Nucleic acid delivery

Abstract

The present invention provides a lipid / dendrimer system that can deliver therapeutic molecules, such as nucleic acids, to mammalian cells and to the human and animal body.For example, the present invention provides a system that allows for the effective delivery of nucleic acids to lymphatic organs, skeletal muscle, 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 present invention can deliver mRNA.

Description

[Technical field]

[0001] The present invention relates to compositions capable of delivering therapeutic molecules, such as nucleic acids, to mammalian cells and the human and animal body. Delivery to specific cell types, such as lymphatic tissue and circulating white blood cells, has been demonstrated. Delivery to specific organs and tissues has also been demonstrated. For example, the present invention provides compositions that allow effective delivery of nucleic acids to the spleen, lymphatic organs, skeletal muscle, brain, and adipose tissue, as well as the lung, tumor tissue, heart, skeletal muscle, adipose tissue, brain, liver, and kidney. Delivery of DNA and RNA is provided. In particular, delivery of mRNA has been demonstrated. [Background technology]

[0002] To realize the full potential of nucleic acid therapeutics as next-generation precision medicines to transform healthcare, key challenges in drug delivery must be addressed. Limitations of current viral and non-viral vector platforms have significantly hindered their development, with many therapies failing at the clinical translation stage due to off-target effects, immune system activation, and challenges with large-scale manufacturing.

[0003] In the context of in vivo delivery of nucleic acids, virus-derived agents are the most powerful vectors, with some making great advances in the clinic (Sheridan et al., 2011). In particular, adeno-associated virus (AAV) is an effective system for in vivo delivery (Wang et al., 2019). However, the application of such systems is limited, as they can only transport DNA less than 5 kb, and cannot transport RNA or larger DNA. Furthermore, despite the advances in viral vectors, issues such as potential random insertion and immunotoxicity are associated with the use of this type of nucleic acid delivery vector. For example, AAV delivery can be highly immunogenic, especially when high doses are required for target tissues other than the liver. AAV delivery systems are also limited for repeated administration. Patients typically acquire immunity to the AAV delivery system, making repeated administration impossible. Finally, production of AAV delivery systems is expensive and difficult to manufacture at large scale and at Good Manufacturing Practice (GMP) grade.

[0004] Considering the shortcomings of viral systems, non-viral vectors have been explored for delivering nucleic acids to cells and tissues in vivo. Various delivery techniques have been developed for the delivery of smaller nucleic acids such as siRNA and antisense oligonucleotides (ASOs). These include bioconjugated oligonucleotide delivery systems in which siRNA or ASOs are conjugated to antibodies or ligands (Benizri et al., 2019). However, siRNA and ASO therapy can only achieve gene silencing or exon skipping, not gene expression. For many diseases, it is advantageous to express functional genes with DNA or mRNA. It is difficult to apply conjugated systems for effective delivery of large genetic payloads such as plasmid DNA or mRNA. Plasmid DNA or mRNA are large negatively charged molecules that cannot easily pass through the negatively charged cell membrane. It is useful if the delivery vehicle encapsulates them and neutralizes the charge for effective delivery.

[0005] Lipid nanoparticles (LNPs) as encapsulating non-viral delivery vehicles have been used, for example, for mRNA delivery in COVID-19 vaccines (Qui et al., 2021). To cultivate the immune system to fight the virus, it is sufficient to transfect a relatively small number of immune and muscle cells locally at the injection site. Thus, RNA-based COVID-19 vaccines are administered intramuscularly. However, widespread diseases can only be effectively treated if many cells of a specific type are transfected, which may require intravenous injection. The surface properties of LNPs currently used in the clinic make them well suited to target the liver when administered intravenously. However, LNPs currently used in the clinic are less suitable for targeting many other tissues.

[0006] Various liposome-based systems have been investigated as well, including uncharged lipids such as dioleoylphosphatidylethanolamine (DOPE) and / or cationic lipids such as 1,2-dioleoyl-3-trimethylammonium-propane 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 in the clinic for nucleic acid delivery and have shown moderate efficacy.

[0007] The addition of peptides to lipid-based vectors has been investigated. In peptide / lipid hybrid systems, peptides and lipids associate with nucleic acids to form nanoparticles that can be internalized by cells. The peptide element can be linear (Kwok et al., 2016) or branched, such as peptide dendrimers (Kwok et al., 2013). However, this previous work on peptide / lipid hybrid systems was only performed for double-stranded plasmid DNA (pDNA) and short interfering RNA (siRNA) in serum-free in vitro conditions that are substantially different from in vivo conditions. It was not clear whether this system could be used for nucleic acid delivery in vivo. Only delivery of short single-stranded oligonucleotides using peptide dendrimer / lipid nanoparticles has been reported in vivo (Saher et al., 2018; Saher et al., 2019), with a low increase of 20-30% in ASO liver delivery observed compared to ASO alone. From this data it can be concluded that peptide dendrimer systems may not be capable of delivering larger nucleic acids such as mRNA to tissues in vivo, the delivery of which has not previously been demonstrated in vivo using peptide dendrimer / lipid hybrid systems.

[0008] The present invention has been made in view of the above considerations. Summary of the Invention

[0009] The development of nucleic acid therapy depends on efficient nucleic acid delivery. The inventors have developed a framework for nucleic acid delivery using peptide dendrimers. These dendrimers are branched peptides that exhibit 1, 2, 3, or 4 amino acid residues between "branch residues" that act as branching units within the dendrimer molecule. The present invention is surprisingly effective in delivering larger nucleic acids (e.g., larger than antisense oligonucleotides, ASOs). While some smaller nucleic acids, such as ASOs, are easily taken up into cells without a vector system, the compositions of the present invention allow for the effective delivery of larger nucleic acids, such as mRNA. This opens up fields such as CRISPR-mediated gene editing and related technologies to clinical applications without the need for viral delivery vectors.

[0010] Since there is still a lack of clinical mRNA or CRISPR cassette (i.e., sgRNA and Cas9 mRNA) delivery system that bypasses the liver, it would be a significant advance to specifically develop tissue-specific mRNA delivery system.The present invention can effectively deliver CRISPR cassette and mRNA in cells to various tissues, especially lung and immune cell-rich tissues (including spleen, lymph node, and bone marrow).This allows the development of mRNA and CRISPR therapy for extrahepatic tissues. The ability to deliver mRNA to immune cells, and even myeloid cells including monocytes, macrophages, neutrophils, and dendritic cells, allows this technology to be developed to treat 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-associated disorders such as Gaucher's disease. The invention also allows mRNA to be delivered to the lungs and / or immune cells, allowing for the treatment of lung-related diseases (e.g., Cystic Fibrosis and Chronic Obstructive Pulmonary Disease).

[0011] The inventors have also found that their dendrimer-based systems are surprisingly versatile. DNA and RNA molecules can be delivered using a range of first-, second-, and third-generation dendrimers. In one example, a vector system based on a dendrimer such as the lipid-associated G1,2,3-KL dendrimer (a "third generation" dendrimer) can improve cell transfection of DNA by 6-10 fold compared to some of the leading commercial reagents such as Lipofectamine 2000. In another example, a vector system based on a dendrimer such as the lipid-associated G1,2-RL,3-LR dendrimer (a "third generation" dendrimer) can improve cell transfection nucleic acids such as mRNA by 10 fold compared to some of the leading commercial reagents such as Lipofectamine 2000. Furthermore, specific organs and tissues can be targeted. As described in more detail below, the inventors have found that dendrimer-based systems are surprisingly robust and versatile, exhibiting high activity in vitro in the presence of serum, and unexpected tissue targeting properties in vivo. Effective delivery of DNA and RNA is discussed below.

[0012] We 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) can transfect HeLa and Neuro2A cells in the presence of serum. (Serum components present a challenge for in vivo DNA delivery, especially systemic delivery, since components such as albumin can interfere with cationic formulations). This unexpected finding led us to investigate the mechanisms underlying cell entry and to evaluate the capabilities of this vector system in vivo. Surprisingly, G1,2,3-RL-based vectors were found to mediate effective delivery of functional nucleic acids to specific tissues after systemic delivery. Liver and skeletal muscle are highly targeted. Endocytic pathway analysis indicates that the G1,2,3-RL DNA complex delivers DNA via both clathrin- and caveolae-mediated endocytosis, and macropinocytosis.

[0013] We found that for mRNA delivery, first or second generation dendrimers (e.g., RHCG1-R, RHCG1,2-R, RHCG1-LR, RHCG1-RL, 2-LR) can effectively transfect cells and mediate similar transfection efficiency as third generation dendrimers (see FIG. 28A showing the transfection efficacy of HeLa cells under full growth medium conditions with serum). This indicates the particular versatility of the hybrid system for RNA delivery.

[0014] The dendrimers used in the present invention are first, second or third generation peptide dendrimers, meaning that they have up to three "layers" of peptide motifs (typically dipeptide motifs) interspersed between "branching" residues such as lysine. A first generation dendrimer has the following structure, shown in the N- to C-terminal orientation, with Lys as the branching unit: (N-terminus-Pep1)2-Lys-(core)-(C-terminus)

[0015] The second generation dendrimer has the following structure, shown in the N-terminus to C-terminus orientation, with Lys as the branching unit: (N-terminus-Pep2)4-Lys2-(Pep1)2-Lys-(core)-(C-terminus)

[0016] The third generation dendrimer has the following structure, shown in the N-terminus to C-terminus orientation, with Lys as the branching unit: (N-terminus-Pep3)8-Lys4-(Pep2)4-Lys2-(Pep1)2-Lys-(core)-(C-terminus)

[0017] A third generation dendrimer is represented diagrammatically in FIG. 31 (with the N-terminus on the left and the C-terminus on the right).

[0018] The circles represent the core sequence. Each triangle represents a branching residue such as lysine. Each rectangle represents a peptide motif. There are two peptide motifs in the first layer of the third generation dendrimer, 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, in underivatized embodiments, the C-terminus is a carboxylic acid, while in other embodiments, as a result of the chemical route used to synthesize the dendrimer, the C-terminus is derivatized to include, for example, a primary amide group, CONH2 (instead of COOH). Functionally significant derivatizations such as targeting moieties (e.g., antibodies, peptide groups, sugar groups, and / or lipid chains) are also envisioned, and these moieties can be attached to the N- and / or C-terminus or at other locations along the dendrimer.

[0019] As described herein, dendrimers can be first, second, or third generation. They can be structurally defined as follows: A first generation dendrimer comprises a core peptide sequence, a first branch residue, and two first peptide motifs. The two first peptide motifs are independently composed of a single amino acid, dipeptide, tripeptide, or tetrapeptide motif. A second generation dendrimer further comprises two second branch residues (e.g., lysine) and four second peptide motifs, one of the second branch residues is covalently bonded to one of the first peptide motifs, the other second branch residue is covalently bonded to the other first peptide motif, and each second branch residue is covalently bonded to two second peptide motifs. The four second peptide motifs are independently composed of a single amino acid, dipeptide, tripeptide, or tetrapeptide motif. The third generation dendrimer further comprises four third branch residues (e.g., lysine) and eight third peptide motifs, each second peptide motif being covalently bound to one of the third branch residues such that each third branch residue is covalently bound to one second peptide motif, and each third branch residue is covalently bound to two third peptide motifs. The eight third peptide motifs are independently comprised of a single amino acid, dipeptide, tripeptide, or tetrapeptide motif. Each of the first, second, and third peptide motifs, if present, may include: (1) amino acids having basic side chains, such as, but not limited to, lysine (K) or arginine (R) or histidine (H); (2) amino acids having acidic side chains, such as, but not limited to, aspartic acid (D) and glutamic acid (E); (3) amino acids having nonpolar side chains, such as, but not limited to, glycine (G), alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), beta-alanine (B), tryptophan (W), proline (P), aminohexanoic acid (X), and cysteine ​​(C); and (4) amino acids having uncharged polar side chains, such as, but not limited to, asparagine (N), glutamine (Q), serine (S), threonine (T), and tyrosine (Y).

[0020] Preferred dendrimers are shown in Table 2 below. Specific examples are specifically described. For example, in a dendrimer in which each peptide motif is an Arg-Leu (RL) dipeptide, the structure can be represented as G1-RL, G1,2-RL, and G1,2,3-RL. In a dendrimer in which each peptide motif is a Lys-Leu (KL) dipeptide, the structure can be represented as G1-KL, G1,2-KL, and G1,2,3-KL. In a dendrimer in which each peptide motif is a Leu-Arg (LR) dipeptide, the structure can be represented as G1-LR, G1,2-LR, and G1,2,3-LR.

[0021] ("G1", "G2", and "G3" refer to the "generation 1", "generation 2", and "generation 3" peptide motifs of the first, second, and third layers, respectively. Each amino acid residue can be an L-amino acid or a D-amino acid. D-amino acids can be indicated using lower case letters in the single letter code. Alternatively, dendrimers in which each amino acid is the D-isoform can be written with "D-" preceding the shorthand representation of the dendrimer.)

[0022] Thus, in a first aspect, the present invention provides a composition for use in medicine, comprising a first, second or third generation peptide dendrimer, a nucleic acid and a lipid. The composition further comprises a nucleic acid and a lipid. 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.

[0023] 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 residues 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, 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.

[0024] In a third aspect, the present invention provides a method of delivering a nucleic acid into a cell of a subject in need of delivery of the nucleic acid, comprising administering a pharma- ceutically effective amount of a composition to the subject. The composition comprises a peptide dendrimer, a nucleic acid, and a lipid, the peptide dendrimer comprising at least a core peptide sequence, a first branching residue, and two first peptide motifs. The branching residues 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.

[0025] In some embodiments, the nucleic acid is RNA. For example, the RNA can be selected from mRNA, ssRNA, dsRNA, sgRNA, crRNA, tracrRNA, lncRNA, siRNA, saRNA, and / or self-amplifying RNA. Preferably, the RNA is mRNA.

[0026] In some embodiments, the nucleic acid is DNA. For example, the DNA may include ssDNA, dsDNA, plasmids, and / or cDNA.

[0027] For the avoidance of doubt, a composition may comprise more than one nucleic acid (e.g., more than one type of RNA molecule). Similarly, a composition may comprise more than one lipid. A composition may comprise more than one peptide dendrimer.

[0028] In some embodiments, the composition comprises an RNA nucleic acid and a DNA nucleic acid. The RNA nucleic acid and the DNA nucleic acid may be part of a single nucleic acid molecule.

[0029] In some embodiments, the nucleic acid comprises a modified nucleic acid. Exemplary nucleic acid modifications are described herein.

[0030] Preferably, the nucleic acid encodes a transgene and is capable of expressing the transgene in the target cell. The transgene may be a protein or a peptide. Additionally or alternatively, the nucleic acid may modulate the expression or activity of an endogenous gene. Modulation may be an increase in expression of the gene and / or the exogenous expression of additional copies of the gene, or modulation may be a decrease in expression of the gene.

[0031] In some embodiments, the regulated endogenous gene is a gene that expresses a protein or peptide.

[0032] In some embodiments, the protein or peptide comprises an antigen, a hormone, a receptor, a chimeric antigen receptor, a transcription factor, and / or a cytokine.

[0033] 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.

[0034] In some embodiments, the composition can be used as a vaccine.

[0035] In some embodiments, the nucleic acid comprises or encodes a self-amplifying RNA.

[0036] In some embodiments, the use comprises treating a genetic disorder in a subject.

[0037] In some embodiments, the nucleic acid expresses a functional version of a gene that is non-functional, down-regulated, inactive, or impaired in a subject.

[0038] In some embodiments, the nucleic acid encodes and / or comprises one or more components of a system for editing a genome or for altering gene expression. For example, the system for editing a genome or for altering gene expression may be a CRISPR / Cas system. The nucleic acid may encode a Cas protein or peptide and / or comprises a sgRNA, a crRNA, and / or a tracrRNA. The nucleic acid may comprise an mRNA encoding a Cas protein or peptide and an RNA sequence comprising an sgRNA. The composition may comprise an mRNA encoding a Cas protein or peptide and another RNA comprising an sgRNA (as separate molecules). In some embodiments, one or more of the sgRNA, crRNA, tracrRNA, and nucleic acid encoding a Cas protein, if present, are part of a single nucleic acid. In some embodiments, one or more of the sgRNA, crRNA, tracrRNA, and nucleic acid encoding a Cas protein, if present, are present on two or more nucleic acids.

[0039] In some embodiments, the compositions target the spleen, lymphatic tissue, skeletal muscle, brain, and adipose tissue, as well as the lung, tumor tissue, heart, skeletal muscle, adipose tissue, brain, liver, and kidney.

[0040] In some embodiments, the composition targets the spleen, lymphatic tissue, lung, and / or bone. In these embodiments, the nucleic acid may be RNA, for example, mRNA.

[0041] In some embodiments, the nucleic acid is delivered to a cell that is a leukocyte, e.g., a B lymphocyte, a T lymphocyte, a monocyte, a neutrophil, a dendritic cell, a macrophage, or a monocyte; a lymph node tissue cell, a bone marrow cell, a fibroblast, a muscle cell, a skeletal muscle cell, an endothelial cell, a liver cell, a stellate cell, a neuron, an astrocyte, a splenocyte, a lung cell, a cardiac muscle cell, a kidney cell, an adipocyte, a stem cell, and / or a tumor cell.

[0042] In some embodiments, the composition is administered to a subject such that the nucleic acid is delivered to immune cells.

[0043] In some embodiments, the nucleic acid expresses an immune molecule or transcription factor in the target cell. The immune molecule may be a T cell receptor, a chimeric antigen receptor, a cytokine, a decoy receptor, an antibody, a costimulatory receptor, a costimulatory ligand, a checkpoint inhibitor, an immunoconjugate, or a tumor antigen.

[0044] In some embodiments, the cell is a B lymphocyte, a T lymphocyte, a monocyte, a neutrophil, a dendritic cell, a macrophage, a monocyte, a myeloid derived suppressor cell (MDSC), a tumor associated macrophage, or a tumor associated neutrophil.

[0045] In some embodiments, the nucleic acid is RNA, e.g., mRNA.

[0046] In some embodiments, the composition is for use in a method of treating cancer in a subject. The cancer may be a hematological cancer, such as leukemia, lymphoma, myeloma, or myelodysplastic syndrome; or lung cancer, cardia cancer, 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, bile duct cancer (Cholangiocarcinoma), gallbladder cancer, neuroendocrine tumors, hepatocellular carcinoma, colorectal cancer, pancreatic cancer, and solid tumors.

[0047] In some embodiments, the composition is for use in a method of treating a pulmonary disease, for example, the composition may be for use in treating chronic obstructive pulmonary disease (COPD) or cystic fibrosis (CF).

[0048] In some embodiments, the composition is for use in a method of treating an autoimmune disease in a subject.

[0049] In some embodiments, the composition is for use in treating Pompe disease, a muscle wasting disease, a myopathy, or a muscular dystrophy, such as Duchenne muscular dystrophy, in a subject. In these embodiments, the nucleic acid may be DNA.

[0050] In some embodiments, the composition is for use in treatment for limb ischemia, such as diabetic limb ischemia, in a subject, and the transgene is hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and / or fibroblast growth factor (FGF).

[0051] In some embodiments, the two first peptide motifs independently consist of a single amino acid, a dipeptide, a tripeptide, or a tetrapeptide motif.

[0052] In some embodiments, the peptide dendrimer further comprises two second branch residues (e.g., lysine) and four second peptide motifs, one of the second branch residues covalently bonded to one of the first peptide motifs and the other second branch residue covalently bonded to the other first peptide motif, and each second branch residue is covalently bonded to two second peptide motifs.

