the spleen. Mice were injected intravenously with either mRNA alone or with NTX1 (NP=0.15:1), G1,2-RL,3-LR (NP=0.15:1), or G1,2-RL,3-LR (NP=8:1). mRNA was labeled with AlexaFluor 488. Spleens were harvested from mice 2 hours after injection for treatment. It is possible to deliver mRNA to a wide variety of immune cells present in the spleen using compositions comprising NTX1, G1,2-RL,3-LR (NP 8:1) or G1,2-RL,3-LR (NP 0.15:1).

Mrna delivery to immune cells in bone marrow. Mice were injected intravenously with either mRNA alone or with NTX1 (NP=0.15:1), G1,2-RL,3-LR (NP=0.15:1), or G1,2-RL,3-LR (NP=8:1). mRNA was labeled with AlexaFluor 488. Bone marrow was harvested from mice 2 hours after injection for treatment.

Mrna is delivered to immune cells in blood. Mice were injected intravenously with either mRNA alone or with NTX1 (NP=0.15:1), G1,2-RL,3-LR (NP=0.15:1), or G1,2-RL,3-LR (NP=8:1). mRNA was labeled with AlexaFluor 488. Blood was harvested from mice 2 hours after injection for treatment. It is possible to deliver mRNA to a wide variety of immune cells present in blood using compositions comprising NTX1, G1,2-RL,3-LR (NP 8:1) or G1,2-RL,3-LR (NP 0.15:1).

Fig. 24.Mrna delivery to immune cells in lymph nodes. Mice were injected intravenously with either mRNA alone or with NTX1 (NP=0.15:1), G1,2-RL,3-LR (NP=0.15:1), or G1,2-RL,3-LR (NP=8:1). mRNA was labeled with AlexaFluor 488. Lymph nodes were harvested from mice 2 hours after injection for treatment.

FIG. 25 in vitro DNA delivery to HeLa and C2C12 cells. HeLa cells were transfected with DNA alone, with G1,2,3-RL (N: P=5:1) and DOTMA/DOPE (w/w=1:1 relative to DNA) and with G1,2,3-RL (N: P=5:1) and DOPG/DOPE (w/w=1:1 relative to DNA) and with DNA (upper panel). C2C12 cells (myogenic cell lines) were transfected with DNA alone, with polyethylenimine and DNA, with G1,2-RL,3-LR (N: p=8:1) and DOTMA/DOPE (w/w=1:1 relative to DNA) and DNA, with NTX2 (N: p=8:1) and DOTMA/DOPE (w/w=1:1 relative to DNA) (lower panel). The DNA used in both experiments encodes a luciferase reporter. NTX2 is a G1,2-RL,3-LR dendrimer conjugated to the muscle targeting peptide ASSLINA ((LR) 8 (KRL) 4 (KRL) 2KGSCGAASSLNIA (Acp) -NH 2).

FIG. 26 aspartate aminotransferase (AST; 26A, upper panel), TNF- α (26A, lower panel), IL-6 (26B, upper panel) and IL-1β (26B, lower panel) levels following administration of mRNA formulations. Mice were treated with mRNA alone or with mRNA, dendrimer and DOTMA/DOPE (w/w=10:1 relative to mRNA). The N:P ratio of dendrimer to mRNA is indicated in the X-axis. All formulations were administered once, except that G1,2,3-RL and G1,2-RL,3-LR were also administered twice. For mice injected with 2 doses, the 1 st dose was injected 24 hours before the 2 nd dose. Plasma from mice was harvested 6 hours after dose 2 for measuring AST or cytokine levels. For mice dosed 1 time, plasma was harvested 6 hours after dosing for measuring AST and cytokine levels. The use of the dendrimers of the present invention to deliver mRNA in vivo does not cause significant hepatotoxicity or significant immune response.

FIG. 27 aspartate aminotransferase (upper left panel), TNF- α (upper right panel), IL-6 (lower left panel) and IL-1β (lower right panel) levels after administration of the DNA formulation. Mice were treated with DNA alone or with G1,2,3-RL and DOTMA/DOPE (w/w=1:1 relative to DNA). All formulations were dosed twice before harvesting plasma samples and measuring AST or cytokine levels. The use of the dendrimers of the present invention to deliver DNA in vivo does not cause significant hepatotoxicity or significant immune response.

FIG. 28 mRNA transfection of peptide dendrimers with lipid systems in vitro. HeLa cells were transfected with mRNA alone or with mRNA in combination with peptide dendrimer (N: P=0.16:1) and DOTMA/DOPE (w/w=10:1 relative to mRNA). The mRNA used expressed the eGFP reporter gene. 24 hours after transfection, cells were harvested to determine eGFP expression. The eGFP expression was normalized to the total amount of protein per transfection. Gene expression levels were calculated by normalizing transfection values of G1,2-RL,3-LR, DOTMA/DOPE and mRNA treated cells. Under the test conditions, the 1 st, 2 nd and 3 rd generation dendrimers with different cores and 1 st, 2 nd and 3 rd generation peptide motifs can efficiently transfect mRNA into cells.

FIG. 29 mRNA transfection of peptide dendrimers with lipid systems in vitro. HeLa cells were transfected with mRNA alone or with mRNA in combination with peptide dendrimers and DOTMA/DOPE (w/w=10:1 relative to mRNA). The N:P ratio of G1,2-RL,3-LR was 0.16:1, while the N:P ratio of all other dendrimers was 8:1. The mRNA used expressed the eGFP reporter gene. 24 hours after transfection, cells were harvested to determine eGFP expression. The eGFP expression was normalized to the total amount of protein per transfection. Gene expression levels were calculated by normalizing transfection values of G1,2-RL,3-LR, DOTMA/DOPE and mRNA treated cells. Under the test conditions, the 1 st, 2 nd and 3 rd generation dendrimers with different cores and 1 st, 2 nd and 3 rd generation peptide motifs can efficiently transfect mRNA into cells.

FIG. 30 luciferase expression in mouse tissues following intravenous administration of compositions comprising G1,2-RL,3-LR or RHGG 1-RL,2-LR with DOTMA/DOPE (w/w=10:1 relative to mRNA) and luciferase-expressing mRNA. The dendrimer composition was injected in an N:P ratio of 0.15:1 or 8:1. Mice were injected with these compositions and after 6 hours tissues were harvested to measure luciferase signals in muscle (gastrocnemius), liver, lung, heart, spleen, kidney, adipose tissue, brain and lymph nodes. The generation 2 dendrimer is capable of targeted delivery of mRNA to the spleen and lung with greater efficiency than the generation 3 dendrimer G1,2-RL, 3-LR.

Figure 31 shows diagrammatically a third generation dendrimer (N-terminal on the left and C-terminal on the right).

Detailed Description

Aspects and embodiments of the present invention will now be discussed with reference to the above figures and the following technical definitions. Additional aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated herein by reference.

Current immunotherapeutic response rates are about 15% to 20%, and improvement in treatment outcome is highly desirable. One strategy is to deliver mRNA to express proteins (such as CEBPA, IRF8, cGAS-STING, SOCS1, and/or SOCS 3) to reverse the immunosuppressive phenotype of myeloid cells in the tumor microenvironment, which would provide an environment that would be more favorable for an immunotherapy response. Another strategy may be to transfer mRNA into immune cells in a tumor to express cytokines (such as IL-2, IL-7, IL-12, IL-15, IL-21 and/or interferon) to activate the immune cells, thereby combating cancer cells. mRNA may also be delivered to macrophages to express chimeric antigen receptors so that the macrophages may be activated to kill tumor cells. Delivery of mRNA to express tumor antigens in antigen presenting cells will help activate the immune system to attack cancer cells. These strategies can be applied to treat all tumors, in particular non-small cell lung cancer, advanced melanoma, prostate cancer, ovarian cancer, breast cancer, lung cancer, cholangiocarcinoma (cholangiocellular carcinoma), gall bladder cancer, neuroendocrine tumors, hepatocellular carcinoma, colorectal cancer, pancreatic cancer, and solid tumors.

Nucleic acid delivery

The compositions of the invention may be used to deliver the nucleic acids described herein to certain tissues of the human or animal body. For example, delivery to the following tissues:

1. skeletal muscle

2. Liver

3. Lung (lung)

4. Heart and method for producing the same

5. White adipose tissue

6. Brown adipose tissue

7. Brain

8. Spleen

9. Bone marrow

10. Joint

11. Kidney and kidney

12. Gastrointestinal tract

13. Tumor(s)

14. Eyes (eyes)

15. Thymus gland

16. Skin of a person

17. Lymph nodes

18. Pancreas gland

19. Adrenal gland

20. Testis

21. Prostate gland

22. Ovary

23. Uterus

24. Bladder of bladder

25. Diaphragm

mRNA transfection

The effect of the algebra of the dendrimers on mRNA delivery was studied by transfecting HeLa cells under complete growth medium conditions. We have demonstrated that dendrimers with 1 or 2 generations (e.g., RHCG1-R, RHCG1,2-R, RHCG1-LR, RHCG1-RL, 2-LR) can effectively transfect cells and mediate similar transfection efficiencies as dendrimers with 3 generations (FIG. 28A). This is indeed very different from DNA transfection, since dendritic polymers with 1 or 2 generations are not as effective as dendritic polymers with 3 generations in complete growth medium for transfection of cells (fig. 4). Interestingly, increasing the N:P ratio of the 1 st or 2 nd generation dendrimers from 0.16 to 8 further improved transfection efficiency (FIG. 29A).

The inventors studied the effect of the core sequence of the dendrimer on transfection and have demonstrated that tripeptide sequences such as GSC (e.g. from G1,2-RL, 3-LR) are effective. Dipeptide core sequences such as KA and YM are also effective. This change in core peptide length did not affect mRNA transfection, indicating that a dendrimer with 2 amino acids in the core would be as good as a dendrimer with 3 amino acids in the core (fig. 28A). Different tripeptide core sequences such as RFW, RYM perform comparably to GSC. Although arginine containing cationic groups was added to the core, this did not increase or decrease transfection. Interestingly, transfection could be improved by 50% when the 3 amino acids at the core were changed from GCS to RHC. This suggests that ionizable groups such as histidine in the core of the dendrimer can enhance transfection (fig. 28A).

The use of a 12 amino acid core sequence (e.g., dendrimer "linear G1,2-RL, 3-LR") demonstrated that transfection efficiency was not affected by the increase in core sequence length (fig. 28A). The results indicate that longer amino acid chains and/or longer structures can be introduced in the core without affecting transfection efficiency. In fact, NTX1, which contains 27 amino acids in the core, is more efficiently transfected than G1,2-RL, 3-RL. This suggests that longer core sequences do enhance transfection. The enhanced transfection from NTX1 is likely due to the presence of cell penetrating peptides within the core sequence.

The inventors explored the effect of the number of amino acids in each generation on transfection. Thus, we tested dendrimers with only 1 amino acid (R), 2 amino acids (RL or LR), 3 amino acids (RLR) and 4 amino acids (LRLR). These studies indicate that dendrimers with 1,2, 3 or 4 amino acids in each generation can still transfect mRNA well into cells (fig. 28A). Even though dendrimers with different numbers of amino acids in each generation were used, it was shown that transfection efficiency was not affected.

Based on the G1,2-RL,3-LR structure, the inventors designed a library of 3-generation dendrimers, in which we replaced basic amino acid R with K, and/or changed hydrophobic amino acid L with acidic amino acids (such as E) and/or amino acids with nonpolar side chains (such as M, F, β -alanine (B), aminocaproic acid (X) and W) and/or amino acids with polar side chains (such as Q, T and Y).

Replacing R within the dendrimer with K reduced mRNA transfection efficiency (fig. 28A). However, in contrast to G1,2-RL,3-LR, changing hydrophobic amino acids to other hydrophobic amino acids and/or amino acids with polar or nonpolar side chains may not affect transfection or may reduce transfection by 30% to 40%. However, all of these dendrimers induced mRNA transfection significantly higher than the mRNA control alone.

The inventors also investigated the effect of amino acids in the L or D form within dendrimers on mRNA transfection. Based on the G1,2-RL,3-LR dendrimers, we found that changing part or all of the amino acids from L to D in each generation of dendrimers did not affect transfection efficiency. Substitution of lysine to diaminobutyric acid within the dendrimer will reduce transfection, although the transfection of the dendrimer is still significantly higher than the mRNA control alone.

The inventors demonstrated that G1-LL,2-RR can be used to deliver mRNA with our formulation protocol where we used a 0.16:1 N:P ratio of G1-LL,2-RR to DOTMA/DOPE (w/w 10:1). Interestingly, the dendrimers have been used to deliver ASO in different formulations in vitro and in vivo (Saher 2018). The formulations used were DOTMA/DOPE (w/w=2:1 relative to ASO) and N:P=20:1G 1-LL,2-RR and ASO. We have tried this formulation (i.e., DOTMA/DOPE (w/w=2:1 relative to mRNA) and N: P=20:1G 1-LL,2-RR and mRNA) at the time of cell transfection, but the transfection efficiency was poor. This formulation (DOTMA/DOPE (w/w=2:1 relative to mRNA) and N: P=20:1 of G1-LL,2-RR and mRNA) produced only 10% of the mRNA transfection efficiency of our improved formulation (DOTMA/DOPE (w/w=10:1 relative to mRNA) and N: P=0.16:1 of G1-LL,2-RR and mRNA).

The present inventors have explored 3-generation dendrimers for mRNA delivery with RL or LR or RL in different generations. We found that most of these dendrimers similarly transfected cells, with G1,2-RL,3-LR being most effective in mRNA transfection. Taken together, our data indicate that dendrimers with hydrophobic and cationic amino acids in each generation will produce efficient mRNA delivery to cells.

Since the 1 st and 2 nd generation dendrimers were more efficiently transfected into cells than G1,2-RL,3-LR, we have selected these dendrimers for further testing of transfection. At various N:P ratios, these dendrimers can generally be transfected better than G1,2-RL, 3-LR. In particular, the transfection effect of RHCG1-R, RHCG1,2-R, RHCG1-LR, RHCG1-RL,2-LR, RHCG1-RLR and G1-LRLR may be 700% to 1815% better than G1,2-RL,3-LR at N: p=4:1. These data indicate that the requirements for DNA and mRNA delivery are quite different. There is a trend for mRNA to be more effective in dendritic polymers with 1 or 2 generation than 3 generation dendritic polymers transfected cells under complete growth medium conditions. However, under complete growth medium conditions, the 3 rd generation dendrimers transfected DNA into cells much better than the 1 st or 2 nd generation dendrimers (FIG. 4).

Particularly preferred certain dendrimers of the present invention for mRNA delivery include RHCG1-R, RHCG1,2-R, RHCG1-LR, RHCG1-RL,2-LR, G1,2-RL,3-LR, RHCG1-RLR, RHCG1,2-RLR, G1-LRL, RHCG1,2-RL,3-LR and G1,2,3-R.

RNA interference

The present invention facilitates therapeutic down-regulation of target gene expression by delivery of nucleic acids. These include RNA interference (RNAi). Small RNA molecules can be used to regulate gene expression.

These include targeted degradation of mRNA by small interfering RNAs (sirnas), post-transcriptional gene silencing (PTG), sequence-specific translational repression of developmental regulation of mRNA by micrornas (mirnas), and targeted transcriptional gene silencing.

RNAi mechanisms and the role of small RNAs in heterochromatin complex targeting and epigenetic gene silencing at specific chromosomal sites have also been demonstrated. Double-stranded RNA (dsRNA) -dependent post-transcriptional silencing, also known as RNA interference (RNAi), is a phenomenon in which dsRNA complexes can target specific homologous genes for silencing within a short period of time. Which acts as a signal that promotes the degradation of mRNA with sequence identity. 21-nt siRNAs are typically long enough to induce gene-specific silencing, but short enough to evade the host response. The expression of the targeted gene product can be greatly reduced, and several molecules of siRNA can induce 90% silencing.

These RNA sequences are referred to in the art as "short or small interfering RNAs" (siRNAs) or "microRNAs" (miRNAs) depending on their source. Both types of sequences can be used to down-regulate gene expression by binding to complementary RNAs and triggering mRNA elimination (RNAi) or preventing translation of mRNA into protein. siRNA is processed from long double stranded RNA and is usually of exogenous origin when found in nature. Micro-interfering RNA (miRNA) is an endogenously encoded small non-coding RNA processed by short hairpin. Both siRNA and miRNA can inhibit translation of mRNA carrying a partially complementary target sequence without RNA cleavage and degrade mRNA carrying a fully complementary sequence.

