Hybrid peptide dendrimer systems and extrahepatic delivery

Abstract

The present invention relates to compositions capable of delivering therapeutic molecules, such as nucleic acids, to mammalian cells and within the human and animal body, as well as methods for their preparation and use. The compositions include nanoparticles comprising peptide dendrimers, nucleic acids, and lipids. The compositions of the present invention find utility in the medical field, such as for treating cancer and autoimmune diseases.

Description

[Technical Field]

[0001] The present invention relates to compositions capable of delivering therapeutic molecules, such as nucleic acids, to mammalian cells and within the human and animal body. [Background technology]

[0002] Nucleic acid therapeutics have the potential to be the next generation of precision medicine, transforming healthcare. However, significant challenges remain. One such challenge is the efficient and safe delivery of nucleic acid therapeutics to patients. Current viral and non-viral vector platforms are often hindered in clinical translation due to off-target effects, immune activation, and difficulties in large-scale vector manufacturing.

[0003] In the context of in vivo delivery of nucleic acids, virus-derived vectors have been extensively studied, and some, including adeno-associated virus (AAV), have made significant progress in the clinic (Sheridan et al., 2011 and Wang et al., 2019). However, the use of these systems is limited to delivering DNA less than 5 kb and cannot transport RNA or larger DNA fragments. Despite advances in these delivery systems, the potential for random insertions and immunotoxins resulting from their use remains. AAV systems can be more immunogenic, particularly when targeting non-liver tissues, which requires the use of higher doses. This tendency to generate an immune response against AAV systems also limits the usefulness of these systems for repeated dosing, as patients typically develop immunity to AAV delivery systems. Finally, AAV delivery systems are expensive and known to be difficult to manufacture at the scale required for therapeutic use and to Good Manufacturing Practice (GMP)-grade standards.

[0004] Non-viral vector systems for nucleic acid delivery to cells and tissues in vivo are also being investigated. These systems include those designed for the delivery of smaller nucleic acids, such as siRNA and antisense oligonucleotides (ASOs). One approach being investigated is bioconjugate oligonucleotide delivery systems, in which siRNA or ASOs are conjugated to antibodies or ligands (Benizri et al., 2019). However, these approaches are limited to gene silencing or exon skipping and cannot be used to express genes in target tissues. Bioconjugate-based delivery systems are difficult to apply to large gene payloads, such as plasmid DNA and mRNA, because each is a large, negatively charged molecule that cannot easily cross the negatively charged plasma membrane of cells.

[0005] To address the difficulty of translocating plasmid DNA and mRNA through the plasma membrane, it may be useful to encapsulate nucleic acids and neutralize their charge for effective delivery. Lipid nanoparticles (LNPs) encompass non-viral delivery vehicles, such as those used to encapsulate and deliver mRNA in COVID-19 vaccines (Qui et al., 2021). These systems may be useful for vaccine delivery because they only require transfection of a small number of muscle and immune cells localized at the delivery site to train the immune system to fight infection. Therefore, RNA-based COVID-19 vaccines are administered intramuscularly. However, for widespread disease, a large number of target cells must be transfected to effectively treat the disease. While LNPs currently used in clinical trials may be well suited to targeting the liver when administered intravenously, they are not suitable for targeting other tissues, making them unsuitable for targeting diseases associated with diseases other than the liver.

[0006] Liposome-based systems have also been investigated using 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).

[0007] To overcome the various drawbacks of the above-mentioned delivery systems, the addition of peptides to lipid-based vectors has been investigated. In these hybrid systems, peptides and lipids associate with nucleic acids to form nanoparticles that can be internalized by cells. The peptide component can be linear (Kwok et al., 2016) or branched, such as peptide dendrimers (Kwok et al., 2013). These peptide / lipid systems were first used to study the delivery of plasmid DNA and siRNA in serum-free in vitro conditions, which are substantially different from in vivo conditions. However, it remains unclear whether these systems can be used to deliver long nucleic acids in vivo. The only reports demonstrating in vivo liver delivery of ASOs using peptide dendrimer / lipid nanoparticles achieved only a modest 20–30% increase in delivery compared to ASOs alone (Saher et al., 2018, Saher et al., 2019). Considering the low delivery rates of ASOs using these systems, it can be concluded that these peptide dendrimer / lipid systems are not suitable or capable of delivering larger nucleic acids, such as mRNA, to tissues in vivo.

[0008] In PCT application WO2022162200, the present inventors recently demonstrated that delivery of long nucleic acids, such as mRNA, can be achieved in vivo using a peptide dendrimer / lipid hybrid system. The present inventors developed a framework for nucleic acid delivery using peptide dendrimers, as disclosed in WO2022162200 (incorporated by reference in its entirety). The dendrimers disclosed in WO2022162200 are branched peptides exhibiting one, two, three, or four amino acid residues between "branch residues" that function as branching units within the dendrimer molecule. WO2022162200 demonstrated that these dendrimer / lipid compositions are surprisingly effective in delivering larger nucleic acids (e.g., larger than antisense oligonucleotides (ASOs)), for example, to extrahepatic tissues. While some smaller nucleic acids, such as ASOs, are easily taken up into cells without a vector system, the compositions of WO2022162200 enable the effective delivery of larger nucleic acids, such as mRNA.

[0009] Despite the advances provided by the compositions disclosed in WO2022162200, there remains a need to develop nucleic acid delivery systems with improved transfection efficiency and cell and tissue specificity to maximize the therapeutic potential of nucleic acid therapeutics.

[0010] The present invention has been devised in light of the above considerations. Summary of the Invention

[0011] The development of nucleic acid therapeutics relies on efficient and targeted nucleic acid delivery. The present inventors have found that peptide dendrimer / lipid-based nucleic acid delivery systems can be modified to improve nucleic acid delivery to specific cells and tissues. One way in which the present inventors have found that cell- and / or tissue-specific nucleic acid delivery can be achieved is by modifying the ratio of peptide dendrimer (also referred to herein as "dendrimer") to nucleic acid and / or the ratio of lipid to nucleic acid in the nanoparticle. Alternatively, targeted delivery can be achieved by including a cell- or tissue-specific targeting motif within the nanoparticle. The location of the targeting motif is not particularly limited; for example, it may be covalently attached to the peptide dendrimer, to a second peptide (which may be further included in the nanoparticle), or to a polymer (which may be further included in the nanoparticle).

[0012] The inventors have also found that peptide dendrimer / lipid nanoparticles containing a combination of two different dendrimers can be more effective in achieving nucleic acid delivery in vitro and in vivo than nanoparticles containing a single peptide dendrimer. For example, combining a "first-generation" peptide dendrimer with a "second-generation" or "third-generation" peptide dendrimer in a single composition can enable more efficient nucleic acid delivery in vitro and in vivo than compositions containing a single first-, second-, or third-generation peptide dendrimer alone. This can be achieved, for example, by varying the number of charged and hydrophobic residues within each peptide dendrimer that can form noncovalent bonds with nucleic acids. Alternatively, or in addition, a mixture of dendrimers can be selected based on the stability of each dendrimer / nucleic acid complex and its ability to form a monodisperse population of nanoparticles. For example, a nanoparticle may comprise one dendrimer that, when used alone, is incapable of forming a monodisperse population of dendrimer / lipid nanoparticles (indicating a relatively high dendrimer-nucleic acid dissociation rate), along with a second dendrimer that, when used alone, is capable of forming a monodisperse population of dendrimer / lipid nanoparticles (indicating a relatively low dendrimer-nucleic acid dissociation rate). Thus, peptide dendrimers may be selected based on the polydispersity index of each single dendrimer / lipid nanoparticle. Additionally, nanoparticles may comprise advantageous combinations of lipids described herein, which may improve transfection efficiency.

[0013] These new findings further open up the field of nucleic acid therapeutics and offer a promising solution to the unsolved problem of providing efficient, safe and targeted vectors for nucleic acid delivery in vivo.

[0014] The present invention may also be useful for ex vivo transfection of cells / tissues derived from patients. For example, the nanoparticles of the present invention may be suitable for more efficient nucleic acid delivery to ex vivo bone marrow or lymphoid cells. This discovery is particularly useful in the field of chimeric antigen receptor (CAR)-based therapeutics, such as CAR-T and CAR-M, aimed at treating various cancers. For example, embodiments in which advantageous lipid combinations exhibit enhanced transfection efficiency find use in in vitro applications.

[0015] Based on the surprising finding in WO2022162200 that peptide dendrimer / lipid nanoparticles containing a single peptide dendrimer can effectively deliver nucleic acid cargo to cells in vivo, the present mixed peptide dendrimer / lipid nanoparticles are also expected to effectively deliver nucleic acid cargo to cells in vivo. Additionally, based on the increased cargo delivery efficiency achieved in vitro using mixed dendrimer nanoparticles compared to single dendrimer nanoparticles, it is further expected that the efficiency of nucleic acid cargo delivery to cells in vivo will also be higher than that exhibited by single dendrimer / lipid nanoparticles. Thus, given the inventors' findings in WO2022162200, mixed dendrimer nanoparticles can effectively deliver nucleic acids, such as DNA and RNA, to various tissues, particularly lungs, as well as immune cell-rich tissues, including the spleen, lymph nodes, and bone marrow. This enables the development of improved DNA- and RNA-based therapies targeted to extrahepatic tissues.

[0016] The present invention can effectively and specifically deliver nucleic acids, including DNA and mRNA, to various cells and tissues, particularly bone marrow and lymphoid cells, as well as muscle and lung tissues, and cancer cells. Nanoparticles can also be adapted to target other organs and tissues. The ability to deliver nucleic acids, particularly mRNA, to immune cells, and even bone marrow cells, including monocytes, macrophages, neutrophils, and dendritic cells, allows this technology to be developed to treat all types of cancer (including solid tumors and hematological cancers, such as myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML)), autoimmune diseases (e.g., diabetes, including type 1 diabetes, rheumatoid arthritis, Crohn's disease, uveitis, inflammatory bowel disease), and other immune cell-related disorders, such as graft-versus-host disease, allograft rejection, acute rejection after transplantation, chronic rejection after transplantation, primary graft dysfunction, chronic granulomatous disease (CGD), or Gaucher disease. The present invention also enables delivery of nucleic acids, including DNA and mRNA, to lung and / or immune cells, allowing for the treatment of lung-related diseases such as cystic fibrosis and chronic obstructive pulmonary disease. Additionally, the present invention enables delivery of nucleic acids, including DNA and mRNA, to muscle tissue and muscle cells, allowing for the treatment of muscular dystrophies (e.g., Duchenne muscular dystrophy, myotonic dystrophy, facioscapulohumeral muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, oculopharyngeal muscular dystrophy, Emery-Dreifuss muscular dystrophy, hereditary muscular dystrophies, congenital muscular dystrophies, and distal muscular dystrophies) and myopathies, including muscle-wasting diseases.