[0053] In some embodiments, the four second peptide motifs independently consist of a single amino acid, a dipeptide, a tripeptide, or a tetrapeptide motif.

[0054] In some embodiments, the peptide dendrimer further comprises four third branch residues (e.g., lysine) and eight third peptide motifs, each second peptide motif being covalently bound to one of the third branch residues such that each third branch residue is covalently bound to one second peptide motif, and each third branch residue is covalently bound to two third peptide motifs.

[0055] In some embodiments, the eight third peptide motifs independently consist of a single amino acid, a dipeptide, a tripeptide, or a tetrapeptide motif.

[0056] Each peptide motif independently comprises naturally occurring L- or D-amino acids and / or non-naturally occurring L- or D-amino acids, such as beta-alanine (B) or aminohexanoic acid (X).

[0057] In some embodiments, the first, second, and / or third peptide motif comprises an amino acid with a basic side chain.

[0058] In some embodiments, the core sequence includes amino acid residues having ionizable groups, such as histidine.

[0059] In some embodiments, the first, second, and / or third peptide motif comprises amino acids having non-polar side chains.

[0060] In some embodiments, the first, second, and / or third peptide motif comprises an amino acid with an acidic side chain.

[0061] In some embodiments, the first, second, and / or third peptide motif comprises amino acids with uncharged polar side chains.

[0062] In some embodiments, the first, second, and third peptide motifs (if present) comprise a) arginine (R) or lysine (K); and / or b) leucine (L), valine (V), histidine (H), or isoleucine (I).

[0063] In some embodiments, the first, second, and / or third peptide motif comprises leucine (L) and / or arginine (R) residues.

[0064] In some embodiments, the peptide dendrimer comprises a structure set forth in Table 2.

[0065] In some embodiments, the peptide dendrimer further comprises a tissue and / or cell targeting motif, which may include a muscle targeting motif, e.g., GAASSLNIA (SEQ ID NO: 1), an integrin targeting motif, e.g., arginine-glycine-aspartic acid (RGD), or a chemical modification, e.g., a chemical modification including mannose glycosylation.

[0066] In some embodiments, the peptide dendrimer further comprises a cell-penetrating peptide. The cell-penetrating peptide may comprise a TAT-derived sequence. The cell-penetrating peptide may comprise the peptide sequence XRXRRBRRXRRBRXB (SEQ ID NO:2), where X is 6-aminohexanoic acid and B is beta-alanine.

[0067] In some embodiments, the peptide dendrimer further comprises an alkyl chain, an alkenyl chain, an antibody or fragment thereof, a sugar, and / or a fatty acid. 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.

[0068] In some embodiments, the alkyl or alkenyl chain contains from about 5 carbons to about 50 carbons, preferably from about 12 to about 30 carbons.

[0069] In some embodiments, the peptide dendrimer comprises a fatty acid conjugated to the C-terminus of the peptide dendrimer. In other embodiments, the peptide dendrimer comprises a fatty acid conjugated to the N-terminus of the peptide dendrimer.

[0070] In some embodiments, the lipids of the composition comprise cationic lipids, neutral lipids, anionic lipids, and / or ionizable lipids.

[0071] 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.

[0072] 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.

[0073] In some embodiments, the lipid comprises dioleoylphosphatidylethanolamine (DOPE) and / or N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). In some embodiments, the lipid comprises dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylglycerol (DOPG).

[0074] In some embodiments, the N / P ratio is from about 0.01:1 to 100:1. For example, the N / P ratio can be from about 0.05:1 to 50:1 or from about 0.1:1 to 30:1. Narrower ranges, such as 0.2:1 to 25:1 and 0.5:1 to 20:1, are also contemplated.

[0075] In some embodiments, the N / P ratio is between 1: 1 and 50: 1. In some embodiments, following administration to a subject, a higher percentage of the composition is observed in the spleen and / or lymph nodes than in the liver.

[0076] In some embodiments, the N / P ratio is between 0.01: 1 and 1: 1. In some embodiments, following administration to a subject, a greater proportion of the composition is observed in the lungs, spleen, and / or lymph nodes than in the liver.

[0077] In some embodiments, the peptide dendrimer, nucleic acid, and lipid form a positively charged particle.

[0078] In other embodiments, the peptide dendrimers, nucleic acids, and lipids form particles that are negatively charged or have a neutral charge.

[0079] In some embodiments, the delivery of nucleic acid to a 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 the delivery of the same nucleic acid to the same tissue or cell type using a lipid-based nucleic acid delivery system. The target cell or tissue is a cell type or organ / tissue as defined herein. For example, the target tissue may be the spleen, lymphoid organs, skeletal muscle, brain, and adipose tissue, as well as the lung, tumor tissue, heart, skeletal muscle, adipose tissue, brain, liver, and kidney. The lipid-based nucleic acid delivery system may be DOTMA / DOPE.

[0080] The composition can be administered to a subject intravenously, intramuscularly, intratumorally, subcutaneously, intradermally, or intraperitoneally.

[0081] 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. Dry compositions can be prepared using lyophilization and / or freeze-drying techniques.

[0082] In certain 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 linked to the first lysine residue; (i) a first lysine residue is covalently linked to two first peptide motifs, each of which is covalently linked to two second lysine residues; (ii) each second lysine residue is covalently linked to two second peptide motifs, each second peptide motif being covalently linked to one of the third lysine residues; (iii) each third lysine residue is covalently linked to two of the third peptide motifs, the first, second, and third peptide motifs being, independently, mono-, di-, tripeptide, or tetra-peptide motifs. Each of the first, second, and third peptide motifs may comprise arginine (R) or lysine (K); and / or b) leucine (L), valine (V), histidine (H), or isoleucine (I). (The term "covalently linked" refers to a direct covalent bond between the described moieties without any intervening atoms. Thus, only mono-, di-, tri-, or tetra-peptide motifs exist between the first lysine residue and each second lysine residue, and only mono-, di-, tri-, or tetra-peptide motifs exist between each second lysine residue and each third lysine residue.)

[0083] In some embodiments, each of the first, second, and third peptide motifs, if present, may include: (1) amino acids having basic side chains, such as, but not limited to, lysine (K) or arginine (R) or histidine (H); (2) amino acids having acidic side chains, such as, but not limited to, aspartic acid (D) and glutamic acid (E); (3) amino acids having nonpolar side chains, such as, but not limited to, glycine (G), alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), beta-alanine (B), tryptophan (W), proline (P), aminohexanoic acid (X), and cysteine ​​(C); and (4) amino acids having uncharged polar side chains, such as, but not limited to, asparagine (N), glutamine (Q), serine (S), threonine (T), and tyrosine (Y).

[0084] Preferably, at least one of the first, second, and third peptide motifs comprises arginine (R). At least two of the first, second, and third peptide motifs may comprise arginine (R). In some embodiments, all of the first, second, and third peptide motifs comprise arginine (R).

[0085] Preferably, at least one of the first, second, and third peptide motifs comprises leucine (L). At least two of the first, second, and third peptide motifs may comprise leucine (L). In some embodiments, all of the first, second, and third peptide motifs comprise leucine (L).

[0086] In some embodiments, each of the first, second, and third peptide motifs is a dipeptide motif. In some embodiments, each of the first, second, and third peptide motifs is a tripeptide motif. In some embodiments, each of the first, second, and third peptide motifs is a tetrapeptide motif. In some embodiments, each of the first, second, and third peptide motifs is independently a mono-, dipeptide, tripeptide, or tetrapeptide motif. Preferably, at least one of the first, second, and third peptide motifs is a dipeptide motif that includes both leucine (L) and arginine (R). In some embodiments, at least two of the first, second, and third peptide motifs are dipeptide motifs that include both leucine (L) and arginine (R). In particularly preferred embodiments, each peptide motif is a dipeptide motif that includes both leucine (L) and arginine (R). Each amino acid residue is independently selected from an L-isoform or a D-isoform.

[0087] In some embodiments, the peptide dendrimer is selected from any one of the peptide dendrimers listed in Table 2.

[0088] In some embodiments, the peptide dendrimer is G1,2,3-RL and comprises a (RL)8(KRL)4(KRL)2K-core.

[0089] In some embodiments, the peptide dendrimer is G1-LR,G2,3-RL and comprises a (RL)8(KRL)4(KLR)2K-core.

[0090] In some embodiments, the peptide dendrimer is G1,2,3-rl and comprises a (rl)8(krl)4(krl)2k-core, where "r" is D-arginine, T is D-leucine, and "k" is D-lysine.

[0091] In some embodiments, the peptide dendrimer is G1,2-RL, G3-LR and comprises a (LR)8(KRL)4(KRL)2K-core.

[0092] In some embodiments, the peptide dendrimer is G1,2-LR,G3-RL and comprises a (RL)8(KLR)4(KLR)2K-core.

[0093] In some embodiments, the peptide dendrimer is G1,2,3-LR and comprises a (LR)8(KLR)4(KLR)2K-core.

[0094] In some embodiments, the peptide dendrimer is RHCG1-R and comprises (R)2KRHC-NH2.

[0095] In some embodiments, the peptide dendrimer is RHCG1,2-R and comprises (R)4(KR)2KRHC-NH2.

[0096] In some embodiments, the peptide dendrimer is RHCG1-LR and comprises (LR)2KRHC-NH2.

[0097] In some embodiments, the peptide dendrimer is RHCG1-RL,2-LR and comprises (LR)4(KRL)2KRHC-NH2.

[0098] In some embodiments, the peptide dendrimer is G1,2-RL,3-LR and comprises (LR)8(KRL)4(KRL)2KGSC-NH2.

[0099] In some embodiments, the peptide dendrimer is RHCG1-RLR and comprises (RLR)2KRHC-NH2.

[0100] In some embodiments, the peptide dendrimer is RHCG1,2-RLR and comprises (RLR)4(KRLR)2KRHC-NH2.

[0101] In some embodiments, the peptide dendrimer is G1-LRLR and comprises (LRLR)2KGSC-NH2.

[0102] In some embodiments, the peptide dendrimer is RHCG1,2-RL,3-LR and comprises (LR)8(KRL)4(KRL)2KRHC-NH2.

[0103] In some embodiments, the peptide dendrimer is G1,2,3-RL and comprises (RL)s(KRL)4(KRL)2KGSC-NH2.

[0104] In some embodiments, the peptide dendrimer is NTX1 and comprises (LR)8(KRL)4(KRL)2KGSCGAASSLNIAXRXRRBRRXRRBRXB-NH2, where X=6-aminohexanoic acid and B=beta-alanine.

[0105] ("G1" refers to the "generation 1" peptide motifs in the first tier; "G2" refers to the "generation 2" peptide motifs in the second tier; and "G3" refers to the "generation 3" peptide motifs in the third tier.)

[0106] Thus, the present invention provides a composition comprising a nucleic acid, a lipid, and a dendrimer as described herein for use in medicine. The dendrimer may be a first generation dendrimer, or a second generation dendrimer, or a third generation dendrimer. Preferably, the N / P ratio, which is the amount of peptide (measured by the number of 1+ charged nitrogen atoms on the peptide, N) relative to the amount of nucleic acid (measured by the number of 1-charged phosphate groups in the backbone, P), is greater than 0.05:1, for example, greater than 0.1:1. (The term N / P ratio can be expressed 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 1:1 or more, e.g., about 2:1 or more, about 2.5:1 or more, about 3:1 or more, about 4:1 or more, about 5:1 or more, 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.

[0107] The lipid component of the composition may include DOTMA, DOPE, DOPC, and / or DOPG.

[0108] The amount of lipid component can be expressed as a weight:weight ratio ("w / w" or "w:w") relative to the amount of nucleic acid in the composition and can be in the range of 1:50 to 50:1. More preferably, the amount (weight) of lipid is 1:1 to 50:1, or 2:1 to 20:1, relative to the amount (weight) of nucleic acid. The lipid:nucleic acid ratio can be at least 2:1. Most preferably, the weight:weight ratio of lipid:nucleic acid is about 10:1. These ratios refer to the weight of the total lipid. As described herein, the composition may include lipids including two or more lipid components, for example, a mixture of two, three, or four lipids. The weight of the lipid component is the total (combined) weight of these lipid components. Preferably, each lipid component is mixed in approximately equal proportions.

[0109] Relatedly, the invention provides pharmaceutical compositions comprising a nucleic acid, a lipid, and a first, second, or third generation dendrimer as described herein, and a pharma- ceutically acceptable excipient.

[0110] The nucleic acids forming part of the compositions of the invention may be antisense oligonucleotides (ASOs). Preferably, the ASOs are at least 20 nucleotides in length, at least 25 nucleotides in length, at least 30 nucleotides in length, at least 35 nucleotides in length, or have a length as specified in relation to the nucleic acids disclosed elsewhere herein, e.g., at least 40 nucleotides in length.

[0111] 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 lncRNA 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 capable of expressing an siRNA or saRNA molecule in a target cell. The nucleic acid may be a DNA plasmid (linear or circular), e.g. a plasmid capable of expressing a transgene in a target cell.

[0112] The transgene may be a viral protein, a bacterial protein, or a protein of a microorganism that is parasitic to a mammal. The composition expressing the viral protein, bacterial protein, or parasitic microorganism protein may be used as a vaccine. For example, an effective amount of the composition may be delivered systemically (e.g., intravenously) to a subject to achieve expression of the viral protein, bacterial protein, or parasitic microorganism protein in the skeletal muscle of the subject and prime an immune response against the viral protein, bacterial protein, or parasitic microorganism protein. Thus, the present invention provides a method of vaccinating a subject and a composition for use in vaccinating a subject. In such an example, the transgene may be expressed in immune cells, such as white blood cells, such as B lymphocytes, T lymphocytes, monocytes, neutrophils, dendritic cells, macrophages, or monocytes; lymph node tissue cells, as described herein.

[0113] The transgene may express a therapeutic protein for use in gene therapy. The gene therapy may be for treating a genetic disorder in a patient. The genetic disorder may be a single gene disorder in a patient, such as muscular dystrophy. In embodiments where the single gene disorder is muscular dystrophy, the transgene may be dystrophin. 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 wasting, the transgene may be follistatin. In embodiments where the disorder is a neuromuscular disease, the transgene may be acid alpha-glucosidase (GAA). The transgene may be expressed in one or more of the tissues disclosed herein, although the expressed protein may be secreted from the tissue into the circulation.

[0114] It is contemplated that the present invention can be used to deliver nucleic acid therapeutics to treat myopathies. It is also contemplated that the present invention can be used to deliver nucleic acid therapeutics to treat muscular dystrophies, such as Duchenne muscular dystrophy, myotonic dystrophy, facioscapulohumeral muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, oculopharyngeal muscular dystrophy, Emery-Dreifuss muscular dystrophy, genetic muscular dystrophies, congenital muscular dystrophies, and distal myopathies.

[0115] The nucleic acid therapy may be for treating a muscle wasting condition such as cachexia. The nucleic acid therapy may be for treating a genetic muscle disorder such as myotonia congenita or other muscle disorder such as familial periodic paralysis. The nucleic acid therapy may be for treating a motor neuron disease such as amyotrophic lateral sclerosis (ALS), spinal-bulbar muscular atrophy (SBMA), or spinal muscular atrophy (SMA). The nucleic acid therapy may be for treating a mitochondrial disease such as Friedreich's ataxia (FA), or a mitochondrial myopathy such as Kearns-Sayre syndrome (KSS), Leigh encephalopathy (subacute necrotizing encephalomyopathy), mitochondrial DNA depletion syndrome, mitochondrial encephalomyopathy, lactic acidosis and stroke-like episode (MELAS), mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), myoclonus epilepsy with ragged red fibers (MERRF), neuropathy, ataxia and retinitis pigmentosa (NARP), Pearson's syndrome, or progressive external opthalmoplegia (PEO). The nucleic acid therapy may be for treating a congenital myopathy, such as cap myopathy, centronuclear myopathy, congenital myopathy with muscle fiber type disproportion, core myopathy, central core disease, multiminicore myopathy, myosin storage myopathy, myotubular myopathy, or nemaline myopathy.The nucleic acid therapy may be for treating distal myopathies such as GNE myopathy / Nonaka myopathy / hereditary inclusion-body myopathy (HIBM), Laing distal myopathy, Markesbery-Griggs late-onset distal myopathy, Miyoshi myopathy, Udd myopathy / cervical muscular dystrophy, VCP myopathy / IBMPFD, vocal cord and pharyngeal distal myopathy, or Welander distal myopathy. The nucleic acid therapy may be for treating endocrine myopathies such as hyperthyroid myopathy or hypothyroid myopathy. The nucleic acid therapy may be for treating inflammatory myopathies such as dermatomyositis, inclusion body myositis, or polymyositis. The nucleic acid therapy may be for treating metabolic myopathies such as acid maltase deficiency (AMD, Pompe disease), carnitine deficiency, carnitine palmitoyltransferase deficiency, debrancher enzyme deficiency (Cori disease, Forbes disease), lactate dehydrogenase deficiency, myoadenylate deaminase deficiency, phosphofructokinase deficiency (Tarui disease), phosphoglycerate kinase deficiency, phosphoglycerate mutase deficiency, or phosphorylase deficiency (McArdle disease). The nucleic acid therapy may be for treating myofibrillar or scapuloperoneal myopathy. The nucleic acid therapy may be for treating a neuromuscular junction disorder such as congenital myasthenic syndrome (CMS), Lambert-Eaton myasthenic syndrome (LEMS), or myasthenia gravis (MG). The nucleic acid therapy may be for treating a peripheral nerve disorder such as Charcot-Marie-Tooth disease (CMT) or giant axonal neuropathy (GAN).The nucleic acid therapy may be for treating cardiovascular diseases such as thromboangiitis obliterans / Buerger's 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 comprehensive severe chronic limb ischemia and diabetic limb ischemia), severe peripheral artery occlusive disease (PAOD), or intermittent claudication / arteriosclerosis. The nucleic acid therapy may be for treating infectious diseases such as COVID-19, HIV, HBV, HCV, Ebola and Marburg viruses, West Nile fever, SARS, avian influenza, HPV, cytomegalovirus, or malaria. The nucleic acid therapy may be for treating cancers such as sarcoma, melanoma, breast cancer, lung cancer, pancreatic cancer, prostate cancer, liver cancer, acute myeloid leukemia, or B-cell lymphoma. The nucleic acid therapy may be for treating allergies such as peanut allergy. The nucleic acid therapy may be for treating multiple sclerosis (MS). The nucleic acid therapy may be for treating myelodysplastic syndrome (MDS).

[0116] Pompe disease is caused by a deficiency in human acid alpha-glucosidase (GAA), a lysosomal enzyme that cleaves terminal alpha 1-4 and alpha 1-6 glucose from glycogen. The compositions of the present invention can be used to treat Pompe disease. The compositions of the present invention, which include a nucleic acid encoding GAA, can be administered to a subject suffering from Pompe disease to deliver the nucleic acid to the target tissue of the subject and express GAA in the target tissues described herein, particularly liver and skeletal muscle. The enzyme can be secreted from the tissue into the circulation.

[0117] Follistatin is an inhibitor of TGF-β superfamily ligands that suppress skeletal muscle growth and promote muscle wasting. The composition of the present invention, which includes a nucleic acid encoding follistatin, can be administered to a subject suffering from a muscle wasting disorder to deliver the nucleic acid to the target tissue of the subject and express follistatin in the target tissues described herein, particularly the liver and skeletal muscle. The protein can be secreted from the tissue into the circulation.