Thus, the present invention provides the use of these sequences in the compositions of the invention for down-regulating the expression of a target gene.

The siRNA ligand is typically double-stranded and, in order to optimize the effect of RNA-mediated down-regulation of target gene function, the length of the siRNA molecule is preferably selected to ensure that RISC complexes that mediate siRNA recognition of mRNA targets correctly recognize the siRNA and to make the siRNA short enough to reduce host response.

miRNA ligands are typically single-stranded and have regions of partial complementarity such that the ligand is capable of forming a hairpin. mirnas are RNA genes that are transcribed from DNA but are not translated into protein. The DNA sequence encoding the miRNA gene is longer than miRNA. The DNA sequence includes a miRNA sequence and a near reverse complement. When the DNA sequence is transcribed into a single-stranded RNA molecule, the miRNA sequence and its reverse complementary base pair form a partially double-stranded RNA fragment. The design of microRNA sequences is discussed in John et al, 2004.

Typically, the RNA ligand intended to mimic the effect of an siRNA or miRNA has between 10 and 40 ribonucleotides (or synthetic analogues thereof), more preferably between 17 and 30 ribonucleotides, more preferably between 19 and 25 ribonucleotides and most preferably between 21 and 23 ribonucleotides. In some embodiments of the invention employing double stranded siRNA, the molecule may have a symmetrical 3 'overhang, e.g., an overhang of one or two (ribose) nucleotides, typically UU or dTdT 3' overhangs. Based on the disclosure provided herein, a skilled artisan can readily design suitable siRNA and miRNA sequences, for example using resources such as the Ambion's online siRNA finder. siRNA and miRNA sequences may be synthetically produced and exogenously added to cause down-regulation of genes or produced using expression systems (e.g., vectors). In a preferred embodiment, the siRNA is synthesized synthetically.

Longer double stranded RNAs can be processed in cells to produce sirnas (see, e.g., myers et al (2003)). Longer dsRNA molecules may have symmetrical 3 'or 5' overhangs (e.g., one or two (ribose) nucleotides), or may have blunt ends. Longer dsRNA molecules may be 25 nucleotides or more. Preferably, the longer dsRNA molecule is 25 to 30 nucleotides long. More preferably, the longer dsRNA molecule is 25 to 27 nucleotides long. Most preferably, the longer dsRNA molecule is 27 nucleotides long.

In one embodiment, the siRNA, longer dsRNA or miRNA is produced endogenously (in the cell) by transcription from the vector. The vector may be introduced into the cell in any manner known in the art. Optionally, tissue-specific promoters may be used to regulate expression of the RNA sequences. In another embodiment, the siRNA, longer dsRNA or miRNA is produced exogenously (in vitro) by transcription from a vector.

Alternatively, siRNA molecules can be synthesized using standard solid phase or solution phase synthesis techniques known in the art. The linkages between nucleotides may be phosphodiester linkages or alternatives, such as linking groups of the formula: p (O) S (thio); p (S) S (dithioate); p (O) NR'2; p (O) R'; p (O) OR6; CO; or CONR'2 (wherein R is H (or salt) or alkyl (1-12C) and R6 is alkyl (1-9C)) is linked to the adjacent nucleotide through-O-or-s-.

Long non-coding RNA

Mammalian genomes are ubiquitously transcribed, producing a large number of transcripts, including thousands of long non-coding RNA molecules (incrnas). It has been demonstrated that IncRNA can regulate chromatin status, transcription, RNA stabilization, and translation of certain genes.

RNA activation (RNAa)

RNA activation (RNAa) is a process mediated by RNA that enhances gene expression through highly regulated and evolutionarily conserved pathways. RNAa can be induced by small activating RNA (saRNA), a type of non-coding RNA consisting of 21 nucleotide dsRNA with 2 nucleotide overhangs at both ends. Although the saRNA mediates gene activation in a sequence-specific manner, the saRNA has the same structure and chemical composition as siRNA. To activate gene expression, the guide strand of the saRNA is loaded onto AGO2 and the complex is then transported into the nucleus. Once inside the nucleus, the guide strand-AGO 2 complex binds directly to the gene promoter or related transcript, thereby recruiting key components (including RNA polymerase II) to initiate gene activation (Kwok et al, 2019).

Antisense oligonucleotides (ASO)

Antisense oligonucleotides (ASOs) are single strands of DNA or RNA complementary to a target sequence. ASO hybridizes to the target nucleic acid. For example, ASOs may be used to target coding or non-coding RNA molecules in cells. After target binding, the ASO/target complex may be enzymatically degraded (e.g., by rnase H).

Messenger RNA (mRNA)

The skilled person knows that messenger RNAs (mrnas) are single-stranded molecules of RNA, the coding sequences of which lead to translation of the coding sequences of the genes into the corresponding amino acid sequences by ribosomes. mRNA is produced during transcription, in which enzymes (RNA polymerase) convert genes into primary transcript mRNA (also known as pre-mRNA). The pre-mRNA will typically still contain introns, i.e., regions that will not continue to encode the final amino acid sequence. These introns are removed during RNA splicing, leaving only the exons, i.e. the region encoding the protein. This exon sequence constitutes the mature mRNA. The mature mRNA is then read by the ribosome, producing the encoded protein. The invention can be used to deliver mRNA molecules to target cells and tissues as a means of inducing expression of a desired protein or peptide. Induction of peptide/protein expression by mRNA delivery is particularly useful when transient expression is desired.

mRNA and IncRNA are typically macromolecules having a negatively charged side and a hydrophobic side. Thus, mRNA and IncRNA will require a balance between hydrophobic and hydrophilic interactions to be encapsulated and delivered to target tissues and cells. This balance between hydrophobic and hydrophilic interactions will be different from, for example, double stranded nucleic acids such as pDNA and siRNA that have charges on both sides. Since mRNA and IncRNA are significantly larger than, for example, ASO, the requirements for encapsulation and delivery may also be different. Thus, the optimal NP ratio and w/w ratio of DOTMA/DOPE for dendrimers for mRNA and IncRNA delivery will be different compared to ASO delivery.

Modified nucleic acids

Modified nucleotide bases may be used in addition to naturally occurring bases, and these modified nucleotide bases may impart advantageous properties to the nucleic acids comprising them.

For example, modified bases can increase the stability of a nucleic acid molecule, thereby reducing the amount required. Providing modified bases may also provide nucleic acid molecules that are more stable or less stable than unmodified nucleic acids.

The term "modified nucleotide base" encompasses nucleotides having covalently modified bases and/or sugars. For example, modified nucleotides include nucleotides having a sugar covalently attached to a low molecular weight organic group that is different from the hydroxyl group at the 3 'position and different from the phosphate group at the 5' position. Thus, modified nucleotides may include 2 'substituted sugars, such as 2' -0-methyl-; 2' -0-alkyl; 2' -0-allyl; 2' -S-alkyl; 2' -S-allyl; 2' -fluoro-; 2' -halo or azido-ribose, carbocyclyl sugar analogs, α -anomer sugar; epimeric sugars such as arabinose, xylose or lyxose, pyranose, furanose and sedoheptulose.

Modified nucleotides are known in the art and include alkylated purines and pyrimidines, acylated purines and pyrimidines, and other heterocycles. These classes of pyrimidines and purines are known in the art and include pseudoisocytosine, N4, N4-desmethylidene cytosine, 8-hydroxy-N6-methyl adenine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyl uracil, dihydrouracil, inosine, N6-isopentyl-adenine, 1-methyladenine, 1-methylpseuduracil, 1-methylguanine, 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine 5-methylaminomethyl uracil, 5-methoxyaminomethyl-2-thiouracil, -D-mannosyl braided glycoside, 5-methoxycarbonylmethyl uracil, 5-methoxyuracil, methyl 2-methylthio-N6-isopentenyl adenine, uracil-5-oxoacetate, pseudouracil, 2-mercaptocytosine, 5-methyl-2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxoacetate methyl uracil, uracil 5-oxoacetic acid, braided glycoside, 2-mercaptocytosine, 5-propyluracil, 5-propylcytosine, 5-ethyluracil, 5-ethylcytosine, 5-butyluracil, 5-pentylhyde, 2, 6-diaminopurine, methylpseuduracil, 1-methylguanine, 1-methylcytosine.

Nucleic acid based vaccines

DNA and RNA vaccines as defined by the World Health Organization (WHO) involve the direct introduction of plasmids containing DNA sequences or RNAs encoding antigens against which an immune response is sought and which is dependent on the in situ generation of target antigens into the appropriate tissues (of the subject to be vaccinated). These methods offer a number of potential advantages over traditional methods, including simultaneous stimulation of B cell and T cell responses, improved vaccine stability, absence of any infectious agents, and relative ease of mass production. As evidence of the principle of DNA vaccination, immune responses in animals have been obtained using genes from a variety of infectious agents including influenza virus, hepatitis b virus, human immunodeficiency virus, rabies virus, lymphocytic choriomeningitis virus, malaria parasites and mycoplasma. In some cases, prevention of disease in animals has also been achieved. However, the value and advantages of DNA vaccines must be assessed on a case-by-case basis, and their suitability will depend on the nature of the factors against which the immunization is to be performed, the nature of the antigen and the type of immune response required for protection.

The field of DNA and RNA vaccination is rapidly evolving. Vaccines currently developed use not only DNA but also adjuvants that assist the DNA in entering cells, targeting it to specific cells or may act as adjuvants to stimulate or direct an immune response. By 2020, WHO indicated that the first nucleic acid vaccine to obtain marketing approval might use plasmid DNA derived from bacterial cells, but in the future other nucleic acid vaccines might use RNA or might use complexes of nucleic acid molecules with other entities. However, as the popularity of covd-19 has exploded in 2020, people have struggled to market the first RNA-based vaccine of covd-19, which was approved for use in the middle and later portions of 2020. Since approval, these RNA-based vaccines have been successfully introduced worldwide to allow the population to gain immunity to COVID-19.

As with other vaccine technologies, intramuscular delivery of DNA vaccines is a common approach (Lim et al 2020). The low replication rate of muscle cells (muscle cells) in skeletal muscle makes them attractive targets for DNA vaccination, as stable expression is independent of genomic integration.

RNA vaccines currently on the market use mRNA encoding an antigen as the payload. The area of increasing the effectiveness of RNA vaccines currently under investigation is the use of self-amplifying RNA. Self-amplifying RNAs share many structural features of mRNA and may include a 5 'cap, a 3' polyadenylation tail, and 5 'and 3' untranslated regions (UTRs). In addition to encoding the antigen of interest, the self-amplifying RNA will also include a system for self-amplification. For example, self-amplifying RNA may also encode RNA-dependent RNA polymerase (RDRA), a promoter, and an antigen of interest.

After translation of RDRA by the subject's translation mechanism, RDRA may bind self-amplified RNA and replicate RNA. Including a system for self-amplification reduces the minimum RNA required in the vaccine and therefore will reduce the likelihood of the subject experiencing side effects.

Medical therapy: gene therapy

The present invention contemplates use in gene therapy regimens. The nucleic acid may be present in a composition that, when introduced into a target cell, results in the expression of a therapeutic gene product (e.g., a transgene). Target cells include muscle cells, liver cells, astrocytes, brain cells (neurons, astrocytes), spleen cells, lung cells, heart muscle cells, kidney cells, adipocytes, stem cells, monocytes, macrophages, dendritic cells, neutrophils, B cells, T cells, myeloid-derived suppressor cells, tumor-associated macrophages, tumor-associated neutrophils, or tumor cells.

In order for gene therapy to be viable, it is desirable to employ such a DNA/RNA transfer system: which (1) directs the therapeutic sequence into the target cell, (2) mediates uptake of the therapeutic nucleic acid into a portion of the target cell population, and (3) is suitable for therapeutic use in vivo.

The nucleic acid-containing compositions of the invention may be stored and administered in a sterile pharmaceutically acceptable carrier. Various sterile solutions may be used for administration of the compositions, including water, PBS, ethanol, lipids, and the like. The concentration of DNA/RNA will be sufficient to provide a therapeutic dose, which will depend on the efficiency of transport into the cell.

Actual delivery of the gene sequences formulated as described above may be performed by a variety of techniques, including direct injection, instillation of the lung and other epithelial surfaces, or by intravenous injection. Administration may be through injection needles, trocars, cannulas, catheters, etc., as bolus injections, multiple dose or prolonged infusion, etc.

Gene editing

The present invention contemplates use in gene editing therapies, including gene editing therapies using techniques well known in the art such as CRISPR/Cas (e.g., CRISPR/Cas9 system), TALENS and zinc finger nucleases.

In some embodiments, the CRISPR/Cas system comprises a Cas nuclease, a CRISPR RNA (crRNA), and a transactivating crRNA (trRNA or tracrRNA). In this system, the crRNA contains a sequence complementary to the target DNA and is used to direct the Cas nuclease to a target site in the genome, and the tracrRNA serves as a binding scaffold for the Cas nuclease required for Cas activity. In some embodiments, the CRISPR/Cas system comprises a Cas nuclease and a single guide RNA (sgRNA) to direct the Cas nuclease to a target site in a target gene. The sgrnas comprise target-specific crrnas fused to scaffold tracrRNA in a single nucleic acid.

In some embodiments, the nucleic acid comprises DNA or mRNA encoding a Cas protein or peptide (e.g., cas9 protein or peptide). In some embodiments, the nucleic acid comprises sgRNA. In some embodiments, the nucleic acid comprises crRNA and/or tracrRNA. In some embodiments, the nucleic acid comprises DNA or mRNA encoding a Cas protein or peptide, crRNA, and tracrRNA. In some embodiments, the nucleic acid comprises DNA or mRNA encoding a Cas protein or peptide, or sgRNA.

CRISPR/Cas systems can also be used to direct repair or modification of target genes. For example, a CRISPR/Cas system can comprise a nucleic acid template to facilitate DNA repair or introduce an exogenous nucleic acid sequence into a target gene by, for example, facilitating homology-directed repair. CRISPR/Cas systems can also be used to introduce targeted modifications into target genomic DNA, for example, using base editing techniques. This can be achieved using a Cas protein fused to a base editor such as cytidine deaminase, as disclosed, for example, in W02017070633A2, incorporated by reference. In another example, the CRISPR/Cas system can be used to "rewrite" nucleic acid sequences in a genome. For example, the CRISPR/Cas system may be a lead editing system (Prime editing system). In such a lead editing system, a fusion protein may be used. For example, the fusion protein can comprise a catalytically impaired Cas domain (e.g., "nickase") and a reverse transcriptase. A catalytically impaired Cas domain may be able to cleave a DNA single strand to create a nicked DNA duplex. The leader editing system may include a leader editing guide RNA (pegRNA) that includes an extended sgRNA that includes a primer binding site and a reverse transcriptase template sequence. Where the DNA duplex nicks under the action of a catalytically compromised Cas, the primer binding site allows the 3' end of the nicked DNA strand to hybridize to the pegRNA, while the RT template serves as a template for synthesizing the edited genetic information.

In some embodiments, the CRISPR/Cas gene editing system may include a nucleic acid template to direct repair of a target gene of interest. In other embodiments, the Cas protein or peptide may include a base editor. In still further embodiments, the CRISPR/Cas system may be a lead editing system.

CRISPR/Cas gene silencing and gene activation

CRISPR/Cas systems have been adapted for gene silencing and activation. Such a system is contemplated for use with the present invention. For example, in some embodiments, the nucleic acid can encode a fusion protein comprising a Cas protein or peptide fused to a transcription repressor or activator. In some embodiments, the Cas protein is catalytically inactive. The fusion protein may be directed by the sgRNA or crRNA to a site of interest in the genome. After the fusion protein binds to the site of interest, a transcriptional repressor or activator may regulate the expression of the gene of interest.

Combination therapy

The compounds of the invention or compounds identified by the methods of the invention can be used to treat tumors and cancers in a subject in need of such treatment. These compounds may be administered alone or in combination with other anticancer agents.

"anticancer agent" refers to any agent useful in the treatment of neoplastic disorders. One class of anticancer agents includes chemotherapeutic agents. "chemotherapy" refers to the administration of one or more chemotherapeutic drugs and/or other agents to a cancer patient by various methods including intravenous, oral, intramuscular, intraperitoneal, intravesical, subcutaneous, transdermal, buccal or inhalation, or in the form of suppositories. Some chemotherapeutic agents are cytotoxic.