[0017] Thus, in one aspect, the present invention provides nanoparticles comprising a peptide dendrimer, a nucleic acid, and a lipid. Those skilled in the art will understand that two or more peptide dendrimers and / or two or more lipids may be included. 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 nanoparticles may target specific cell types or tissues, such as bone marrow cells, lymphoid cells, muscle cells, lung cells, and / or cancer cells, and / or bone marrow tissue, lymphoid tissue, muscle tissue, lung tissue, and / or tumor tissue. The nanoparticles may target bone marrow, pancreas, neural tissue, kidney tissue, heart tissue, liver tissue, eye, joint, or prostate, or stem cells, pancreatic cells, neural cells, kidney cells, cardiac cells, liver cells, eye cells, synovial cells, or prostate cells. As used herein, bone marrow tissue includes bone marrow tissue and cells of the myeloid lineage. As used herein, lymphoid tissue includes any organ of the lymphatic system, including the spleen, thymus, lymph nodes, and bone marrow. The nanoparticles find use in vivo, ex vivo, and in vitro. The nanoparticles can transfect target cells or tissues, such as bone marrow, lymph, muscle, lung, or cancer cells, in vitro with an efficiency of at least 10%, at least 12.5%, at least 15%, at least 17.5%, at least 20%, at least 25%, at least 30%, 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%, or at least 85%. Transfection efficiency can be determined by any standard method in the art, such as fluorescence microscopy, real-time PCR (qPCR), a plasmid reporting system, a reporter gene assay, flow cytometry, Western blot, or immunofluorescence staining. Such methods are described in Chong, Z., et al. (2021) or as described in Example 1.Preferably, flow cytometry is used to determine the percentage of transfection efficiency of a cell population after treatment with nanoparticles containing fluorescently labeled nucleic acids. Flow cytometry is used to determine nanoparticle uptake. Preferably, the transfection efficiency is measured after transfection of a 600 μL volume of cell population in a 24-well plate in serum-containing medium at 37° C. for 4 hours. Preferably, the nanoparticle population is added to the wells at a dose containing 1.5 μg of nucleic acid. Alternatively, flow cytometry is used to determine the percentage of transfection efficiency of a cell population after treatment with nanoparticles containing nucleic acids expressing a reporter gene. Flow cytometry is used to determine reporter gene expression. In this case, reporter gene expression is measured after transfection of a 600 μL volume of cell population in a 24-well plate in serum-containing medium at 37° C. for 24 hours. Preferably, the nanoparticle population is added to the wells at a dose containing 1.5 μg of nucleic acid. Some modifications to the test protocol may be made, such as using a larger or smaller cell volume within the range of 300–1,200 μL. In some embodiments, 12-, 48-, or 96-well plates may be used instead of 24-well plates. For example, 120,000 macrophages may be used in a 24-well plate, or 240,000 macrophages may be used in a 12-well plate. 60,000 C2c12 cells may be used in a 24-well plate. Transfection times may vary; for example, incubation with the nanoparticle population may be 1 or 2 hours. Longer incubation times, such as 16 or 24 hours, may be used, particularly when flow cytometry is used to determine reporter gene expression. Slightly larger amounts, such as 3 μg of nanoparticles, may also be used.

[0018] In another aspect, the present invention provides nanoparticles comprising a peptide dendrimer, a nucleic acid, and a lipid for use in medicine. 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 nanoparticles target bone marrow, lymphatic, muscle, lung, and / or cancer cells, and / or bone marrow, lymphatic, muscle, lung tissue, and / or tumors. The nanoparticles can transfect target cells or tissues, such as bone marrow, lymphatic, muscle, lung, and / or cancer cells, in vitro with an efficiency of at least 10%, at least 12.5%, at least 15%, at least 17.5%, at least 20%, at least 25%, at least 30%, 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%, or at least 85%.

[0019] In a further aspect, the present invention provides nanoparticles comprising a peptide dendrimer, a nucleic acid, and a lipid for use in treating cancer, autoimmune disease, lung disease, and / or myopathy. 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 nanoparticles may target bone marrow, lymphatic, muscle, and / or lung cells, and / or bone marrow, lymphatic, muscle, lung tissue, and / or tumors. The nanoparticles may target bone marrow, pancreas, nervous tissue, kidney tissue, heart tissue, liver tissue, eye, joint, or prostate, or stem cells, pancreatic cells, nervous cells, kidney cells, cardiac cells, liver cells, ocular cells, synovial cells, or prostate cells. The nanoparticles can transfect target cells or tissues in vitro, e.g., bone marrow, lymphatic, muscle, lung and / or cancer cells, with an efficiency of at least 10%, at least 12.5%, at least 15%, at least 17.5%, at least 20%, at least 25%, at least 30%, 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%.

[0020] In a further aspect, the present invention provides a method for treating cancer, autoimmune disease, lung disease, and / or myopathy, comprising administering to a patient or subject nanoparticles 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 residues may be lysine, 2,4-diaminobutyric acid, ornithine, or diaminopropionic acid. The nanoparticles may target bone marrow, lymphatic, muscle, lung, and / or cancer cells, and / or bone marrow, lymphatic, muscle, lung tissue, and / or tumors. The nanoparticles may target bone marrow, pancreas, nervous tissue, kidney tissue, heart tissue, liver tissue, eye, joint, or prostate, or stem cells, pancreatic cells, nervous cells, kidney cells, heart cells, liver cells, eye cells, synovial cells, or prostate cells. The nanoparticles can transfect target cells or tissues in vitro, e.g., bone marrow, lymphatic, muscle, lung and / or cancer cells, with an efficiency of at least 10%, at least 12.5%, at least 15%, at least 17.5%, at least 20%, at least 25%, at least 30%, 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%.

[0021] In a further aspect, the present invention provides nanoparticles comprising a peptide dendrimer, a nucleic acid, and a lipid selected from Table 1. For example, the nanoparticles of the present invention have the structure (RHL)(KRHL)KGSC-NH, (HL)(KRF)(KRF)KGSC-NH, (LR)(KRL)KRHA-NH, (LR)(KRL)(KRL)KGSC-NH, (LR)(KRL)KRHC-NH, (LR)(KRL)KRHCR-(Acp)-RR-(β-A)-RR-(Acp)-RR-(β-A)- R-(Acp)-(β-A)-NH2, (LR)4(KRL)2KRHCGAASSLNIA-(Acp)-NH2, (LR)4(KRL)2KRHCGAASSLNIA-(Acp)-R-(Acp )-RR-(β-A)-RR-(Acp)-RR-(β-A)-R-(Acp)-(β-A)-NH2, (LR)8(KRL)4(KRL)2KRHCR-(Acp)-RR-(β-A)-RR-(Ac p)-RR-(β-A)-R-(Acp)-(β-A)-NH2, (LRLR)2KGSC-NH2, (HR)2KK-NH2, (RFI)4(KKE)2KRG-NH2, (SYR)4(KLRF) 2KER-NH2, (RL)4(KHGD)2KLR-NH2, (HVR)4(KHVR)2KVR-NH2, (LHR)4(KRHL)2KGSC-NH2, (RLRL)2KLRL-NH2, (LR )(KRL)(KRL)KGSCGAASSLNIA(Acp)-NH2, (LR)(KRL)(KRL)KGSCHHHHHHGAASSLNIA(Acp)-NH2, (LR)(KRL)KGSGGSGGSGGSC[(SS)-α-D-thiomannose], (Ac-EEEE)KGSGGSGGSC[(SS)-α-D-thiomannose].In some embodiments, the nanoparticles have the structure (RHL)4(KRHL)2KGSC-NH2, (HL)8(KRF)4(KRF)2KGSC-NH2, (LR)4(KRL)2KRHA-NH2, (LR)4(KRL)2KRHC R-(Acp)-RR-(β-A)-RR-(Acp)-RR-(β-A)-R-(Acp)-(β-A)-NH2, (LR)8(KRL)4(KRL)2KRHCR-(Acp)-RR-(β-A)-RR-(Acp)-RR- The compound may include dendrimers having (β-A)-R-(Acp)-(β-A)-NH2, (HR)2KK-NH2, (RFI)4(KKE)2KRG-NH2, (SYR)4(KLRF)2KER-NH2, (RL)4(KHGD)2KLR-NH2, or (HVR)4(KHVR)2KVR-NH2, (LHR)4(KRHL)2KGSC-NH2, (RLRL)2KLRL-NH2, (LR)4(KRL)2KRHC-NH2, (LRLR)2KGSC.

[0022] In a further aspect, the present invention provides a composition comprising a first peptide dendrimer, a second peptide dendrimer, a nucleic acid, and a lipid. The first and second peptide dendrimers comprise 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.

[0023] In another aspect, the present invention provides a composition for use in medicine, the composition comprising a first peptide dendrimer, a second peptide dendrimer, a nucleic acid, and a lipid. The first and second peptide dendrimers comprise 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.

[0024] In another aspect, the present invention provides a composition comprising a first peptide dendrimer, a second peptide dendrimer, a nucleic acid, and a lipid for use in a method for treating cancer and / or an autoimmune disease. The first and second peptide dendrimers comprise 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.

[0025] In another aspect, the present invention provides a method of treating a patient with a composition comprising a first peptide dendrimer, a second peptide dendrimer, a nucleic acid, and a lipid. The first and second peptide dendrimers comprise at least a core peptide sequence, a first branching residue, and two first peptide motifs. The branching residues can be lysine, 2,4-diaminobutyric acid, ornithine, or diaminopropionic acid. The method can include treating a patient with cancer and / or an autoimmune disease.

[0026] Those skilled in the art will understand that these compositions comprising a first peptide dendrimer, a second peptide dendrimer may comprise three or more dendrimers, such as three or four or more dendrimers.

[0027] Relatedly, the present invention provides compositions comprising the nanoparticles of the present invention. The present invention further provides pharmaceutical compositions comprising the nanoparticles of the present invention and a pharmaceutically acceptable excipient. The pharmaceutical compositions may be used in medicine. The pharmaceutical compositions may be for use in treating cancer, autoimmune diseases, pulmonary diseases, and / or myopathies. Methods of treating cancer, autoimmune diseases, pulmonary diseases, and / or myopathies are also provided, comprising administering the pharmaceutical composition to a patient or subject. In some embodiments, the composition or pharmaceutical composition is contained within a liquid. In other embodiments, the composition or pharmaceutical composition is provided as a dry composition, e.g., a dry powder. The dry composition may be prepared using lyophilization and / or freeze-drying techniques.

[0028] The nanoparticles of the present invention may also comprise bone marrow, lymphatic, muscle, lung, or cancer cell targeting motifs. For example, muscle cell targeting motifs may comprise the peptide motifs ASSLNIA (SEQ ID NO: 1), PYDQLRH (SEQ ID NO: 2), or KAMHQMQ (SEQ ID NO: 3). Muscle cell targeting motifs may comprise variants of the peptide motifs ASSLNIA (SEQ ID NO: 1), PYDQLRH (SEQ ID NO: 2), or KAMHQMQ (SEQ ID NO: 3). For example, variant muscle cell targeting motifs may comprise one, two, or three amino acid substitutions, deletions, or additions to the peptide motifs ASSLNIA (SEQ ID NO: 1), PYDQLRH (SEQ ID NO: 2), or KAMHQMQ (SEQ ID NO: 3), provided that the variants retain the ability to target the nanoparticles to muscle cells.