[0118] Accordingly, the present invention provides methods for treating such disorders and compositions for use in such treatments.

[0119] The core peptide motif of the dendrimer is a single amino acid residue or a short peptide motif such as a dipeptide or tripeptide motif. The core sequence may include 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 include non-naturally occurring amino acids (L- and / or D-isomer), such as beta-alanine (B) and / or aminohexanoic acid (X). When the core is a tripeptide motif, the core may comprise glycine (G), serine (S), and either cysteine ​​(C) or alanine (A). Preferably, the core comprises an ionizable residue 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. The tripeptide motif may comprise alanine (A), lysine (K), and leucine (L). For example, the core sequence may be KLA. The core peptide may be covalently linked to a further moiety, for example a cell-specific targeting peptide, or may be derivatized with a lipid molecule. One, some, or all of the amino acids of the dendrimer, e.g., the core peptide motif, may be covalently linked to further moieties, e.g., antibodies, cell-specific targeting peptides, sugar ligands, e.g., glucose, mannose, galactose, and GalNAc (or glycans containing same), and / or lipid substituents. One skilled in the art can readily select further moieties that do not adversely affect solubility or nucleic acid binding characteristics.

[0120] The lipid component of the composition may comprise a mixture of lipids, including cationic lipids. For example, the lipid component may comprise dioleoylphosphatidylethanolamine (DOPE) and N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). The DOPE:DOTMA ratio can be readily determined for optimal properties for a given application, but will typically be in the range of 1:5 to 5:1.

[0121] Preferably, the range is 3:1 to 1:3 or 2:1 to 1:2. Most preferably, the DOPE:DOTMA ratio is 1:1. In other embodiments, the lipid comprises 1,2-dioleoyl-3-trimethylammonium-propane chloride (DOTAP), for example, as the sole lipid or in combination with DOPE.

[0122] In other embodiments, the lipid component of the composition may include other lipids in addition to (or instead of) DOPE and DOTMA. Exemplary lipid components are shown below.

[0123] Cationic lipids: N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) 1,2-dioleoyl-3-trimethylammonium-propane chloride (DOTAP) 2,3-dioleyloxy-N-(2[spermine-carboxamido]ethyl)-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) 3p-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-chol)

[0124] Neutral lipids: 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) cholesterol

[0125] Anionic lipids: 1,2-Dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG)

[0126] Ionizable lipids: DLin-DMA DLin-KC2-DMA DLin-MC3-DMA

[0127] Other potential lipids include 4-(2-aminoethyl)-morpholino-cholesterol-hemisuccinate (MoChol), cholesterol hemisuccinate (CHEMS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), dimyristoylphosphatidylcholine (DMPC), dipa Examples of phosphatidylcholine include dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), cholesterol-(3-imidazol-1-yl propyl)carbamate (CHIM), dimethyldioctadecylammonium bromide (DDAB), dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylglycerol (DOPG), and cholesterol sulfate (chol-SO4).

[0128] 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. Additionally, lipids may be derivatized via linkage to PEG groups, such as PEG2000.

[0129] The present invention provides a method of delivering a nucleic acid into a target cell, comprising contacting the target cell with a composition of the present invention. The target cell may be a muscle cell, a liver cell, a stellate cell, a neuron, an astrocyte, a splenocyte, a lung cell, a cardiomyocyte, a kidney cell, an adipocyte, a myeloid-derived suppressor cell (MDSC), a tumor-associated macrophage or a tumor-associated neutrophil, a stem cell, or a tumor cell. The method may be performed in vitro. The cell may be obtained from a patient, and the cell may be administered to the patient after the method is performed, i.e., the method may be ex vivo. Alternatively, the method may be performed in vivo as part of a medical use or treatment. The method includes cell entry via clathrin-mediated endocytosis, caveolae-mediated endocytosis, and / or micropinocytosis. Such in vivo treatment may include administering the composition to a subject to deliver the nucleic acid to a particular target tissue(s), for example, the muscle and / or liver of the subject. The composition may be administered as a single dose, or as two or more doses.

[0130] The present invention also provides novel dendrimers such as those shown in Table 2. In some embodiments, the dendrimers are third generation dendrimers, e.g., G1,2,3-LR dendrimers, G1,2,3-rl dendrimers, G1-LR,G2,3-RL dendrimers, G1,2-RL,G3-LR dendrimers, and G1,2-LR,G3-RL dendrimers, which collectively are peptide dendrimers 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 linked to the first lysine residue, wherein: (i) the first lysine residue is covalently linked to the two first dipeptide motifs and two first dipeptide motifs; Each of the dipeptide motifs of (i) is covalently linked to two second lysine residues, (ii) each of the second dipeptide motifs is covalently linked to two second dipeptide motifs, each of the second peptide motifs is covalently linked to one of the third lysine residues, and (iii) each of the third lysine residues is covalently linked to two of the third dipeptide motifs, wherein the first, second, and third dipeptide motifs each comprise leucine (L) and arginine (R), and at least one of the first, second, and third dipeptide motifs consists of a leucine-arginine (LR) dipeptide motif (the "LR" motif is written according to the 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 can form part of the compositions of the invention and can be used in other aspects of the invention disclosed herein.

[0131] The present invention includes combinations of the described aspects and preferred features except where such combinations are expressly not permitted or explicitly avoided. [Brief description of the drawings]

[0132] Next, embodiments and experiments illustrating the principles of the present invention will be described with reference to the accompanying drawings. [Figure 1] Comparison of transfection efficiency of dendrimers with different lysine and leucine configurations for DNA delivery. Changing the position of lysine and leucine within a generation does not significantly change the transfection efficiency in (A) HeLa or (B) Neuro2A cells. Structural modification of dendrimer G1,2,3-KL to its variants, such as G1,2,3-K (a version of G1,2,3-KL with all leucines removed) and replacement of all L-amino acids with D-type does not significantly change the transfection efficiency in (C) HeLa or (D) Neuro2A cells. There is no significant difference in the case of G0,1,2,3-KL (a version of G1,2,3-KL of generation 0 with KL motifs) in (E) HeLa or (F) Neuro2A cells. Conditions: 1×104 cells were transfected with 0.25 μg pCI-Luc. Error bars refer to the mean ± SEM for experiments performed in triplicate. D / D stands for DOTMA / DOPE. [Diagram 2] Comparison of transfection efficiency of dendrimers with different generations (G1, G2, and G3) and cationic residues (e.g., KL vs. RL) for DNA delivery. (A) Transfection efficiency in HeLa cells, (B) Transfection efficiency in Neuro2A cells. Cells transfected with luciferase expression plasmid (pCI-Luc) in the presence of peptide dendrimers with D / D (measured 24 hours after transfection). Luminescence values ​​were normalized by dividing by the similar value for cells treated with D / D DNA complex (w / w 1:1, 0.25 μg) to obtain the % of transfection. Condition: 1×104 cells were transfected with 0.25 μg pCI-Luc. Error bars refer to the mean ± SEM for experiments performed in triplicate. * indicates p<0.05. D / D is DOTMA / DOPE. [Diagram 3]Comparison of transfection efficiency of G1,2,3-RL with other commercially available transfection reagents including DOTMA / DOPE, polyethyleneimine, and Lipofectamine 2000 for DNA delivery. (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 transfected with luciferase expression plasmid (pCI-Luc) in the presence of G1,2,3-RL with D / D (measured 24 hours after transfection). Luminescence values ​​were normalized by dividing by the similar value for cells treated with D / D DNA complex to obtain % transfection. Conditions: 1×104 cells were transfected with 0.25 μg pCI-Luc for 4 hours in serum-free or 10% serum. Error bars refer to the mean ± SEM for experiments performed in triplicate. G1,2,3-RL mediates significantly more potent transfection than other reagents in HeLa and Neuro2A cells. * indicates p<0.05, *** indicates p<0.001, **** indicates p<0.0001. D / D is DOTMA / DOPE. [Figure 4]Comparison of transfection efficiency of dendrimers with different generations (G1, G2, and G3) and cationic residues (e.g., KL vs. RL) for DNA delivery in serum conditions. (A) Transfection efficiency in HeLa cells, (B) Transfection efficiency in Neuro2A cells. Cells transfected with luciferase expression plasmid (pCI-Luc) in the presence of peptide dendrimers with D / D (measured 24 hours after transfection). Luminescence values ​​were normalized by dividing by the similar value for cells treated with D / D DNA complex (w / w 1:1, 0.25 μg) to obtain the % of transfection. Conditions: 1×104 cells were transfected with 0.25 μg pCI-Luc for 4 hours in 10% serum. Error bars refer to the mean ± SEM for experiments performed in triplicate. G1,2,3-RL (N / P: 5-20) mediates significantly more potent transfection than other groups in both HeLa and Neuro2A cells. * indicates p<0.05. G1,2,3-RL (N / P: 10, 20) mediates significantly more potent transfection than other groups. *** indicates p<0.001. D / D is DOTMA / DOPE. [Diagram 5] Cellular uptake pathways of G1,2,3-RL-D / D-DNA nanoparticles. Chlorpromazine inhibits clathrin-mediated endocytosis. Genistein and rottlerin inhibit caveolae-mediated endocytosis and micropinocytosis, respectively. [Figure 6] Follistatin expression in skeletal muscle following intravenous administration of a composition containing G1,2,3-RL with D / D and plasmid DNA expressing follistatin. Mice were injected twice with the G1,2,3-RL complex, tissues were harvested, and follistatin mRNA expression levels were assayed by qPCR. [Figure 7] Body weight of mice following intravenous administration of the disclosed compositions. [Figure 8]Luciferase expression in mouse tissues after intravenous administration of a composition containing G1,2,3-RL or G1,2,3-LR with DOTMA / DOPE (w / w=1:1 to DNA) and luciferase-expressing minicircle DNA. The composition was injected at a NP ratio of 20:1. Mice were injected with the composition, and tissues were harvested 24 hours later to measure luciferase signals in heart, kidney, liver, lung, spleen, and skeletal muscle. [Figure 9] Luciferase expression in mouse skeletal muscle after intramuscular administration of a composition comprising G1,2-RL,3-LR with DOTMA / DOPE (w / w=1:1 to DNA) and luciferase-expressing minicircle DNA. The composition was injected at a NP ratio of 20:1. Mice were injected with the composition, and skeletal muscle tissue was harvested 48 hours later to measure luciferase signal. [Figure 10] Luciferase expression in mouse tissues after intravenous administration of compositions containing 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 mRNA expressing luciferase. Each dendrimer composition was injected at NP ratios of 8:1 and 0.15:1. Mice were injected with the compositions and tissues were harvested 6 hours later to measure luciferase signals in muscle (gastrocnemius), liver, lung, heart, spleen, kidney, adipose tissue, brain, cervical lymph nodes, and inguinal lymph nodes. Compositions containing G1,2,3-RL and G1,2-RL,3-LR, when injected at NP ratio of 0.15:1, can deliver mRNA to immune cell-rich tissues including lung, spleen, and lymph nodes. G1,2-RL,3-LR injected with NP8:1 can target delivery of mRNA to the spleen. [Figure 11]Luciferase expression in mouse tissues after intravenous administration of compositions containing 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 mRNA expressing luciferase. Each dendrimer composition was injected at NP ratios of 8:1 and 0.15:1. Mice were injected with the compositions and tissues were harvested 6 hours later to measure luciferase signals in muscle (gastrocnemius), liver, heart, kidney, adipose tissue, and brain. [Figure 12] Comparison of luciferase expression in mouse tissues after intravenous administration of compositions containing G1,2,3-RL or G1,2-RL,3-LR with DOTMA / DOPE (w / w=10:1 relative to mRNA) and mRNA expressing luciferase at an NP ratio of 8:1. Mice were injected with the compositions and 6 hours later tissues were harvested to measure luciferase signal in muscle (gastrocnemius), liver, lung, heart, spleen, kidney, adipose tissue, brain, cervical lymph node, and inguinal lymph node (Figure 12A, upper panel). The same data is presented to allow comparison between lung, cervical lymph node, and inguinal lymph node (Figure 12A, lower panel) and muscle (gastrocnemius), liver, heart, kidney, adipose tissue, and brain (Figure 12B). A composition comprising G1,2-RL,3-LR injected at an NP ratio of 8:1 can deliver mRNA to immune cell-rich tissues with higher efficiency compared to G1,2,3-RL injected at an NP ratio of 8:1. [Figure 13] Luciferase expression in mouse tissues after intravenous administration of two doses of a composition containing G1,2,3-RL with DOTMA / DOPE (w / w=10:1 relative to mRNA) and mRNA expressing luciferase. The dendrimer composition was injected at a NP ratio of 0.15:1. Mice were injected with the composition and 6 hours later tissues were harvested to measure luciferase signals in muscle (gastrocnemius), liver, lung, heart, spleen, kidney, adipose tissue, brain, cervical lymph node, and inguinal lymph node. [Figure 14]Luciferase expression in mouse tissues after intravenous administration of compositions containing G1,2,3-RL, G1,2-RL,3-LR, or NTX1 with DOTMA / DOPE (w / w=10:1 relative to mRNA) and mRNA expressing luciferase. Each dendrimer composition was injected at an NP ratio of 0.15:1. Mice were injected with the compositions and tissues were harvested 6 hours later to measure luciferase signals in muscle (gastrocnemius), liver, lung, heart, spleen, kidney, adipose tissue, brain, cervical lymph node, and inguinal lymph node. A composition containing NTX1 with a skeletal muscle targeting domain and a cell-penetrating peptide domain can deliver mRNA to immune cell-rich tissues with higher efficiency compared to compositions containing G1,2,3-RL and G1,2-RL,3-LR. [Figure 15] Luciferase expression in mouse tissues after intravenous administration of a composition containing G1,2,3-RL, G1,2-RL,3-LR, or NTX1 with DOTMA / DOPE (w / w=10:1 relative to mRNA) and mRNA expressing luciferase, compared to mice injected with a composition containing DOTMA / DOPE (w / w=10:1 relative to mRNA) and mRNA expressing luciferase without dendrimer. Each dendrimer composition was injected at a NP ratio of 0.15:1. Mice were injected with the composition and tissues were harvested 6 hours later to measure luciferase signals in muscle (gastrocnemius), liver, lung, heart, spleen, kidney, adipose tissue, brain, cervical lymph node, and inguinal lymph node. [Figure 16]Luciferase expression in mouse tissues after intravenous administration of a composition containing G1,2-RL,3-LR with DOTMA / DOPE (w / w=10:1 relative to mRNA) and luciferase-expressing mRNA, compared to mice injected with a composition containing DOTMA / DOPE (w / w=10:1 relative to mRNA) and luciferase-expressing mRNA without dendrimer. The dendrimer composition was injected at a NP ratio of 8:1. Mice were injected with the composition and 6 hours later 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. The compositions containing NTX1, G1,2,3-RL, and G1,2-RL,3LR each show increased delivery of mRNA to all tissues compared to compositions containing mRNA alone or compositions containing DOTMA / DOPE and mRNA. [Figure 17]Luciferase and eGFP expression in HeLa or C2C12 cells after transfection with dendrimer compositions or commercial transfection reagents. HeLa cells were transfected with 1) luciferase mRNA alone, a composition comprising G1,2-RL,3-LR (NP=0.16:1), DOTMA / DOPE (w / w=10:1 relative to mRNA) and luciferase mRNA, or a composition comprising Lipofectamine 2000™ and luciferase mRNA (top 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-MC3-DMA:Cholesterol:DSPC:DMG-PEG lipid nanoparticles and eGFP mRNA (top right panel). Luciferase mRNA alone, G1,2-RL,3-LR (NP=8:1), DOTMA / DOPE (w / w=10:1 relative to mRNA), and a composition comprising luciferase mRNA, Lipofectamine 2000™ and luciferase mRNA, or polyethylenimine and luciferase mRNA (lower panel) were transfected into C2C12 cells. Luciferase and eGFP expression was measured 24 hours after transfection. The peptide dendrimers of the present invention are more efficient in transfecting HeLa cells in vitro compared to commercially available transfection reagents such as Lipofectamine 2000 and LNP. [Figure 18]RNA transfection efficiency in HeLa cells after transfection with fluorescently labeled sgRNA and Cas9 mRNA or G1,2-RL,3-LR together with fluorescently labeled sgRNA and Cas9 mRNA at NP ratios of 0.15:1 or 8:1. sgRNA was labeled with TM-rhodamine and Cas9 mRNA was labeled with Cy5 fluorophore. Cells were transfected for 2 hours and assayed by flow cytometry. 100% of HeLa cells exposed to the composition containing G1,2-RL,3LR were double positive for sgRNA and Cas9 mRNA, demonstrating extremely high transfection efficiency. [Figure 19] RNA transfection efficiency in HeLa cells after transfection with tracrRNA (trRNA), crRNA, and Cas9 mRNA, or G1,2-RL,3-LR together with tracrRNA, crRNA, and Cas9 mRNA at NP ratios of 0.15:1 or 8:1. TracrRNA was labeled with ATTO 55 fluorophore. crRNA was labeled with fluorescein and Cas9 mRNA was labeled with Cy5 fluorophore. Cells were transfected for 2 hours and assayed by flow cytometry. [Figure 20] RNA transfection efficiency in HeLa cells after transfection with either tracrRNA (trRNA), crRNA, or Cas9 mRNA. Cells were transfected with G1,2-RL,3-LR or not. tracrRNA was labeled with ATTO 55 fluorophore. crRNA was labeled with fluorescein and Cas9 mRNA was labeled with Cy5 fluorophore. Cells were transfected for 2 hours and assayed by flow cytometry. [Figure 21]mRNA delivery to immune cells in the spleen. Mice were intravenously injected with either mRNA alone or mRNA and either 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 AlexaFluor488. Spleens were harvested from mice 2 hours after injection for processing. Delivery of mRNA to a wide range of immune cells present in the spleen is possible using a composition containing NTX1, G1,2-RL,3-LR (NP8:1), or G1,2-RL,3-LR (NP0.15:1). [Figure 22] mRNA delivery to immune cells in bone marrow. Mice were intravenously injected with mRNA alone or with either mRNA and 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 AlexaFluor488. Bone marrow was harvested from mice 2 hours after injection for processing. [Diagram 23] Delivery of mRNA to immune cells in blood. Mice were intravenously injected with either mRNA alone or mRNA and either 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 AlexaFluor488. Blood was collected from mice 2 hours after injection for processing. Delivery of mRNA to a wide range of immune cells present in blood is possible using a composition containing NTX1, G1,2-RL,3-LR (NP8:1), or G1,2-RL,3-LR (NP0.15:1). [Figure 24] mRNA delivery to immune cells in lymph nodes. Mice were intravenously injected with mRNA alone or with either mRNA and 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 AlexaFluor488. Lymph nodes were harvested from mice 2 hours after injection for processing. [Diagram 25]In vitro DNA delivery into HeLa and C2C12 cells. HeLa cells were transfected with either DNA alone, G1,2,3-RL (N:P=5:1) and DOTMA / DOPE (w / w=1:1 to DNA) with DNA, or G1,2,3-RL (N:P=5:1) and DOPG / DOPE (w / w=1:1 to DNA) with DNA (top panel). C2C12 cells (a muscle-derived cell line) were transfected with either DNA alone, polyethylenimine with DNA, G1,2-RL,3-LR (N:P=8:1) and DOTMA / DOPE (w / w=1:1 to DNA) with DNA, or NTX2 (N:P=8:1) and DOTMA / DOPE (w / w=1:1 to DNA) with DNA (bottom panel). The DNA used in both experiments encodes a luciferase reporter gene. NTX2 is a G1,2-RL,3-LR dendrimer conjugated to the muscle-targeting peptide ASSLINA ((LR)8(KRL)4(KRL)2KGSCGAASSLNIA(Acp)-NH2). [Figure 26] Levels of aspartate transaminase (AST, 26A, top panel), TNF-α (26A, bottom panel), IL-6 (26B, top panel), and IL-1β (26B, bottom panel) after administration of mRNA formulations. Mice were treated with either mRNA alone or mRNA, dendrimer, and DOTMA / DOPE (w / w=10:1 relative to mRNA). The N:P ratio of dendrimer to mRNA is shown on the X-axis. All formulations were administered once, except for G1,2,3-RL and G1,2-RL,3-LR, which were administered twice. For mice injected with two doses, the first dose was injected 24 hours before the second dose. Plasma of mice was collected 6 hours after the second dose for measurement of AST or cytokine levels. For mice administered once, plasma was collected 6 hours after administration for measurement of AST and cytokine levels. The use of the dendrimers of the present invention to deliver mRNA in vivo does not cause significant liver toxicity or significant immune responses. [Figure 27]Levels of aspartate transaminase (top left panel), TNF-α (top right panel), IL-6 (bottom left panel), and IL-1β (bottom right panel) after administration of DNA formulations. Mice were treated with either DNA alone or G1,2,3-RL and DOTMA / DOPE (w / w=1:1 to DNA). Plasma samples were taken after two doses of all formulations and AST or cytokine levels were measured. The use of the dendrimers of the present invention to deliver DNA in vivo does not cause significant liver toxicity or significant immune responses. [Figure 28] Lipid-based mRNA transfection of peptide dendrimers in vitro. HeLa cells were transfected with either mRNA alone or with peptide dendrimers (N:P=0.16:1) and DOTMA / DOPE (w / w=10:1 relative to mRNA). The mRNA used expressed the eGFP reporter gene. After 24 hours of transfection, cells were harvested and assayed for eGFP expression. eGFP expression was normalized to the total amount of protein for each transfection. Gene expression levels were calculated by normalizing the transfection values ​​of G1,2-RL,3-LR, DOTMA / DOPE, and mRNA-treated cells. First-, second-, and third-generation dendrimers with various cores and first-, second-, and third-generation peptide motifs could effectively transfect cells with mRNA under the conditions tested. [Figure 29]Lipid-based mRNA transfection of peptide dendrimers in vitro. HeLa cells were transfected with either mRNA alone or 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, whereas the N:P ratio of all other dendrimers was 8:1. The mRNAs used expressed the eGFP reporter gene. After 24 hours of transfection, cells were harvested and assayed for eGFP expression. eGFP expression was normalized with the total amount of protein for each transfection. Gene expression levels were calculated by normalizing the transfection values ​​of G1,2-RL,3-LR, DOTMA / DOPE, and mRNA-treated cells. First-, second-, and third-generation dendrimers with various cores and first-, second-, and third-generation peptide motifs can effectively transfect cells with mRNA under the conditions tested. [Diagram 30] Luciferase expression in mouse tissues after intravenous administration of compositions containing G1,2-RL,3-LR or RHCG1-RL,2-LR with DOTMA / DOPE (w / w=10:1 relative to mRNA) and mRNA expressing luciferase. The dendrimer compositions were injected at either an N:P ratio of 0.15:1 or 8:1. Mice were injected with the compositions and 6 hours later 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 can target mRNA delivery to the spleen and lungs with higher efficiency than the third generation dendrimer G1,2-RL,3-LR. [Diagram 31] A third generation dendrimer is represented diagrammatically (with the N-terminus on the left and the C-terminus on the right). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0133] Aspects and embodiments of the present invention are described with reference to the figures identified above and the technical definitions that follow. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated by reference.