Cytotoxic chemotherapeutic agents trigger cell death through non-receptor mediated mechanisms or means. Cytotoxic chemotherapeutic agents trigger cell death by interfering with functions necessary for cell division, metabolism, or cell survival. Due to this mechanism of action, cells that grow rapidly (meaning proliferate or divide) or are metabolically active will be killed preferentially over cells that do not grow rapidly or are metabolically active. The state of division or utilization of energy (which is the metabolic activity supporting cell function) of different cells in the body determines the dose of chemotherapeutic agent that triggers cell death. Cytotoxic chemotherapeutic agents are non-exclusively related to alkylating agents, antimetabolites, plant alkaloids, topoisomerase inhibitors, antineoplastic agents and arsenic trioxide, carmustine, fludarabine, idaara-C, myalotang, GO, dichloromethyl diethylamine, cyclophosphamide, gemcitabine, bendamustine, systemic irradiation, cytarabine, etoposide, melphalan, pravastatin and radiation.

Anticancer agents also include protein kinase inhibitors, which may be useful in the treatment of a wide variety of cancers, including blood and lung cancers. Protein kinases generally promote cell proliferation, survival and migration, and are often constitutively overexpressed or active in cancer. Inhibitors of protein kinases are therefore common drug targets in cancer treatment. Examples of kinase inhibitors for clinical use include crizotinib, ceritinib, aprtinib, bragg tinib, bosutinib, dasatinib, imatinib, nilotinib, ponatinib, vemurafenib, dabrafenib, ibrutinib, palbociclib, sorafenib and rebaudinib.

Anticancer agents also include agents for immunotherapy, including antibodies. Immunotherapy may elicit, amplify, reduce or suppress an immune response, depending on the specific disease context. For example, tumor cells expressing PDL1 ligand inhibit the normal immune response of a subject by binding to PD-1 receptor expressed on T cells. In this way, tumor cells resist immune-induced apoptosis and promote tumor progression. anti-PD-1 and anti-PDL 1 antibodies have been successfully used clinically to inhibit this immune checkpoint and promote immune cell-mediated killing of tumor cells. Other examples of immunotherapy include oncolytic virus therapy, T cell therapy, and cancer vaccines.

Pharmaceutical composition

The pharmaceutical compositions provided herein may include one or more pharmaceutically acceptable excipients, such as solvents, solubilizers, suspending agents, buffers, isotonic agents, antioxidants, or antimicrobial preservatives. By "pharmaceutically acceptable" is meant "generally regarded as safe" molecular entities and compositions, for example, which are physiologically tolerable and generally do not produce allergies or similar adverse reactions, such as gastric discomfort, etc., when administered to a human. In some embodiments, the term refers to molecular entities and compositions approved by regulatory bodies of the federal or state government, such as the GRAS list specified in federal food, drug and cosmetic laws, clauses 204(s) and clause 409, which are subject to prior review and approval by the FDA or a similar list, U.S. pharmacopoeia, or another recognized pharmacopoeia for use in animals, more particularly humans. The excipients of the composition do not adversely affect the stability, bioavailability, safety and/or efficacy of the active ingredient when used. Thus, the skilled artisan will appreciate that there is no incompatibility between any of the components of the dosage form in the compositions provided. The excipient may be selected from the group consisting of buffers, tonicity agents, chelating agents, antioxidants, antimicrobial agents and preservatives.

Expression of therapeutic products

The nucleic acid delivered by the compositions of the invention may exhibit therapeutic effects (e.g., by acting directly to down-regulate or up-regulate a target gene), or it may express a gene product (which may be a therapeutic protein or a therapeutic nucleic acid) via an expression cassette comprising a coding sequence operably linked to a promoter. In the present specification, the term "operably linked" may include the case where the selected nucleotide sequence and the regulatory nucleotide sequence are covalently linked in such a way that expression of the coding sequence is placed under the influence or control of the regulatory sequence. Thus, a regulatory sequence is operably linked to a selected nucleotide sequence if it is capable of affecting the transcription of the coding sequence that forms part or all of the selected nucleotide sequence. Where appropriate, the resulting transcript may then be translated into the desired protein or polypeptide.

Route of administration

Compositions according to aspects of the invention may be formulated for administration by a variety of routes including, but not limited to, intravenous, parenteral, intra-arterial, intramuscular, intratumoral, subcutaneous, oral and intranasal. The composition may be administered to a human or animal subject by injection, for example by intravenous, intra-arterial, intramuscular, intradermal, subcutaneous or intratumoral injection.

A subject

The subject to be treated may be any animal or human. The subject is preferably a mammal, more preferably a human. The subject may be a non-human mammal, but is more preferably a human. The subject may be male or female. The subject may be a patient. The therapeutic use may be for humans or animals (veterinary use).

***

The features disclosed in the foregoing description, or the following claims, or the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for attaining the disclosed result, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes may be made to the described embodiments without departing from the spirit and scope of the invention.

For the avoidance of any doubt, any theoretical explanation provided herein is intended to enhance the reader's understanding. The inventors do not wish to be bound by any of these theoretical explanations.

Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Throughout this specification (including the claims which follow), unless the context requires otherwise, the words "comprise" and variations such as "comprises", "comprising" and "comprising" will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. The term "about" in relation to a numerical value is optional and means, for example, +/-10%.

Examples

Example 1-Gene delivery Using a combination of peptide dendrimers displaying cationic and hydrophobic residues with lipids Feeding the articles

Introduction to the invention

Cationic lipid-based nucleic acid delivery systems are one of the most studied, most effective non-viral vector platforms described so far, and rational design and development of peptide vectors with natural amino acids is particularly attractive for therapeutic applications due to the non-toxic nature of the amino acids. The present inventors have developed a structural framework for nucleic acid delivery using peptide dendrimers. The structural framework relates to a tri-layer peptide (or dipeptide) motif bound to a lysine residue. The inventors have found that the distribution of cationic amino acid residues (Lys or Arg) in each generation (layer) makes the peptide dendrimer more efficient in transfection than dendrimers with charges localized only on the surface (Kwok et al 2013). Using the solid phase peptide dendrimer synthesis procedure, the inventors can precisely manipulate the position of each amino acid residue incorporated into the dendrimer scaffold. This allows for better control of the structure and function of the dendrimer, which is generally not possible with previously investigated systems such as polymers or other dendrimers, where the modification is mainly made on the surface of the molecule.

The peptide dendrimer/lipid vehicle shows high transfection efficiency, good reproducibility of results and low toxicity.

The inventors hypothesize that (1) the dendritic backbone, (2) the dendrimer compactness, (3) the dendrimer size, (4) the dendrimer building block chirality, and (5) the amino acid composition within the dendrimer may be important parameters to improve gene transfer. The inventors explored structural features by generating a new dendrimer library based on potent G1,2,3-KL with alternating LysLeu motifs, and found that transfection activity was generation-dependent, with 3 generations being optimal for transfection. It is important to obtain a uniform charge distribution in each generation, however the exact location of the charge within each generation is not important. Some activity was observed when dendrimers of different chiralities and those with different numbers of amino acids within each generation were used. However, changing the cationic amino acid within the dendrimer from Lys to Arg (i.e., from LysLeu to ArgLeu motif) improved transfection under serum conditions. This allows in vivo gene transfer by systemic delivery, which is associated with improved stability of the dendrimer-DNA complex. The G1,2,3-RL complex is effective in delivering genes to a group of tissues.

Results

The role of molecular recognition units in determining the efficacy of nucleic acid delivery

In order to evaluate the effect of fine tuning the molecular recognition units of the dendrimer on DNA packaging and delivery while keeping the balance of hydrophobicity and cationic properties constant, the inventors studied dendrimers with different (1) backbones, (2) compactability, (3) size, (4) chirality in the building blocks, and (5) amino acid composition based on derivatives of G1,2, 3-KL. dendrimer-DNA complexes with DOTMA/DOPE (D/D). Transfection efficiency in widely used human cervical cancer HeLa and mouse neuroblastoma Neuro2A cells was evaluated using certain dendrimers in the following table:

table 1. Exemplary dendrimers. G1,2,3-KL; g1,2-KL; G1-KL; g1,2,3-LR; G1-LR, G2.3-RL; g1,2,3-rl; g1,2-RL, G3-LR; g1,2-LR, G3-RL; g1,2,3-RL; g1,2, -RL; G1-RL; g1,2-LK, G3-KL; g1,2,3-LK; ala-G1,2,3-KL; g1,2, 3-K; g0,1,2,3-KL; and D-G1,2.3-KL (also denoted as "G1, 2,3-k 1") has alternating charge (Lys or Arg) and hydrophobic (Leu) residue distributions throughout the dendrimer. G1,2,3-K has only a single amino acid in each generation of peptide motifs. NH present at C-terminal of core sequence ₂ The group is the result of a peptide synthesis process. Molecular Weight (MW) does not include a counterion (trifluoroacetate).

The in vivo and in vitro transfection efficiencies of mRNA and DNA were also assessed using certain dendrimers in the following table and are discussed in the examples below:

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table 2. Additional exemplary dendrimers. The NH2 group present at the C-terminus of the core sequence is the result of the peptide synthesis process. X=6-aminocaproic acid, b=β -alanine, dab=2, 4-diaminobutyric acid. Lowercase letters refer to amino acids of the D-type, and uppercase letters refer to amino acids of the L-type. And 1 is D-leucine. Glycine has no form D or L.

The hydrodynamic size, polydispersity index and zeta potential of a set of dendrimers disclosed in tables 1 and 2 were determined.

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Table 3. Hydrodynamic size and zeta potential of peptide dendrimers, lipids and mRNA complexes. The N: P ratio of the dendrimer was 0.16:1, and the w/w ratio of lipid DOTMA/DOPE to mRNA was 10:1. The NH2 group present at the C-terminus of the core sequence is the result of the peptide synthesis process. X=6-aminocaproic acid, b=β -alanine, dab=2, 4-diaminobutyric acid. Lowercase letters refer to amino acids of the D-type, and uppercase letters refer to amino acids of the L-type. And 1 is D-leucine. Glycine has no form D or L.

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Table 4. Hydrodynamic size and zeta potential of peptide dendrimers, lipids and mRNA complexes. The N: P ratio of the dendrimer was 8:1, and the w/w ratio of lipid DOTMA/DOPE to mRNA was 10:1. The NH group present at the C-terminus of the core sequence is the result of the peptide synthesis process. X=6-aminocaproic acid, b=β -alanine, dab=2, 4-diaminobutyric acid. Lowercase letters refer to amino acids of the D-type, and uppercase letters refer to amino acids of the L-type. And 1 is D-leucine. Glycine has no form D or L.

Changes in dendritic structure, symmetry, topology and chirality

Various dendritic structures provide different amino acid distributions within the scaffold and the number of amino acids between branching points to give structures that exhibit varied compactness and size (see, e.g., table 1 above).

To investigate the effect of topology changes, some or all of the dendrimers with reversed KL positions (G1, 2-LK,3-KL and G1,2, 3-LK) were compared to G1,2, 3-KL. Compared to G1,2,3-KL, G1,2,3-LK did not differ significantly in DNA binding and transfection (fig. 1A, B). For G1,2,3-K, where the total charge is the same as G1,2,3-KL, but the size and molecular weight is reduced (MW/charge ratio G1,2,3-KL is 1.5-fold lower for G1,2, 3-K), the transfection efficacy is slightly reduced; unlike G1,2,3-KL, it was not as effective (FIG. 1C, D). There is a trend that G1,2,3-K is more effective at lower N/P ratio (2.5) than G1,2,3-KL, and increasing the N/P ratio decreases the transfection effect.

To investigate the effect of chirality, G1,2,3-KL dendrimers with D-type amino acids (denoted D-G1,2,3KL) were synthesized. Substitution of the dendrimer L-amino acid to D-form did not have any significant effect on transfection (fig. 1C, D).

The importance of the amino acid sequence at the focus of transfection was investigated by testing G0,1,2,3-KL and Ala-G1,2, 3-KL. The GlySerCys sequence in focus (constant in all dendrimers studied previously) was replaced with LysLeuAla with additional charged residues. This change did not affect the transfection results (fig. 1E, F).

Taken together, the above results indicate that the charge distribution within each generation of dendrimer is a determinant of transfection efficacy. Further changes in the optimal structure may slightly alter transfection activity, but the overall effect is not important, provided that the charge distribution itself is not altered. This observation is consistent with our previous observation that the charge is concentrated only in the outer generations, rather than being uniformly distributed in each generation, resulting in a significant decrease in transfection efficiency.

Comparison of transfection efficiency of KL and RL dendrimers with different generations

Algebra from G1 to G2 to G3 based on KL repeat units in delivery efficiency was explored. The KL units were also replaced with RL repeat units to compare the effect of protonated basic groups with different pKa. Interestingly, we observed a relationship between each generation and transfection in cells (fig. 2). The generation dependence of transfection revealed that the first generation dendrimers G1-KL or G1-RL did not transfect HeLa or Neuro2A cells with lysine or arginine as charged residues. This is probably due to the fact that the G1 peptide is not physically large enough to completely encapsulate the plasmid DNA to form stable nanoparticles, as shown by the complex stability assay (data not shown).

However, for the second generation, arginine-containing G1,2-RL showed higher transfection efficiency (N/P ratio of 10 or 20 in HeLa cells and N/P ratio of 20 in Neuro2A cells) compared to G1,2-KL (fig. 2). This transfection advantage of G1,2-RL over G1,2-KL is consistent with the ability of G1,2-RL to form more stable transfection complexes with DNA than G1,2-KL (data not shown). For the corresponding optimal activity, the optimal N/P ratio for the G2 dendrimer is significantly higher than the optimal N/P ratio for the G3 dendrimer. Our results also show that G1,2-KL transfects HeLa cells with higher efficiency than in Neuro2A cells. This highlights the fact that the activity of certain transfectants may depend on the cell, since different components such as proteoglycans and the like and their concentration on the cell surface may have an effect on the binding of the transfection complex and the final gene delivery to the cell.

In summary, the G3 dendrimers were transfected efficiently in both HeLa and Neuro2A cells. Our third generation dendrimers were 2 to 600-fold better in HeLa and Neuro2A cells than some of the widely used commercial agents such as Polyethylenimine (PEI), lipofectin (also known as DOTMA/DOPE) and Lipofectamine 2000 (fig. 3).

In the presence of serum, the G1,2, 3-RL-dendrimer/DOTMA/DOPE formulation was up to 800 times more efficient than DOTMA/DOPE alone (transfection with D/D alone is defined as 100%). G1,2,3-KL produced a 200-fold improvement in transfection efficiency in HeLa cells compared to D/D alone (FIG. 4A). In Neuro2A cells, G1,2,3-KL and G1,2,3-RL were 340 and 1500 fold more active than DOTMA/DOPE alone, respectively (fig. 4B). This observation can be explained by the fact that: RL dendrimers bind DNA more strongly than KL dendrimers, so they are more stable and resistant to attack from charged components in serum.

Transfection reagent mediated cytotoxicity is an important parameter for in vivo applications. The toxicity of the formulation containing the dendrimer was compared to that of the control. Most modifications of dendrimers did not induce higher toxicity to HeLa cells, where cell viability was comparable to G1,2,3-KL (about 40% or higher) and commercial reagent Lipofectamine 2000 (about 20%) and similar to the supplementation of lipids (D/D, about 40%). Increasing algebra from 1 to 3 did not increase toxicity. The actual mechanism of toxicity caused by dendrimer formulations is not yet clear, however, part of the toxicity from the formulation is due to the addition of D/D as shown previously and also from the D/D control alone. Indeed, most lipid-dendrimer-DNA complexes did not mediate higher toxicity compared to D/D controls, whereas most dendrimer complexes caused higher cell viability compared to the widely used lipofectamine 2000. The toxicity of the dendrimer complex is not directly related to transfection efficiency, as ineffective G1-KL or RL formulations also induced 40% of cell death. In Neuro2A cells, most dendrimer-DNA complexes are not as toxic (80% or more of cell viability) as they are in HeLa cells. In summary, the cell viability after transfection was significantly higher than the reference reagent Lipofectamine 2000 (-45%).

Uptake mechanism

The uptake mechanism of the composition of the present invention was studied. Chlorpromazine is used to inhibit clathrin-mediated endocytosis. Genistein and coarse bran Chai Gusu are also used; these agents have been reported to inhibit cell-mediated endocytosis and microcellular potion, respectively.