[0029] In some embodiments, the targeting motif is a lung cell targeting motif. In some embodiments, the lung cell targeting motif may comprise the peptide sequence CGFECVRQCPERC (SEQ ID NO: 4). The lung cell targeting motif may comprise a variant of the peptide sequence CGFECVRQCPERC (SEQ ID NO: 4). For example, the variant lung cell targeting motif may comprise one, two, or three amino acid substitutions, deletions, or additions to the peptide sequence CGFECVRQCPERC (SEQ ID NO: 4), provided that the variant retains the ability to target the nanoparticle to lung cells.

[0030] In another example, the targeting motif is a cancer cell targeting motif. In some embodiments, the cancer cell targeting motif is a peptide comprising an RGD integrin targeting peptide motif. In some embodiments, the cancer cell targeting motif is a peptide comprising an ACDCRGDCFCG (SEQ ID NO: 5) integrin targeting peptide motif. The cancer cell targeting motif may comprise a variant of the peptide sequence CGFECVRQCPERC (SEQ ID NO: 5). For example, a variant cancer cell targeting motif may comprise one, two, or three amino acid substitutions, deletions, or additions to the peptide sequence CGFECVRQCPERC (SEQ ID NO: 5), provided that the variant retains the ability to target the nanoparticle to cancer cells. In another example, the cancer cell targeting motif may be a maltotriose sugar. Maltotriose sugars may be used as maltotriose, which can bind to GLUT receptors that are highly expressed on cancer cells (Sakamaki, Y., et al. (2021)).

[0031] The myeloid cell targeting motif may comprise a sugar. The myeloid cell targeting motif may comprise a mannose sugar for targeted delivery to macrophages. In another example, the myeloid cell targeting motif may be maltotriose. Maltotriose is a trisaccharide consisting of three glucose molecules linked by α-1,4 glycosidic bonds. Mannose sugars, mannose glycosylation, and / or maltotriose may be used to target nanoparticles to macrophages with an M2 phenotype. For example, the targeting motif may comprise a mannose sugar or maltotriose, each of which can bind to the CD206 receptor expressed on M2 phenotype macrophages. Other sugars may be selected for conjugation with peptide dendrimers to target other tissues and cell types. For example, N-acetylgalactosamine (GalNAc) may be selected for hepatocyte targeting (Holland et al. (2021)).

[0032] In some embodiments, the cell targeting motif targets lymphocytes, e.g., T cells. The cell targeting motif may be a CD3, CD4, or CD8 conjugate, e.g., an antibody that specifically binds to one of these markers. In some embodiments, an anti-CD3 antibody is used. This may be conjugated to a negatively charged polymer such as PGA or a glutamic acid-containing peptide.

[0033] Nanoparticles may be targeted for delivery to lung tissue. Lung cells that may be targeted include alveolar macrophages, ciliated cells, epithelial cells, basal cells, secretory cells, club cells, alveolar cells, fibroblasts, and / or endothelial cells. In some embodiments, targeting to tissues such as the lung may be achieved without additional ligands or peptide motifs. For example, by adjusting the lipid:nucleic acid ratio, as described herein, lung targeting can be dramatically enhanced. Furthermore, adjusting the surface charge of the nanoparticles can also affect tissue targeting specificity, as described herein.

[0034] The nanoparticles of the present invention may also include a motif for targeting bone marrow, pancreas, nerve tissue, kidney tissue, heart tissue, liver tissue, eye, joint, or prostate, or stem cells, pancreatic cells, nerve cells, kidney cells, heart cells, liver cells, eye cells, synovial cells, or prostate cells. For example, the targeting motif may include a motif for targeting bone marrow or stem cells, such as an antibody that specifically binds to CD34, and / or a lipid containing a bisphosphonate (BP) group (Xue et al., 2022, incorporated herein by reference in its entirety). Those skilled in the art will understand that such BP-containing lipids can be conjugated to any component of the nanoparticle, such as a peptide and / or dendrimer component.

[0035] In some embodiments, the targeting motif is a pancreatic targeting peptide, such as a glucagon-like peptide-1 (GLP-1) homolog. Jones et al., 2018 (incorporated herein by reference in its entirety) discloses "exendin-4" and related peptides. Exendin-4 has the amino acid sequence HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO: 9). Such peptides, as well as variants and binding fragments thereof, can be used as pancreatic targeting peptides of the present invention. Preferably, variants have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% amino acid identity to SEQ ID NO: 9, for example, over at least 20 consecutive amino acids. Binding fragments preferably have at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, or at least 20 consecutive amino acids of SEQ ID NO: 9.

[0036] In some embodiments, the targeting motif is a kidney targeting peptide such as (KKEEE)3K (Wischnjow et al., 2016, incorporated herein by reference in its entirety). KKEEE is SEQ ID NO: 10.

[0037] In some embodiments, the targeting motif is a neural tissue / cell targeting peptide, such as YTIWMPENPRPGTPCDIFTNSRGKRASNGGGG (SEQ ID NO: 11) or KSVRTWNEIIPSKGCLRVGGRCHPHVNGGG (SEQ ID NO: 12) (Wang et al., 2021, incorporated herein by reference in its entirety). In some embodiments, the neural targeting peptide comprises a Phe-Arg-Trp (FRW) motif (Tang et al., 2019, incorporated herein by reference in its entirety).

[0038] In some embodiments, the targeting motif is a cardiac targeting or cardiac cell targeting peptide, such as APWHLSSQYSRT (SEQ ID NO: 13) (Zahid et al., 2018, incorporated herein by reference in its entirety).

[0039] In some embodiments, the targeting motif is a liver-targeting or hepatocyte-targeting moiety, such as an asialoglycoprotein receptor-binding sugar moiety. An exemplary asialoglycoprotein receptor conjugate is an N-acetylgalactosamine (Gal-NAc) sugar moiety (Cui et al., 2021, incorporated herein by reference in its entirety). Those skilled in the art will understand that such sugar moieties can be conjugated to any component of the nanoparticle, such as a peptide and / or dendrimer component. In other embodiments, the liver-targeting or hepatocyte-targeting moiety is vitamin A (Senoo et al., 2010, incorporated herein by reference in its entirety).

[0040] In some embodiments, the targeting motif is an ocular targeting or ocular cell targeting moiety, such as CARSKNKDC (SEQ ID NO: 14) (Vahatupa et al., 2021, incorporated herein by reference in its entirety).

[0041] In some embodiments, the targeting motif is a joint-targeting or synovial cell-targeting moiety, such as an antibody that specifically binds to CD44 and / or a hyaluronan sugar (Gorantla et al., 2021, incorporated herein by reference in its entirety).

[0042] In some embodiments, the targeting motif is a prostate-targeting moiety, such as PKRGFQD (SEQ ID NO: 15) or SNTRVAP (SEQ ID NO: 16) (Mandelin et al., 2015, incorporated herein by reference in its entirety).

[0043] In some embodiments, the nanoparticle further comprises a cell-penetrating peptide. The cell-penetrating peptide may comprise a sequence derived from TAT. The cell-penetrating peptide may comprise the peptide sequence XRXRRBRRXRRBRXB (SEQ ID NO: 6), where X is 6-aminohexanoic acid and B is beta-alanine. The cell-penetrating peptide may comprise a variant of the peptide sequence XRXRRBRRXRRBRXB (SEQ ID NO: 6). For example, the variant may include one, two, or three amino acid substitutions, deletions, or additions to the peptide sequence XRXRRBRRXRRBRXB (SEQ ID NO: 6), provided that the variant retains biological activity.

[0044] In some embodiments, the bone marrow, lymphoid, muscle, or lung cell targeting motif may be an antibody. "Antibody" includes fragments or derivatives thereof, or synthetic antibodies or synthetic antibody fragments. Antibody fragments, such as Fab and Fab2 fragments, may also be used as recombinant antibodies and antibody fragments. Single chain Fv (scFv) antibodies may also be used. The variable heavy chain (V) of an antibody may be a H ) and variable light chain (V L ) domain is involved in antigen recognition, a fact first recognized by early protease digestion experiments. Further confirmation was found by the "humanization" of rodent antibodies, in which variable domains of rodent origin are fused to constant domains of human origin, resulting in antibodies that retain the antigen specificity of rodent-derived antibodies (Morrison et al (1984) Proc. Natl. Acad. Sd. USA 81, 6851-6855). "ScFv molecules" are molecules in which the V H and V L" refers to a molecule in which the partner domains are covalently linked, for example, by a flexible oligopeptide. In some embodiments, the antibody may be a T cell-specific antibody, for example, an anti-CD3 antibody (e.g., clone OKT3, BioXCell, catalog number BE0001-2). In some embodiments, the antibody may be an anti-CD4 antibody (e.g., clone OKT4, BioXCell, catalog number BE0003-2), an anti-CD8 antibody (clone OKT8, BioXCell, catalog number BE0004-2), or an anti-CD28 antibody (e.g., clone 9.3, BioXCell, catalog number BE0248).

[0045] In some embodiments, the cell and / or tissue targeting motif may be covalently attached to a polymer or lipid. In some embodiments, the cell and / or tissue targeting motif may be covalently attached to a polymer or lipid contained within the nanoparticle. The polymer or lipid may be positively charged. Alternatively, the polymer or lipid may be a negatively or neutrally charged polymer. The polymer or lipid may be selected from polyglutamic acid (PGA), poly(acrylic acid), alginic acid, polyethylene glycol (PEG), cholesteryl hemisuccinate / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, a neutrally charged zwitterionic polymer or lipid, or a glutamic acid-containing peptide. PGA, poly(acrylic acid), alginate, PEG, cholesteryl hemisuccinate / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, neutrally charged zwitterionic polymer or lipid, or glutamic acid-containing peptide may comprise an additional targeting domain, e.g., an antibody or target-binding fragment thereof, as described herein. A cell and / or tissue targeting motif, e.g., an antibody, may be conjugated to one or more glutamic acid residues. PGA is a peptide containing a region composed primarily of glutamic acid residues, preferably containing at least two consecutive glutamic acid residues. (Other amino acid residues may be present within the PGA molecule.) In some embodiments, PGA is a peptide containing a (EXXEXX...) region with a glutamic acid residue at every third position, totaling 2-50, 2-40, 2-30, 2-20, or 2-10 amino acid residues. PGA may be linear or dendritic (branched) PGA. The PGA can be derivatized at its N-terminus and / or C-terminus(s). For example, the C-terminus can be derivatized with an amine group (NH). The linear PGA can be a short PGA. A short PGA is any PGA molecule having fewer than 100, fewer than 75, fewer than 50, or fewer than 20 glutamic acid residues. In embodiments where the PGA is linear, the nanoparticles preferably do not contain PBAE polymer. For example, the PGA can contain 2 to 100, 2 to 75, 2 to 50, 2 to 40, 2 to 30, 2 to 20, or 2 to 10 glutamic acid residues.In another embodiment, the PGA may be a dendritic PGA. A dendritic PGA is a peptide dendrimer containing two or more branches, each with at least two consecutive glutamic acid residues. The dendritic PGA may be a first-, second-, or third-generation peptide dendrimer. In some embodiments, the dendritic PGA is a short dendritic PGA. The short dendritic PGA may contain 2-100, 2-75, 2-50, 2-40, 2-30, 2-20, or 2-10 glutamic acid residues. Short dendritic PGAs may prove beneficial because they may provide better flexibility for "wrapping" over nanocarriers and thus may provide better binding / coating of nanoparticles compared to linear PGAs. PGA is a peptide polymer. Thus, in embodiments comprising PGA molecules, the PGA may be referred to as a "polymer" or a "second peptide." In some embodiments, the nanoparticles comprise PGA as the only peptide. In these embodiments, the nanoparticles are first formed with only lipids and nucleic acids and then coated with PGA.