[0134] Current immunotherapy response rates are around 15-20%, and there is an urgent need to improve treatment outcomes. One strategy is to deliver mRNA to express proteins (such as CEBPA, IRF8, cGAS-STING, SOCS1, and / or SOCS3) to revert the immunosuppressive phenotype of myeloid cells in the tumor microenvironment, which provides a more favorable environment for immunotherapy to be responsive. Another strategy could be to transfer mRNA into immune cells in the tumor to express cytokines (such as IL-2, IL-7, IL-12, IL-15, IL-21, and / or interferons) to activate the immune cells to fight cancer cells. It is also possible to deliver mRNA to macrophages to express chimeric antigen receptors so that the macrophages can be activated to kill tumor cells. Delivering mRNA to express tumor antigens in antigen-presenting cells helps activate the immune system to attack cancer cells. These strategies can be applied to treat all tumors, especially non-small cell lung cancer, advanced melanoma, prostate cancer, ovarian cancer, breast cancer, lung cancer, bile duct cancer (Cholangiocarcinoma), gallbladder cancer, neuroendocrine tumors, hepatocellular carcinoma, colorectal cancer, pancreatic cancer, and solid tumors.

[0135] Nucleic Acid Delivery The compositions of the invention can be used to deliver the nucleic acids described herein to specific tissues of the human or animal body, for example to the following tissues: 1. Skeletal Muscle 2. Liver 3. Lungs 4. Heart 5.White adipose tissue 6.Brown adipose tissue 7. Brain 8. Spleen 9. Bone Marrow 10. Joints 11. Kidneys 12.Gastrointestinal tract 13. Tumor 14.Eye 15.Thymus 16.Skin 17. Lymph nodes 18. Pancreas 19. Adrenal Glands 20. Testes 21. Prostate 22. Ovaries 23. Uterus 24. Bladder 25. Diaphragm

[0136] mRNA transfection The effect of dendrimer generation number on mRNA delivery was studied by transfecting HeLa cells in full growth medium conditions. We showed that first or second generation dendrimers (e.g., RHCG1-R, RHCG1,2-R, RHCG1-LR, RHCG1-RL, 2-LR) can effectively transfect cells and mediate similar transfection efficiency as third generation dendrimers (Figure 28A). This is indeed very different from DNA transfection, since first or second generation dendrimers do not transfect cells as well as third generation dendrimers in full growth medium (Figure 4). Interestingly, increasing the N:P ratio of generation 1 or generation 2 dendrimers from 0.16 to 8 further increases the transfection efficiency (Figure 29A).

[0137] We have studied the effect of the dendrimer core sequence on transfection and shown that tripeptide sequences such as GSC (e.g., derived 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 does not affect mRNA transfection, suggesting that dendrimers with two amino acids in the core transfect similarly to dendrimers with three amino acids in the core (Figure 28A). Different tripeptide core sequences such as RFW, RYM perform comparably to GSC. An arginine containing a cationic group is added to the core, but this arginine does not improve or decrease transfection. Interestingly, when three amino acids are changed in the core from GCS to RHC, transfection can be improved by 50%. This suggests that ionizable groups such as histidine in the dendrimer core may improve transfection (Figure 28A).

[0138] The use of a 12 amino acid core sequence (e.g., the dendrimer "Linear GI,2-RL,3-LR") demonstrates that the transfection effect is not affected by the increase in the length of the core sequence (Figure 28A). This result indicated that it is possible to introduce longer amino acid chains and / or longer structures into the core without affecting the transfection efficiency. Indeed, NTX1, which contains 27 amino acids in the core, transfects more effectively than G1,2-RL,3-RL. This demonstrates that a longer core sequence can indeed enhance transfection. The enhanced transfection from NTX1 is likely due to the presence of a cell-penetrating peptide in the core sequence.

[0139] We investigated the effect of the number of amino acids in a generation on transfection. Thus, we tested dendrimers with only one amino acid (R), two amino acids (RL or LR), three amino acids (RLR), and four amino acids (LRLR). These studies showed that dendrimers with one, two, three, or four amino acids in each generation were still able to transfect mRNA well into cells (Figure 28A). It was shown that the transfection efficiency was not affected when dendrimers with different numbers of amino acids in each generation were used.

[0140] Based on the G1,2-RL,3-LR structure, we designed a library of third generation dendrimers in which the basic amino acid R was replaced by K and / or the hydrophobic amino acid L was changed to an acidic amino acid such as E and / or an amino acid with a non-polar side chain such as M, F, beta-alanine (B), aminohexanoic acid (X), and W, and / or an amino acid with a polar side chain such as Q, T, and Y.

[0141] Substituting R for K in the dendrimer reduces mRNA transfection efficiency (Figure 28A). However, changing the hydrophobic amino acids to other hydrophobic amino acids and / or amino acids with polar or non-polar side chains may have no effect on transfection or may reduce transfection by 30-40% compared to G1,2-RL,3-LR. However, all of these dendrimers are able to induce significantly higher mRNA transfection than the mRNA alone control.

[0142] We also investigated the effect of L- or D-type amino acids within the dendrimer on mRNA transfection. Based on the G1,2-RL,3-LR dendrimer, we found that changing some or all of the amino acids from L-type to D-type in each generation of the dendrimer did not affect transfection efficiency. Substituting diaminobutyric acid for lysine within the dendrimer reduced transfection, but the transfection of this dendrimer was still significantly higher than the mRNA-only control.

[0143] We have demonstrated that G1-LL,2-RR can be used to deliver mRNA using our formulation protocol, where G1-LL,2-RR was used with DOTMA / DOPE (w / w 10:1) at an N:P ratio of 0.16:1. Interestingly, this dendrimer was used to deliver ASO in different formulations in vitro and in vivo (Saher 2018). The formulations used were DOTMA / DOPE (w / w=2:1 for ASO) and N:P=20:1, G1-LL,2-RR vs. ASO. We have attempted this to transfect cells (i.e., DOTMA / DOPE (w / w=2:1 for mRNA) and N:P=20:1, G1-LL,2-RR vs. mRNA), but transfection efficiency is low. The mRNA transfection efficiency of this formulation (DOTMA / DOPE (w / w=2:1 relative to mRNA) and N:P=20:1, G1-LL,2-RR to mRNA) was only 10% of that of our improved formulation (DOTMA / DOPE (w / w=10:1 relative to mRNA) and N:P=0.16:1, G1-LL,2-RR to mRNA).

[0144] We investigated three generations of dendrimers with either RL or LR or rl in different generations for mRNA delivery. We found that most of these dendrimers transfected cells similarly, with G1,2-RL,3-LR being the most effective in mRNA transfection. Overall, our data suggested that dendrimers with hydrophobic and cationic amino acids in each generation provided effective mRNA delivery to cells.

[0145] Since there are first and second generation dendrimers that transfect cells more effectively than G1,2-RL,3-LR, we selected these dendrimers for further testing on transfection. These dendrimers can generally transfect better than G1,2-RL,3-LR at various N:P ratios. In particular, RHCG1-R, RHCG1,2-R, RHCG1-LR, RHCG1-RL,2-LR, RHCG1-RLR, and G1-LRLR with N:P=4:1 can transfect 700%-1815% better than G1,2-RL,3-LR. These data demonstrated that the requirements for DNA and mRNA delivery are very different. For mRNA, first or second generation dendrimers tend to transfect cells better than third generation dendrimers in full growth medium conditions. However, generation 3 dendrimers transfect cells with DNA better than either generation 1 or generation 2 dendrimers under full growth medium conditions (Figure 4).

[0146] Specific dendrimers of the invention that are particularly preferred 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-RL.

[0147] RNA interference The present invention promotes therapeutic downregulation of target gene expression by delivery of nucleic acids. These include RNA interference (RNAi). Small RNA molecules may be used to regulate gene expression.

[0148] These include targeted degradation of mRNA by small interfering RNA (siRNA), post-transcriptional gene silencing (PTGs), developmentally regulated sequence-specific translational repression of mRNA by micro-RNA (miRNA), and targeted transcriptional gene silencing.

[0149] The role of the RNAi machinery and small RNAs in targeting heterochromatin complexes and epigenetic gene silencing at specific chromosomal loci has also been demonstrated. Double-stranded RNA (dsRNA)-dependent posttranscriptional silencing, also known as RNA interference (RNAi), is a phenomenon in which dsRNA complexes can target specific genes of homology for silencing in a short period of time. This acts as a signal to promote the degradation of mRNAs with sequence identity. 21-nt siRNAs are generally long enough to induce gene-specific silencing, yet short enough to avoid a host response. The reduction in expression of the target gene product can be extensive, with 90% silencing induced by a few molecules of siRNA.

[0150] In the art, these RNA sequences are referred to as "short or small interfering RNA" (siRNA) or "microRNA" (miRNA), depending on their origin. Both types of sequences can be used to downregulate gene expression by binding to complementary RNA and inducing mRNA elimination (RNAi) or halting the translation of mRNA into protein. siRNAs are derived by processing of long double-stranded RNAs and, when found in nature, are typically of exogenous origin. Microinterfering RNAs (miRNAs) are endogenously encoded small non-coding RNAs that are derived by processing of short hairpins. Both siRNAs and miRNAs can inhibit the translation of mRNAs with partially complementary target sequences without RNA cleavage and degrade mRNAs with fully complementary sequences.

[0151] Thus, the present invention provides for the use of these sequences in the compositions of the invention to downregulate the expression of target genes.

[0152] To optimize the efficacy of RNA-mediated downregulation of target gene function, the siRNA ligand is typically double-stranded, and the length of the siRNA molecule is preferably selected to ensure accurate recognition of the siRNA by the RISC complex, which mediates siRNA recognition of the mRNA target, and such that the siRNA is short enough to reduce the host response.

[0153] miRNA ligands are typically single-stranded and have a partially complementary region that allows the ligand to form a hairpin. miRNAs are RNA genes that are transcribed from DNA but are not translated into protein. The DNA sequence that codes for the miRNA gene is longer than the miRNA. This DNA sequence contains the miRNA sequence and an approximate reverse complement. When this DNA sequence is transcribed into a single-stranded RNA molecule, the miRNA sequence and its reverse complement base pairs to form a partially double-stranded RNA segment. The design of microRNA sequences is discussed in John et al, 2004.

[0154] Typically, RNA ligands intended to mimic the effect of siRNA or miRNA have 10-40 ribonucleotides (or synthetic analogs thereof), more preferably 17-30 ribonucleotides, more preferably 19-25 ribonucleotides, and most preferably 21-23 ribonucleotides. In some embodiments of the invention using double-stranded siRNA, the molecules may have, for example, symmetric 3' overhangs of one or two (ribo)nucleotides, typically UU of dTdT 3' overhangs. Based on the disclosure provided herein, one of skill in the art can readily design appropriate siRNA and miRNA sequences using resources such as, for example, Ambion's online siRNA finder. siRNA and miRNA sequences can be synthetically produced and exogenously added to cause gene downregulation, or can be produced using an expression system (e.g., vector). In a preferred embodiment, the siRNA is synthetically synthesized.

[0155] The longer double-stranded RNA may be processed intracellularly to produce siRNA (see, for example, Myerset et al (2003)). The longer dsRNA molecule may have, for example, symmetric 3' or 5' overhangs of one or two (ribo)nucleotides, or may have blunt ends. The longer dsRNA molecule may be 25 nucleotides or more. Preferably, the longer dsRNA molecule is 25-30 nucleotides long. More preferably, the longer dsRNA molecule is 25-27 nucleotides long. Most preferably, the longer dsRNA molecule is 27 nucleotides long.

[0156] In one embodiment, siRNA, longer dsRNA, or miRNA is produced endogenously (intracellularly) by transcription from a vector. The vector may be introduced into a cell by any method known in the art. If necessary, the expression of the RNA sequence can be controlled using a tissue-specific promoter. In a further embodiment, siRNA, longer dsRNA, or miRNA is produced exogenously (in vitro) by transcription from a vector.

[0157] Alternatively, siRNA molecules can be synthesized using standard solid-phase or liquid-phase synthesis techniques known in the art. The linkage between nucleotides can be a phosphodiester bond or alternative, for example, a linkage group of the formula P(O)S, (thioate); 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 attached to adjacent nucleotides via -O- or -S-.

[0158] Long non-coding RNA Mammalian genomes are extensively transcribed, producing a vast number of transcripts, including thousands of long non-coding RNA molecules (lncRNAs), which have been shown to be capable of regulating the chromatin state, transcription, RNA stability, and translation of specific genes.

[0159] RNA activation (RNAa) RNA activation (RNAa) is a process mediated by RNA to enhance gene expression through a highly regulated and evolutionarily conserved pathway. RNAa can be induced by small activating RNAs (saRNAs), a class of non-coding RNAs consisting of 21-nucleotide dsRNA with two-nucleotide overhangs at both ends. Despite the fact that saRNAs mediate gene activation in a sequence-specific manner, saRNAs have identical structural and chemical components to siRNAs. To activate gene expression, the guide strand of saRNA is loaded into AGO2, which then transports the complex to the nucleus. Once inside the nucleus, the guide strand-AGO2 complex directly binds to gene promoters or associated transcripts and recruits key components, including RNA polymerase II, to initiate gene activation (Kwok et al. 2019).

[0160] Antisense Oligonucleotides (ASOs) Antisense oligonucleotides (ASOs) are single strands of DNA or RNA that are complementary to a target sequence. ASOs hybridize with target nucleic acids. For example, ASOs can be used to target coding or non-coding RNA molecules in cells. After target binding, ASO / target complexes can be enzymatically degraded, for example, by RNase H.

[0161] Messenger RNA (mRNA) Those skilled in the art are aware that messenger RNA (mRNA) is a single-stranded molecule of RNA that takes the coding sequence of a gene to be translated by ribosomes into the corresponding amino acid sequence. mRNA is generated during the transcription process, where an enzyme (RNA polymerase) converts a gene into the primary transcript mRNA (also known as pre-mRNA). This pre-mRNA usually still contains introns, which are regions that do not code for the final amino acid sequence. These are removed during the process of RNA splicing, leaving only exons, which are regions that code for proteins. This exon sequence constitutes the mature mRNA. The mature mRNA is then read by ribosomes, thereby producing the encoded protein. The present 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.

[0162] mRNA and lncRNA are typically large molecules with negatively charged and hydrophobic sides. Therefore, mRNA and lncRNA 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 is different from double-stranded nucleic acids such as pDNA and siRNA, which have charges on both sides. Since mRNA and lncRNA are significantly larger than, for example, ASOs, the requirements for encapsulation and delivery are likely to be different. Therefore, the optimal NP ratio of dendrimers and w / w ratio of DOTMA / DOPE for mRNA and lncRNA delivery are different compared to ASO delivery.

[0163] modified nucleic acid Modified nucleotide bases can be used in addition to the naturally occurring bases and can impart advantageous properties to nucleic acids containing them.

[0164] For example, modified bases can increase the stability of a nucleic acid molecule, thereby reducing the amount required. The provision of modified bases can also provide a nucleic acid molecule that is more or less stable than an unmodified nucleic acid.

[0165] The term "modified nucleotide base" encompasses nucleotides having covalently modified bases and / or sugars. For example, modified nucleotides include nucleotides having sugars covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position. Thus, modified nucleotides can also include 2'-substituted sugars, such as 2'-O-methyl-; 2'-O-alkyl; 2'-O-allyl; 2'-S-alkyl; 2'-S-allyl; 2'-fluoro-; 2'-halo- or azido-ribose, carbocyclic sugar analogs, α-anomeric sugars; epimeric sugars, such as arabinose, xylose or lyxose, pyranose sugars, furanose sugars, and sedoheptulose.

[0166] 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-ethanocytosine, 8-hydroxy-N6-methyladenine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentyl-adenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl- Examples of such uracil include 2-thiouracil, -D-mannosyl euosine, 5-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methyl ester, psueouracil, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, queosine, 2-thiocytosine, 5-propyluracil, 5-propylcytosine, 5-ethyluracil, 5-ethylcytosine, 5-butyluracil, 5-pentyluracil, 5-pentylcytosine, and 2,6,diaminopurine, methylpseudouracil, 1-methylguanine, and 1-methylcytosine.