The inventors observed that the addition of chlorpromazine significantly inhibited transfection by 80% (fig. 5B), suggesting that clathrin-mediated endocytosis plays an important role in DNA delivery through dendrimer systems. Genistein and coarse bran Chai Gusu inhibited transfection by about 50% (fig. 5D, F). This suggests that the peptide dendrimer system mediates cellular uptake through at least three different pathways, with clathrin-mediated endocytosis as a key pathway for internalization. The ability to trigger different cellular internalization pathways will allow different cell types to ingest the nanoparticle and will therefore explain the fact that the dendrimer system delivers DNA cargo in vivo.

Discussion of the invention

The inventors have previously reported the effect of charge distribution on transfection of third generation dendrimers based on RL and KL (i.e. a combination of hydrophobic and cationic groups) (Kwok et al, 2013). Providing uniform distribution over the third generation yields an effective transfection reagent (in combination with DOTMA/DOPE), whereas the concentration of charge on the surface only (third generation) significantly reduces DNA transfection. This study of the third generation dendrimer variants disclosed herein (e.g., table 1 above) shows that charge distribution in the dendrimer structure is more important than the topology and chirality of the amino acids within the scaffold for efficient transfection.

The inventors have demonstrated that algebra is important for transfection, with the trend that increasing algebra increases transfection efficiency. The improved transfection efficiency is consistent with the improved stability of the complex. For example, G1,2,3-RL is a highly efficient dendrimer for transfection and in vivo gene delivery under serum conditions (data not shown).

The G1,2,3-RL, DOTMA/DOPE complex delivers DNA for functional gene expression in all tissues assayed by systemic administration. Gene expression was particularly high in liver and skeletal muscle, and no toxicity was observed. Delivery of DNA to all different tissues met the following observations: the G1,2,3-RL and DOTMA/DOPE complexes can mediate cellular internalization via clathrin-mediated endocytosis, litter-mediated endocytosis and megapotion, a cargo uptake process expressed at different levels in all cell types (data not shown).

The biodistribution of G1,2,3-RL, DOTMA/DOPE, DNA composition is different from other systems described in the art. For example, lipid-based, peptide-based systems and dendrimer PAMAM scaffolds were observed to deliver DNA primarily to the lung and spleen after IV administration. PEI and amphiphilic peptide deliver DNA efficiently to the lung and liver, but toxicity was observed with PEI. In contrast, the present invention provides an effective means of delivery to muscle without toxicity. The relatively low gene expression in the kidneys and spleen means that the compositions of the invention are not cleared by the renal and reticuloendothelial systems. Thus, high gene expression in the liver suggests delivery mainly to hepatocytes and astrocytes (data not shown).

The compositions of the invention may also functionally deliver nucleic acids to the brain. Although the pathway through or around the Blood Brain Barrier (BBB) is not yet defined, transcytosis (i.e., the process of endocytosis of cargo by endothelial cells at one end of the barrier and exocytosis at the other end) is one possibility. Transcytosis has been reported to deliver cargo by other synthetic systems such as double-headed amphiphilic molecule systems that bypass the BBB.

Surprisingly, the compositions of the present invention effectively deliver DNA to skeletal muscle despite the fact that most non-viral gene delivery systems show ineffective delivery of the target (data not shown). Skeletal muscle expresses only caveolin 3 (Cav 3), which may indicate that the delivery system of the invention interacts with Cav3 components to mediate efficient cellular internalization.

The peptide dendrimers of the present invention represent a versatile platform, providing efficient and potentially target-tailored transfection reagents that have a large chemical space in terms of amino acid composition, dendrimer topology, and possible modifications at the C-and N-termini. The peptide dendrimer/DOTMA/DOPE system can bypass biological barriers to deliver DNA plasmids to the nucleus in vivo without any other adjuvant. It is contemplated that cell-specific targeting domains, peptides, or other molecules that aid endosomal escape and/or nuclear localization may be conjugated to dendrimers to further improve cell/tissue targeted delivery and gene expression.

Materials and methods

G1,2,3-KL ((KL) was obtained after preparative RP-HPLC as a foamy colourless solid ₈ (KKL) ₄ (KKL) ₂ KGSC-NH ₂ ) (72.3 mg, 10.3. Mu. Mol, 9%). Analytical RP-HPLC: t is t _R =1.47 min (a/D100/0 to 0/100 in 2.2 min, λ=214 nm). MS (esi+): c (C) ₂₁₈ H ₄₂₂ N ₆₀ O ₃₉ S calculated/found 4540.1/4539.0[ M] ⁺ 。

Obtaining a foamy colorless solid after preparative RP-HPLCG1,2-KL ((KL) in bulk form ₄ (KKL) ₂ KGSC-NH ₂ ) (90.5 mg, 27.9. Mu. Mol, 41%). Analytical RP-HPLC: t is t _R =1.36 min (a/D100/0 to 0/100 in 2.2 min, λ=214 nm). MS (esi+): c (C) ₉₆ H ₁₈₂ N ₂₈ O ₁₉ S calculated/measured 2096.8/2096.0[ M] ⁺ 。

G1-KL ((KL) was obtained after preparative RP-HPLC as a foamy colourless solid ₂ KGSC-NH ₂ ) (46.0 mg, 41.7. Mu. Mol, 60%). Analytical RP-HPLC: t is t _R =1.23 min (a/D100/0 to 0/100 in 2.2 min, λ=214 nm). MS (esi+): c (C) ₃₈ H ₇₄ N ₁₂ O ₉ S calculated/found 875.1/875.4[ M] ⁺ 。

G1,2,3-RL ((RL) was obtained after preparative RP-HPLC as a foamy colorless solid ₈ (KRL) ₄ (KRL) ₂ KGSC-NH ₂ ) (14.6 mg, 2.0. Mu. Mol, 2%). Analytical RP-HPLC: t is t _R =1.50 min (a/D100/0 to 0/100 in 2.2 min, λ=214 nm). HRMS (nsi+): c (C) ₂₁₈ H ₄₂₂ N ₈₈ O ₃₉ S calculated/found 4932.3/4931.4[ M] ⁺ ；5106.3/5105.7[M+TFA+Na+K] ⁺ ；5220.3/5219.9[M+2TFA+Na+K] ⁺ ；5616.4/5616.3[M+6TFA] ⁺ ；5730.4/5730.3[M+7TFA] ⁺ ；5844.5/5844.3[M+8TFA] ⁺ ；5958.5/5958.3[M+9TFA] ⁺ ；6072.5/6072.3[M+10TFA] ⁺ 。

G1,2-RL ((RL) was obtained after preparative RP-HPLC as a foamy colorless solid ₄ (KRL) ₂ KGSC-NH ₂ ) (143.2 mg, 42.1. Mu. Mol, 38%). Analytical RP-HPLC: t is t _R =1.45 min (a/D100/0 to 0/100 in 2.2 min, λ=214 nm). MS (esi+): c (C) ₉₆ H ₁₉₀ N ₄₀ O ₁₉ S calculated/found 2264.9/2264.0[ M] ⁺ 。

G1-RL ((RL) was obtained after preparative RP-HPLC as a foamy colorless solid ₂ KGSC-NH ₂ ) (78.5 mg, 56.6. Mu. Mol, 51%). Analytical RP-HPLC: t is t _R =1.37 min (a/D100/0 to 0/100 in 2.2 min, λ=214 nm). MS (esi+): c (C) ₃₈ H ₇₄ N ₁₆ O ₉ s calculated/measured 931.2/931.2[ M ]] ⁺ ；1045.2/1045.4[M+1 TFA] ⁺ 。

G1,2-LK,3-KL ((KL) in the form of a foamy colourless solid is obtained after preparative RP-HPLC ₈ (KLK) ₄ (KLK) ₂ KCGSC-NH ₂ ) (56.7 mg, 8.1. Mu. Mol, 7%). Analytical RP-HPLC: t is t _R =1.47 min (a/D100/0 to 0/100 in 2.2 min, λ=214 nm). MS (esi+): c (C) ₂₁₈ H ₄₂₂ N ₆₀ O ₃₉ S calculated/found 4540.1/4539.0[ M] ⁺ 。

G1,2,3-LK ((LK)) is obtained after preparative RP-HPLC as a foamy colorless solid ₈ (KLK) ₄ (KLK) ₂ KGSC-NH ₂ ) (32.4 mg, 4.6. Mu. Mol, 4%). Analytical RP-HPLC: t is t _R =1.41 min (a/D100/0 to 0/100 in 2.2 min, λ=214 nm). MS (esi+): c (C) ₂₁₈ H ₄₂₂ N ₆₀ O ₃₉ S calculated/found 4540.1/4539.0[ M] ⁺ 。

From TentaGel S after preparative RP-HPLCResin (500 mg,0.22mmol G-1) Ala-G1,2,3-KL ((LysLeu)) was obtained ₈ (LysLysLeu) ₄ (LysLysLeu) ₂ LysGlySerAla-NH ₂ ) Ala-G1,2,3-KL (36.6 mg, 5.2. Mu. Mol, 5%) was in the form of a foamy colorless solid. Analytical RP-HPLC: tr=1.46 min (100% a to 100% d in 2.2 min, λ=214 nm); MS (esi+): c (C) ₂₁₈ H ₄₂₂ N ₆₀ O ₃₉ Actual value/calculated value 4508.0/4508.1[ M] ⁺ 。

G1,2,3-K ((K) is obtained after preparative RP-HPLC as a foamy colourless solid ₈ (KK) ₄ (KK) ₂ KGSC-NH ₂ ) (79.9 mg, 14.6. Mu. Mol, 12%). Analytical RP-HPLC: t is t _R =1.17 min (a/D100/0 to 0/100 in 2.2 min, λ=214 nm). MS (esi+): c (C) ₁₃₄ H ₂₆₈ N ₄₆ O ₂₅ S calculated/found 2955.9/2956.0[ M] ⁺ 。

G0,1,2,3-KL ((KL) in the form of a foamy colourless solid is obtained after preparative RP-HPLC ₈ (KKL) ₄ (KKL) ₂ KKLC-NH ₂ ) (54.2 mg, 7.5. Mu. Mol, 6%). Analytical RP-HPLC: t is t _R =1.47 min (a/D100/0 to 0/100 in 2.2 min, λ=214 nm). MS (esi+): c (C) ₂₂₅ H ₄₃₇ N ₆₁ O ₃₈ S calculated/found 4637.3/4638.0[ M ]] ⁺ 。

D-G1,2,3-KL ((KL) in the form of a foamy colourless solid is obtained after preparative RP-HPLC ₈ (kkl) ₄ (kkl) ₂ kgsc-NH ₂ ) (28.3 mg, 4.0. Mu. Mol, 5%). Analytical RP-HPLC: t is t _R =1.44 min (a/D100/0 to 0/100 in 2.2 min, λ=214 nm). MS (esi+): c (C) ₂₁₈ H ₄₂₂ N ₆₀ O ₃₉ S calculated/measured 4540.1/4540.0[ M] ⁺ It contains a small amount of impurities.

DNA transfection

Cell lines, transfection reagents and plasmids. At 5% CO ₂ And in a humid atmosphere at 37℃HeLa cells were maintained in RPMI medium containing 10% (v/v) FCS and 1% (v/v) P/S. Plasmid pCI-Luc was derived from plasmid pCI (Promega, southampton, UK) into which the luciferase gene was inserted. Branched PEI (25 kDa) was purchased from Sigma Aldrich. Lipofectamine 2000 (L2000) and Lipofectin (DOTMA: DOPE, 1:1 (w/w)) were obtained from Invitrogen. PEI, L2000 and lipofectin were used as positive control transfection agents according to the manufacturer's instructions.

Transfection procedure. 24 hours prior to transfection, heLa cells (10,000 cells in 100. Mu.L/well) were seeded in 96-well plates to achieve 70% confluency. Plasmid transfection complexes were formed by mixing dendrimers (100. Mu.L, from 60. Mu.g to 105. Mu.g depending on the N/P ratio) with lipofectin (4. Mu.g; 100. Mu.L). These mixtures were incubated with pCI-Luc (4. Mu.g; 100. Mu.L) at different N/P ratios in OptiMEM for 30 minutes at 25 ℃. The transfection control complexes (PEI, lipofectin or L2000) (100. Mu.L total in OptiMEM) were mixed with pCI-Luc (4. Mu.g; 100. Mu.L) at the corresponding manufacturer's recommended concentrations. For transfection in serum-free medium, optiMEM was added to dilute the complexes before overlaying the DNA complexes on the cells, so that each complex contained 0.25. Mu.g of DNA in a total volume of 100. Mu.L in one well of a 96-well plate. For transfection in serum medium, complete growth medium was added to dilute the complexes before overlaying the DNA complexes on the cells, such that each complex contained 0.25 μg DNA in a total volume of 100 μl in one well of a 96-well plate. After removal of complete medium from the cells, the complex is added to the plate. Plates were incubated for 4 hours at 37 ℃. The transfection solution was then replaced with complete growth medium for 24 hours before luciferase activity was assayed.

Transgenic expression assays. Cells were washed twice with PBS and incubated with reporter lysis buffer (20. Mu.L, promega) at 4℃for 20 min, then at-80℃overnight. After thawing the cells at room temperature, luciferase assay buffer (100. Mu.L, promega; prepared according to the manufacturer's protocol) was added to each well. The luminescent product was measured by Relative Light Unit (RLU) in a FLUOstar Optima photometer (BMG Labtech).

And (5) measuring the protein content. The protein content of each cell lysate (20. Mu.L) was determined by mixing it with Bio-Rad protein assay reagent (180. Mu.L, berle company of Hemer Hempstead, UK), hemer Hempstead, UK, hemerger. After 10 minutes of incubation, absorbance at 590nm was measured and converted to protein concentration using a BSA standard curve. RLU per mg protein represents luciferase activity. The ratio of these two values is the activity per protein unit (in RLU/mg). The values shown in the transfection figures are presented after normalization with the DOTMA/DOPE control transfection experiment and are shown as percentages.

Cell viability. Cells were transfected as described in the "transfection procedure". After 24 hours of transfection, the medium was removed and the cells were washed twice with PBS. Thereafter, the cells were dried at room temperature for 1 hour (to allow nuclear stain penetration). Crystal violet solution (50. Mu.L of stock solution supplied by Sigma Aldrich) was added to the cells. They were incubated at room temperature for 15 minutes. After washing with distilled water (5 times), the cells were dried at room temperature for 30 minutes. Then MeOH (200 μl) was added and the suspension was incubated for 1 hour at room temperature. The relative amount of crystal violet stain retained by living cells was determined by absorbance of methanol solution at 550 nm.

Fluorescence quenching assay

PicoGreen (England) was added to the DNA and diluted to a final DNA concentration of 0.002. Mu.g/. Mu.L in TE buffer (10 mM Tris/HCl, pH 7.5;1mM EDTA). PicoGreen was added to the DNA at a ratio of 1:150 (v/v) so that 0.2. Mu.g of DNA was contained per 100. Mu.L of solution. The mixture was incubated at room temperature for 10 minutes. During incubation, different amounts of transfection reagent were diluted in TE buffer. Then, 50. Mu.L of lipofectin (0.2. Mu.g) and 50. Mu.L of dendrimer (the amount varies between 0.625:1 and 40:1 depending on the N/P ratio selected) were added to each well of the flat bottom 96 well plate. Then, 100. Mu.L of DNA-PicoGreen solution (0.2. Mu.g of DNA) was added to each well. As a control, 100pL of DNA-PicoGreen solution (0.2 pg of DNA) was added to 100pL of TE buffer. After incubation for 30 minutes at room temperature, 100 μl of TE buffer was added to each well. The PicoGreen signal was then detected with a fluorescence reader (FLUOstar Optima, BMG laboratories technology Co.) with excitation wavelength of 485nm and emission wavelength of 520nm. PicoGreen signals from the complexes were normalized to DNA controls to yield the percentage of PicoGreen signals detected.