[0046] In some embodiments, the polymer is a glutamic acid-containing peptide. The glutamic acid-containing peptide has a domain containing at least two glutamic acid (E) residues, preferably at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine E residues. Preferably, E residues comprise at least 10%, at least 15%, at least 20%, at least 25%, or at least 30% of the amino acid residues in the glutamic acid-rich domain. Generally, the glutamic acid-rich domain may contain at least four amino acids (e.g., divided into two dipeptide motifs within a given generation of the dendrimer), or at least six, at least eight, at least ten, or at least twelve amino acid residues. Preferably, E residues are present at a frequency of every second, third, or fourth amino acid residue (EXEX...), (EXXEXX...), or (EXXXEXXX...) in the glutamic acid-containing domain. Most preferably, E residues occur at a frequency of every third amino acid residue within a glutamic acid-rich domain spanning at least 10 amino acid residues. Preferably, the glutamic acid-containing peptide contains a total of 2-50, 2-40, 2-30, 2-20, or 2-10 amino acid residues. The glutamic acid-containing peptide may be a first-, second-, or third-generation peptide dendrimer or a linear sequence. A cell and / or tissue targeting motif, e.g., an antibody, may be conjugated to one or more glutamic acid residues. In some examples of the present invention, such glutamic acid-containing peptides containing several glutamic acid residues are referred to as "PGAs." In embodiments comprising a glutamic acid-containing peptide, the glutamic acid-containing peptide may be referred to as a "polymer" or a "second peptide." In other embodiments, the nanoparticle comprises a glutamic acid-containing peptide, which is the only peptide. In these embodiments, the nanoparticle may be initially formed with only lipids and nucleic acid and then coated with the glutamic acid-containing peptide.

[0047] In embodiments where the lipid to which the targeting motif is attached is PEG, this may be used as a "linker." In these embodiments, the PEG lipid may be covalently attached to the peptide dendrimer or lipid (in addition to being attached to the cell and / or tissue targeting motif).

[0048] In some embodiments, the cell and / or tissue targeting motif is a mannose sugar covalently attached to the polymer. In embodiments where the polymer is a peptide, the mannose may be linked at the C-terminus and / or N-terminus. In other embodiments where the polymer is a peptide, the mannose may be linked at other positions along the peptide. In some embodiments, the mannose sugar is covalently attached to a PGA molecule. In some embodiments, the mannose sugar is covalently attached to a PGA molecule containing 2-100, 2-75, 2-50, 2-40, 2-30, 2-20, or 2-10 glutamic acid residues. Mannose-derivatized PGA molecules are particularly preferred for targeting CD206-expressing cells, such as certain lymphoid or endothelial cells, dendritic cells, neutrophils, and macrophages, particularly N2 neutrophils and M2 macrophages, respectively.

[0049] In some embodiments, the cell and / or tissue targeting motif is an antibody covalently linked to a polymer or lipid. In some embodiments, the antibody is covalently linked to a PGA molecule. In some embodiments, the antibody is covalently linked to a PGA molecule comprising 2-100, 2-75, 2-50, 2-40, 2-30, 2-20, or 2-10 glutamic acid residues. The antibody may be, for example, an anti-CD3 antibody.

[0050] In some embodiments, the cell and / or tissue targeting motif may be covalently attached to a linear peptide. The linear peptide is not, for example, part of a peptide dendrimer. When the targeting motif comprises a peptide sequence, the peptide sequence may be contiguous with the linear peptide amino acid sequence. In some embodiments, the linear peptide comprises an ASSLNIA (SEQ ID NO: 1), PYDQLRH (SEQ ID NO: 2), or KAMHQMQ (SEQ ID NO: 3) muscle targeting motif. The muscle cell targeting motif may comprise a variant of the peptide motif ASSLNIA (SEQ ID NO: 1), PYDQLRH (SEQ ID NO: 2), or KAMHQMQ (SEQ ID NO: 3). For example, a variant muscle cell targeting motif may comprise one, two, or three amino acid substitutions, deletions, or additions to the peptide motif ASSLNIA (SEQ ID NO: 1), PYDQLRH (SEQ ID NO: 2), or KAMHQMQ (SEQ ID NO: 3), provided that the variant retains the ability to target the nanoparticle to muscle cells. In some embodiments, the linear peptide comprises an RGD or ACDCRGDCFCG (SEQ ID NO: 5) integrin targeting peptide motif. The cancer cell targeting motif may comprise a variant of the peptide sequence CGFECVRQCPERC (SEQ ID NO: 5). For example, the variant cancer cell targeting motif may comprise one, two, or three amino acid substitutions, deletions, or additions to the peptide sequence CGFECVRQCPERC (SEQ ID NO: 5), provided that the variant retains the ability to target the nanoparticle to cancer cells. In some embodiments, the linear peptide comprises a CGFECVRQCPERC (SEQ ID NO: 4) lung targeting motif. The lung cell targeting motif may comprise a variant of the peptide sequence CGFECVRQCPERC (SEQ ID NO: 4). For example, the variant lung cell targeting motif may comprise one, two, or three amino acid substitutions, deletions, or additions to the peptide sequence CGFECVRQCPERC (SEQ ID NO: 4), provided that the variant retains the ability to target the nanoparticle to lung cells. A linear peptide may comprise one or more units of a targeting motif, for example, a linear peptide may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more targeting motifs.In some embodiments, the linear peptide comprises an ASSLNIA (SEQ ID NO: 1), PYDQLRH (SEQ ID NO: 2), or KAMHQMQ (SEQ ID NO: 3) muscle-targeting motif, an RGD or ACDCRGDCFCG (SEQ ID NO: 5) integrin-targeting peptide motif, or a CGFECVRQCPERC (SEQ ID NO: 4) lung-targeting peptide motif. In some embodiments, the linear peptide comprises the lung-targeting peptide sequence CGFECVRQCPERC (SEQ ID NO: 4). When the targeting motif is a sugar, the linear peptide may comprise one or more glycosylations. For example, the linear peptide may comprise one or more mannose glycosylations.

[0051] In some embodiments, a cell and / or tissue targeting motif may be covalently attached to the peptide dendrimer of the nanoparticle. The cell and / or tissue targeting motif may be covalently attached to the C-terminus and / or N-terminus of the peptide dendrimer. The peptide dendrimer core may comprise the cell and / or tissue targeting motif. The cell and / or tissue targeting motif may be attached to the outermost layer of the peptide motif. When the targeting motif is a sugar, the peptide dendrimer may comprise one or more glycosylations. For example, the peptide dendrimer may comprise one or more mannose glycosylations. For example, the peptide dendrimer may have the structure mannose-G1-RL,2-LR. In some embodiments, the peptide dendrimer may have the structure mannose-G1-EEEE. For example, the peptide dendrimer may have the structure mannose-G1-RL,2-LR. In some embodiments, the peptide dendrimer may have the structure mannose-G1-EEEE. In some embodiments, the peptide dendrimer may have the structure (LR)4(KRL)2KGSGGSGGSGGSC[(SS)-α-D-thiomannose]. In some embodiments, the peptide dendrimer may have the structure (Ac-EEEE)2KGSGGSGGSC[(SS)-α-D-thiomannose], which may be derivatized at its C-terminus, for example, with an amine group (NH2). In some embodiments, the cell and / or tissue targeting motif is an antibody. In some embodiments, the antibody is an anti-CD3 antibody.

[0052] In some embodiments, the nanoparticles comprise a second peptide comprising a cell and / or tissue targeting motif. The second peptide may be a linear peptide or a dendritic peptide. In embodiments in which the second peptide is a dendritic peptide, it may be referred to herein as a "second peptide dendrimer." Thus, in some embodiments, the nanoparticles comprise a second peptide dendrimer covalently linked to a cell and / or tissue targeting motif. The cell and / or tissue targeting motif may be covalently linked to the C-terminus and / or N-terminus of the second peptide dendrimer. The second peptide dendrimer core may comprise a cell and / or tissue targeting motif. The cell and / or tissue targeting motif may be attached to the outermost layer of the peptide motif of the second peptide dendrimer. When the targeting motif is a sugar, the second peptide dendrimer may comprise one or more glycosylations. For example, the second peptide dendrimer may comprise one or more mannose glycosylations. In some embodiments, the second peptide dendrimer may have the structure mannose-G1-RL,2-LR. In some embodiments, the second peptide dendrimer may have the structure mannose-G1-EEEE. For example, the second peptide dendrimer may have the structure mannose-G1-RL,2-LR. In some embodiments, the peptide second dendrimer may have the structure mannose-G1-EEEE. In some embodiments, the second peptide dendrimer has the structure (Ac-EEEE)2KGSGGSGGSC[(SS)-α-D-thiomannose]. In some embodiments, the second peptide dendrimer has the structure (Ac-EEEE)2KGSGGSGGSC[(SS)-α-D-thiomannose] (where "Ac" represents acetylation at the N-terminus of the peptide dendrimer). In some embodiments, the cell and / or tissue targeting motif is an antibody. In some embodiments, the antibody is an anti-CD3 antibody.

[0053] In some embodiments, nanoparticles are targeted to specific tissues and / or cell types, which is achieved by selecting the dendrimer:nucleic acid NP ratio and / or lipid:nucleic acid w / w ratio. For example, higher NP ratios favor splenic targeting, and some lung targeting is also seen at lipid:nucleic acid w / w ratios of approximately 10:1. Lung targeting can be enhanced by increasing the lipid:nucleic acid w / w ratio, for example, to approximately 23:1.

[0054] 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 interspersed between "branching" residues such as lysine. First generation dendrimers have the following structure, shown in the N- to C-terminal direction, with Lys as the branching unit: (N-terminus-Pep1)2-Lys-(core)-(C-terminus)

[0055] The second generation dendrimer has the following structure, shown in the N- to C-terminal direction, with Lys as the branching unit: (N-terminus-Pep2)4-Lys2-(Pep1)2-Lys-(core)-(C-terminus)

[0056] The third generation dendrimer has the following structure, shown in the N- to C-terminal direction, with Lys as the branching unit: (N-terminus-Pep3)8-Lys4-(Pep2)4-Lys2-(Pep1)2-Lys-(core)-(C-terminus)

[0057] A third generation dendrimer is illustrated in Figure 1 (N-terminus on the left and C-terminus on the right).