[0167] Nucleic Acid-Based Vaccines DNA vaccines, as defined by the World Health Organisation (WHO), and RNA vaccines rely on the in situ production of target antigens, involving the direct introduction into the appropriate tissues (of the subject to be vaccinated) of plasmids containing DNA sequences or RNA encoding the antigen against which an immune response is sought. These approaches offer many potential advantages over traditional approaches, including stimulation of both B and T cell responses, improved vaccine stability, the absence of any infectious agents, and the relative ease of large-scale manufacture. As proof of 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, protection from disease in animals has also been obtained. However, the value and benefits of DNA vaccines must be evaluated on a case-by-case basis, and their applicability depends on the nature of the agent against which it is immunised, the nature of the antigen, and the type of immune response required for protection.

[0168] The field of DNA and RNA vaccination is rapidly evolving. Vaccines currently being developed not only use DNA, but also include adjuncts that can assist the DNA to enter cells and target it to specific cells, or act as adjuvants to stimulate or direct an immune response. As of 2020, the WHO noted that while the first nucleic acid vaccines approved for commercial use will likely use plasmid DNA derived from bacterial cells, in the future, other vaccines may use RNA or a complex of nucleic acid molecules and other entities. However, with the onset of the COVID-19 pandemic in 2020, a concerted effort was made to bring the first RNA-based COVID-19 vaccines to market, and these were approved for use in mid-to-late 2020. Since being approved, these RNA-based vaccines have been successfully deployed around the world to immunize populations against COVID-19.

[0169] Intramuscular delivery of DNA vaccines is a common approach, in common with other vaccine technologies (Lim et al., 2020). This makes them an attractive target for DNA vaccination due to the low replication rate of myocytes (muscle cells) in skeletal muscles, as stable expression does not depend on genomic integration.

[0170] Currently commercially available RNA vaccines use mRNA encoding antigens as the payload. An area currently being explored to increase the efficacy of RNA vaccines is the use of self-amplifying RNA. Self-amplifying RNA shares many of the structural attributes of mRNA and may include a 5' cap, a 3' polyA tail, and 5' and 3' untranslated regions (UTRs). In addition to encoding an antigen of interest, the self-amplifying RNA also contains a system for self-amplification. For example, the self-amplifying RNA may also encode an RNA-dependent RNA polymerase (RDRA), a promoter, and the antigen of interest.

[0171] Upon translation of RDRA by the subject translation machinery, RDRA can associate with self-amplifying RNA and replicate the RNA. Including a system for self-amplification reduces the minimum RNA required in a vaccine, and as a result, reduces the likelihood that a subject will experience side effects.

[0172] Medical Therapy: Gene Therapy The present invention contemplates use in gene therapy regimens. The nucleic acid may be in a composition that, when introduced into a target cell, results in expression of a therapeutic gene product, e.g., a transgene. Target cells include muscle cells, liver cells, stellate cells, brain cells (neurons, astrocytes), splenocytes, lung cells, cardiomyocytes, 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.

[0173] For gene therapy to be practical, it is desirable to employ a DNA / RNA transfer system that (1) directs therapeutic sequences to target cells, (2) mediates uptake of the therapeutic nucleic acid into a portion of the target cell population, and (3) is suitable for use in vivo for therapeutic applications.

[0174] The nucleic acid-containing composition of the present invention can be stored and administered in a sterile pharma- ceutically acceptable carrier. A variety of sterile solutions can be used, including water, PBS, ethanol, lipids, etc. The concentration of DNA / RNA is sufficient to provide a therapeutic dose, which depends on the efficiency of delivery to cells.

[0175] The actual delivery of the gene sequences formulated as described above can be accomplished by a variety of techniques including direct injection, instillation of the lung and other epithelial surfaces, or intravenous injection. Administration can be by needle, trocar, cannula, catheter, etc., as a bolus, multiple doses, or continuous infusion, etc.

[0176] 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., the CRISPR / Cas9 system), TALENS and zinc finger nucleases.

[0177] In some embodiments, the CRISPR / Cas system comprises a Cas nuclease, a crispr RNA (crRNA), and a trans-activating crRNA (trRNA or tracrRNA). In this system, the crRNA comprises a sequence complementary to the target DNA and serves 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 the target gene. The sgRNA comprises a target-specific crRNA fused to the scaffold tracrRNA in a single nucleic acid.

[0178] In some embodiments, the nucleic acid comprises DNA or mRNA encoding a Cas protein or peptide, such as a Cas9 protein or peptide. In some embodiments, the nucleic acid comprises an sgRNA. In some embodiments, the nucleic acid comprises a crRNA and / or a tracrRNA. In some embodiments, the nucleic acid comprises DNA or mRNA encoding a Cas protein or peptide, a crRNA, and a tracrRNA. In some embodiments, the nucleic acid comprises DNA or mRNA encoding a Cas protein or peptide and an sgRNA.

[0179] CRISPR / Cas systems can also be used to direct the repair or modification of target genes. For example, CRISPR / Cas systems can include nucleic acid templates for facilitating DNA repair or for introducing exogenous nucleic acid sequences into target genes, for example by facilitating homologous recombination 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 accomplished using a Cas protein fused to a base editor, such as cytidine deaminase, as disclosed, for example, in WO 2017 / 070633(A2) (incorporated by reference). In another example, CRISPR / Cas systems can be used to "rewrite" nucleic acid sequences in a genome. For example, CRISPR / Cas systems can be prime editing systems. In such prime editing systems, fusion proteins can be used. For example, the fusion protein can include a catalytically impaired Cas domain (e.g., "nickase") and a reverse transcriptase. The catalytically impaired Cas domain may be capable of cleaving a single strand of DNA to generate a nicked DNA duplex. The prime editing system may include a prime editing guide RNA (pegRNA) that includes an extended sgRNA that includes a primer binding site and a reverse transcriptase template sequence. Upon nicking of the DNA duplex by the catalytically impaired 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 the synthesis of the edited genetic information.

[0180] In some embodiments, the CRISPR / Cas gene editing system may include a nucleic acid template that directs the repair of a target gene of interest. In other embodiments, the Cas protein or peptide may include a base editor. In yet further embodiments, the CRISPR / Cas system may be a primed editing system.

[0181] CRISPR / Cas gene silencing and gene activation CRISPR / Cas systems have been adapted for use in gene silencing and activation. Such systems are envisioned for use in the present invention. For example, in some embodiments, the nucleic acid may encode a fusion protein comprising a Cas protein or peptide fused to a transcriptional repressor or activator. In some embodiments, the Cas protein is catalytically inactive. The fusion protein can be directed to a site of interest in the genome by either the sgRNA or the crRNA. Upon binding of the fusion protein to the site of interest, the transcriptional repressor or activator can control expression of the gene of interest.

[0182] Combination therapy The compounds of the present invention or compounds identified by the methods of the present invention can be used to treat tumors and cancer in subjects in need of such treatment. The compounds may be administered alone or in combination with other anti-cancer agents.

[0183] "Anti-cancer agent" refers to any agent useful in the treatment of a neoplastic condition. One class of anti-cancer agents includes chemotherapeutic agents. "Chemotherapy" refers to the administration of one or more chemotherapeutic 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 a suppository. Some chemotherapeutic agents are cytotoxic.

[0184] Cytotoxic chemotherapeutic agents induce cell death through mechanisms or means that are not receptor-mediated. Cytotoxic chemotherapeutic agents induce cell death by interfering with cell division, metabolism, or functions necessary for cell survival. Due to this mechanism of action, cells that are growing (meaning they are proliferating or dividing) or are metabolically active die preferentially over cells that are not. The status of different cells in the body as division or energy use (which is metabolic activity that supports the function of the cells) determines the dose of chemotherapeutic agent that will cause cell death. Cytotoxic chemotherapeutic agents non-exclusively relate to alkylating agents, antimetabolites, plant alkaloids, topoisomerase inhibitors, antineoplastic agents, and arsenic trioxide, carmustine, fludarabine, IDA ara-C, myalotang, GO, mustargen, cyclophosphamide, gemcitabine, bendamustine, total body radiation, cytarabine, etoposide, melphalan, pentostatin, and radiation.

[0185] Anticancer drugs also include protein kinase inhibitors, which can be used to treat a wide range of cancers, including blood and lung cancers.Protein kinases typically promote cell proliferation, survival, and migration, and are often constitutively overexpressed or active in cancer.Therefore, protein kinase inhibitors are common drug targets in the treatment of cancer.Examples of kinase inhibitors for clinical use include crizotinib, ceritinib, alectinib, brigatinib, bosutinib, dasatinib, imatinib, nilotinib, ponatinib, vemurafenib, dabrafenib, ibrutinib, palbociclib, sorafenib, and ribociclib.

[0186] Anti-cancer drugs also include agents for use in immunotherapy, including antibodies. Immunotherapy can induce, amplify, reduce, or suppress immune responses depending on the specific disease situation. For example, tumor cells expressing PDL1 ligands suppress normal immune responses in subjects by binding to the 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-PDL1 antibodies have been successfully used in clinics to inhibit this immune checkpoint and promote immune cell-mediated death of tumor cells. Other examples of immunotherapy include oncolytic virus therapy, T cell therapy, and cancer vaccines.

[0187] Pharmaceutical Compositions The pharmaceutical compositions provided herein may include one or more pharma- ceutically acceptable excipients, such as solvents, dissolution enhancers, suspending agents, buffers, tonicity agents, antioxidants, or antimicrobial preservatives. "Pharmaceutically acceptable" refers to molecular entities and compositions that are "generally regarded as safe", e.g., those that are physiologically tolerated and typically do not produce allergic or similar adverse reactions, such as stomach upset, when administered to humans. In some embodiments, the term refers to molecular entities and compositions that have been approved by a U.S. federal or state government regulatory agency as GRAS listed under Sections 204(s) and 409 of the Federal Food, Drug and Cosmetic Act, which are subject to premarket review and approval by the FDA or a similar listing, the U.S. Pharmacopeia, or another generally recognized pharmacopoeia for use in animals, more specifically in humans. When used, the excipients of the composition do not adversely affect the stability, bioavailability, safety, and / or efficacy of the active ingredient. Thus, one of ordinary skill in the art will appreciate that a composition is provided in which there is no incompatibility between any of the components of the dosage form. The excipient may be selected from the group consisting of a buffering agent, a tonicity agent, a chelating agent, an antioxidant, an antimicrobial agent, and a preservative.

[0188] Therapeutic Product Expression The nucleic acid delivered by the composition of the present invention may exhibit a therapeutic effect (e.g., by acting to directly down-regulate or up-regulate a target gene) or may express a gene product (which may be a therapeutic protein or a therapeutic nucleic acid) via an expression cassette that includes a coding sequence operably linked to a promoter. As used herein, the term "operably linked" may include a situation in which a selected nucleotide sequence and a control nucleotide sequence are covalently linked in such a manner that the expression of the coding sequence is under the influence or control of the control sequence. Thus, a control sequence is operably linked to a selected nucleotide sequence if the control sequence is capable of effecting the transcription of the coding sequence that forms part or all of the selected nucleotide sequence. If appropriate, the resulting transcript may then be translated into a desired protein or polypeptide.

[0189] Route of administration Compositions according to aspects of the invention can be formulated for administration by several routes, including, but not limited to, intravenous, parenteral, intraarterial, intramuscular, intratumoral, subcutaneous, oral, and intranasal. The compositions can be administered by injection into a human or animal subject, for example, by intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intratumoral injection.

[0190] 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 more preferably a human. The subject may be male or female. The subject may be a patient. The therapeutic use may be in humans or animals (veterinary use). ***

[0191] The features disclosed in the foregoing description, the following claims, or the accompanying drawings, expressed in a particular form, or as means for performing a disclosed function, or as a method or process for obtaining a disclosed result, may, as appropriate, be utilized separately or in any combination of such features to realize the invention in diverse forms thereof.

[0192] While the present invention has been described in conjunction with the exemplary embodiments set forth above, many equivalent modifications and variations will become apparent to those skilled in the art upon receipt of this disclosure. Accordingly, the exemplary embodiments of the present invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the present invention.

[0193] For the avoidance of doubt, any theoretical explanations provided herein are provided for the purpose of enhancing the understanding of the reader, and the inventors do not wish to be bound by any of these theoretical explanations.

[0194] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0195] Throughout this specification, including the claims, unless the context requires otherwise, the terms "comprise" and "include," as well as variations such as "comprises," "comprising," and "including," 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 should be noted that as used in this 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 reference to numerical values ​​is optional and means, for example, + / - 10%. EXAMPLES

[0196] Example 1 - Gene delivery using peptide dendrimers displaying cationic and hydrophobic residues in combination with lipids Introduction Cationic lipid-based nucleic acid delivery systems are among the most studied and efficient non-viral vector platforms described to date, and the rational design and development of peptide vectors with natural amino acids is particularly attractive for therapeutic applications due to the non-toxic nature of amino acids. We have developed a structural framework for nucleic acid delivery using peptide dendrimers. The structural framework comprises a three-layer peptide (or dipeptide) motif linked to lysine residues. We have found that the distribution of cationic amino acid residues (Lys or Arg) in each generation (layer) results in peptide dendrimers that transfect more efficiently than dendrimers with charges localized only on the surface (Kwok et al, 2013). Using a solid-phase peptide dendrimer synthesis procedure, we can precisely engineer the position of every amino acid residue incorporated within the dendritic scaffold. This allows for greater control of dendrimer structure and function, which was not usually possible with previously studied systems such as polymers or other dendrimers, where modifications were primarily performed on the surface of the molecule. The peptide dendrimer / lipid vectors demonstrated high transfection efficiency, good reproducibility of results, and low toxicity.

[0197] We hypothesized that (1) dendritic backbone, (2) dendrimer compactness, (3) dendritic size, (4) dendrimer building block chirality, and (5) amino acid composition within the dendrimer may be important parameters for improving gene transfer. We investigated structural attributes by generating a new dendrimer library based on the effective G1,2,3-KL with alternating LysLeu motifs and found that transfection activity was generation dependent, with three generations being optimal for transfection. Although achieving a uniform charge distribution in each generation is important, the exact location of the charge within each generation is not critical. Some activity was observed when dendrimers with different chirality and different numbers of amino acids within each generation were used. However, changing the cationic amino acid from Lys to Arg (i.e., LysLeu to ArgLeu motif) within the dendrimer improved transfection in serum conditions. This allows in vivo gene transfer via systemic delivery and correlates with the improved stability of the dendrimer-DNA complex. The G1,2,3-RL complex is effective in gene delivery to a panel of tissues.

[0198] result The role of molecular recognition units in determining the efficacy of nucleic acid delivery To evaluate the effect of fine-tuning the molecular recognition units of dendrimers for DNA packaging and delivery while keeping a constant balance of hydrophobic and cationic properties, we investigated dendrimers with different (1) backbones, (2) compactness, (3) size, (4) chirality in building blocks, and (5) amino acid composition based on derivatives of G1,2,3-KL. Dendrimer-DNA is complexed with DOTMA / DOPE (D / D). Transfection efficiency in the widely used human cervical cancer HeLa cells and mouse neuroblastoma Neuro2A cells was evaluated using specific dendrimers from the table below.

[0199] [Table 1] 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 designated "G1,2,3-kl") have alternating charge (Lys or Arg) and hydrophobic (Leu) residue distribution throughout the dendrimer. G1,2,3-K has only one amino acid in each generation peptide motif. The NH2 group present at the C-terminus of the core sequence is a result of the peptide synthesis method. The molecular weight (MW) does not include the counter ion (trifluoroacetate).

[0200] In vivo and in vitro transfection efficiencies for mRNA and DNA delivery were also evaluated using specific dendrimers from the table below and are discussed in the Examples below.

[0201] [Table 2] Table 2. Further exemplary dendrimers. The NH2 group present at the C-terminus of the core sequence is the result of peptide synthesis methods. X=6-aminohexanoic acid, B=beta-alanine, Dab=2,4-diaminobutyric acid. Lower case letters denote D-amino acids and upper case letters denote L-amino acids. I is D-leucine. Glycine does not exist in D or L form.

[0202] The hydrodynamic size, polydispersity index, and zeta potential were determined for the set of dendrimers disclosed in Tables 1 and 2.

[0203] [Table 3] Table 3. Hydrodynamic size and zeta potential of peptide dendrimer, lipid, and mRNA complexes. The dendrimer had an N:P ratio of 0.16:1 and the lipid DOTMA / DOPE had a w / w ratio of 10:1 to the mRNA. The NH2 group present at the C-terminus of the core sequence is the result of the peptide synthesis method. X=6-aminohexanoic acid, B=beta-alanine, Dab=2,4-diaminobutyric acid. Lower case letters denote D-amino acids and upper case letters denote L-amino acids. l is D-leucine. Glycine does not exist in D or L form.

[0204] [Table 4] Table 4. Hydrodynamic size and zeta potential of peptide dendrimer, lipid, and mRNA complexes. The dendrimer had an N:P ratio of 8:1 and the lipid DOTMA / DOPE had a w / w ratio of 10:1 to the mRNA. The NH group present at the C-terminus of the core sequence is a result of the peptide synthesis method. X=6-aminohexanoic acid, B=beta-alanine, Dab=2,4-diaminobutyric acid. Lower case letters denote D-amino acids and upper case letters denote L-amino acids. l is D-leucine. Glycine does not exist in D or L form.

[0205] Variations in dendritic structure, symmetry, topology, and chirality The various dendritic structures provide different distributions of amino acids within the scaffold and the number of amino acids between branch points, resulting in structures exhibiting a range of compactness and size (see, for example, Table 1 above).

[0206] To investigate the effect of topology variation, dendrimers in which some or all KL positions are reversed (G1,2-LK,3-KL and G1,2,3-LK) are compared with G1,2,3-KL. G1,2,3-LK had no significant difference in DNA binding and transfection compared to G1,2,3-KL (Figure 1A,B). For G1,2,3-K, which has the same total charge as G1,2,3-KL but a reduced size and molecular weight (MW per charge of G1,2,3-K is 1.5-fold lower than G1,2,3-KL), transfection efficiency is somewhat reduced and it is not as effective as G1,2,3-KL (Figure 1C,D). G1,2,3-K transfected better at a lower N / P ratio of 2.5, but increasing the N / P ratio tends to be less effective when compared to G1,2,3-KL.

[0207] To investigate the effect of chirality, we synthesized a G1,2,3-KL dendrimer with D-amino acids (denoted D-G1,2,3KL). Substituting the L-amino acids of the dendrimer with D-amino acids did not significantly affect transfection (Figure 1C,D).

[0208] The importance of the amino acid sequence at the focal spot on transfection was studied by testing G0,1,2,3-KL and Ala-G1,2,3-KL. The GlySerCys sequence at the focal spot, which was constant in all dendrimers previously studied, was replaced by LysLeuAla, which carries an additional charged residue. The change did not affect the transfection outcome (Figure 1E, F).

[0209] Overall, the above results indicate that the charge distribution in each generation of the dendrimer is a determining factor for transfection efficiency. Further variations in the optimal structure may slightly alter the transfection activity, but the overall effect is not significant unless the charge distribution itself is changed. This observation is consistent with our previous observation that the charges are only concentrated in the outer generations and are not distributed evenly across each generation, resulting in a drastic decrease in transfection efficiency.