Complex dissociation assay

PicoGreen (Invitrogen) was added to the DNA and diluted in TE buffer (10 mM Tris/HCl, pH 7.5;1mM EDTA) to a final DNA concentration of 0.002. Mu.g/. Mu.L (PicoGreen was added to the DNA in a ratio of 1:150 (v/v) so that 0.2. Mu.g of DNA was contained per 100. Mu.L of solution). The mixture was incubated at room temperature for 10 minutes. During incubation, different amounts of transfection reagent were diluted in TE buffer. Then, 50. Mu.L of lipofectin (0.2. Mu.g) and 50. Mu.L of dendrimer (the amount varies between 0.625:1 and 40:1 depending on the N/P ratio selected) were added to each well of the flat bottom 96 well plate. Then, 100. Mu.L of DNA-PicoGreen solution (0.2. Mu.g of DNA) was added to each well. As a control, 100. Mu.L of DNA-PicoGreen solution (0.2. Mu.g of DNA) was added to 100. Mu.L of TE buffer. After incubation for 30 min at room temperature, heparin (0.2 to 1.4U/mL, sigma Aldrich) diluted in 100. Mu.L of TE buffer at different concentrations was added to the DNA complex or DNA alone (0.2. Mu.g, total volume 200. Mu.L). After incubation for 30 minutes at room temperature, fluorescence signals from PicoGreen were recorded using a microplate reader (FLUOstar Optima, BMG laboratories technology Co.) with excitation wavelength of 485nm and emission wavelength of 520nm. DNA controls were used to normalize the signal.

In vivo luciferase DNA expression assay

The mice were injected with dendrimer, lipid and DNA formulations, and tissues were isolated and snap frozen for luciferase expression analysis at 24 hours and 48 hours after intravenous and intramuscular injection, respectively.

Tissue was homogenized with 1x reporter lysis buffer supplemented with protease inhibitors. Depending on the tissue, 1.5 to 3 times of lysis buffer was used for tissue homogenization. After homogenization, the lysate was centrifuged and the supernatant was used to determine luciferase expression and protein content. Luciferase expression levels were measured with a luminometer using a luciferase assay kit (plagmatogram) according to the manufacturer's instructions. Protein content was measured at 595nm absorbance using a burle (Biorad) protein assay kit according to the manufacturer's instructions.

In vivo luciferase mRNA expression assay

Peptide dendrimer-lipid-mRNA particles

The peptide dendrimers are diluted to the corresponding concentrations and mixed with mRNA at the desired NP ratio (such as np=0.15:1 or 8:1). The solution was incubated at room temperature for 10 minutes. Lipids such as DOTMA/DOPE are diluted to the corresponding concentrations and mixed with dendrimer-mRNA complexes at the desired NP ratio (such as np=4.7:1). The solution was incubated at room temperature for 20 minutes. When needed, the solution was further diluted prior to injection.

lipid-mRNA pellet control

DOTMA/DOPE was diluted to the corresponding concentration and mixed with dendrimer-mRNA complexes. The solution was incubated at room temperature for 20 minutes. When needed, the solution was further diluted prior to injection.

Mouse and delivery route

All animal procedures performed in our study were compliant with uk law and were ethically approved. Female CD-1 mice (n=39) of 6 to 8 weeks old passed a 1 week adaptation period after arrival. All mice were weighed and intravenous delivery of the formulation (100 μl) was performed via the tail vein using a 30G insulin syringe (BD biosciences).

Bioluminescence imaging (BLI)

All mice were imaged 6 hours after injection. The BLI was performed using an IVIS luminea II (Perkin Elmer) imaging system. D-luciferin (30 mg/mL, xenolight, perkin Elmer) was administered to mice at a dose of 150 mg/kg. Mice were anesthetized in a chamber containing 5% isoflurane for 6 minutes after receiving D-luciferin and then placed on a heated imaging platform while maintaining 2.5% isoflurane. Mice were imaged 10 minutes after administration of D-luciferin, and exposure time was set to ensure that the acquired signals were within the effective detection range (open filter, binning 8, f-stop) 1. The bioluminescence signal was quantified by measuring the photon flux (photons/s) in a defined region of interest (ROI) using the Living Image software (perkin elmer). After whole body imaging in vivo, mice were euthanized, heart blood was collected, and tissues were extracted for imaging ex vivo. Here, each tissue was placed in individual wells of a 24-well imaging plate (black face, ai Bende company (Eppendorf)) containing 0.3 mg/mLD-luciferin in PBS. The imaging plate was centered on the imaging platform (luminea II system) and the signals were measured using the acquisition setup detailed above. Finally, the tissue was placed in a storage vial and flash frozen in liquid nitrogen.

Endocytic assay

Cells were pre-incubated with each inhibitor (chlorpromazine, genistein and coarse bran Chai Gusu) for 30 minutes at 37 ℃. Subsequently, transfection was performed at 37℃for a further 4 hours in the presence of inhibitors, as described in the transfection section.

In vivo study

Induction of FST expression using compositions comprising DNA

Mice were injected on day 1 and day 3 with follistatin-expressing DNA alone or with G1,2,3-RL and D/D lipids. On day 5, tissues were harvested for RNA extraction. cDNA was then synthesized from mRNA for qPCR. The relative expression levels of Follistatin (FST) in mouse skeletal muscle are shown in fig. 6.

Immunogenicity ofResistance/toleranceSusceptance

Body weight was monitored during the above-described DNA delivery type IV protocol. No weight loss was observed (see fig. 7).

Mice that received no or only intravenous injection of DNA or intravenous injection of DNA, G1,2,3-RL, and D/D formulations were also monitored for aspartate Aminotransferase (AST) and alanine Aminotransferase (ALT) levels on days 1 and 3. All groups remained at AST levels substantially below 200U/L and ALT levels remained below 100U/L. ( These levels of AsT and ALT are much lower than threshold levels (greater than 7000U/L) in disease models of liver injury (induced by carbon tetrachloride). (Bonnet et al, 2008) )

Cytokine levels in mice were also measured to check for immunogenicity. Serum was collected on day 5 for measuring AST and ALT levels. No substantial increase was observed. For IFN-gamma, the level remained below 2pg/mL without significant increase (other non-viral systems could raise IFN-gamma to 60,000pg/mL (Bonnet et al, 2008)). For TNF- α, the level remained below 20pg/mL without significant increase (lipopolysaccharide could raise TNF- α to 5000pg/mL (Bonnet et al, 2008)). For IL-6, the levels of the mice group to which the compositions of the invention were administered remained below 20pg/mL without significant increase (other non-viral systems could raise IL-6 levels to 15000pg/mL (Bonnet et al, 2008)). For IL-1-beta and IL-10, the levels were also kept very low without significant increase.

Induction of luciferase expression using compositions comprising DNA

Mice were injected with dendrimer, lipid and DNA formulations prepared for detection of luciferase expression following injection. Tissues were isolated and snap frozen for luciferase expression analysis at 24 hours and 48 hours after intravenous and intramuscular injection, respectively.

Intravenous injection of a composition comprising both G1,2,3-RL and G1,2,3-LR delivered DNA targeted to skeletal muscle as determined by luciferase expression, with G1,2,3-LR exhibiting the most efficient delivery to skeletal muscle (fig. 8).

Intramuscular injection of compositions comprising G1,2-RL,3-LR was also able to deliver DNA to skeletal muscle as determined by luciferase expression (fig. 9).

Induction of luciferase expression using compositions comprising mRNA

Mice were injected with compositions comprising DOTMA/DOPE, mRNA encoding luciferase and G1,2,3-RL (FIG. 10, top panel; "G1, 2,3-RL, lipid and mRNA preparation") or G1,2-RL,3-LR (FIG. 10, bottom panel; "G1, 2,3-RL, lipid and mRNA preparation"). Each formulation was prepared and injected at NP ratios of 8:1 and 0.15:1. Mice were prepared for detection of luciferase expression following injection.

Altering the NP ratio of the G1,2,3-RL or G1,2-RL,3-LR formulations resulted in a change in mRNA biodistribution in the mouse tissue. NP ratios of 0.15:1 target a wide variety of immune cell tissues including lung, spleen and lymph nodes, in contrast np=8:1 specifically targeted the spleen and to a lesser extent the lymph nodes (fig. 10). This suggests that altering the NP ratio of the composition may alter the tissue distribution of the composition. For example, increasing the NP ratio may improve targeting to lymphoid organs.

G1,2,3-RL and G1,2-RL,3-LR, lipids and mRNA formulations can also successfully deliver mRNA to tissues including muscle, liver, heart, kidney and adipose tissue (fig. 11). In particular, G1,2-RL,3-LR, lipid and mRNA preparations with NP ratios of 8:1 or 0.15:1 were able to induce significant expression in gastrocnemius and quadriceps tissues, liver, heart, kidney and adipose tissue.

Comparison of luciferase expression was performed between mice injected with compositions comprising G1,2,3-RL, DOTMA/DOPE and mRNA and mice injected with compositions comprising G1,2-RL.3-LR, DOTMA/DOPE and mRNA. Increased delivery of G1,2-RL,3-LR to all test tissues was observed relative to G1,2,3-RL (fig. 12). This suggests that the selection of dendrimer sequences may be used to target specific tissues.

Repeated dosing experiments

To determine if the composition can be repeatedly dosed and remain delivered to the tissue, a comparison was made between mice receiving single or repeated doses (2 doses, 24 hours apart) of DOTMA/DOPE, mRNA and peptide dendrimer. Mice receiving either of the compositions showed increased luciferase expression compared to mRNA alone. In general, luciferase expression in mice receiving both doses showed about the same degree of luciferase expression or increased luciferase expression compared to mice receiving a single dose. Luciferase expression was measured 6 hours after treatment.

Tissue/cell targeting peptide-dendrimer fusion proteins

Comparison of luciferase expression was performed between compositions comprising DOTMA/DOPE, mRNA and one of G1,2,3-RL, G1,2-RL,3-LR or NXT1 (FIG. 14). The NXTI dendrimers include G1,2-RL,3-LR dendrimers having a core peptide sequence comprising a muscle targeting peptide and a cell penetrating peptide. NXT1 is more effective in mediating mRNA delivery to a variety of tissues, including muscle, than G1,2,3-RL and G1,2-RL, 3-LR. Surprisingly, NXT1 also delivered mRNA more effectively to spleen and lymph nodes than G1,2,3-RL and G1,2-RL, 3-LR. NXT1 and G1,2-RL,3-LR showed similar efficiency in delivering mRNA to the lung.

Dendrimers increase mRNA delivery to tissues compared to lipid alone

To determine whether the presence of dendrimers improves mRNA delivery to tissue, mice were injected with a composition comprising any of the following: i) Individual mRNA; ii) mRNA and DOTMA/DOPE; iii) G1,2,3-RL, DOTMA/DOPE and mRNA; iv) G1,2-RL,3-LR, DOTMA/DOPE and mRNA or v) NTX1, mRNA and DOTMA/DOPE, and mice were ready for detection of luciferase expression after injection. The NP ratio in each of the dendrimer compositions was 0.15:1. As shown in fig. 15, the presence of G1,2,3-RL, G1,2-RL,3-LR and NTX1 increased the efficiency of mRNA delivery to lung, spleen and lymph nodes compared to mRNA alone or mRNA alone and DOTMA/DOPE.

Comparisons were also made between mice injected with compositions comprising i) mRNA alone, ii) mRNA, G1,2-RL,3-LR and DOTMA/DOPE, or iii) mRNA and DOTMA/DOPE. The NP ratio in this experiment was 8:1. This suggests that mRNA delivery to the spleen is significantly enhanced in the presence of dendrimers as compared to mRNA alone or mRNA and lipid. Furthermore, luciferase expression in lung and lymph nodes was reduced in compositions comprising dendrimers compared to lipids alone. The data further indicate that G1,2-RL,3-LR improves the specificity of mRNA delivery to the spleen compared to DOTA/DOPE alone.

Dendritic polymer: comparison of lipid delivery System with commercially available lipid delivery System

Use of a composition comprising G1,2-RL,3-LR and DOTMA/DOPE (NP=0.16:1) or commercially available Lipofectamine ^TM 2000, heLa cells were transfected with mRNA encoding luciferase. As shown in the upper left panel of FIG. 17, with a commercially available transfection reagent Lipofectamine ^TM 2000, the composition comprising the NP ratio = 0.16:1G 1,2-RL,3-LR and DOTMA/DOPE increased the transfection efficiency in vitro by about one order of magnitude. Similarly, compared to mRNA transfected with DLin-MC 3-DMA: cholesterol: DSPC: DMG-PEG lipid nanoparticle delivery system, NP ratio = 0.16:1,2-RL,3-LR and DOTMA/DOPE, heLa cells transfected with mRNA encoding eGFP increased eGFP expression approximately 4-fold (fig. 17, upper right panel).

C2C12 cells were transfected with mRNA alone, with G1,2-RL,3-LR (N: p=8:1) and DOTMA/DOPE (w/w=10:1 relative to mRNA) with mRNA, with polyethylenimine with mRNA, and with Lipofectamine 2000 with mRNA for 24 hours. As shown in the lower panel of fig. 17, dendrimer formulations improved mRNA delivery to C2C12 cells compared to commercially available lipid-based transfection reagents.

In vitro CRISPR assay

Label is used according to the manufacturing instructionsThe RNA was labeled with a nucleic acid labeling kit (Mi Lusi Bio Inc. (Mirus Bio LLC)). Briefly, single guide RNAs (sgrnas) consisting of CRISPR RNA (crrnas) fused to tracerRNA (trRNA) were labeled with TM-rhodamine fluorophores, while Cas9 mRNA was labeled with Cy5 fluorophores. crRNA and trRNA were labeled with fluorescein and ATTO 550 fluorophores, respectively.

HeLa cells were transfected with different combinations of labeled RNA with either (1) G1,2-RL,3-LR and DOTMA/DOPE at NP ratio=0.15:1 or (2) G1,2-RL,3-LR and DOTMA/DOPE at NP ratio=8:1. The lipid to RNA ratio was 10:1 w/w for all compositions and the DOTMA to DOPE weight ratio was 1:1. Cells were incubated with each formulation for 2 hours, then digested with trypsin and fixed. The percentage of RNA uptake in the cells was determined by flow cytometry. Pure RNA without formulation was used as control.

About 100% of HeLa cells exposed to the sgrna+cas9 mRNA formulation were observed to be double positive for the sgRNA and Cas9 mRNA, demonstrating that the G1,2-RL,3-LR dendrimer was a highly efficient transfection reagent (fig. 18).

In dendrimer and lipid formulations comprising crRNA, trRNA, and Cas9 mRNA, the dendrimer formulation can mediate the delivery of all three components in about 4% of cells (fig. 19). The percentage of positive cells is different in dendrimer and lipid formulations that contain only one of crRNA, trRNA, or Cas9 mRNA. In compositions comprising trRNA or Cas9 mRNA, about 100% of the cells are positive for nucleic acid. In contrast, only 4% of cells were positive for crRNA when exposed to compositions containing crRNA alone (fig. 20). Taken together, the data indicate that while the G1,2-RL,3-LR dendrimers can successfully mediate the delivery of relatively short RNAs (e.g., 36 nucleotides in crRNA used in this study) to cells, successful delivery of RNAs increases as the length of the RNA to be transfected increases (the trRNA and Cas9 mRNA used in this study are 67 and 5421 nucleotides in length, respectively).

Immune cell targeting

Fifteen 5 to 7 week old female CD-1 mice (Charles River laboratory UK) were given 1mg/kg of either Alexa Fluor 488-labeled mRNA alone or Alexa Fluor 488-labeled mRNA with NTX1 and DOTMA/DOPE or Alexa Fluor 488-labeled mRNA with G1,2-RL,3-LR and DOTMA/DOPE via intravenous injection, respectively, using a 5ml/kg dose volume. Group 1 received vehicle and group 5 received mRNA alone as a control. All groups of mice were slaughtered 2 hours after IV administration. At euthanasia, whole blood, spleen, inguinal lymph node and femur were collected from all mice. White Blood Cells (WBCs) were obtained from EDTA-treated whole blood, spleen, inguinal lymph nodes and bone marrow for flow cytometry analysis. Briefly, bone Marrow (BM) was flushed from the femur and spleen and lymph nodes were treated to single cell suspensions by a 70 μm filter. Erythrocytes in blood, BM and spleen were lysed and WBCs isolated from all tissues were then stained with different antibody sets to identify specific subpopulations of immune cells. Staining was performed in a 96-well plate format prior to analysis of the samples using an aceenovocyte 3005 flow cytometer.