[0058] In Figure 1, circles represent core sequences. Each triangle represents a branch 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 described herein. For example, in underivatized embodiments, the C-terminus is a carboxylic acid, while in other embodiments, the C-terminus is derivatized to include, for example, a primary amide group CONH2 (instead of COOH) as a result of the chemical route used to synthesize the dendrimer. Functionally significant derivatizations, such as targeting moieties (e.g., antibodies, peptide groups, sugar groups, and / or lipid chains), are also contemplated and may be attached to the N- and / or C-termini or other positions along the dendrimer. The N-terminus of the peptide dendrimers disclosed herein may be derivatized, for example, acetylated.

[0059] As described herein, dendrimers can be first-generation, second-generation, 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, each bound to a first branch residue. The two first peptide motifs independently consist 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, where one of the second branch residues is covalently bound to one of the first peptide motifs, the other second branch residue is covalently bound to the other first peptide motif, and each second branch residue is covalently bound to two second peptide motifs. The four second peptide motifs independently consist 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, where each second peptide motif is 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, and the eight third peptide motifs independently consist 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), 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).

[0060] The core peptide motif of the dendrimer can be a single amino acid residue or a short peptide motif, such as a dipeptide or tripeptide motif. The core sequence can 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 can 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, it may contain glycine (G), serine (S), and either cysteine (C) or alanine (A). Preferably, the core contains an ionizable residue such as histidine (H). The core sequence may contain arginine (R), histidine (H), and cysteine (C). The core sequence may contain 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 contain alanine (A), lysine (K), and leucine (L). For example, the core sequence may be KLA. The core peptide may be covalently linked to an additional moiety, such as a cell-specific targeting peptide, or may be derivatized with a lipid molecule. One, some, or all of the amino acids of the dendrimer, e.g., the core peptide motif, may be covalently attached to additional moieties, e.g., antibodies, cell-specific targeting peptides, sugar ligands such as glucose, mannose, galactose, and GalNAc (or glycans containing same), and / or lipid substituents, etc. One skilled in the art can readily select additional moieties that do not adversely affect solubility or nucleic acid binding properties.

[0061] Preferred dendrimers are presented in Table 1 below. Certain examples are specifically described. For example, in a dendrimer in which each peptide motif is an Arg-His-Leu (RHL) tripeptide, the structure may be designated G1-RHL, G1,2-RHL, and G1,2,3-RHL. In dendrimers in which the peptide motifs are not the same across generations, e.g., in a third-generation dendrimer in which two first peptide motifs and four second peptide motifs are Arg-Leu (RL) dipeptides and eight third peptide motifs are Leu-Arg (LR) dipeptides, the structure may be designated G1,2-RL,3-LR. In dendrimers in which the peptide motifs are not the same across second-generation dendrimers, e.g., in a dendrimer in which two first peptide motifs are Arg-Leu (RL) dipeptides and four second peptide motifs are Leu-Arg (LR) dipeptides, the structure may be designated G1-RL,2-LR. In dendrimers in which each peptide motif is an Arg-Leu (RL) dipeptide, the structure may be designated G1-RL, G1,2-RL, and G1,2,3-RL. In dendrimers in which each peptide motif is a Lys-Leu (KL) dipeptide, the structure may be designated G1-KL, G1,2-KL, and G1,2,3-KL. In dendrimers in which each peptide motif is a Leu-Arg (LR) dipeptide, the structure may be designated G1-LR, G1,2-LR, and G1,2,3-LR.

[0062] "G1," "G2," and "G3" refer to the "first generation," "second generation," and "third generation" peptide motifs of the first, second, and third tiers, respectively. Each amino acid residue can be an L-amino acid or a D-amino acid. D-amino acids may be designated using lowercase letters in the single-letter code. Alternatively, dendrimers in which each amino acid is the D-isoform may be described by adding a preceding "D-" before the short-form designation of the dendrimer.

[0063] Peptide dendrimers having specific cores are also described herein. For example, in dendrimers where a defined peptide core, e.g., an Arg-His-Cys peptide core, is intended, this structure may be depicted as RHCG1,2-RL. According to the nomenclature of peptide dendrimers disclosed herein, in the preceding examples, it will be understood that "G" refers to the "generation" of the peptide motif and not a glycine residue. In contrast, in a peptide dendrimer having a structure depicted as GSCG1,2-RL,3-LR, it will be understood that in this context the first "G" refers to a glycine residue, while the second "G" refers to the "generation" of the peptide motif. In other examples, where a defined core is intended, the core sequence is underlined and the generation "G" is left ununderlined (e.g., GSC G1,2-RL, 3-LR).

[0064] 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, or in addition, the alkyl or alkenyl chain may be conjugated to the N-terminus of the peptide dendrimer.

[0065] 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.

[0066] 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.

[0067] Preferably, the N / P ratio, which is the amount of peptide (measured by the number of mono-charged nitrogen atoms on the peptide, N) to the amount of nucleic acid (measured by the number of mono-charged phosphate groups in the backbone, P), is greater than 0.05:1, e.g., greater than 0.1:1. (The term N / P ratio may 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 0.6:1, or about 0.6:1, or at least 0.6:1. In some embodiments, the N / P ratio is at least or greater than 1:1, e.g., about 2:1 or greater, about 2.5:1 or greater, about 3:1 or greater, about 4:1 or greater, about 5:1 or greater, about 10:1, or up to 20:1. In some embodiments, the N / P ratio is about 5:1, about 8:1, about 10:1, or about 20:1. In some embodiments, the N / P ratio ranges from about 0.01:1 to 100:1, from about 2:1 to about 20:1, or from about 2.5:1 to about 10:1.

[0068] In some embodiments, the peptide dendrimer has the structure G1,2-RL, 3-LR; G1-RL, 2-LR; G1,2-RHL; G1-LRLR; G1,2-RF, 3-HL; or G1-R. In some embodiments, the peptide dendrimer has the structure GSC G1,2-RL, 3-LR; RHC G1-RL, 2-LR; GSC G1,2-RHL; GSC G1-LRLR; GSC G1,2-RF, 3-HL; or GSC Has G1-R.

[0069] In some embodiments, the peptide dendrimer has the structure G1,2-RL, 3-LR; G1-RL, 2-LR; G1,2-RHL; G1-LRLR; G1,2-RF, 3-HL; or G1-R, with an N:P ratio of 0.05:1 to 20:1, e.g., the N:P ratio can be 0.16:1 or 0.6:1 for delivery of nanoparticles to bone marrow cells. In some embodiments, the peptide dendrimer has the structure GSC G1,2-RL, 3-LR; RHC G1-RL, 2-LR; GSC G1,2-RHL; GSC G1-LRLR; GSC G1,2-RF, 3-HL; GSC G1-R, with an N:P ratio of 0.05:1 to 20:1, for example, the N:P ratio may be 0.16:1 or 0.6:1 for delivery of nanoparticles to macrophages. In some embodiments, the peptide dendrimer has the structure GSC G1,2-RL, 3-LR, with an N:P ratio of 0.16:1 for delivery of nanoparticles to macrophages. In some embodiments, the peptide dendrimer has the structure RHC G1-RL, 2-LR, with an N:P ratio of 8:1 for delivery of nanoparticles to macrophages. In some embodiments, the peptide dendrimer has the structure GSC G1,2-RHL, with an N:P ratio between 5:1 for delivery of nanoparticles to macrophages.

[0070] Typically, compositions containing nanoparticles containing first-generation peptide dendrimers, lipids, and nucleic acids do not form monodisperse populations of nanoparticles when the nanoparticles have a dendrimer:nucleic acid NP ratio greater than about 2:1, e.g., an N:P ratio of about 8:1. However, using higher NP ratios allows for control over certain nanoparticle properties. Furthermore, the ability to form monodisperse populations is important for the development of pharmaceutical compositions because it limits batch-to-batch variability, allowing for more clearly defined nanoparticle characterization and consistent results in vitro and in vivo. The size distribution of nanoparticles in a population can be expressed by the polydispersity index (PDI) of the population. A PDI of 1 indicates a completely polydisperse population of nanoparticles, while a PDI of 0 indicates a completely monodisperse population of nanoparticles. Therefore, the PDI can be used as a measure of the uniformity of the nanoparticle population in a composition. A PDI of 0.35 or less is believed to provide a monodisperse population of nanoparticles suitable for the development of suitable pharmaceutical compositions, for example. However, for nanoparticles used for in vitro or ex vivo transfection of cells, the PDI may be higher than 0.35; for example, the PDI may be 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, or 0.3 or less. For brevity and clarity, as used herein, when a PDI value is given for a particular peptide dendrimer or mixture of peptide dendrimers, this PDI value refers to a population of nanoparticles comprising the peptide dendrimer(s), nucleic acid, and lipid. For example, if the peptide dendrimer RHCG1-R has a PDI of 0.566, this PDI value refers to nanoparticles comprising RHCG1-R, nucleic acid, and lipid. The PDI of a particular nanoparticle formulation can be measured by any standard method in the art, such as using a Zetasizer Advance Series-Pro according to the manufacturer's instructions and the conditions disclosed in Example 3.

[0071] A heterogeneous or non-monodisperse population of nanoparticles (e.g., the PDI of the nanoparticles is greater than 0.35) may indicate that the binding of the peptide dendrimers to the nucleic acids in the nanoparticles is unstable (i.e., the peptide dendrimers and nucleic acids have low binding affinity and dissociate easily). In contrast, a homogeneous or monodisperse population of nanoparticles may indicate that the binding of the peptide dendrimers to the nucleic acids in the nanoparticles is stable (i.e., the peptide dendrimers and nucleic acids bind with high affinity and do not dissociate easily).

[0072] Without wishing to be bound by any particular theory, it is proposed that nanoparticles containing first-generation peptide dendrimers tend to form less uniform nanoparticle populations (and therefore have a "high" PDI greater than 0.35) compared to populations of nanoparticles containing second- or third-generation peptide dendrimers because the first-generation dendrimers have fewer cationic groups per peptide dendrimer. Because there are fewer cationic groups to interact with the anionic groups on the nucleic acid, first-generation peptide dendrimer / nucleic acid complexes may form unstable complexes that may dissociate at a relatively high rate compared to second- or third-generation dendrimer / nucleic acid complexes, resulting in a heterogeneous mixture of nanoparticles in solution. It is believed that the inclusion of second- or third-generation peptide dendrimers in combination with first-generation peptide dendrimers may balance the relatively stable associations required to form uniform particles (provided by the second- or third-generation peptide dendrimers) with the relatively unstable associations required to efficiently release the nucleic acid once delivered to cells (provided by the first-generation peptide dendrimers).

[0073] The present inventors have also found that transfection efficiency can be improved by using nanoparticles comprising two peptide dendrimers that, when used alone in nanoparticles comprising nucleic acids and lipids, can preferably form a homogeneous nanoparticle population. That is, improved transfection efficiency can be achieved even when each of the peptide dendrimers individually forms a monodisperse population of nanoparticles in solution. For example, the first and second peptide dendrimers can be independently selected from second- or third-generation peptide dendrimers that, when used individually, form a monodisperse nanoparticle population.