[0210] Comparison of transfection efficiency of different generations of KL and RL dendrimers We investigated the number of generations from G1 to G2 to G3 based on the KL repeat unit on the delivery efficiency. We also replaced the KL unit with the RL repeat unit to compare the impact of protonated basic groups with different pKa. Interestingly, we observed a relationship between generation and transfection in cells (Figure 2). The generation dependency on transfection reveals that the first generation dendrimers G1-KL or G1-RL do not transfect HeLa or Neuro2A cells with lysine or arginine as the charged residue. This may be 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).

[0211] However, for the second generation, the arginine-containing G1,2-RL shows higher transfection efficiency compared to G1,2-KL (at N / P ratios of 10 or 20 in HeLa cells and at N / P ratios of 20 in Neuro2A cells) (Figure 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). The optimal N / P ratio of the G2 dendrimer is significantly higher than that of the G3 dendrimer for the respective best activity. Our results also showed that G1,2-KL transfected HeLa cells more efficiently than Neuro2A cells. This highlights the fact that the activity of certain transformants may be cell-dependent due to different components, such as proteoglycans, and their concentrations on the cell surface that may affect transfection complex binding and ultimately gene delivery to the cells.

[0212] Overall, the G3 dendrimers transfect efficiently in both HeLa and Neuro2A cells. Our third generation dendrimers transfected HeLa and Neuro2A cells 2-600 times better than some widely used commercial reagents such as Polyethylenimine (PEI), Lipofectin (also known as DOTMA / DOPE), and Lipofectamine 2000 (Figure 3).

[0213] 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 using D / D alone is defined as 100%). G1,2,3-KL improved the transfection efficiency by 200 times compared to D / D alone in HeLa cells (Figure 4A). In Neuro 2A cells, the activity of G1,2,3-KL and G1,2,3-RL was 340- and 1500-fold higher, respectively, than DOTMA / DOPE alone (Figure 4B). This observation can be explained by the fact that RL dendrimers bind DNA more strongly than KL dendrimers and therefore they are more stable and resistant to attack from charged components in serum.

[0214] Cytotoxicity mediated by transfection reagents is an important parameter for in vivo applications. The toxicity of dendrimer-containing formulations was compared to that of the controls. Most of the dendrimer modifications did not induce higher toxicity to HeLa cells, and cell viability was comparable to G1,2,3-KL (about 40% or more) and the commercial reagent Lipofectamine 2000 (about 20%), and similar to the supplemented lipid (D / D, about 40%). Increasing the number of generations from 1 to 3 did not increase toxicity. Although the actual mechanism of toxicity caused by the dendrimer formulations is not well known, some of the toxicity from the formulations is due to the addition of D / D, as previously shown, and also arose from the D / D alone control. Indeed, most of the lipid-dendrimer-DNA complexes did not mediate higher toxicity compared to the D / D control, while most of the dendrimer complexes resulted in higher cell viability compared to the widely used Lipofectamine 2000. The toxicity of the dendrimer complexes was not directly related to the transfection efficiency, since the ineffective G1-KL or RL formulations also induced 40% cell death. In Neuro2A cells, most dendrimer-DNA complexes were less toxic than in HeLa cells (cell viability above 80%). Overall, cell viability after transfection was significantly higher than that of the reference reagent Lipofectamine 2000 (about 45%).

[0215] Capture Mechanism The uptake mechanism of the composition of the present invention was investigated. Chlorpromazine was used to inhibit clathrin-mediated endocytosis. Genistein and rottlerin were also used. These drugs have been reported to inhibit caveolae-mediated endocytosis and micropinocytosis, respectively.

[0216] We observed that the addition of chlorpromazine significantly inhibited transfection by 80% (Figure 5B), suggesting that clathrin-mediated endocytosis plays an important role in DNA delivery by the dendrimer system. Genistein and rottlerin inhibited transfection by about 50% (Figure 5D, F). This suggested that the peptide dendrimer system mediates cellular uptake via at least three different pathways, with clathrin-mediated endocytosis being a key pathway for internalization. The ability to trigger different cellular internalization pathways would allow the uptake of nanoparticles by different cell types, thus explaining the fact that this dendrimer system delivers DNA cargo in vivo.

[0217] Consideration We have previously reported the effect of charge distribution of RL and KL (i.e., a combination of hydrophobic and cationic groups) based third generation dendrimers on transfection (Kwok et al, 2013). Providing a uniform distribution across the three generations resulted in an efficient transfection reagent (in combination with DOTMA / DOPE), but the concentration of charge only on the surface (third generation) significantly reduced DNA transfection. This study on the third generation dendrimer variants disclosed herein (e.g., Table 1 above) shows that charge distribution in the dendritic structure is more important for efficient transfection than the topology and chirality of amino acids within the scaffold.

[0218] We demonstrate that the generation number is important for transfection, and increasing the generation number tends to enhance transfection efficiency. Improved transfection efficiency is consistent with improved complex stability. For example, G1,2,3-RL is a highly effective dendrimer for transfection and in vivo gene delivery in serum conditions (data not shown).

[0219] Upon systemic administration, G1,2,3-RL-DOTMA / DOPE complexes deliver DNA for functional gene expression in all tissues assayed. Gene expression was particularly high in liver and skeletal muscle, and no toxicity was observed. Delivery of DNA to all different tissues is consistent with the observation that G1,2,3-RL and DOTMA / DOPE complexes can mediate cellular internalization via clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis, cargo uptake processes expressed at different levels in all cell types (data not shown).

[0220] The biodistribution of the G1,2,3-RL, DOTMA / DOPE, DNA compositions 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 lungs and spleen after IV administration. PEI and amphiphilic peptides effectively delivered DNA to the lungs and liver, but toxicity was observed with the use of PEI. In contrast, the present invention provides an effective means of delivery to muscle without toxicity. The relatively low gene expression in the kidney and spleen means that the compositions of the present invention are not subject to clearance by the kidney and reticuloendothelial system. Thus, the high gene expression in the liver suggests delivery primarily to hepatocytes and stellate cells (data not shown).

[0221] The compositions of the present invention can also functionally deliver nucleic acids to the brain. Although the route through or around the blood brain barrier (BBB) ​​is uncertain, transcytosis is possible, a process in which endothelial cells endocytose cargo at one end of the barrier and exocytose it at the other end. Transcytosis has been reported to deliver cargo by bypassing the BBB with other synthetic systems, such as bolaamphiphile systems.

[0222] Surprisingly, the composition of the present invention effectively delivers DNA to skeletal muscle, despite the fact that most non-viral gene delivery systems show ineffective delivery to this target (data not shown). Skeletal muscle expresses only caveolin 3 (Cav3), which may suggest that the delivery system of the present invention interacts with the Cav3 component to mediate effective cellular internalization.

[0223] The peptide dendrimers of the present invention represent a versatile platform that provides efficient and potentially targeted transfection reagents with a large chemical space available in terms of amino acid composition, dendrimer topology, and possible modifications on the C-terminus and N-terminus. The peptide dendrimer / DOTMA / DOPE system can circumvent biological barriers and deliver DNA plasmids to cell nuclei in vivo without other adjuvants. It is envisioned that cell-specific targeting domains, peptides, or other molecules that aid in endosomal escape and / or nuclear localization can be conjugated to the dendrimers to further improve cell / tissue targeted delivery and gene expression.

[0224] Materials and Methods After preparative RP-HPLC, G1,2,3-KL((KL)8(KKL)4(KKL)2KGSC-NH2) was obtained as a foamy colorless solid (72.3 mg, 10.3 μmol, 9%). Analytical RP-HPLC: R =1.47 minutes (A / D 100 / 0~0 / 100 in 2.2 minutes, λ=214nm). MS(ESI+):C 218 H 422 N60 O 39 S Calculated value / Measured value: 4540.1 / 4539.0 [M] + .

[0225] After preparative RP-HPLC, G1,2-KL((KL)4(KKL)2KGSC-NH2) was obtained as a foamy colorless solid (90.5 mg, 27.9 μmol, 41%). Analytical RP-HPLC: R =1.36 minutes (A / D 100 / 0~0 / 100 in 2.2 minutes, λ=214nm). MS(ESI+):C 96 H 182 N 28 O 19 S Calculated value / Measured value: 2096.8 / 2096.0 [M] + .

[0226] After preparative RP-HPLC, G1-KL ((KL)2KGSC-NH2) was obtained as a foamy colorless solid (46.0 mg, 41.7 μmol, 60%). Analytical RP-HPLC: R =1.23 minutes (A / D 100 / 0~0 / 100 in 2.2 minutes, λ=214nm). MS(ESI+):C 38 H 74 N 12 O9S calculated value / measured value: 875.1 / 875.4[M] + .

[0227] After preparative RP-HPLC, G1,2,3-RL((RL)8(KRL)4(KRL)2KGSC-NH2) was obtained as a foamy colorless solid (14.6 mg, 2.0 μmol, 2%). Analytical RP-HPLC: R =1.50 minutes (A / D 100 / 0~0 / 100 in 2.2 minutes, λ=214nm). HRMS(NSI+):C 218 H 422 N 88 O 39 S Calculated value / Measured value: 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] + .

[0228] After preparative RP-HPLC, G1,2-RL((RL)4(KRL)2KGSC-NH2) was obtained as a foamy colorless solid (143.2 mg, 42.1 μmol, 38%). Analytical RP-HPLC: R =1.45 minutes (A / D 100 / 0~0 / 100 in 2.2 minutes, λ=214nm). MS(ESI+):C 96 H 190 N 40 O 19 S Calculated value / Measured value: 2264.9 / 2264.0 [M] + .

[0229] After preparative RP-HPLC, G1-RL((RL)2KGSC-NH2) was obtained as a foamy colorless solid (78.5 mg, 56.6 μmol, 51%). Analytical RP-HPLC: R =1.37 minutes (A / D 100 / 0~0 / 100 in 2.2 minutes, λ=214nm). MS(ESI+):C 38 H 74 N 16 O9S calculated value / measured value: 931.2 / 931.2[M] + ;1045.2 / 1045.4[M+1TFA] + .

[0230] After preparative RP-HPLC, G1,2-LK,3-KL((KL)8(KLK)4(KLK)2KCGSC-NH2) was obtained as a foamy colorless solid (56.7 mg, 8.1 μmol, 7%). Analytical RP-HPLC: R =1.47 minutes (A / D 100 / 0~0 / 100 in 2.2 minutes, λ=214nm). MS(ESI+):C 218 H 422 N 60 O 39 S Calculated value / Measured value: 4540.1 / 4539.0 [M] +.

[0231] After preparative RP-HPLC, G1,2,3-LK, ((LK)8(KLK)4(KLK)2KGSC-NH2) was obtained as a foamy colorless solid (32.4 mg, 4.6 μmol, 4%). Analytical RP-HPLC: R =1.41 minutes (A / D 100 / 0~0 / 100 in 2.2 minutes, λ=214nm). MS(ESI+):C 218 H 422 N 60 O 39 S Calculated value / Measured value: 4540.1 / 4539.0 [M] + .

[0232] Ala-G1,2,3-KL ((LysLeu)8(LysLysLeu)4(LysLysLeu)2LysGlySerAla-NH2) was obtained from TentaGel S RAM® resin (500 mg, 0.22 mmol g-1) and Ala-G1,2,3-KL was obtained as a foamy colorless solid after preparative RP-HPLC (36.6 mg, 5.2 μmol, 5%). Analytical RP-HPLC: tR=1.46 min (100% A:100% D over 2.2 min, λ=214 nm). MS (ESI+): C 218 H 422 N 60 O 39 Actual value / calculated value: 4508.0 / 4508.1[M] + .

[0233] After preparative RP-HPLC, G1,2,3-K((K)8(KK)4(KK)2KGSC-NH2) was obtained as a foamy colorless solid (79.9 mg, 14.6 μmol, 12%). Analytical RP-HPLC: R =1.17 minutes (A / D 100 / 0~0 / 100 in 2.2 minutes, λ=214nm). MS(ESI+):C 134 H 268 N 46 O 25 S Calculated value / Actual value: 2955.9 / 2956.0 [M] + .

[0234] After preparative RP-HPLC, G0,1,2,3-KL((KL)8(KKL)4(KKL)2KKLC-NH2) was obtained as a foamy colorless solid (54.2 mg, 7.5 μmol, 6%). Analytical RP-HPLC: R =1.47 minutes (A / D 100 / 0~0 / 100 in 2.2 minutes, λ=214nm). MS(ESI+):C 225 H 437 N 61 O 38 S Calculated value / Measured value: 4637.3 / 4638.0 [M] + .

[0235] After preparative RP-HPLC, D-G1,2,3-KL((kl)8(kkl)4(kkl)2kgsc-NH2) was obtained as a foamy colorless solid (28.3 mg, 4.0 μmol, 5%). Analytical RP-HPLC: R =1.44 minutes (A / D 100 / 0~0 / 100 in 2.2 minutes, λ=214nm). MS(ESI+):C 218 H 422 N 60 O 39 S Calculated value / Measured value: 4540.1 / 4540.0 [M] + Contains small amounts of impurities

[0236] DNA transfection Cell lines, transfection reagents, and plasmids. HeLa cells were maintained in RPMI medium containing 10% (v / v) FCS and 1% (v / v) P / S in a humidified atmosphere at 5% CO2 and 37 °C. 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.

[0237] Transfection procedure. 24 h prior to transfection, HeLa cells were seeded in 96-well plates (10,000 cells in 100 μL / well) and allowed to reach 70% confluence. Plasmid transfection complexes were formed by mixing dendrimers (100 μL, 60 μg-105 μg depending on N / P ratio) with lipofectin (4 μg; 100 μL). These mixtures were incubated with pCI-Luc (4 μg; 100 μL) at different N / P ratios in OptiMEM for 30 min at 25 °C. Transfection control complexes (PEI, lipofectin or L2000) (total 100 μL in OptiMEM) were mixed with pCI-Luc (4 μg; 100 μL) at the respective manufacturer's recommended concentrations. For transfection in serum-free medium, before the DNA complexes were layered on the cells, OptiMEM was added to dilute the complexes so that each complex contained 0.25 μg of DNA in a total volume of 100 μL in one well of a 96-well plate. For transfection in serum medium, before the DNA complexes were layered on the cells, complete growth medium was added to dilute the complexes so that each complex contained 0.25 μg of DNA in a total volume of 100 μL in one well of a 96-well plate. After removing complete medium from the cells, the complexes were added to the plate. The plate was incubated at 37° C. for 4 hours. The transfection solution was then replaced with complete growth medium for 24 hours before assaying luciferase activity.

[0238] Transgene expression assay. Cells were washed twice with PBS and incubated with reporter lysis buffer (20 μL, Promega) at 4°C for 20 min, then at -80°C overnight. After cells were thawed at room temperature, luciferase assay buffer (100 μL, Promega; prepared according to the manufacturer's protocol) was added to each well. Luminescence production was measured by relative light units (RLU) in a FLUOstar Optima luminometer (BMG Labtech).

[0239] Protein content determination. The protein content of each cell lysate was determined by mixing the lysate (20 μL) with Bio-Rad Protein Assay Reagent (180 μL, Bio-Rad, Hemel Hempstead, UK). After 10 min of incubation, the absorbance at 590 nm was measured and converted to protein concentration using a BSA standard curve. RLU per mg of protein represented the luciferase activity. The ratio of these two values ​​is the activity per unit of protein (RLU / mg). Values ​​shown in the transfection figures are expressed after normalization to a control transfection experiment with DOTMA / DOPE and are shown as percentages.

[0240] Cell viability. Cells were transfected as described under "Transfection procedure". 24 h after transfection, the medium was removed and the cells were washed twice with PBS. Afterwards, the cells were dried for 1 h at room temperature (to allow the nuclear stain to penetrate). Crystal violet solution (50 μL of stock solution supplied by Sigma-Aldrich) was added to the cells. They were incubated for 15 min at room temperature. After washing with distilled water (5 times), the cells were dried for 30 min at room temperature. MeOH (200 μL) was then added and the suspension was incubated for 1 h at room temperature. The relative amount of crystal violet stain retained by the viable cells was determined by the absorbance of the methanol solution at 550 nm.

[0241] Fluorescence quenching assay PicoGreen (Invitrogen) was added to the DNA and TE buffer (10 mM Tris / HCl, pH 7.5; 1 mM EDTA) was added to a final DNA concentration of 0.002 μg / μL. PicoGreen was added to the DNA in a ratio of 1:150 (v / v), so that every 100 μL of solution contained 0.2 μg of DNA. The mixture was incubated at room temperature for 10 min. During the incubation, different amounts of transfection reagent were diluted in TE buffer. Then, 50 μL of lipofectin (0.2 μg) and 50 μL of dendrimer (amount depends on the selected N / P ratio, which varied from 0.625:1 to 40:1) were added per well of a flat-bottom 96-well plate. Afterwards, 100 μL of DNA-PicoGreen solution (0.2 μg DNA) was added per well. As a control, 100 μL of DNA-PicoGreen solution (0.2 μg DNA) was added to 100 μL of TE buffer. After 30 min incubation at room temperature, 100 μL of TE buffer was added to each well. PicoGreen signal was then detected using a fluorescent plate reader (FLUOstar Optima, BMG Labtech) with excitation at 485 nm and emission at 520 nm. PicoGreen signal from the complex was normalized to the DNA control to obtain the percentage of detected PicoGreen signal.

[0242] Complex dissociation assay PicoGreen (Invitrogen) was added to the DNA and diluted in TE buffer (10 mM Tris / HCl, pH 7.5; 1 mM EDTA) to a final DNA concentration of 0.002 μg / μL (PicoGreen was added to DNA in a ratio of 1:150 (v / v) so that every 100 μL of solution contained 0.2 μg of DNA). The mixture was incubated for 10 min at room temperature. During the incubation, different amounts of transfection reagent were diluted in TE buffer. Then, 50 μL of lipofectin (0.2 μg) and 50 μL of dendrimer (amount depends on the selected N / P ratio, which varied from 0.625:1 to 40:1) were added per well of a flat-bottom 96-well plate. Afterwards, 100 μL of DNA-PicoGreen solution (0.2 μg DNA) was added per well. As a control, 100 μL of DNA-PicoGreen solution (0.2 μg DNA) was added to 100 μL of TE buffer. After 30 min of incubation at room temperature, different concentrations of heparin (0.2-1.4 U / mL, Sigma-Aldrich) diluted in 100 μL of TE buffer were added to the DNA complex or DNA alone (0.2 μg, total volume 200 μL). After 30 min of incubation at room temperature, the fluorescent signal from PicoGreen was recorded at excitation 485 nm and emission 520 nm using a microplate reader (FLUOstar Optima, BMG Labtech). The signal was normalized using the DNA control.

[0243] In vivo luciferase DNA expression assay Dendrimer, lipid, and DNA formulations were injected into mice, and tissues were isolated and snap frozen for luciferase expression analysis 24 and 48 hours after intravenous and intramuscular injection, respectively.

[0244] Tissues were homogenized in 1x reporter lysis buffer supplemented with protease inhibitors. Depending on the tissue, 1.5-3x lysis buffer was used for tissue homogenization. After homogenization, lysates were centrifuged and the supernatants were used for assaying luciferase expression and protein content. Luciferase expression levels were measured in a luminometer using a luciferase assay kit (Promega) according to the manufacturer's instructions. Protein content was measured at absorbance at 595 nm using a Biorad protein assay kit according to the manufacturer's instructions.