NTX1 and G1,2-RL,3-LR mediate uptake of mRNA in immune cells present in the spleen (fig. 21). mRNA formulated with dendrimers and lipids is taken up by (A) B cells, (B) T cells, (C) monocytes and macrophages, (D) neutrophils and (E) dendritic cells. In particular, dendrimers and lipid formulations can deliver mRNA to monocytes and macrophages, neutrophils and dendritic cells within a relatively short period of time after injection (2 hours after injection). NTX1 (np=0.15:1) and G1,2-RL,3LR (np=0.15:1 and 8:1) mediate mRNA delivery to about 5% or more of monocytes/macrophages, neutrophils and dendritic cells. Considering the short period of time from injection to tissue harvest, it is reasonable to assume that the delivery of mRNA to each immune cell type analyzed will increase over time.

NTX1 and G1,2-RL,3-LR mediate uptake of mRNA in immune cells present in bone marrow (fig. 22). mRNA formulated with dendrimers and lipids is taken up by (A) monocytes and macrophages, (B) dendritic cells, (C) CD38+ cells, (D) B cells and (E) neutrophils. In particular, dendrimers and lipid formulations can deliver mRNA to monocytes and macrophages, neutrophils and dendritic cells within a relatively short period of time after injection (2 hours after injection). Considering the short period of time from injection to tissue harvest, it is reasonable to assume that the delivery of mRNA to each immune cell type analyzed will increase over time.

In vitro DNA delivery

The upper panel of FIG. 25 shows luciferase expression in HeLa cells transfected with DNA alone, with G1,2,3-RL (N: P=5:1) and DOTMA/DOPE (w/w=1:1 relative to DNA) and with G1,2,3-RL (N: P=5:1) and DOPG/DOPE (w/w=1:1 relative to DNA). Compositions comprising G1,2,3-RL and DOTMA/DOPE or DOPG/DOPE are capable of successful transfection of HeLa cells in vitro.

Similarly, the lower panel of fig. 25 shows luciferase expression in C2C12 cells (myogenic cell lines) transfected with DNA alone, with polyethylenimine and DNA, with G1,2-RL,3-LR (N: p=8: 1) and DOTMA/DOPE (w/w=1: 1 relative to DNA) and with NTX2 (N: p=8: 1) and DOTMA/DOPE (w/w=1: 1 relative to DNA) and DNA. NTX2 is a G1,2-RL,3-LR dendrimer conjugated to the muscle targeting peptide ASSLINA ((LR) 8 (KRL) 4 (KRL) 2KGSCGAASSLNIA (Acp) -NH 2). Both dendrimers were able to successfully transfect C2C12 cells in vitro. Dendrimers comprising muscle targeting domains further increase transfection efficiency compared to dendrimers without muscle targeting domains.

In vivo toxicity assay

Mice were injected with mRNA preparations 1 or 2 times. For 2 IV injections, mice were injected 24 hours prior to the 2 nd injection. At 6 hours after IV injection 2, mice were harvested for plasma and AST levels and other cytokine levels were measured. For mice injected 1 time, plasma was harvested 6 hours after injection for measuring AST levels and other cytokine levels.

Aspartate Aminotransferase (AST) is a biomarker that can be used as a measure of liver health and also as a marker to determine liver toxicity of an agent. All dendrimer-lipid-mRNA compositions tested showed no significant increase in AST levels compared to compositions containing DOTMA/DOPE alone, and indeed showed a decrease in AST levels compared to mRNA alone (fig. 26A, upper panel). These results indicate that the dendrimer compositions tested did not exhibit any significant hepatotoxicity.

TNF- α, IL-6 and IL-1β were also measured in mice receiving injections of various dendrimer-lipid-mRNA formulations. As shown in the lower panel of fig. 26A and the upper and lower panels of fig. 26B, none of the dendrimer compositions tested significantly increased the plasma levels of TNF- α, IL-6 or IL-1β, respectively, compared to the mRNA or DOTMA/DOPE and mRNA compositions alone. These data indicate that the dendrimer composition did not elicit a significant immune response in mice exposed to the composition. This further demonstrates the safety of dendrimer compositions for delivery of mRNA to in vivo tissues.

Mice were also injected with DNA alone or with formulations comprising G1,2,3-RL, DNA and DOTMA/DOPE compositions, and plasma levels of AST, TNF-alpha, IL-6 and IL-1β were measured after 24 hours. As shown in fig. 27, injection of the dendrimer-containing formulation did not cause a significant increase in plasma levels of AST (upper left), TNF- α (upper right), IL-6 (lower left) or IL-1β (lower right). These data indicate that DNA delivery using the dendrimers of the present invention is safe and does not cause liver damage or adverse immune reactions during the process.

Comparison of transfection efficiency of peptide dendrimers

The effect of the algebra of the dendrimers on mRNA delivery was studied by transfecting HeLa cells under complete growth medium conditions. We have demonstrated that dendrimers with 1 or 2 generations (e.g., RHCG1-R, RHCG1,2-R, RHCG1-LR, RHCG1-RL, 2-LR) can effectively transfect cells and mediate similar transfection efficiencies as dendrimers with 3 generations (FIG. 28). This is indeed very different from DNA transfection, since dendritic polymers with 1 or 2 generations are not as effective as dendritic polymers with 3 generations in complete growth medium for transfection of cells (fig. 4). Interestingly, increasing the N:P ratio of the 1 st or 2 nd generation dendrimers from 0.16 to 8 further improved transfection efficiency (FIG. 29).

We have studied the effect of the core sequence of the dendrimer on transfection. We have changed the core sequence GSC of 3 amino acids from G1,2-RL,3-LR to only two amino acids, such as KA and YM. This change did not affect mRNA transfection, indicating that a dendrimer with 2 amino acids in the core would be as good as a dendrimer with 3 amino acids in the core. Next, we substituted the core sequence of 3 amino acids with different amino acids, such as RFW, RYM, compared to GSC. Although arginine containing cationic groups was added to the core, this did not increase or decrease transfection. Interestingly, when the 3 amino acids at the core were changed from GCS to RHC, transfection was improved by 50%. This suggests that ionizable groups such as histidine in the core of the dendrimer can enhance transfection (fig. 28A).

We introduced 12 amino acids in the core of the dendrimer (linear G1,2-RL, 3-LR) and transfection efficiency was not affected (fig. 28A). The results indicate that longer amino acid chains and/or longer structures can be introduced in the core without affecting transfection efficiency. In fact, NTX1, which contains 27 amino acids in the core, is more efficiently transfected than G1,2-RL, 3-RL. This suggests that longer core sequences do enhance transfection. The enhanced transfection from NTX1 is likely due to the presence of cell penetrating peptides within the core sequence.

We also explored the effect of the number of amino acids in each generation on transfection. Thus, we tested dendrimers with only 1 amino acid (R), 2 amino acids (RL or LR), 3 amino acids (RLR) and 4 amino acids (LRLR). Our transfection studies showed that dendrimers with 1,2, 3 or 4 amino acids in each generation can still transfect mRNA well into cells (fig. 28A). We even tested dendrimers with different numbers of amino acids in each generation, and the results showed that transfection efficiency was not affected.

Based on the G1,2-RL,3-LR structure, we have designed libraries of 3-generation dendrimers, where we have replaced basic amino acid R with K, and/or hydrophobic amino acid L with acidic amino acids (such as E) and/or amino acids with nonpolar side chains (such as M, F, β -alanine (B), aminocaproic acid (X) and W) and/or amino acids with polar side chains (such as Q, T and Y).

Replacing R within the dendrimer with K reduced mRNA transfection efficiency (fig. 28A). However, in contrast to G1,2-RL,3-LR, changing hydrophobic amino acids to other hydrophobic amino acids and/or amino acids with polar or nonpolar side chains may not affect transfection or may reduce transfection by 30% to 40%. The underlying model is not yet clear and will be studied further. However, all of these dendrimers induced mRNA transfection significantly higher than the mRNA control alone.

We have also studied the effect of amino acids in the L or D form within dendrimers on mRNA transfection. Based on the G1,2-RL,3-LR dendrimers, we found that changing part or all of the amino acids from L to D in each generation of dendrimers did not affect transfection efficiency. Substitution of lysine to diaminobutyric acid within the dendrimer will reduce transfection, although the transfection of the dendrimer is still significantly higher than the mRNA control alone.

We have demonstrated that G1-LL,2-RR can be used to deliver mRNA using our formulation protocol where we use a G1-LL,2-RR to DOTMA/DOPE (w/w 10:1) ratio of 0.16:1N:P. Interestingly, the dendrimers have been used to deliver ASO in different formulations in vitro and in vivo (Saher 2018). The formulations used were DOTMA/DOPE (w/w=2:1 relative to ASO) and N:P=20:1G 1-LL,2-RR and ASO. We have tried this formulation (i.e., DOTMA/DOPE (w/w=2:1 relative to mRNA) and N: P=20:1G 1-LL,2-RR and mRNA) at the time of cell transfection, but the transfection efficiency was poor. This formulation (DOTMA/DOPE (w/w=2:1 relative to mRNA) and N: P=20:1 of G1-LL,2-RR and mRNA) produced only 10% of the mRNA transfection efficiency of our improved formulation (DOTMA/DOPE (w/w=10:1 relative to mRNA) and N: P=0.16:1 of G1-LL,2-RR and mRNA).

We have explored 3-generation dendrimers with RL or LR or RL in different generations for mRNA delivery. We found that most of these dendrimers similarly transfected cells, with G1,2-RL,3-LR being most effective in mRNA transfection. Taken together, our data indicate that dendrimers with hydrophobic and cationic amino acids in each generation will produce efficient mRNA delivery to cells.

Since the 1 st and 2 nd generation dendrimers were more efficiently transfected into cells than G1,2-RL,3-LR, we have selected these dendrimers for further testing of transfection. We demonstrate that these dendrimers can generally be transfected better than G1,2-RL,3-LR at various N:P ratios. In particular, the transfection effect of RHCG1-R, RHCG1,2-R, RHCG1-LR, RHCG1-RL,2-LR, RHCG1-RLR and G1-LRLR may be 700% to 1815% better than G1,2-RL,3-LR at N: p=4:1. These data indicate that the requirements for DNA and mRNA delivery are quite different. There is a trend for mRNA to be more effective in dendritic polymers with 1 or 2 generation than 3 generation dendritic polymers transfected cells under complete growth medium conditions. However, under complete growth medium conditions, the 3 rd generation dendrimers transfected DNA into cells much better than the 1 st or 2 nd generation dendrimers (FIG. 4).

Figure 30 shows that the 2 nd generation dendrimer comprising RHC peptide cores delivered at an NP ratio of 8:1 delivered mRNA primarily targeted to the spleen (upper panel) and to a lesser extent to the lung (lower panel). At an NP ratio of 0.15:1, relative to 8:1, mRNA delivery to the lung is increased. Modulation of mRNA expression

mRNA expression can be regulated by injecting mice with a composition comprising PEG2000 lipids. Mice injected with compositions comprising G1,2-RL,3-LR, DOTMA/DOPE/PEG2000 and mRNA had reduced luciferase expression compared to mice injected with compositions comprising G1,2-RL,3-LR, DOTMA/DOPE and mRNA.

Numbered paragraphs

The following numbered paragraphs set forth certain features and combinations of features of the invention.

1. A composition for use in medicine, wherein the composition comprises a peptide dendrimer, a nucleic acid, and a lipid, wherein the peptide dendrimer comprises: a first lysine residue and two first peptide motifs; two second lysine residues and four second peptide motifs; four third lysine residues and eight third peptide motifs; and a core peptide sequence covalently bonded to the first lysine residue,

(i) Wherein the first lysine residue is covalently bonded to the two first peptide motifs, which are each covalently bonded to the two second lysine residues;

(ii) Wherein each second lysine residue is covalently bonded to two second peptide motifs, wherein each second peptide motif is covalently bonded to one of the third lysine residues, respectively; and

(iii) Wherein each third lysine residue is covalently bonded to both of the third peptide motifs, wherein the first peptide motif, the second peptide motif and the third peptide motif independently consist of a mono-or dipeptide motif, and wherein the first peptide motif, the second peptide motif and the third peptide motif each comprise a) arginine (R) or lysine (K); and/or b) leucine (L), valine (V), histidine (H) or isoleucine (I), wherein each amino acid residue is independently selected from the L-isoform or the D-isoform.

2. The composition for use according to paragraph 1, wherein at least two of the first peptide motif, the second peptide motif and the third peptide motif comprise arginine (R).

3. The composition for use according to paragraph 1 or paragraph 2, wherein at least two of the first peptide motif, the second peptide motif and the third peptide motif comprise leucine (L).

4. The composition for use according to any one of the preceding paragraphs, wherein the first peptide motif, the second peptide motif and the third peptide motif are each a dipeptide motif.

5. The composition for use according to paragraph 4, wherein the first peptide motif, the second peptide motif and the third peptide motif each comprise leucine (L) and arginine (R).

6. The composition for use according to any one of the preceding paragraphs, wherein the nucleic acid comprises a double stranded region.

7. The composition for use according to paragraph 6, wherein the nucleic acid is a DNA plasmid.

8. The composition for use according to any one of the preceding paragraphs, wherein the nucleic acid is or encodes an mRNA molecule or an antisense oligonucleotide (ASO).

9. The composition for use according to any of the preceding paragraphs, wherein the nucleic acid comprises a CRISPR sequence.

10. The composition for use according to paragraph 6, wherein the nucleic acid is an siRNA or a saRNA molecule.

11. A composition for use according to paragraph 7, wherein the DNA plasmid can express an siRNA or an SARNA molecule in a target cell.

12. The composition for use of paragraph 7 or paragraph 8, wherein the nucleic acid can express a transgene in a target cell.

13. The composition for use of paragraph 12 wherein the transgene is a viral protein, a bacterial protein or a protein of a microorganism parasitic to a mammal.

14. A composition for use according to paragraph 13, for use as a vaccine.

15. The composition for use according to paragraph 12 for use in gene therapy of a genetic disorder in a patient.

16. The composition for use according to paragraph 15, wherein the genetic disorder causes muscular dystrophy in the patient.

17. The composition for use according to paragraph 15, wherein the genetic disorder causes myopathy in the patient.

18. The composition of paragraph 12 for use in a method of treating diabetic limb ischemia wherein the transgene is Hepatocyte Growth Factor (HGF), vascular Endothelial Growth Factor (VEGF) and/or Fibroblast Growth Factor (FGF).

19. The composition for use according to any one of the preceding paragraphs, wherein the core peptide sequence consists of a tripeptide motif.

20. The composition according to paragraph 18, wherein the tripeptide motif comprises glycine (G), serine (S), and cysteine (C) and/or alanine (a).

19. The composition for use according to any one of the preceding paragraphs, wherein the lipid comprises dioleoyl phosphatidylethanolamine (DOPE) and/or N- [1- (2, 3-dioleyloxy) propyl ] -N, N-trimethylammonium chloride (DOTMA).

20. The composition for use according to any of the preceding paragraphs, wherein the lipid comprises dioleoyl phosphatidylethanolamine (DOPE) and dioleoyl phosphatidylglycerol (DOPG).

21. The composition for use according to any one of the preceding paragraphs, wherein the use comprises delivering the nucleic acid to a muscle of the patient.

22. A pharmaceutical composition comprising a composition according to any one of the preceding paragraphs and a pharmaceutically acceptable excipient.

23. A method of delivering a nucleic acid into a target cell, the method comprising contacting the target cell with the composition of any one of paragraphs 1-20, wherein the target cell is a muscle cell, a liver cell, an astrocyte, a neuron, an astrocyte, a spleen cell, a lung cell, a cardiac muscle cell, a kidney cell, an adipocyte, or a tumor cell.

24. A peptide dendrimer, the peptide dendrimer comprising: a first lysine residue and two first peptide motifs; two second lysine residues and four second peptide motifs; four third lysine residues and eight third peptide motifs; and a core peptide sequence covalently bonded to the first lysine residue,

(i) Wherein the first lysine residue is covalently bonded to the two first peptide motifs, which are each covalently bonded to the two second lysine residues;

(ii) Wherein each second lysine residue is covalently bonded to two second peptide motifs, wherein each second peptide motif is covalently bonded to one of the third lysine residues, respectively; and

(iii) Wherein each third lysine residue is covalently bonded to both of the third peptide motifs, wherein the first peptide motif, the second peptide motif and the third peptide motif each consist of (i) a leucine-arginine (LR) dipeptide motif or (ii) an arginine-leucine (RL) motif; wherein at least one of the first peptide motif, the second peptide motif and the third peptide motif is (i) leucine-arginine (LR), wherein each amino acid residue is independently selected from the L-isoform or the D-isoform.