[0074] Thus, in some embodiments, a first peptide dendrimer may be selected based on the polydispersity index (PDI) of a reference peptide dendrimer / lipid nanoparticle comprising the first peptide dendrimer at an NP ratio of 8:1. Similarly, a second peptide dendrimer may be selected based on the PDI of a reference peptide dendrimer / lipid nanoparticle comprising the second peptide dendrimer at an NP ratio of 8:1. In some embodiments, when a first peptide dendrimer is used in a first reference nanoparticle comprising the first peptide dendrimer, nucleic acid, and lipid, the first reference nanoparticle has a higher PDI than the PDI of a second reference nanoparticle comprising the second peptide dendrimer, nucleic acid, and lipid, and the reference nanoparticles are prepared at an NP ratio of 8:1.

[0075] In some embodiments, the PDI of the nanoparticles comprising a first peptide dendrimer, a second peptide dendrimer, a nucleic acid, and a lipid is 0.6, 0.55, 0.5, 0.45, 0.4, 0.39, 0.38, 0.37, 0.36, 0.35, 0.34, 0.33, 0.32, 0.31, 0.30, or less. In some embodiments, the PDI of the nanoparticles comprising a first peptide dendrimer, a second peptide dendrimer, a nucleic acid, and a lipid is 0.35 or less.

[0076] In some embodiments, the PDI of the first dendrimer is greater than about 0.20, about 0.21, about 0.22, about 0.23, about 0.24, about 0.25, about 0.26, about 0.27, about 0.28, about 0.29, about 0.30, about 0.31, about 0.32, about 0.33, about 0.34, about 0.35, about 0.36, about 0.37, about 0.38, about 0.39, or about 0.40. In some embodiments, the PDI of the second dendrimer is less than about 0.40, about 0.39, about 0.38, about 0.37, about 0.36, about 0.35, about 0.34, about 0.33, about 0.32, about 0.31, about 0.30, about 0.29, about 0.28, about 0.27, about 0.26, about 0.25, about 0.24, about 0.23, about 0.22, about 0.21, or about 0.20. In some embodiments, the PDI of the first peptide dendrimer is greater than about 0.25, about 0.26, about 0.27, about 0.28, about 0.29, or about 0.30, and the PDI of the second peptide dendrimer is less than about 0.25, about 0.24, about 0.23, about 0.22, about 0.21, or about 0.20. In some embodiments, the PDI of the first peptide dendrimer is greater than about 0.40 and the PDI of the second peptide dendrimer is less than about 0.40. In some embodiments, the PDI of the first peptide dendrimer is greater than about 0.35 and the PDI of the second peptide dendrimer is less than about 0.35. In some embodiments, the PDI of the first peptide dendrimer is greater than about 0.30 and the PDI of the second peptide dendrimer is less than about 0.30. In some embodiments, the PDI of the first peptide dendrimer is greater than about 0.30 and the PDI of the second peptide dendrimer is less than about 0.30. In some embodiments, the PDI of the first peptide dendrimer is greater than about 0.20 and the PDI of the second peptide dendrimer is less than about 0.20. In some embodiments, the PDI of the first peptide dendrimer is greater than about 0.10 and the PDI of the second peptide dendrimer is less than about 0.10. In some embodiments, the first peptide dendrimer has a PDI greater than about 0.10 and the second peptide dendrimer has a PDI less than about 0.20.

[0077] In some embodiments, the first peptide dendrimer has a PDI that is at least about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.10, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, or about 0.9 higher than the PDI of the second peptide dendrimer. In some embodiments, the first peptide dendrimer has a PDI that is 0.05-0.9, 0.06-0.8, 0.07-0.6, 0.08-0.5, 0.09-0.4, about 0.1-0.3, 0.12-0.2, or 0.13-0.15 higher than the PDI of the second peptide dendrimer.

[0078] In some embodiments, when a first peptide dendrimer is used in a first reference nanoparticle consisting of a first peptide dendrimer, a nucleic acid, and a lipid, the first reference nanoparticle has a PDI that is 0.05 to 0.9, 0.06 to 0.8, 0.07 to 0.6, 0.08 to 0.5, 0.09 to 0.4, 0.1 to 0.3, 0.12 to 0.2, or 0.13 to 0.15 higher than the PDI of a second reference nanoparticle consisting of a second peptide dendrimer, a nucleic acid, and a lipid.

[0079] The transfection efficiency and PDI of nanoparticles containing two peptide dendrimers can be optimized by varying the relative levels of each peptide dendrimer in the nanoparticle. For example, the molar ratio of nitrogen contributed by each of the first and second peptide dendrimers in the nanoparticle can be selected from 1:4 to 4:1, 1:3 to 3:1, or 1:2 to 2:1. In some examples, the molar ratio of nitrogen contributed by each of the first and second peptide dendrimers is 1:4, 1:3, 1:2, 1:1, 2:1, 2:1, 3:1, or 4:1.

[0080] In embodiments where the composition comprises a first peptide dendrimer and a second peptide, the first peptide dendrimer may be a first generation peptide dendrimer comprising a core peptide sequence, a first branching unit, and two first peptide motifs. In some embodiments, the first peptide dendrimer may comprise a cell or tissue targeting motif.

[0081] In other such embodiments, the first peptide dendrimer is a second-generation peptide dendrimer comprising a core peptide sequence, a first branching unit and two first peptide motifs, at least two second branching units, and four second peptide motifs. One of the second branching residues is covalently linked to one of the first peptide motifs, and the other second branching residue is covalently linked to the other first peptide motif. Each of the second branching residues is covalently linked to two second peptide motifs. Each of the first branching unit and two second branching units may be independently selected from lysine, 2,4-diaminobutyric acid, ornithine, or diaminopropionic acid.

[0082] In other such embodiments, the first peptide dendrimer is a third-generation peptide dendrimer comprising a core peptide sequence, a first branching unit and two first peptide motifs, at least two second branching units and four second peptide motifs, and at least four third branching residues and eight third peptide motifs. One of the second branching residues is covalently bound to one of the first peptide motifs, and the other second branching residue is covalently bound to the other first peptide motif. Each of the second branching residues is covalently bound to two second peptide motifs. Each second peptide motif is covalently bound to one of the third branching residues, such that each third branching residue is covalently bound to one second peptide motif. Each third branching residue is covalently bound to two third peptide motifs. Each of the first branching unit, two second branching units, and four third branching units may be independently selected from lysine, 2,4-diaminobutyric acid, ornithine, or diaminopropionic acid.

[0083] The first peptide dendrimer may be a first-generation peptide dendrimer comprising a core peptide sequence, a first branching unit, and two first peptide motifs. Alternatively, the first peptide dendrimer may be a second-generation peptide dendrimer comprising a core peptide sequence, a first branching unit, two first peptide motifs, at least two second branching units, and four second peptide motifs. One of the second branching residues is covalently bound to one of the first peptide motifs, and the other second branching residue is covalently bound to the other first peptide motif. Each of the second branching residues is covalently bound to two second peptide motifs. Each of the first branching unit and two second branching units may be independently selected from lysine, 2,4-diaminobutyric acid, ornithine, or diaminopropionic acid.

[0084] In some embodiments, the second peptide dendrimer is a second-generation peptide dendrimer comprising a core peptide sequence, a first branching unit and two first peptide motifs, at least two second branching units, and four second peptide motifs. One of the second branching residues is covalently linked to one of the first peptide motifs, and the other second branching residue is covalently linked to the other first peptide motif. Each of the second branching residues is covalently linked to two second peptide motifs. Each of the first branching unit and two second branching units may be independently selected from lysine, 2,4-diaminobutyric acid, ornithine, or diaminopropionic acid.

[0085] In some embodiments, the second peptide dendrimer is a third-generation peptide dendrimer comprising a core peptide sequence, a first branching unit and two first peptide motifs, at least two second branching units and four second peptide motifs, and at least four third branching residues and eight third peptide motifs. One of the second branching residues is covalently bound to one of the first peptide motifs, and the other second branching residue is covalently bound to the other first peptide motif. Each of the second branching residues is covalently bound to two second peptide motifs. Each second peptide motif is covalently bound to one of the third branching residues, such that each third branching residue is covalently bound to one second peptide motif. Each third branching residue is covalently bound to two third peptide motifs. Each of the first branching unit, two second branching units, and four third branching units may be independently selected from lysine, 2,4-diaminobutyric acid, ornithine, or diaminopropionic acid.

[0086] In some embodiments, the second peptide dendrimer is a first generation peptide dendrimer comprising a core peptide sequence, a first branching unit, and two first peptide motifs.

[0087] In some embodiments, the first peptide dendrimer is a first-generation peptide dendrimer and the second peptide dendrimer is a first-generation peptide dendrimer. In some embodiments, the first peptide dendrimer is a first-generation peptide dendrimer and the second peptide dendrimer is a second-generation peptide dendrimer. In some embodiments, the first peptide dendrimer is a first-generation peptide dendrimer and the third peptide dendrimer is a third-generation peptide dendrimer.

[0088] In some embodiments, the first peptide dendrimer is a second-generation peptide dendrimer and the second peptide dendrimer is a first-generation peptide dendrimer. In some embodiments, the first peptide dendrimer is a second-generation peptide dendrimer and the second peptide dendrimer is a second-generation peptide dendrimer. In some embodiments, the first peptide dendrimer is a second-generation peptide dendrimer and the third peptide dendrimer is a third-generation peptide dendrimer.

[0089] In some embodiments, the first peptide dendrimer is a third-generation peptide dendrimer and the second peptide dendrimer is a first-generation peptide dendrimer. In some embodiments, the first peptide dendrimer is a third-generation peptide dendrimer and the second peptide dendrimer is a second-generation peptide dendrimer. In some embodiments, the first peptide dendrimer is a third-generation peptide dendrimer and the third peptide dendrimer is a third-generation peptide dendrimer.

[0090] In some embodiments, the peptide motifs of the first and second peptide dendrimers are independently selected from single amino acid, dipeptide, tripeptide, or tetrapeptide motifs. For the avoidance of doubt, the two first peptide motifs of the first or second peptide dendrimer, the four second peptide motifs of the first or second peptide dendrimer, and the eight third peptide motifs of the first or second peptide dendrimer are independently selected from single amino acid, dipeptide, tripeptide, or tetrapeptide motifs.

[0091] Each peptide motif of the first and second peptide dendrimers independently comprises a naturally occurring L- or D-amino acid and / or a non-naturally occurring L- or D-amino acid, e.g., beta-alanine (B) or aminohexanoic acid (X or Acp). For the avoidance of doubt, the two first peptide motifs of the first or second peptide dendrimer, the four second peptide motifs of the first or second peptide dendrimer, and the eight third peptide motifs of the first or second peptide dendrimer independently comprise a naturally occurring L- or D-amino acid and / or a non-naturally occurring L- or D-amino acid, e.g., beta-alanine (B) or aminohexanoic acid (X or Acp).

[0092] In some embodiments, the first, second and / or third peptide motif of the first and / or second peptide dendrimer, if present, comprises an amino acid with a basic side chain.