[0245] In vivo luciferase mRNA expression assay Peptide dendrimer-lipid-mRNA particles Peptide dendrimers were diluted to respective concentrations and mixed with mRNA for desired NP ratios such as NP=0.15:1 or 8:1. The solutions were incubated at room temperature for 10 minutes. Lipids such as DOTMA / DOPE were diluted to respective concentrations and mixed with dendrimer-mRNA complexes for desired NP ratios such as NP=4.7:1. The solutions were incubated at room temperature for 20 minutes. If necessary, the solutions are further diluted before injection.

[0246] Lipid-mRNA particle control DOTMA / DOPE was diluted to the respective concentrations and mixed with the dendrimer-mRNA complex. The solution was incubated at room temperature for 20 minutes. If necessary, the solution was further diluted before injection.

[0247] Mice and delivery route All animal procedures performed in our study were in accordance with UK law and included ethical approval. Female CD-1 mice (n=39), 6-8 weeks old, were acclimated for 1 week upon arrival. All mice were weighed and formulations were delivered intravenously (100 μl) via the tail vein using a 30G insulin syringe (BD biosciences).

[0248] Bioluminescence imaging (BLI) All mice were imaged 6 hours after injection. BLI was performed using an IVIS Lumina II (Perkin Elmer) imaging system. Mice were administered D-luciferin (30 mg / mL, XenoLight, Perkin Elmer) at a dose of 150 mg / kg. Mice were anesthetized 6 minutes after D-luciferin administration in a chamber containing 5% isoflurane and then placed on a heated imaging platform while maintained at 2.5% isoflurane. Mice were imaged 10 minutes after D-luciferin administration with exposure times set to ensure that the resulting signal was within the effective detection range (open filter, binning 8, f-stop 1). Bioluminescence signals were quantified by measuring the photon flux (photons / second) in a defined region of interest (ROI) using Living Image software (Perkin Elmer). After in vivo whole-body imaging, mice were euthanized, cardiac blood was collected, and tissues were extracted for ex vivo imaging. Here, each tissue was placed into an individual well of a 24-well imaging plate (black surface, Eppendorf) containing 0.3 mg / mL D-luciferin in PBS. The imaging plate was placed in the center of the imaging platform (Lumina II system) and the signal was measured using the acquisition settings detailed above. Finally, the tissues were placed into storage vials and flash frozen in liquid nitrogen.

[0249] Endocytosis experiments Cells were preincubated with each inhibitor (chlorpromazine, genstein, and rottlerin) for 30 min at 37° C. Transfection was then carried out in the presence of inhibitors for an additional 4 h at 37° C. as described in the transfection section.

[0250] In vivo studies Induction of FST Expression Using Compositions Containing DNA Mice were injected with DNA expressing follistatin alone or with DNA expressing follistatin together with G1,2,3-RL and D / D lipids on days 1 and 3. On day 5, tissues were harvested for RNA extraction. cDNA was then synthesized from the mRNA for qPCR. The relative expression levels of follistatin (FST) in mouse skeletal muscle are shown in Figure 6.

[0251] Immunogenicity / tolerability Body weight was monitored during the type of IV protocol for DNA delivery discussed above, and no weight loss was observed (see FIG. 7).

[0252] Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were also monitored on days 1 and 3 in mice that received no injections or mice that received intravenous injections of DNA alone or DNA, G1,2,3-RL and D / D formulations. For all groups, AST levels remained substantially below 200 U / L and ALT levels below 100 U / L. (These levels of AST and ALT are much lower than the threshold levels of over 7000 U / L in disease models of liver injury (induced by carbon tetrachloride) (Bonnet et al. 2008)).

[0253] Cytokine levels were also measured in mice to check for immunogenicity. Serum was taken on day 5 for measurement of AST and ALT levels. No substantial increase was observed. For IFN-gamma, levels remained below 2 pg / mL and there was no significant increase (other non-viral systems were able to increase IFN-gamma up to 60,000 pg / mL (Bonnet et al 2008)). For TNF-alpha, levels remained below 20 pg / mL and there was no significant increase (lipopolysaccharide was able to increase TNF-alpha up to 5000 pg / mL (Bonnet et al 2008)). For IL-6, levels remained below 20 pg / mL and there was no significant increase (other non-viral systems were able to increase IL-6 levels up to 15000 pg / mL (Bonnet et al 2008)). For IL-1beta and IL-10, levels remained very low and were not significantly increased.

[0254] Induction of luciferase expression using compositions containing DNA Mice were injected with the dendrimer, lipid, and DNA formulations prepared to detect luciferase expression after injection. Twenty-four and 48 hours after intravenous and intramuscular injection, respectively, tissues were isolated and snap frozen for luciferase expression analysis.

[0255] A composition containing both G1,2,3-RL and G1,2,3-LR injected intravenously targeted delivery of DNA to skeletal muscle as determined by luciferase expression, with G1,2,3-LR demonstrating the most effective delivery to skeletal muscle (Figure 8).

[0256] A composition containing G1,2-RL,3-LR injected intramuscularly is also able to deliver DNA to skeletal muscle, as determined by luciferase expression (Figure 9).

[0257] Induction of luciferase expression using compositions containing mRNA Mice were injected with compositions containing DOTMA / DOPE, mRNA encoding luciferase, and either G1,2,3-RL (Figure 10, upper panel; "G1,2,3-RL, lipid, and mRNA formulation") or G1,2-RL,3-LR (Figure 10, lower panel; "G1,2,3-RL, lipid, and mRNA formulation"). Each formulation was prepared and injected at NP ratios of 8:1 and 0.15:1. Mice were prepared for detection of luciferase expression after injection.

[0258] Varying the NP ratio of either the G1,2,3-RL or G1,2-RL,3-LR formulations alters the biodistribution of mRNA in mouse tissues. A NP ratio of 0.15:1 targets a broad range of immune cell tissues, including lung, spleen, and lymph nodes, compared to NP=8:1, which specifically targets the spleen and to a lesser extent lymph nodes (Figure 10). This demonstrates that tissue distribution of a composition can be altered by changing the NP ratio of the composition. For example, increasing the NP ratio can improve targeting to lymphatic organs.

[0259] G1,2,3-RL and G1,2-RL,3-LR, lipid, and mRNA formulations can also successfully deliver mRNA to tissues including muscle, liver, heart, kidney, and adipose tissue (Figure 11). In particular, G1,2-RL,3-LR, lipid, and mRNA formulations could induce significant expression in gastrocnemius and quadriceps tissues, liver, heart, kidney, and adipose tissue at either NP ratios of 8:1 or 0.15:1.

[0260] A comparison of luciferase expression was performed between mice injected with a composition comprising G1,2,3-RL, DOTMA / DOPE, and mRNA and mice injected with a composition comprising G1,2-RL.3-LR, DOTMA / DOPE, and mRNA. Increased delivery to all tissues tested was observed for G1,2-RL,3-LR compared to G1,2,3-RL (Figure 12). This demonstrates that the selection of dendrimer sequences can be used to target specific tissues.

[0261] Repeated dose experiment To determine whether the compositions can be administered repeatedly and maintain delivery to tissues, a comparison was made between mice receiving either a single dose or repeated doses (two doses 24 hours apart) of DOTMA / DOPE, mRNA and peptide dendrimer. Mice administered two doses of either composition showed increased luciferase expression compared to mRNA alone. Typically, luciferase expression in mice receiving two doses showed either approximately the same luciferase expression or increased luciferase expression compared to mice receiving a single dose. Luciferase expression was assayed 6 hours after treatment.

[0262] Tissue / cell-targeted peptide-dendrimer fusion proteins A comparison of luciferase expression was performed between compositions containing DOTMA / DOPE, mRNA, and one of G1,2,3-RL, G1,2-RL,3-LR, or NXT1 (Figure 14). NXTI dendrimers contain G1,2-RL,3-LR dendrimers with a core peptide sequence that includes a muscle targeting peptide and a cell penetrating peptide. NXT1 is more effective than G1,2,3-RL and G1,2-RL,3-LR in mediating mRNA delivery to many tissues, including muscle. Surprisingly, NXT1 is also more effective in delivering mRNA to the spleen and lymph nodes compared to G1,2,3-RL and G1,2-RL,3-LR. NXT1 and G1,2-RL,3-LR show similar efficiency in delivering mRNA to the lung.

[0263] Dendrimers increase mRNA delivery to tissues compared to lipids alone To determine whether the presence of dendrimers improves the delivery of mRNA to tissues, mice were injected with compositions containing either: i) mRNA alone; 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 prepared for detection of luciferase expression after injection. The NP ratio in each dendrimer composition was 0.15:1. As demonstrated in Figure 15, the presence of either G1,2,3-RL, G1,2-RL,3-LR, and NTX1 improves the efficiency of mRNA delivery to the lung, spleen, and lymph nodes compared to either mRNA alone or mRNA and DOTMA / DOPE alone.

[0264] Comparisons were also made between mice injected with compositions containing either 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 demonstrates that mRNA delivery to the spleen is significantly enhanced in the presence of dendrimer compared to mRNA alone or mRNA and lipid. Furthermore, luciferase expression in the lungs and lymph nodes is reduced in compositions containing dendrimer compared to lipid alone. This data further demonstrates that G1,2-RL,3-LR improves the specificity of mRNA delivery to the spleen compared to DOTA / DOPE alone.

[0265] Comparison of dendrimer-lipid delivery systems to commercially available lipid delivery systems HeLa cells were transfected with mRNA encoding luciferase using either a composition containing G1,2-RL,3-LR and DOTMA / DOPE (NP=0.16:1) or the commercially available Lipofectamine™ 2000. As shown in the top left panel of FIG. 17, a composition containing G1,2-RL,3-LR and DOTMA / DOPE at a NP ratio of 0.16:1 increases in vitro transfection efficiency by about an order of magnitude compared to the commercially available transfection reagent Lipofectamine™ 2000. Similarly, HeLa cells transfected with mRNA encoding eGFP using G1,2-RL,3-LR and DOTMA / DOPE at a NP ratio of 0.16:1 increased eGFP expression by about 4-fold compared to mRNA transfected using the DLin-MC3-DMA:cholesterol:DSPC:DMG-PEG lipid nanoparticle delivery system (FIG. 17, top right panel).

[0266] C2C12 cells were transfected for 24 hours with either mRNA alone, G1,2-RL,3-LR (N:P=8:1) and DOTMA / DOPE (w / w=10:1 relative to mRNA) and mRNA, DOTMA / DOPE (w / w=10:1 relative to mRNA) and mRNA, polyethyleneimine and mRNA, and Lipofectamine 2000 and mRNA. As shown in the lower panel of Figure 17, the dendrimer formulation improves the delivery of mRNA into C2C12 cells compared to commercially available lipid-based transfection reagents.

[0267] In vitro CRISPR experiments RNA was labeled using Label IT® Nucleic Acid Labeling Kit (Mirus Bio LLC) according to the manufacturer's instructions. Briefly, single guide RNA (sgRNA) consisting of CRISPR RNA (crRNA) fused to tracerRNA (trRNA) was labeled with TM-rhodamine fluorophore, and Cas9 mRNA was labeled with Cy5 fluorophore. crRNA and trRNA were tagged with fluorescein and ATTO 550 fluorophore, respectively.

[0268] HeLa cells were transfected with different combinations of labeled RNA and either formulation: (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 in all compositions was 10:1 w / w, with DOTMA:DOPE at a weight ratio of 1:1. Cells were incubated with each formulation for 2 hours, followed by trypsinization and fixation. The percentage of RNA uptake in cells was assayed by flow cytometry. RNA alone without formulation was used as a control.

[0269] Approximately 100% of HeLa cells exposed to the sgRNA+Cas9 mRNA formulation were found to be double positive for sgRNA and Cas9 mRNA, demonstrating that the G1,2-RL,3-LR dendrimer is a highly effective transfection reagent (Figure 18).

[0270] In dendrimer and lipid formulations containing crRNA, trRNA and Cas9 mRNA, the dendrimer formulation can mediate delivery of all three components in about 4% of cells (Figure 19). In dendrimer and lipid formulations containing only one of crRNA, trRNA or Cas9 mRNA, the percentage of positive cells varies. In compositions containing either trRNA or Cas9 mRNA, about 100% of cells are positive for the nucleic acid. In contrast, when exposed to compositions containing only crRNA, only 4% of cells are positive for crRNA (Figure 20). Taken together, this data demonstrates that while the G1,2-RL,3-LR dendrimer can successfully mediate delivery of relatively short RNAs (e.g., the crRNA used in this study was 36 nucleotides long) to cells, the success of RNA delivery increases as the length of the transfected RNA increases (the trRNA and Cas9 mRNA used in this study were 67 and 5421 nucleotides long, respectively).

[0271] Immune cell targeting Fifteen 5-7 week old female CD-1 mice (Charles River UK) were administered 1 mg / kg of either mRNA labeled with Alexa Fluor488 alone, or with NTX1 and DOTMA / DOPE, or with G1,2-RL,3-LR and DOTMA / DOPE, or with G1,2-RL,3-LR and DOTMA / DOPE, respectively, by intravenous injection using a dose volume of 5 ml / kg. Group 1 received vehicle and group 5 received only mRNA as a control. Mice in all groups were sacrificed 2 hours after IV administration. At the time of euthanasia, whole blood, spleen, inguinal lymph nodes, and femurs 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 femurs, and spleen and lymph nodes were processed into single cell suspensions by passing them through a 70 μm filter. After lysis of red blood cells in blood, BM, and spleen, WBCs isolated from all tissues were stained with different antibody panels to identify specific subpopulations of immune cells. After staining was performed in a 96-well plate format, samples were analyzed using an ACEA Novocyte 3005 flow cytometer.

[0272] NTX1 and G1,2-RL,3-LR mediated the uptake of mRNA in immune cells present in the spleen (Figure 21). mRNA formulated with dendrimers and lipids was taken up by (A) B cells, (B) T cells, (C) monocytes and macrophages, (D) neutrophils, and (E) dendritic cells. Notably, dendrimer and lipid formulations can deliver mRNA to monocytes and macrophages, neutrophils, and dendritic cells within a relatively short time from injection (2 hours after injection). NTX1 (NP=0.15:1) and G1,2-RL,3LR (NP=0.15:1 and 8:1) mediate the delivery of mRNA to approximately 5% or more of monocytes / macrophages, neutrophils, and dendritic cells. Given the short time between injection and tissue harvest, it is reasonable to assume that the delivery of mRNA to each of the immune cell types analyzed increases over time.

[0273] NTX1 and G1,2-RL,3-LR mediated the uptake of mRNA in immune cells present in bone marrow (Figure 22). mRNA formulated with dendrimers and lipids was taken up by (A) monocytes and macrophages, (B) dendritic cells, (C) CD38+ cells, (D) B cells, and (E) neutrophils. Notably, the dendrimer and lipid formulations are able to deliver mRNA to monocytes and macrophages, neutrophils, and dendritic cells within a relatively short time from injection (2 hours after injection). Given the short time period between injection and tissue harvest, it is reasonable to assume that the delivery of mRNA to each of the immune cell types analyzed increases over time.

[0274] In vitro DNA delivery The top panel of Figure 25 shows luciferase expression in HeLa cells transfected with either DNA alone, G1,2,3-RL (N:P=5:1) and DOTMA / DOPE (w / w=1:1 relative to DNA) with DNA, and G1,2,3-RL (N:P=5:1) and DOPG / DOPE (w / w=1:1 relative to DNA) with DNA. Compositions containing G1,2,3-RL and either DOTMA / DOPE or DOPG / DOPE can successfully transfect HeLa cells in vitro.

[0275] Similarly, the bottom panel of FIG. 25 shows luciferase expression in C2C12 cells (a muscle-derived cell line) transfected with either DNA alone, polyethylenimine and DNA, G1,2-RL,3-LR (N:P=8:1) and DOTMA / DOPE (at w / w=1:1 to DNA) and DNA, and NTX2 (N:P=8:1) and DOTMA / DOPE (at w / w=1:1 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)-NH2). Both dendrimers can successfully transfect C2C12 cells in vitro. Dendrimers containing the muscle-targeting domain further increase transfection efficiency compared to dendrimers without the muscle-targeting domain.

[0276] In vivo toxicity assay Mice were injected once or twice with the mRNA formulation. For two IV injections, mice were injected 24 hours before the second injection. Six hours after the second IV injection, mouse plasma was collected and AST and other cytokine levels were measured. For mice injected once, plasma was collected six hours after injection for AST and other cytokine levels.

[0277] Aspartate transaminase (AST) is a biomarker that can be used as a measure of liver health and can also be used as a marker to determine the liver toxicity of drugs. All dendrimer-lipid-mRNA compositions tested did not show a significant increase in AST levels compared to compositions containing DOTMA / DOPE alone, and in fact showed a decrease in AST levels compared to mRNA alone (Figure 26A, upper panel). These results indicate that the dendrimer compositions tested do not show significant liver toxicity.

[0278] TNF-α, IL-6 and IL-1β were also measured in mice injected with the various dendrimer-lipid-mRNA formulations. As shown in the bottom panel of FIG. 26A and the top and bottom panels of FIG. 26B, none of the dendrimer compositions tested significantly increased plasma levels of TNF-α, IL-6 or IL-1β compared to mRNA alone or DOTMA / DOPE and mRNA compositions, respectively. These data demonstrate that the dendrimer compositions do not elicit a significant immune response in mice exposed to the compositions. This further demonstrates the safety of the dendrimer compositions for use in delivering mRNA to tissues in vivo.

[0279] Mice were also injected with either DNA or a formulation containing G1,2,3-RL, DNA and DOTMA / DOPE compositions, and plasma levels of AST, TNF-α, IL-6 and IL-1β were measured 24 hours later. As shown in Figure 27, injection of the formulation containing the dendrimer did not cause a significant increase in plasma levels of either AST (top left), TNF-α (top right), IL-6 (bottom left), or IL-1β (bottom right). These data indicate that delivery of DNA using the dendrimers of the present invention is safe and does not cause liver damage or adverse immune reactions during.

[0280] Comparison of peptide dendrimer transfection efficiency The effect of the dendrimer generation number on mRNA delivery was studied by transfecting HeLa cells in full growth medium conditions. We showed that first or second generation dendrimers (e.g., RHCG1-R, RHCG1,2-R, RHCG1-LR, RHCG1-RL, 2-LR) can effectively transfect cells and mediate similar transfection efficiency as third generation dendrimers (Figure 28). This is indeed very different from DNA transfection, since first or second generation dendrimers do not transfect cells as well as third generation dendrimers in full growth medium (Figure 4). Interestingly, increasing the N:P ratio of generation 1 or generation 2 dendrimers from 0.16 to 8 further increases the transfection efficiency (Figure 29).

[0281] We studied the effect of the dendrimer core sequence on transfection. We changed the three amino acid core sequence GSC from G1,2-RL,3-LR to only two amino acids such as KA and YM. This change does not affect mRNA transfection, suggesting that dendrimers with two amino acids in the core transfect similarly to dendrimers with three amino acids in the core. We then replace the three amino acid core sequence with different amino acids such as RFW, RYM compared to GSC. An arginine containing a cationic group is added to the core, but this arginine does not improve or decrease transfection. Interestingly, when three amino acids are changed in the core from GCS to RHC, transfection improves by 50%. This suggests that ionizable groups such as histidine in the dendrimer core may improve transfection (Figure 28A).

[0282] We introduced 12 amino acids into the core of the dendrimer (linear G1,2-RL,3-LR), but the transfection effect was not affected (Figure 28A). This result indicates that it is possible to introduce longer amino acid chains and / or longer structures into the core without affecting the transfection efficiency. In fact, NTX1, which contains 27 amino acids in the core, transfects more effectively than G1,2-RL,3-RL. This demonstrates that a longer core sequence can indeed enhance transfection. The enhanced transfection from NTX1 is likely due to the presence of a cell-penetrating peptide in the core sequence.