25. A composition comprising a nucleic acid, a lipid, and a peptide dendrimer according to paragraph 24.

26. A composition for use in medicine according to paragraph 25.

27. Use of the composition of paragraph 25 for delivering the nucleic acid to a cell in vitro or ex vivo.

28. A composition or peptide dendrimer according to any one of paragraphs 1 to 22 or 24 to 26 for use in the treatment of pompe disease, muscle wasting disease or muscular dystrophy, such as duchenne muscular dystrophy.

Reference to the literature

In order to more fully describe and disclose the present invention and the prior art to which the present invention pertains, a number of publications are cited above. The complete citations for these references are provided below. The entire contents of each of these references are incorporated herein.

Kwok et al, comparative structural and functional studies of nanoparticle formulations for DNA and siRNA delivery (Comparative structural and functional studies of nanoparticle formulations for DNA and siRNA delivery); nano medicine: nanotechnology, biology and medicine (Nanomedicine: nanotechnology, biology and Medicine) 7;210-219 (2011).

Kwok et al, peptide dendrimer/lipid hybrid systems are effective DNA transfection reagents: the Structure-activity relationship highlights the role of charge distribution in dendrimer generations (Peptide Dendrimer/Lipid Hybrid Systems Are Efficient DNA Transfection Reagents: structure-Activity Relationships Highlight the Role of Charge Distribution Across Dendrimer Generations); journal of american society of chemistry (ACS Nano) 7;54668-4682 (2013).

Kwok et al, linear oligolysine peptides compared to the system of formulations of siRNA and plasmid DNA (Systematic Comparisons of Formulations of Linear Oligolysine Peptides with siRNA and Plasmid DNA); chemical biology and drug design (Chem Biol Drug Des), 87:747-763 (2016).

Kwok et al, developed small activating RNAs as therapeutic agents: current challenges and prospects (Developing small activating RNA as a therapeutic: current challenges and promises), "therapeutic agent delivery (Therapeutic delivery), 10 (3): 151-164 (2019)

Saher et al, novel polypeptide-dendrimer/lipid/oligonucleotide ternary complexes that can achieve efficient cellular uptake and enhance splice switching activity (Novel peptide-dendrimer/lipid/oligonucleotide ternary complexes for efficient cellular uptake and improved splice-switching activity); journal of European pharmacy and biopharmacology (Eur J Pharmaceutics and Biopharmaceutics), 132:29-40 (2018).

Saher et al, sugar and polymeric excipients enhance the uptake and splice switching activity of peptide-Dendrimer/Lipid/oligonucleotide formulations (Sugar and Polymer Excipients Enhance Uptake and Splice-Switching Activity of Peptide-Dendrimer/Lipid/Oligonucleotide Formulations), "pharmacy", 11 (12): 666 (2019)

Sherdan et al, gene therapy found its own localization (Gene therapy finds its niche), "Nat-Biotechnology" (Nat Biotechnol), 29 (2), 121-8 (2011).

Braum, a Non-viral vector for muscle-mediated gene therapy (Non-viral Vector for Muscle-Mediated Gene Therapy); chapter 9 muscle gene therapy (Chapter 9 Muscle Gene Therapy), schpringer natural swiss company (Springer Nature Switzerland AG), d.dutan, j.r.mendell (edit), 157-178 (2019).

Ren et al, DOTMA has a structural basis of high intravenous transfection activity in mice (Structural basis of DOTMA for its high intravenous transfection activity in mouse); gene Therapy 7, 764-768 (2000).

John et al, human microrna target (Human MicroRNA Targets); public science library Biology, 11 (2), 1862-1879 (2004).

Myers et al, recombinant Dicer efficiently converts large dsRNA into siRNA (Recombinant Dicer efficiently converts large dsRNAs into siRNAs suitable for gene silencing) suitable for gene silencing; natural-biotechnology (Nature Biotechnology), 21:324-328 (2003).

Lim et al, engineering nano-delivery systems (Engineered Nanodelivery Systems to Improve DNA Vaccine Technologies) that improve DNA vaccine technology; pharmacy, 12 (1) 30 (2020).

Luo et al, arginine-functionalized peptide dendrimers as potential gene delivery vehicles (Arginine functionalized peptide dendrimers as potential gene delivery vehicles), "Biomaterials" (Biomaterials), 33, 4917-4927 (2012).

Bolnet et al, linear polyethylenimine (L-PEI) mediated systemic delivery of DNA or siRNA did not induce an inflammatory response (Systemic Delivery of DNA or siRNA Mediated by Linear Polyethylenimine (L-PEI) Does Not Induce an Inflammatory Response), drug study (Pharmaceutical Res), 25, 2972 (2008).

Wang et al, adeno-associated viral vectors as a platform for gene therapy delivery (Adeno-associated virus vector as a platform for gene therapy delivery), natural reviews: drug discovery (Nat Rev Drug Discov), 18 (5): 358-378 (2019).

Philippidis, fourth boy died in An Si Talai AT132 clinical trial (Fourth Boy Dies in Clinical Trial of Astellas' AT 132), human gene therapy (Human Gene Therapy), 32, 19-20 (2021). Qiu et al, developed biodegradable lipid nanoparticles for intracellular mRNA delivery and genome editing (Developing Biodegradable Lipid Nanoparticles for Intracellular mRNA Delivery and Genome Editing), review of chemical research (acc. Chem. Res), 54 (21), 4001-4011 (2021).

Benizri et al, bioconjugated oligonucleotides: recent developments and therapeutic applications (Bioconjugated Oligonucleotides: recent Developments and Therapeutic Applications), "bioconjugate chemistry" (Bioconjug Chem), 30 (2): 366-383. (2019).

Jasinski et al, influence of the size and shape of RNA nanoparticles on biodistribution (The Effect of Size and Shape of RNA Nanoparticles on Biodistribution), molecular therapy (Mol Ther), 26 (3), 784-792 (2018).

Huang et al, using 3DNA nanocarriers to deliver therapeutic agents targeting the mRNA binding protein HuR can inhibit ovarian tumor growth (Delivery of Therapeutics Targeting the mRNA-Binding Protein HuR Using 3DNA Nanocarriers Suppresses Ovarian Tumor Growth), "Cancer Research", 76 (6), 1549-1559 (2016).

For standard molecular biology techniques, see Sambrook, j., russel, d.w., guidelines for molecular cloning experiments (Molecular Cloning, A Laboratory Manual), 3 rd edition, 2001, new York cold spring harbor (Cold Spring Harbor, new York): cold spring harbor laboratory Press (Cold Spring Harbor Laboratory Press).

Claims (167)

1. A composition for use in medicine, the composition comprising a peptide dendrimer, a nucleic acid and a lipid,

Wherein the peptide dendrimer comprises at least: a core peptide sequence, a first branching residue and two first peptide motifs, wherein the branching residue is lysine, 2, 4-diaminobutyric acid, ornithine or diaminopropionic acid,

and wherein the nucleic acid comprises a nucleic acid of at least 30, at least 35, at least 40, at least 45, or at least 50 nucleotides.

2. The composition for use according to claim 1, wherein the nucleic acid is RNA.

3. The composition for use according to claim 2, wherein the RNA is selected from mRNA, ssRNA, dsRNA, sgRNA, crRNA, tracrRNA, incRNA, siRNA, saRNA and/or self-amplifying RNA.

4. The composition for use according to claim 3, wherein the RNA is mRNA.

5. The composition for use according to claim 1, wherein the nucleic acid is DNA.

6. The composition for use according to claim 5, wherein the DNA comprises ssDNA, dsDNA, plasmid and/or cDNA.

7. The composition for use according to any one of the preceding claims, wherein the composition comprises RNA nucleic acids and DNA nucleic acids.

8. The composition for use according to claim 7, wherein the RNA nucleic acid and DNA nucleic acid are part of a single nucleic acid molecule.

9. The composition for use according to any one of the preceding claims, wherein the nucleic acid comprises a modified nucleic acid.

10. The composition for use according to any one of the preceding claims, wherein the nucleic acid encodes a transgene and is capable of expressing the transgene in a target cell.

11. The composition for use according to any one of the preceding claims, wherein the use relates to the treatment of a disease in the subject by modulating the expression or activity of an endogenous gene.

12. The composition for use according to claim 11, wherein the modulation is an increase in expression of the gene and/or an increase in exogenous expression of an additional copy of the gene.

13. The composition for use according to claim 11, wherein the modulation is a decrease in expression of the gene.

14. The composition for use according to any one of claims 11 to 13, wherein the endogenous gene is translated into a protein or peptide.

15. The composition for use according to claim 14, wherein the protein or peptide comprises an antigen, hormone, receptor, chimeric antigen receptor, transcription factor and/or cytokine, such as IL-2, IL-7, IL-12, IL-15, IL-21 and/or interferon.

16. The composition for use according to claim 10, wherein the transgene comprises a tumor antigen, a viral protein, a bacterial protein or a protein of a microorganism parasitic to a mammal.

17. The composition for use according to any one of the preceding claims, wherein the composition is a vaccine.

18. The composition for use according to claim 17, wherein the nucleic acid comprises or encodes self-amplifying RNA.

19. The composition for use according to any one of claims 1 to 16, wherein the use comprises treating a genetic disorder in the subject.

20. The composition for use according to claim 19, wherein the nucleic acid expresses a functional version of a gene that is nonfunctional, down-regulated, inactivated or impaired in the subject.

21. The composition for use according to any one of the preceding claims, wherein the nucleic acid encodes and/or comprises one or more components of a system for editing a genome or a system for altering gene expression.

22. The composition for use according to claim 21, wherein the system for editing a genome or the system for altering gene expression is a CRISPR/Cas system.

23. The composition for use according to claim 21 or 22, wherein the nucleic acid encodes a Cas protein or peptide and/or comprises sgRNA, crRNA and/or tracrRNA.

24. The composition for use of claim 23, wherein the nucleic acid comprises mRNA encoding a Cas protein or peptide and an RNA sequence comprising sgRNA.

25. The composition for use according to claim 24, wherein the mRNA encoding Cas protein or peptide and the sgRNA-containing RNA sequence are separate molecules.

26. The composition for use of claim 23 or 24, wherein when present, one or more of the sgRNA, the crRNA, the tracrRNA, and the nucleic acid encoding Cas protein is part of a single nucleic acid.

27. The composition for use of claim 23 or 24, wherein when present, one or more of the sgRNA, the crRNA, the tracrRNA, and the nucleic acid encoding Cas protein are present on two or more nucleic acids.

28. The composition for use according to any one of the preceding claims, wherein the composition targets spleen, lymphoid tissue, lung, bone, thymus, liver, tumor tissue, cardiac tissue, skeletal muscle, kidney, adipose tissue and/or brain.

29. The composition for use according to claim 28, wherein the composition targets spleen, lymphoid tissue, lung and/or bone.

30. The composition for use according to claim 29, wherein the nucleic acid is RNA, such as mRNA.

31. The composition for use according to any one of the preceding claims, wherein the use comprises administering the composition to a subject such that the nucleic acid is delivered to cells that are leukocytes, such as B lymphocytes, T lymphocytes, monocytes, neutrophils, dendritic cells, macrophages or monocytes; lymph node tissue cells, myeloid cells, fibroblasts, muscle cells, skeletal muscle cells, endothelial cells, liver cells, astrocytes, neurons, astrocytes, spleen cells, lung cells, myocardial cells, kidney cells, adipocytes, stem cells and/or tumor cells.

32. The composition for use according to any one of the preceding claims, wherein the use comprises administering the composition to a subject such that the nucleic acid is delivered to immune cells.

33. The composition for use according to claim 31 or 32, wherein the nucleic acid expresses an immune molecule or transcription factor in the cell.

34. The composition for use according to claim 33, wherein the immune molecule is a T cell receptor, chimeric antigen receptor, cytokine, decoy receptor, antibody, co-stimulatory receptor, co-stimulatory ligand, checkpoint inhibitor, immunoconjugate or tumor antigen.

35. The composition for use according to any one of claims 31 to 34, wherein the cell is a B lymphocyte, a T lymphocyte, a neutrophil, a dendritic cell, a macrophage, a monocyte, a myeloid-derived suppressor cell (MDSC), a tumor-associated macrophage or a tumor-associated neutrophil.

36. The composition for use according to claim 35, wherein the nucleic acid is RNA, such as mRNA.

37. The composition for use according to any one of the preceding claims, wherein the composition is for use in a method of treating cancer in the subject.

38. The composition for use according to claim 37, wherein the cancer is a blood cancer, such as leukemia, lymphoma, myeloma, myelodysplastic syndrome; or lung cancer, cardiac carcinoma, sarcoma or liver cancer.

39. The composition for use according to claim 37 or claim 38, wherein the method comprises administering an anticancer agent.

40. The composition for use according to any one of the preceding claims, wherein the composition is for use in a method of treating a pulmonary disease or an autoimmune disease in the subject.

41. The composition for use according to any one of claims 19 to 28, wherein the method is the treatment of pompe disease, muscle wasting disease, myopathy or muscular dystrophy, such as duchenne muscular dystrophy, in the subject.

42. The composition for use according to claim 41, wherein the nucleic acid is DNA.

43. The composition for use according to any one of claims 1 to 31, wherein the method is to treat limb ischemia, such as diabetic limb ischemia, in the subject, and wherein the transgene is Hepatocyte Growth Factor (HGF), vascular Endothelial Growth Factor (VEGF), and/or Fibroblast Growth Factor (FGF).

44. The composition for use according to any one of the preceding claims, wherein the two first peptide motifs independently consist of a single amino acid, dipeptide, tripeptide or tetrapeptide motif.

45. The composition for use according to any of the preceding claims, wherein the peptide dendrimer further comprises two second branching residues (e.g. lysine) and four second peptide motifs,

Wherein one of the second branching residues is covalently bonded to one of the first peptide motifs and the other second branching residue is covalently bonded to the other first peptide motif, and wherein each second branching residue is covalently bonded to two second peptide motifs.

46. The composition for use according to claim 45, wherein the four second peptide motifs independently consist of single amino acid, dipeptide, tripeptide or tetrapeptide motifs.

47. The composition for use according to claim 45 or 46, wherein the peptide dendrimer further comprises four third branching residues (e.g.lysine) and eight third peptide motifs,

wherein each second peptide motif is covalently bonded to one of the third branch residues, respectively, such that each third branch residue is covalently bonded to one second peptide motif, and wherein each third branch residue is covalently bonded to two third peptide motifs.

48. The composition for use according to claim 47, wherein said eight third peptide motifs independently consist of single amino acid, dipeptide, tripeptide or tetrapeptide motifs.

49. The composition for use according to any one of claims 44 to 48, wherein the first, second and/or third peptide motif comprises an amino acid having a basic side chain.

50. The composition for use according to any one of claims 44 to 49, wherein the first, second and/or third peptide motif comprises an amino acid having a non-polar side chain.

51. The composition for use according to any one of claims 44 to 50, wherein the first, second and/or third peptide motif comprises an amino acid having an acidic side chain.

52. The composition for use according to any one of claims 44 to 51, wherein the first, second and/or third peptide motif comprises an amino acid having an uncharged polar side chain.

53. The composition for use according to any one of claims 44 to 52, wherein the first, second and/or third peptide motif comprises leucine (L) and/or arginine (R) residues.

54. The composition for use according to any one of claims 44 to 53, wherein the core sequence comprises at least two amino acids.

55. The composition for use according to any one of claims 44 to 54, wherein the core sequence comprises up to 30 amino acids.

56. The composition for use according to any one of claims 44 to 54, wherein the core sequence comprises an ionizable amino acid, such as histidine.

57. The composition for use according to any one of the preceding claims, wherein the peptide dendrimer comprises the structures listed in table 2.

58. The composition for use according to any one of the preceding claims, wherein the peptide dendrimer further comprises a tissue and/or cell targeting motif.

59. The composition for use according to claim 58, wherein the tissue or cell targeting motif comprises a muscle targeting motif such as GAASSLNIA (SEQ ID NO: 1), an integrin targeting motif such as arginine-glycine-aspartic acid or a chemical modification such as comprising mannose glycosylation.

60. The composition for use according to any one of the preceding claims, wherein the peptide dendrimer further comprises a Cell Penetrating Peptide (CPP).

61. The composition for use according to claim 60, wherein the cell penetrating peptide comprises the peptide sequence xrxrxrrrxrrbrxb (SEQ ID NO: 2), wherein X is 6-aminocaproic acid and B is β -alanine.