[0093] In some embodiments, the core sequence of the first and / or second peptide dendrimer comprises amino acid residues having ionizable groups, such as histidine.

[0094] In some embodiments, the first, second and / or third peptide motif of the first and / or second peptide dendrimer, if present, comprises amino acids with non-polar side chains.

[0095] In some embodiments, the first, second and / or third peptide motif of the first and / or second peptide dendrimer, if present, comprises an amino acid with an acidic side chain.

[0096] In some embodiments, the first, second and / or third peptide motif of the first and / or second peptide dendrimer, when present, comprises amino acids with uncharged polar side chains.

[0097] In some embodiments, each of the first, second, and third peptide motifs, if present, may comprise: (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).

[0098] Preferably, at least one of the first, second, and third peptide motifs (if present) of the first and / or second peptide dendrimer comprises leucine (L), arginine (R), and / or histidine (H). At least two of the first, second, and third peptide motifs (if present) may comprise leucine (L), arginine (R), and / or histidine (H). In some embodiments, all of the first, second, and third peptide motifs (if present) of the first and / or second peptide dendrimer comprise leucine (L), arginine (R), and / or histidine (H).

[0099] 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).

[0100] In some embodiments, the core peptide sequence of the first and / or second peptide dendrimer comprises the amino acid sequence RHC, GSA, or GSC.

[0101] In some embodiments, the peptide dendrimer, nucleic acid, and lipid form a positively charged particle.

[0102] In other embodiments, the peptide dendrimers, nucleic acids and lipids form particles that have a negative or neutral charge.

[0103] The lipid component of the nanoparticles may comprise a mixture of lipids, including a cationic lipid. For example, the lipid component may comprise dioleoylphosphatidylethanolamine (DOPE) and N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). The w / w ratio of DOPE:DOTMA can be easily determined for optimal properties for a given application, but typically ranges from 1:10 to 10:1, 1:8 to 8:1, or 1:5 to 5:1. 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.

[0104] In other embodiments, the lipid component of the composition may include other lipids in addition to (or instead of) DOPE and DOTMA, such as DODAP, DOTAP, and / or DORI (N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium bromide). Exemplary lipid components are shown below:

[0105] Cationic lipids: N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) 1,2-Dioleoyl-3-trimethylammonium propane chloride (DOTAP) N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium bromide (DORI) 2,3-Dioleyloxy-N-(2[spermine-carboxamido]ethyl)-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-chol)

[0106] Neutral lipids: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) cholesterol

[0107] Anionic lipids: 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG)

[0108] Ionizable lipids: 1,2-Dioleoyloxy-3-(dimethylamino)propane (DODAP) DLin-DMA DLin-KC2-DMA DLin-MC3-DMA SM-102 ALC-0315

[0109] Other possible lipids include 4-(2-aminoethyl)-morpholino-cholesterol-hemisuccinate, (MoChol) cholesterol hemisuccinate (CHEMS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), cholesterol-(3-imidazol-1-ylpropyl)carbamate (CHIM), dimethyldioctadecylammonium bromide (DDAB), dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylglycerol (DOPG), cholesterol sulfate (chol-SO4).

[0110] It is contemplated that any of the above-mentioned 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.

[0111] In some embodiments, the lipids of the nanoparticles comprise cationic lipids, neutral lipids, anionic lipids, and / or ionizable lipids.

[0112] In some embodiments, the lipid of the nanoparticle comprises a saturated fatty acid. Additionally or alternatively, the lipid of the composition may comprise an unsaturated fatty acid.

[0113] 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.

[0114] 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).

[0115] The lipid component of the nanoparticles may include DOTMA, DOPE, DOPC and / or DOPG.

[0116] 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 nanoparticle, which can range from 1:50 to 50:1. More preferably, the amount (weight) of lipid relative to the amount (weight) of nucleic acid is 1:1 to 50:1, or 2:1 to 25:1, or at least about 0.5:1, at least about 1:1, at least about 2.5:1, or at least about 5:1. The lipid:nucleic acid ratio can be at least 2:1. These ratios refer to the weight of total lipid. As described herein, nanoparticles may contain lipids containing two or more lipid components, for example, a mixture of two, three, or four lipids. The weight of a lipid component is the total (total) weight of these lipid components. Preferably, each lipid component is mixed in approximately equal proportions.

[0117] In some embodiments, the lipid:nucleic acid (w / w) ratio can be selected to target the nanoparticles to lung tissue. For example, the nanoparticles may contain a lipid:nucleic acid (w / w) ratio of 2:1 to 40:1 to target the nanoparticles to the lung and / or spleen. In a further example, the nanoparticles may contain a lipid:nucleic acid (w / w) ratio of 10:1 or 23:1. A lipid:nucleic acid (w / w) ratio of 10:1 targets both the spleen and lung, with the spleen being targeted more strongly than the lung. A lipid:nucleic acid (w / w) ratio of 23:1 targets both the spleen and lung, with the lung being targeted more strongly than the spleen.

[0118] The peptide dendrimer may comprise a cell-penetrating peptide, an endosomal escape peptide, a nuclear localization motif, and / or a fatty acid. The cell-penetrating peptide, the endosomal escape peptide, the nuclear localization motif, and / or the fatty acid may be conjugated to the C-terminus of the peptide dendrimer. Alternatively, or in addition, the cell-penetrating peptide, the endosomal escape peptide, the nuclear localization motif, and / or the fatty acid may be conjugated to the N-terminus of the peptide dendrimer.

[0119] Transfection efficiency may be increased following targeted delivery of nanoparticles to cells or tissues by including dendrimers with one or more charged, hydrophobic, and / or ionizable amino acids, such as arginine, aspartic acid, cysteine, glutamic acid, histidine, lysine, leucine, and tyrosine, by any of the mechanisms described above (e.g., by including a targeting motif, selecting an appropriate lipid:nucleic acid w / w ratio, and / or selecting an appropriate dendrimer:lipid N:P ratio). In some embodiments, the ionizable amino acid is histidine.

[0120] In some embodiments, the core peptide sequence comprises amino acids such as arginine, aspartic acid, cysteine, glutamic acid, histidine, lysine, and tyrosine, etc. In some embodiments, the core peptide sequence comprises the ionizable amino acid histidine.

[0121] For example, the inclusion of histidine in the core and / or peptide motif of one or both peptide dendrimers may confer extracellular stability to the nanoparticle but aid in the intracellular release of nucleic acids from the nanoparticle. For example, the five-membered imidazole ring of histidine contains two nitrogen atoms, which can form hydrogen bonds and provide stability to the nanoparticle. However, when exposed to the acidic endosomal environment, protonation of histidine may lead to endosomal swelling, dissolution, and release of the nucleic acid contained within the nanoparticle.

[0122] In some embodiments, the nucleic acid is RNA. For example, the RNA may be selected from mRNA, circular RNA (circRNA), ssRNA, dsRNA, sgRNA, crRNA, tracrRNA, lncRNA, siRNA, saRNA, and / or self-amplifying RNA.

[0123] In some embodiments, the nucleic acid is DNA. For example, the DNA may include ssDNA, dsDNA, plasmids, and / or cDNA.

[0124] For the avoidance of doubt, a nanoparticle 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.

[0125] In some embodiments, the nanoparticles comprise RNA and DNA nucleic acids, which may be part of a single nucleic acid molecule.

[0126] In some embodiments, the nucleic acid comprises a modified nucleic acid. Exemplary nucleic acid modifications are described herein.

[0127] The nucleic acid may encode a transgene, and the transgene may be expressed in the target cell. The transgene may be a protein or 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 exogenous expression of additional copies of the gene, or modulation may be a decrease in gene expression.

[0128] In some embodiments, the nucleic acid expresses a target transgene identified in Table A and the nanoparticle composition is for use in treating the corresponding disease identified in Table A.

[0129] [Table 1]

[0130] In some embodiments, the regulated endogenous gene is a gene that expresses a protein or peptide.

[0131] 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.

[0132] In some embodiments, the nucleic acid encodes a CAR that specifically binds to carcinoembryonic antigen (CEA) or CEA cell adhesion molecule 7 (CEACAM7).

[0133] In some embodiments, the nucleic acid encodes one or more transcription factors selected from interferon regulatory factor 5 (IRF5), activated IRF5, inhibitor of nuclear factor kappa B kinase subunit beta (IKK2), or CCAAT enhancer-binding protein alpha (CEBPA).

[0134] In some embodiments, the nucleic acid encodes an anti-CEA or anti-CEACAM7 CAR and one or more transcription factors selected from interferon regulatory factor 5 (IRF5), activated IRF5, inhibitor of nuclear factor kappa B kinase subunit beta (IKK2), or CCAAT enhancer-binding protein alpha (CEBPA).

[0135] In some embodiments, the nucleic acid encodes activated IRF5 according to SEQ ID NO: 7 or encodes activated IRF5 according to SEQ ID NO: 8. Activated IRF5 is particularly preferred in embodiments where the nucleic acid therapy is for use in treating cancer.

[0136] In some embodiments, the transgene comprises a tumor antigen, a viral protein, a bacterial protein, or a protein of a microorganism parasitic on a mammal.

[0137] In some embodiments, the nucleic acid comprises or encodes a self-amplifying RNA.

[0138] In some embodiments, the use comprises treating a genetic disorder in a subject.

[0139] In some embodiments, the nucleic acid expresses a functional form of a gene that is non-functional, down-regulated, inactive, or impaired in the subject.

[0140] In some embodiments, the nucleic acid encodes and / or includes 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 include an sgRNA, crRNA, and / or tracrRNA. The nucleic acid may include an RNA sequence comprising an mRNA encoding a Cas protein or peptide and an sgRNA. The composition may include an mRNA encoding a Cas protein or peptide and another RNA (as separate molecules) comprising an sgRNA. In some embodiments, one or more of the nucleic acids encoding the sgRNA, crRNA, tracrRNA, and Cas protein, when present, are part of a single nucleic acid. In some embodiments, one or more of the nucleic acids encoding the sgRNA, crRNA, tracrRNA, and Cas protein, when present, are present on two or more nucleic acids.

[0141] It is contemplated that the present invention may be used to deliver nucleic acid therapies for treating myopathies. It is also contemplated that the present invention may be used to deliver nucleic acid therapies for treating 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, hereditary muscular dystrophies, congenital muscular dystrophies, and distal muscular dystrophy.