[0283] We also investigate the effect of the number of amino acids in a generation on transfection. Therefore, we tested dendrimers with only one amino acid (R), two amino acids (RL or LR), three amino acids (RLR), and four amino acids (LRLR). Our transfection studies show that dendrimers with one, two, three, or four amino acids in each generation are still able to transfect mRNA well into cells (Figure 28A). We further tested dendrimers with different numbers of amino acids in each generation and showed that transfection efficiency is not affected.

[0284] Based on the G1,2-RL,3-LR structure, we designed a library of third generation dendrimers in which the basic amino acid R was replaced by K and / or the hydrophobic amino acid L was changed to an acidic amino acid such as E and / or an amino acid with a non-polar side chain such as M, F, beta-alanine (B), aminohexanoic acid (X), and W, and / or an amino acid with a polar side chain such as Q, T, and Y.

[0285] Substituting R with K in the dendrimer reduces mRNA transfection efficiency (Figure 28A). However, changing the hydrophobic amino acids to other hydrophobic amino acids and / or amino acids with polar or non-polar side chains may have no effect on transfection or may reduce transfection by 30-40% compared to G1,2-RL,3-LR. The underlined patterns are unclear and further investigations will be performed. However, all of these dendrimers are able to induce significantly higher mRNA transfection than the mRNA only control.

[0286] We also investigated the effect of L- or D-type amino acids within the dendrimer on mRNA transfection. Based on the G1,2-RL,3-LR dendrimer, we found that changing some or all of the amino acids from L-type to D-type in each generation of the dendrimer did not affect transfection efficiency. Substituting diaminobutyric acid for lysine within the dendrimer reduced transfection, but the transfection of this dendrimer was still significantly higher than the mRNA-only control.

[0287] We have demonstrated that G1-LL,2-RR can be used to deliver mRNA using our formulation protocol, where G1-LL,2-RR was used with DOTMA / DOPE (w / w 10:1) at an N:P ratio of 0.16:1. Interestingly, this dendrimer was used to deliver ASO in different formulations in vitro and in vivo (Saher 2018). The formulations used were DOTMA / DOPE (w / w=2:1 for ASO) and N:P=20:1, G1-LL,2-RR vs. ASO. We have attempted this to transfect cells (i.e., DOTMA / DOPE (w / w=2:1 for mRNA) and N:P=20:1, G1-LL,2-RR vs. mRNA), but transfection efficiency is low. The mRNA transfection efficiency of this formulation (DOTMA / DOPE (w / w=2:1 relative to mRNA) and N:P=20:1, G1-LL,2-RR to mRNA) was only 10% of that of our improved formulation (DOTMA / DOPE (w / w=10:1 relative to mRNA) and N:P=0.16:1, G1-LL,2-RR to mRNA).

[0288] We investigated third generation dendrimers with either RL or LR or rl in different generations for mRNA delivery. We found that most of these dendrimers transfected cells similarly, with G1,2-RL,3-LR being the most effective in mRNA transfection. Overall, our data suggested that dendrimers with hydrophobic and cationic amino acids in each generation provided effective mRNA delivery to cells.

[0289] Since there are first and second generation dendrimers that transfect cells more effectively than G1,2-RL,3-LR, we selected these dendrimers for further testing on transfection. We showed that these dendrimers can generally transfect better than G1,2-RL,3-LR at various N:P ratios. In particular, RHCG1-R, RHCG1,2-R, RHCG1-LR, RHCG1-RL,2-LR, RHCG1-RLR, and G1-LRLR with N:P=4:1 can transfect 700%-1815% better than G1,2-RL,3-LR. These data demonstrated that the requirements for DNA and mRNA delivery are very different. For mRNA, first or second generation dendrimers tend to transfect cells better than third generation dendrimers in full growth medium conditions. However, generation 3 dendrimers transfect cells with DNA better than either generation 1 or generation 2 dendrimers under full growth medium conditions (Figure 4).

[0290] Figure 30 shows that generation 2 dendrimers containing an RHC peptide core delivered at an NP ratio of 8:1 target mRNA delivery primarily to the spleen (top panel) and to a lesser extent to the lung (bottom panel). At an NP ratio of 0.15:1, mRNA delivery to the lung is increased compared to an NP ratio of 8:1.

[0291] Regulation of mRNA expression mRNA expression can be regulated by injecting mice with a composition comprising PEG2000 lipid. Mice injected with a composition comprising G1,2-RL,3-LR, DOTMA / DOPE / PEG2000, and mRNA showed reduced luciferase expression compared to mice injected with a composition comprising G1,2-RL,3-LR, DOTMA / DOPE, and mRNA.

[0292] Numbered Sections The following numbered paragraphs refer to particular features and combinations of features of the present invention. 1. A composition for use in medicine, the composition comprising a peptide dendrimer, a nucleic acid and a lipid, 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 linked to the first lysine residue; (i) a first lysine residue is covalently linked to two first peptide motifs, each of which is covalently linked to two second lysine residues; (ii) each second lysine residue is covalently linked to two second peptide motifs, each second peptide motif being covalently linked to one of the third lysine residues; (iii) each third lysine residue is covalently linked to two of the third peptide motifs, the first, second, and third peptide motifs independently consisting of mono- or dipeptide motifs, each of the first, second, and third peptide motifs comprising a) arginine (R) or lysine (K), and / or b) leucine (L), valine (V), histidine (H), or isoleucine (I), each amino acid residue independently selected from the L-isoform or the D-isoform. 2. The composition for use according to item 1, wherein at least two of the first, second and third peptide motifs comprise arginine (R). 3. The composition for use according to paragraph 1 or 2, wherein at least two of the first, second and third peptide motifs comprise leucine (L). 4. The composition for use according to any one of items 1 to 3, wherein each of the first, second, and third peptide motifs is a dipeptide motif. 5. The composition for use according to paragraph 4, wherein each of the first, second and third peptide motifs comprises leucine (L) and arginine (R). 6. The composition for use according to any one of items 1 to 5, wherein the nucleic acid comprises a double-stranded region. 7. The composition for use according to item 6, wherein the nucleic acid is a DNA plasmid. 8. A composition for use according to any one of items 1 to 7, wherein the nucleic acid is or codes for an mRNA molecule or an antisense oligonucleotide (ASO). 9. The composition for use according to any one of claims 1 to 8, wherein the nucleic acid comprises a CRISPR sequence. 10. The composition for use according to item 6, wherein the nucleic acid is a siRNA or saRNA molecule. 11. The composition for use according to item 7, wherein the DNA plasmid is capable of expressing an siRNA or saRNA molecule in a target cell. 12. The composition for use according to paragraph 7 or 8, wherein the nucleic acid is capable of expressing a transgene in a target cell. 13. The composition for use according to item 12, wherein the transgene is a viral protein, a bacterial protein, or a protein of a microorganism parasitic to a mammal. 14. The composition for use according to clause 13, for use as a vaccine. 15. The composition for use according to paragraph 12, for use in gene therapy for a genetic disorder in a patient. 16. The composition for use according to item 15, wherein the genetic disorder causes muscular dystrophy in the patient. 17. The composition for use according to item 15, wherein the genetic disorder causes myopathy in the patient. 18. The composition according to item 12, for use in a method for treating diabetic limb ischemia, wherein the introduced gene 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 items 1 to 18, 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 items 1 to 18, wherein the lipid comprises dioleoylphosphatidylethanolamine (DOPE) and / or N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). 20. The composition for use according to any one of items 1 to 19, wherein the lipid comprises dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylglycerol (DOPG). 21. A composition for use according to any one of claims 1 to 20, wherein the use comprises delivery of a nucleic acid to the muscle of a patient. 22. A pharmaceutical composition comprising the composition according to any one of items 1 to 21 and a pharma- ceutically acceptable excipient. 23. A method for delivering a nucleic acid into a target cell, comprising contacting the target cell with the composition according to any one of paragraphs 1 to 20, wherein the target cell is a muscle cell, a liver cell, a stellate cell, a neuron, an astrocyte, a splenocyte, a lung cell, a cardiomyocyte, a kidney cell, an adipocyte, or a tumor cell. 24. A 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 linked to the first lysine residue, (i) a first lysine residue is covalently linked to two first peptide motifs, each of which is covalently linked to two second lysine residues; (ii) each second lysine residue is covalently linked to two second peptide motifs, each second peptide motif being covalently linked to one of the third lysine residues; (iii) a peptide dendrimer, wherein each third lysine residue is covalently linked to two of the third peptide motifs, and the first, second, and third peptide motifs each consist of either (i) a leucine-arginine (LR) dipeptide motif, or (ii) an arginine-leucine (RL) motif, and at least one of the first, second, and third peptide motifs is (i) leucine-arginine (LR), and 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 the peptide dendrimer according to item 24. 26. The composition according to item 25 for use in medicine. 27. Use of the composition according to paragraph 25 for delivering a nucleic acid to a cell in vitro or ex vivo. 28. The 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, a muscle wasting disease, or a muscular dystrophy, such as Duchenne muscular dystrophy.

[0293] References A number of publications have been cited above in order to more fully describe and disclose the invention and the state of the art to which it pertains. Full citations for these references are set forth below. Each of these references is incorporated herein in its entirety.

[0294] Kwok et al. Comparative structural and functional studies of nanoparticle formulations for DNA and siRNA delivery; Nanomedicine: Nanotechnology, Biology and Medicine 7; 210-219 (2011).

[0295] Kwok et al.Peptide Dendrimer / Lipid Hybrid Systems Are Efficient DNA Transfection Reagents:Structure-Activity Relationships Highlight the Role of Charge Distribution Across Dendrimer Generations;ACS Nano 7;54668-4682(2013)。

[0296] Kwok et al.Systematic Comparisons of Formulations of Linear Oligolysine Peptides with siRNA and Plasmid DNA;Chem Biol Drug Des 87:747-763(2016)。

[0297] Kwok et al.Developing small activating RNA as a therapeutic:current challenges and promises Therapeutic delivery 10(3):151-164(2019)

[0298] Saher et al.Novel peptide-dendrimer / lipid / oligonucleotide ternary complexes for efficient cellular uptake and improved splice-switching activity;Eur J Pharmaceutics and Biopharmaceutics 132:29-40(2018)。

[0299] Saher et al.Sugar and Polymer Excipients Enhance Uptake and Splice-Switching Activity of Peptide-Dendrimer / Lipid / Oligonucleotide Formulations.Pharmaceutics.11(12):666(2019)

[0300] Sheridan et al.Gene therapy finds its niche.Nat Biotechnol 29(2),121-8(2011)。

[0301] Braum.Non-viral Vector for Muscle-Mediated Gene Therapy;Chapter 9 Muscle Gene Therapy,Springer Nature Switzerland AG D.Duan,J.R.Mendell(eds.)157-178(2019)。

[0302] Ren et al.Structural basis of DOTMA for its high intravenous transfection activity in mouse;Gene Therapy 7,764-768(2000)。

[0303] John et al.Human MicroRNA Targets;PLoS Biology,11(2),1862-1879(2004)。

[0304] Myers et al.Recombinant Dicer efficiently converts large dsRNAs into siRNAs suitable for gene silencing;Nature Biotechnology 21:324-328(2003)。

[0305] Lim et al.Engineered Nanodelivery Systems to Improve DNA Vaccine Technologies;Pharmaceutics 12(1)30(2020)。

[0306] Luo et al.Arginine functionalized peptide dendrimers as potential gene delivery vehicles.Biomaterials 33,4917-4927(2012)。

[0307] Bonnet et al.Systemic Delivery of DNA or siRNA Mediated by Linear Polyethylenimine(L-PEI)Does Not Induce an Inflammatory Response.Pharmaceutical Res.25,2972(2008)。

[0308] Wang et al.Adeno-associated virus vector as a platform for gene therapy delivery.Nat Rev Drug Discov.18(5):358-378(2019)。

[0309] Philippidis.Fourth Boy Dies in Clinical Trial of Astellas’ AT132.Human Gene Therapy.32,19-20(2021).Qiu et al.Developing Biodegradable Lipid Nanoparticles for Intracellular mRNA Delivery and Genome Editing.Acc.Chem.Res.54(21),4001-4011(2021)。

[0310] Benizri et al.Bioconjugated Oligonucleotides:Recent Developments and Therapeutic Applications.Bioconjug Chem.30(2):366-383.(2019)。

[0311] Jasinski et al.The Effect of Size and Shape of RNA Nanoparticles on Biodistribution.Mol Ther.26(3),784-792(2018)。

[0312] Huang et al.Delivery of Therapeutics Targeting the mRNA-Binding Protein HuR Using 3DNA Nanocarriers Suppresses Ovarian Tumor Growth.Cancer Research.76(6),1549-1559(2016)。

[0313] For standard molecular biology techniques,see Sambrook,J.,Russel,D.W.Molecular Cloning,A Laboratory Manual.3 ed.2001,Cold Spring Harbor,New York:Cold Spring Harbor Laboratory Press

Claims

1. A pharmaceutical composition for use in medicine comprising a peptide dendrimer, a nucleic acid and a lipid, the peptide dendrimer comprises at least a core peptide sequence, a first branching residue, and two first peptide motifs, the branching residue being lysine, 2,4-diaminobutyric acid, ornithine, or diaminopropionic acid; The pharmaceutical composition, 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. A pharmaceutical composition comprising a peptide dendrimer, a nucleic acid, and a lipid, the peptide dendrimer comprising at least a core peptide sequence, a first branching residue, and two first peptide motifs, the branching residues being lysine, 2,4-diaminobutyric acid, ornithine, or diaminopropionic acid; The pharmaceutical composition, 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.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the single stranded nucleic acid is RNA, optionally wherein the RNA is selected from mRNA, ssRNA, sgRNA, crRNA, tracrRNA, lncRNA, siRNA, and / or self-amplifying RNA, optionally wherein the RNA comprises a modified nucleic acid, and further optionally wherein the pharmaceutical composition comprises two or more RNAs.

4. 3. The pharmaceutical composition of claim 1 or 2, wherein the nucleic acid is DNA or the single stranded nucleic acid is ssDNA, optionally comprising a modified nucleic acid.

5. said nucleic acid or single-stranded nucleic acid encodes a transgene and is capable of expressing said transgene in a target cell or said nucleic acid or single-stranded nucleic acid is capable of modulating the expression or activity of an endogenous gene, optionally via the exogenous expression of a further copy of said gene; and / or 5. The pharmaceutical composition of any one of claims 1 to 4, wherein the protein or peptide comprises an antigen, a hormone, a receptor, a chimeric antigen receptor, a transcription factor, and / or a cytokine such as IL-2, IL-7, IL-12, IL-15, IL-21, and / or an interferon.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition is a vaccine.

7. 7. The pharmaceutical composition according to any one of claims 1 to 6, wherein said nucleic acid or 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, optionally wherein said system for editing a genome or a system for altering gene expression is a CRISPR / Cas system.

8. 8. The pharmaceutical composition of any one of claims 1 to 7, wherein the pharmaceutical composition is suitable for targeting the nucleic acid to the spleen, lymphatic tissue, lung, thymus, bone, liver, cardiac tissue, tumor tissue, skeletal muscle, kidney, adipose tissue, and / or brain.

9. A pharmaceutical composition according to any one of claims 1 to 8 for use in the treatment of cancer in a subject, comprising The cancer is optionally a blood cancer, such as leukemia, lymphoma or myeloma, or prostate cancer, ovarian cancer, cardiac cancer, sarcoma, liver cancer, colon cancer, lung cancer, pancreatic cancer, or a solid tumor; or For use in the treatment of a genetic disorder in a subject, optionally wherein said nucleic acid expresses a functional version of a gene that is non-functional, down-regulated, inactive or impaired in said subject; or For use in treating a pulmonary disease or an autoimmune disease in a subject; or for use in the treatment of Pompe disease, a muscle wasting disease, myopathy, or muscular dystrophy, such as Duchenne muscular dystrophy, in a subject; or A pharmaceutical composition for use in treating limb ischemia, such as diabetic limb ischemia, in a subject, wherein the introduced gene is hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and / or fibroblast growth factor (FGF).

10. the pharmaceutical composition delivers the nucleic acid or single-stranded nucleic acid to a subject's white blood cells, e.g., B lymphocytes, T lymphocytes, monocytes, neutrophils, dendritic cells, macrophages, or monocytes; lymph node tissue cells, bone marrow cells, fibroblasts, muscle cells, skeletal muscle cells, liver cells, endothelial cells, myeloid derived suppressor cells (MDSCs), tumor associated macrophages or tumor associated neutrophils, stellate cells, neurons, astrocytes, splenocytes, lung cells, cardiac muscle cells, kidney cells, stem cells, adipocytes, or tumor cells; and / or 10. The pharmaceutical composition of any one of claims 1 to 9, wherein the pharmaceutical composition is administered to a subject such that the nucleic acid is delivered to an immune cell, optionally wherein the nucleic acid expresses an immune molecule or a transcription factor in the cell, and further optionally wherein the immune molecule is a T cell receptor, a cytokine, a decoy receptor, an antibody, a co-stimulatory receptor, a co-stimulatory ligand, a checkpoint inhibitor, an immunoconjugate, or a tumor antigen.

11. the two first peptide motifs independently consist of a single amino acid, dipeptide, tripeptide, or tetrapeptide motif; Optionally, the peptide dendrimer further comprises two second branch residues (e.g., lysine) and four second peptide motifs, one of the second branch residues being covalently bonded to one of the first peptide motifs and the other of the second branch residues being covalently bonded to the other of the first peptide motifs, each second branch residue being covalently bonded to two second peptide motifs; further optionally, said four second peptide motifs independently consist of monopeptide, dipeptide, tripeptide, or tetrapeptide motifs; Further optionally, said peptide dendrimer further comprises four third branch residues (e.g., lysine) and eight third peptide motifs, each second peptide motif being covalently bound to one of said third branch residues, such that each third branch residue is covalently bound to one second peptide motif, and each third branch residue is covalently bound to two third peptide motifs; 11. The pharmaceutical composition of any one of claims 1 to 10, further optionally wherein the eight third peptide motifs independently consist of a monopeptide, a dipeptide, a tripeptide, or a tetrapeptide motif.

12. the first, second and / or third peptide motif, when present, comprises amino acids with non-polar side chains and / or further comprises amino acids with acidic or basic side chains; 12. The pharmaceutical composition of any one of claims 1 to 11, optionally wherein the first, second and third peptide motifs, if present, comprise leucine (L), lysine (K), valine (V), histidine (H), isoleucine (I), glycine (G), aspartic acid (D), phenylalanine (F) and / or arginine (R) residues.

13. 13. The pharmaceutical composition of any one of claims 1 to 12, wherein the peptide dendrimer comprises a cell penetrating peptide, optionally the cell penetrating peptide comprises a sequence derived from TAT or the sequence XRXRRBRRXRRBRXB (SEQ ID NO: 2), where X is 6-aminohexanoic acid and B is beta-alanine.

14. the lipids include cationic lipids, neutral lipids, anionic lipids, and / or ionizable lipids; Optionally, the lipid is dioleoylphosphatidylethanolamine (DOPE) and / or N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). and / or dioleoylphosphatidylglycerol (DOPG).

15. 15. The pharmaceutical composition of any one of claims 1 to 14, wherein after administration to a subject, a higher proportion of the pharmaceutical composition is observed in the lungs, spleen, and / or lymph nodes than in the liver.

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