62. The composition for use according to claim 60, wherein said cell penetrating peptide comprises a TAT derivative sequence.

63. The composition for use according to any one of the preceding claims, wherein the peptide dendrimer further comprises an alkyl chain, an alkenyl chain, an antibody or fragment thereof, a targeting peptide, a sugar, a cell penetrating peptide, an endosomal escape peptide, a nuclear localization motif, and/or a fatty acid.

64. The composition for use according to claim 63, wherein said alkyl or alkenyl chain is conjugated to said core peptide sequence.

65. The composition for use according to claim 63 or 64, wherein the alkyl or alkenyl chain, antibody or fragment thereof, the targeting peptide, saccharide, targeting peptide, cell penetrating peptide, endosomal escape peptide, nuclear localization motif and/or fatty acid is conjugated to the C-terminus of the peptide dendrimer.

66. The composition for use according to any one of claims 63 to 65, wherein the alkyl or alkenyl chain, antibody or fragment thereof, targeting peptide, saccharide, targeting peptide, cell penetrating peptide, endosomal escape peptide, nuclear localization motif and/or fatty acid is conjugated to the N-terminus of the peptide dendrimer.

67. The composition for use according to any one of claims 63 to 66, wherein the alkyl or alkenyl chain comprises from about 5 carbons to about 50 carbons, preferably from about 12 to about 30 carbons.

68. The composition for use according to any one of the preceding claims, wherein the lipid comprises a cationic lipid, a neutral lipid, an anionic lipid and/or an ionizable lipid.

69. The composition for use according to any one of the preceding claims, wherein the lipid comprises a saturated fatty acid.

70. The composition for use according to any one of the preceding claims, wherein the lipid comprises an unsaturated fatty acid.

71. The composition for use of claim 69 or 70 wherein the lipid comprises 1, 2,3, 4, 5 or 6 fatty acid chains.

72. The composition for use according to any one of the preceding claims, wherein the lipid comprises dioleoyl phosphatidylethanolamine (DOPE) and/or N- [1- (2, 3-dioleyloxy) propyl ] -N, N-trimethylammonium chloride (DOTMA).

73. The composition for use according to any one of the preceding claims, wherein the lipid comprises dioleoyl phosphatidylethanolamine (DOPE) and dioleoyl phosphatidylglycerol (DOPG).

74. The composition for use according to any one of the preceding claims, wherein the N/P ratio is between about 0.01:1 and 100:1.

75. The composition for use according to claim 74, wherein said N/P ratio is between 1:1 and 50:1.

76. The composition for use according to claim 75, wherein upon administration to a subject a higher proportion of the composition is observed in the spleen and/or lymph nodes than in the liver.

77. The composition for use according to claim 75, wherein said N/P ratio is between 0.01:1 and 1:1.

78. The composition for use according to claim 77, wherein upon administration to a subject a higher proportion of the composition is observed in the lung, spleen and/or lymph nodes than in the liver.

79. The composition for use according to any one of the preceding claims, wherein the peptide dendrimer, the nucleic acid and the lipid form positively charged particles.

80. The composition for use according to any one of the preceding claims, wherein the peptide dendrimer, the nucleic acid and the lipid form negatively charged particles or neutral charged particles.

81. The composition for use according to any one of claims 28 to 32, wherein delivery of the nucleic acid to the target tissue or cell is increased by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 75%, 85%, 90%, 95%, 100% compared to delivering the same nucleic acid to the same tissue or cell type using a lipid-based nucleic acid delivery system.

82. The composition for use according to claim 81, wherein the target tissue is spleen, lymph node, lung, liver or bone.

83. The composition for use of claim 81 or 82, wherein the lipid-based nucleic acid delivery system is DOTMA/DOPE.

84. The composition for use according to any one of the preceding claims, wherein the administration is performed intravenously, intramuscularly, intratumorally, subcutaneously, intradermally or intraperitoneally.

85. The composition for use according to any one of the preceding claims, wherein the composition is contained within a liquid.

86. A composition comprising a peptide dendrimer, a nucleic acid, and a lipid, wherein the peptide dendrimer comprises at least: a core peptide sequence, a first branching residue and two first peptide motifs, wherein the branching residue is lysine, 2, 4-diaminobutyric acid, ornithine or diaminopropionic acid;

wherein the nucleic acid comprises a single stranded nucleic acid of at least 30, at least 35, at least 40, at least 45 or at least 50 nucleotides.

87. The composition of claim 86, wherein the single stranded nucleic acid is RNA.

88. The composition of claim 87, wherein the RNA is selected from mRNA, ssRNA, sgRNA, crRNA, tracrRNA, incRNA and/or self-amplifying RNA.

89. The composition of claim 88, wherein the RNA is mRNA.

90. The composition of any one of claims 86-89, wherein the composition comprises two or more RNAs.

91. The composition of claim 86, wherein said single stranded nucleic acid is ssDNA.

92. The composition of any one of claims 86-91, wherein the composition comprises an RNA nucleic acid and a DNA nucleic acid.

93. The composition of claim 92, wherein the RNA nucleic acid and DNA nucleic acid are part of a single nucleic acid molecule.

94. The composition of any one of claims 86-93, wherein the single stranded nucleic acid comprises a modified nucleic acid.

95. The composition of any one of claims 86-94, wherein said single stranded nucleic acid encodes a transgene and is capable of expressing said transgene in a target cell.

96. The composition of any one of claims 86-95, wherein said single stranded nucleic acid is capable of modulating expression or activity of an endogenous gene.

97. The composition of claim 96, wherein said modulation is an increase in expression of said gene and/or an increase in exogenous expression of an additional copy of said gene.

98. The composition of claim 96, wherein said modulation is a decrease in expression of said gene.

99. The composition of any one of claims 96-98, wherein the endogenous gene is translated into a protein or peptide.

100. The composition of claim 99, wherein the protein or peptide comprises an antigen, hormone, receptor, chimeric antigen receptor, transcription factor, and/or cytokine, such as IL-2, IL-7, IL-12, IL-15, IL-21, and/or interferon.

101. The composition of claim 95, wherein the transgene comprises a viral protein, a bacterial protein, and/or a protein of a microorganism parasitic to a mammal.

102. The composition of any one of claims 86-101, wherein the composition is a vaccine.

103. The composition of claim 108, wherein the single stranded nucleic acid comprises or encodes a self-activating RNA.

104. The composition of any one of claims 86-100, wherein the use comprises treating a genetic disorder in the subject.

105. The composition of claim 104, wherein the nucleic acid expresses a functional version of a gene that is nonfunctional, down-regulated, inactivated or impaired in the subject.

106. The composition of any one of claims 86 to 105, wherein said single stranded nucleic acid encodes and/or comprises one or more components of a system for editing a genome or a system for altering gene expression.

107. The composition of claim 106, wherein the system for editing a genome or the system for altering gene expression is a CRISPR/Cas system.

108. The composition of claim 106 or 107, wherein the single stranded nucleic acid encodes a Cas protein or peptide and/or comprises sgRNA, crRNA, and/or tracrRNA.

109. The composition of claim 108, wherein the single stranded nucleic acid comprises mRNA encoding a Cas protein or peptide and sgRNA.

110. The composition of claim 108 or 109, wherein when present, one or more of the sgRNA, the crRNA, the tracrRNA, and the nucleic acid encoding Cas protein is part of a single nucleic acid.

111. The composition of claim 108 or 109, wherein when present, one or more of the sgrnas, the crrnas, the tracrRNA, and the nucleic acid encoding Cas protein are present on two or more nucleic acids.

112. The composition of any one of claims 86-111, wherein the composition is adapted to target the nucleic acid to spleen, lymphoid tissue, lung, bone, liver, heart tissue, tumor tissue, skeletal muscle, kidney, adipose tissue, and/or brain.

113. The composition of claim 112, wherein the composition is suitable for targeting the nucleic acid to spleen, lymphoid tissue, lung and/or bone.

114. The composition of claim 113, wherein the nucleic acid is RNA, such as mRNA.

115. The composition of any one of claims 86 to 114 for use in a method of treating cancer in a subject.

116. The composition for use according to claim 115, wherein the cancer is a blood cancer, such as leukemia, lymphoma or myeloma, cardiac carcinoma, sarcoma or liver cancer.

117. The composition for use according to claim 115 or claim 116, wherein the method comprises administering an anticancer agent.

118. The composition of any one of claims 86 to 114 for use in a method of treating a pulmonary disease or an autoimmune disease in a subject.

119. The composition of any one of claims 86 to 114, for use in treating pompe disease, muscle wasting disease, myopathy, or muscular dystrophy, such as duchenne muscular dystrophy, in a subject.

120. The composition for use according to claim 119, wherein the nucleic acid is DNA.

121. The composition for use of any one of claims 86-120, wherein the composition delivers the nucleic acid to a leukocyte in the subject, such as a B lymphocyte, T lymphocyte, monocyte, neutrophil, dendritic cell, macrophage or monocyte; lymph node tissue cells, myeloid cells, fibroblasts, muscle cells, skeletal muscle cells, liver cells, astrocytes, neurons, astrocytes, spleen cells, lung cells, myocardial cells, kidney cells, fat cells or tumor cells.

122. The composition for use of any one of claims 86-121, wherein the use comprises administering the composition to a subject such that the nucleic acid is delivered to an immune cell.

123. The composition for use of claim 121 or 122, wherein the nucleic acid expresses an immune molecule or transcription factor in the cell.

124. The composition for use of claim 123, wherein the immune molecule is a T cell receptor, cytokine, decoy receptor, antibody, co-stimulatory receptor, co-stimulatory ligand, checkpoint inhibitor, immunoconjugate or tumor antigen.

125. The composition for use of any one of claims 121-124, wherein the cell is a B lymphocyte, a T lymphocyte, a monocyte, a neutrophil, a dendritic cell, a macrophage or a monocyte.

126. The composition for use according to claim 125, wherein the nucleic acid is RNA, such as mRNA.

127. The composition of any one of claims 86 to 97 or the composition for use of any one of claims 115 to 126, wherein the two first peptide motifs independently consist of a single amino acid, dipeptide, tripeptide or tetrapeptide motif.

128. The composition of claim 127 or composition for use, wherein the peptide dendrimer further comprises two second branching residues (e.g., lysine) and four second peptide motifs, wherein one of the second branching residues is covalently bonded to one of the first peptide motifs and the other second branching residue is covalently bonded to the other first peptide motif, and wherein each second branching residue is covalently bonded to two second peptide motifs.

129. The composition or composition for use of claim 128, wherein the four second peptide motifs independently consist of mono-, di-, tri-, or tetrapeptide motifs.

130. The composition or composition for use of claim 127 or 128, wherein the peptide dendrimer further comprises four third branching residues (e.g., lysine) and eight third peptide motifs,

wherein each second peptide motif is covalently bonded to one of the third branch residues, respectively, such that each third branch residue is covalently bonded to one second peptide motif, and wherein each third branch residue is covalently bonded to two third peptide motifs.

131. The composition or composition for use of claim 130, wherein the eight third peptide motifs independently consist of mono-, di-, tri-, or tetrapeptide motifs.

132. The composition of any one of claims 127 to 131, or the composition for use, wherein said first, second and/or third peptide motifs comprise amino acids having basic side chains.

133. The composition of any one of claims 127 to 132, or the composition for use, wherein said first peptide motif, said second peptide motif and/or said third peptide motif comprises an amino acid having a non-polar side chain.

134. The composition of any one of claims 127 to 133, or the composition for use, wherein said first, second and/or third peptide motifs comprise amino acids having acidic side chains.

135. The composition or composition for use of any of claims 127-134, wherein the first, second and/or third peptide motifs comprise amino acids having uncharged polar side chains.

136. The composition or composition for use of any one of claims 127-135, wherein the first, second, and/or third peptide motifs comprise leucine (L) and/or arginine (R) residues.

137. The composition of any one of claims 86-136, or the composition for use, wherein the peptide dendrimer comprises the structures listed in table 2.

138. The composition of any one of claims 86 to 137, or the composition for use, wherein the peptide dendrimer comprises tissue and/or cell targeting motifs.

139. The composition of claim 138 or composition for use, wherein the tissue targeting motif comprises a muscle targeting motif such as GAASSLNIA (SEQ ID NO: 1), an integrin targeting motif such as arginine-glycine-aspartic acid or a chemical modification, such as comprising mannose glycosylation.

140. The composition of any one of claims 86 to 139, or the composition for use, wherein the peptide dendrimer comprises a cell penetrating peptide.

141. The composition for use of claim 140, wherein the cell penetrating peptide comprises a TAT derivative sequence.

142. The composition of claim 140 or composition for use, wherein the cell penetrating peptide comprises the peptide sequence xrxrxrrrxrrbrxb (SEQ ID NO: 2), wherein X is 6-aminocaproic acid and B is β -alanine.

143. The composition of any one of claims 86 to 142, or the composition for use, wherein the peptide dendrimer comprises alkyl chains, alkenyl chains, antibodies or fragments thereof, sugars and/or fatty acids.

144. The composition or composition for use of claim 143, wherein the alkyl or alkenyl chain is conjugated to the core peptide sequence.

145. The composition for use of claim 144, wherein the alkyl or alkenyl chain is conjugated to the C-terminus of the peptide dendrimer.

146. The composition for use of claim 144, wherein the alkyl or alkenyl chain is conjugated to the N-terminus of the peptide dendrimer.

147. The composition or composition for use of any of claims 143-146, wherein the alkyl or alkenyl chain comprises about 5 carbons to about 50 carbons, preferably about 12 to about 30 carbons.

148. The composition of claim 143, or composition for use, wherein the peptide dendrimer comprises fatty acids conjugated to the C-terminus of the peptide dendrimer.

149. The composition for use of claim 143, wherein the peptide dendrimer comprises fatty acids conjugated to the N-terminus of the peptide dendrimer.

150. The composition for use of any one of claims 86-149, wherein the lipid comprises a cationic lipid, a neutral lipid, an anionic lipid, and/or an ionizable lipid.

151. The composition for use of any one of claims 86-150, wherein the lipid comprises a saturated fatty acid.

152. The composition for use of any one of claims 86-151, wherein the lipid comprises an unsaturated fatty acid.

153. The composition for use of claim 151 or 152, wherein the lipid comprises 1, 2, 3, 4, 5, or 6 fatty acid chains.

154. The composition or composition for use of any of claims 86-153, wherein the lipid comprises dioleoyl phosphatidylethanolamine (DOPE) and/or N- [1- (2, 3 dioleyloxy) propyl ] -N, N-trimethylammonium chloride (DOTMA).

155. The composition or composition for use of any of claims 86-154, wherein the lipid comprises dioleoyl phosphatidylethanolamine (DOPE) and dioleoyl phosphatidylglycerol (DOPG).

156. The composition or composition for use of any of claims 86-155, wherein the N/P ratio is between about 0.01:1 and 100:1.

157. The composition or composition for use of claim 156, wherein the N/P ratio is between 1:1 and 50:1.

158. The composition of claim 157, or the composition for use, wherein upon administration to a subject, a higher proportion of the composition is observed in the spleen and/or lymph nodes than in the liver.

159. The composition or composition for use of claim 156, wherein the N/P ratio is between 0.01:1 and 1:1.

160. The composition or composition for use of claim 159, wherein upon administration to a subject, a higher proportion of the composition is observed in the lung, spleen and/or lymph nodes than in the liver.

161. The composition of any one of claims 86-160, wherein after contacting a target cell with the composition in vitro, the delivery of the nucleic acid to the target cell is increased by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 75%, 85%, 90%, 95%, 100% as compared to delivering the same nucleic acid to the same cell type using a lipid-based nucleic acid delivery system.

162. The composition for use of claim 161, wherein the target cell is a cancer cell or an immune cell.

163. The composition for use of claim 161 or 162, wherein the lipid-based nucleic acid delivery system is Lipofectamine 2000.

164. The composition or composition for use of any one of claims 86-163, wherein the peptide dendrimer, the nucleic acid and the lipid form positively charged particles.

165. The composition or composition for use of any of claims 86-164, wherein the composition is contained within a liquid.

166. The composition of any one of claims 86-165, wherein the composition is a powder composition.

167. A method of delivering a nucleic acid into cells of a subject in need of said delivery, the method comprising administering to the subject a pharmaceutically effective amount of the composition of any one of claims 1-165.