[0142] The nucleic acid therapy may be for treating muscle-wasting conditions such as cachexia. The nucleic acid therapy may be for treating other muscle disorders, such as genetic muscle disorders such as myotonia congenita or familial periodic paralysis. The nucleic acid therapy may be for treating motor neuron diseases 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 syndrome (subacute necrotizing encephalomyopathies), mitochondrial DNA depletion syndrome, mitochondrial encephalomyopathies, lactic acidosis, and stroke-like episodes (MELAS), mitochondrial neurogastrointestinal encephalomyopathies (MNGIE), myoclonic epilepsy with ragged-red fibers (MERRF), neuropathy, ataxia, and retinitis pigmentosa (NARP), Pearson syndrome, or progressive external ophthalmoplegia (PEO). The nucleic acid therapy may be for treating a congenital myopathy, such as cap myopathy, centronuclear myopathy, congenital fiber inequality, core myopathy, central core disease, multiminicore disease, myosin storage myopathy, myotubular myopathy, or nemaline myopathy. The nucleic acid therapy may be for treating a distal myopathy, such as GNE myopathy / Nonaka myopathy / hereditary inclusion body myopathy (HIBM), Laing distal myopathy, Markesbery-Griggs late-onset distal myopathy, Miyoshi myopathy, Udd myopathy / tibial muscular dystrophy, VCP myopathy / IBMPFD, distal vocal cord and pharyngeal myopathy, or Welander distal myopathy. The nucleic acid therapy may be for treating an endocrine myopathy, such as hyperthyroid myopathy or hypothyroid myopathy. The nucleic acid therapy may be for treating an inflammatory myopathy, 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, debranching 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 myopathy or scapuloperoneal myopathy. The nucleic acid therapy may be for treating neuromuscular junction disorders, such as congenital myasthenic syndrome (CMS), Lambert-Eaton myasthenic syndrome (LEMS), or myasthenia gravis (MG). The nucleic acid therapy may be for treating peripheral nerve diseases, 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 (ALS, also referred to as critical limb ischemia and foot ulcers), peripheral arterial disease, lower limb ischemia, critical limb ischemia (also known as comprehensive severe chronic limb ischemia and diabetic limb ischemia), severe peripheral arterial occlusive disease (PAOD), or intermittent claudication / arteriosclerosis. The nucleic acid therapy may be for treating cancer, such as sarcoma, melanoma, breast cancer, lung cancer, pancreatic cancer, prostate cancer, liver cancer, acute myeloid leukemia or B-cell lymphoma, prostate cancer, or anal cancer. The nucleic acid therapy may be for treating allergies, such as peanut allergies. The nucleic acid therapy may be for treating multiple sclerosis (MS). The nucleic acid therapy may be for treating myelodysplastic syndrome (MDS).

[0143] Pompe disease is caused by a deficiency of human acid α-glucosidase (GAA), a lysosomal enzyme that cleaves terminal α1-4 and α1-6 glucose from glycogen. The compositions of the present invention may be used to treat Pompe disease. The compositions of the present invention, which contain a nucleic acid encoding GAA, may 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 the liver and skeletal muscle. The enzyme may be secreted from the tissue into the circulation.

[0144] 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 tissue described herein, particularly skeletal muscle. The protein can be secreted from the tissue into the circulation.

[0145] Accordingly, the present invention provides methods for treating such disorders, and compositions for use in such methods of treatment.

[0146] In some embodiments, the first peptide dendrimer comprises a structure set forth in Table 7. In some embodiments, the first peptide dendrimer comprises the structure G1-LRLR. In some embodiments, the first peptide dendrimer comprises the structure GSC In some embodiments, the first peptide dendrimer comprises the structure G1-R. In some embodiments, the first peptide dendrimer comprises the structure G1-LRLR. RHC In some embodiments, the first peptide dendrimer comprises the structure G1-RLR. In some embodiments, the first peptide dendrimer comprises the structure G1-RLR. RHC In some embodiments, the first peptide dendrimer comprises the structure G1,2-R. In some embodiments, the first peptide dendrimer comprises the structure G1,2-R. RHCIn some embodiments, the first peptide dendrimer comprises the structure G1,2-LR. In some embodiments, the first peptide dendrimer comprises the structure RHCG1,2-LR.

[0147] In some embodiments, the second peptide dendrimer comprises a structure set forth in Table 8. In some embodiments, the second peptide dendrimer comprises the structure G1,2-RL, G3-LR. In some embodiments, the second peptide dendrimer comprises the structure GSC In some embodiments, the second peptide dendrimer comprises the structure G1-RL, G2-LR. In some embodiments, the second peptide dendrimer comprises the structure RHC In some embodiments, the second peptide dendrimer comprises the structure G1,2-KL. In some embodiments, the second peptide dendrimer comprises the structure G1,2-KL. GSC In some embodiments, the second peptide dendrimer comprises the structure G1,2-KL. In some embodiments, the second peptide dendrimer comprises the structure G1,2-RL. In some embodiments, the second peptide dendrimer comprises the structure G1,2-R. In some embodiments, the second peptide dendrimer comprises the structure RHC In some embodiments, the second peptide dendrimer comprises the structure G1,2-RLR. In some embodiments, the second peptide dendrimer comprises the structure G1,2-RLR. RHC In some embodiments, the second peptide dendrimer comprises the structure G1,2-RLR. In some embodiments, the second peptide dendrimer comprises the structure G1,2-LRLR. In some embodiments, the second peptide dendrimer comprises the structure GSC In some embodiments, the second peptide dendrimer comprises the structure G1,2-LRLR. In some embodiments, the second peptide dendrimer comprises the structure G1,2-RHL. In some embodiments, the second peptide dendrimer comprises the structure GSC In some embodiments, the second peptide dendrimer comprises the structure G1-RHL, 2-LHR. In some embodiments, the second peptide dendrimer comprises the structure G1-RHL, 2-LHR. GSC Includes G1-RHL and 2-LHR.

[0148] In some embodiments, the first peptide dendrimer comprises the structure G1-LRLR and the second peptide dendrimer comprises the structure G1,2-RL,3-LR. In some embodiments, the first peptide dendrimer comprises the structure GSC G1-LRLR, and the second peptide dendrimer has the structure GSC In some embodiments, the first peptide dendrimer comprises the structure G1-R and the second peptide dendrimer comprises the structure G1-RL, 2-LR. In some embodiments, the first peptide dendrimer comprises the structure G1-R and the second peptide dendrimer comprises the structure G1-RL, 2-LR. In some embodiments, the first peptide dendrimer comprises the structure G1-R and the second peptide dendrimer comprises the structure G1-RL, 2-LR. In some embodiments, the first peptide dendrimer comprises the structure RHC G1-R, and the second peptide dendrimer comprises the structure RHC In some embodiments, the first peptide dendrimer comprises the structure G1,2-R and the second peptide dendrimer comprises the structure G1-RL,2-LR. In some embodiments, the first peptide dendrimer comprises the structure RHC G1,2-R, and the second peptide dendrimer comprises the structure RHC In some embodiments, the first peptide dendrimer comprises the structure G1-RLR and the second peptide dendrimer comprises the structure G1-RL, 2-LR. In some embodiments, the first peptide dendrimer comprises the structure G1-RLR and the second peptide dendrimer comprises the structure G1-RL, 2-LR. RHC G1-RLR, and the second peptide dendrimer has the structure RHC In some embodiments, the first peptide dendrimer comprises the structure G1-RL, 2-LR. In some embodiments, the first peptide dendrimer comprises the structure G1-LRLR and the second peptide dendrimer comprises the structure G1-RL, 2-LR. In some embodiments, the first peptide dendrimer comprises the structure GSC G1-LRLR, and the second peptide dendrimer has the structure RHC Includes G1-RL and 2-LR.

[0149] In some embodiments, the molar ratio of nitrogen contributed by each of the first peptide dendrimer and the second peptide dendrimer is about 0.1:10, 1:10, 1:2, 1:1, 2:1, 10:1, or 10:0.1. In some embodiments, the molar ratio of nitrogen contributed by each of the first peptide dendrimer and the second peptide dendrimer is 1:2, 1:1, or 2:1.

[0150] In some embodiments, delivery of nucleic acid to a tissue or cell using a composition comprising a first and a second peptide dendrimer is increased by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, or 300% compared to delivery of the same nucleic acid to the same tissue or cell type using a composition comprising only a lipid, a nucleic acid, and either the first or the second peptide dendrimer. Cells or tissues are cell types or organs / tissues as defined herein. For example, the tissue may be lung, tumor tissue, heart, skeletal muscle, adipose tissue, brain, liver and kidney, in addition to spleen, lymphoid organs, skeletal muscle, brain and adipose tissue.

[0151] In some embodiments, the first and / or second peptide dendrimers further comprise a cell-penetrating peptide. The cell-penetrating peptide may comprise a sequence derived from TAT. The cell-penetrating peptide may comprise the peptide sequence XRXRRBRRXRRBRXB (SEQ ID NO: 1), where X is 6-aminohexanoic acid and B is beta-alanine.

[0152] In some embodiments, the first and / or second 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.

[0153] 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.

[0154] In some embodiments, the first and / or second peptide dendrimer comprises a fatty acid conjugated to the C-terminus of the peptide dendrimer, hi other embodiments, the peptide dendrimer comprises a fatty acid conjugated to the N-terminus of the peptide dendrimer.

[0155] In some embodiments, the lipids of the composition comprise cationic lipids, neutral lipids, anionic lipids, and / or ionizable lipids.

[0156] In some embodiments, the lipid of the composition comprises saturated fatty acids. Additionally or alternatively, the lipid of the composition may comprise unsaturated fatty acids.

[0157] 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.

[0158] 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), dioleoylphosphatidylglycerol (DOPG), DMG-PEG, and / or N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium bromide (DORI).

[0159] In some embodiments, the peptide dendrimer, nucleic acid, and lipid form a positively charged particle.

[0160] In other embodiments, the peptide dendrimers, nucleic acids and lipids form particles that have a negative or neutral charge.

[0161] The lipid component of the composition may include DOTMA, DOPE, DOPC and / or DOPG.

[0162] The lipid-based nucleic acid delivery system may be DOTMA / DOPE.

[0163] 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, which may range from 1:50 to 50:1. More preferably, the amount (weight) of lipid relative to the amount (weight) of nucleic acid is 1:1 to 50:1, or 2:1 to 25:1. The lipid:nucleic acid ratio can be at least 2:1. The lipid:nucleic acid weight:weight ratio is approximately 10:1 to 25:1. These ratios refer to the weight of total lipid. As described herein, the composition may contain lipids comprising two or more lipid components, for example, a mixture of two, three, or four lipids. The weight of a lipid component is the total (total) weight of these lipid components. Preferably, each lipid component is mixed in approximately equal proportions.

[0164] Preferably, the N / P ratio, which is the amount of peptide (measured by the number of mono-charged nitrogen atoms on the peptide, N) to the amount of nucleic acid (measured by the number of mono-charged phosphate groups in the backbone, P), is greater than 0.05:1, e.g., greater than 0.1:1. (The term N / P ratio may 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 or greater than 1:1, e.g., about 2:1 or greater, about 2.5:1 or greater, about 3:1 or greater, about 4:1 or greater, about 5:1 or greater, about 10:1, or up to 20:1. In some embodiments, the N / P ratio is about 5:1, about 8:1, about 10:1, or about 20:1. In some embodiments, the N / P ratio is in the range of about 0.01:1 to 100:1, about 2:1 to about 20:1, or about 2.5:1 to about 10:1. The first and / or second peptide dendrimer may comprise a cell-penetrating peptide, an endosomal escape peptide, a nuclear localization motif, and / or a fatty acid. The cell-penetrating peptide, the endosomal escape peptide, the nuclear localization motif, and / or the fatty acid may be conjugated to the C-terminus of the first and / or second peptide dendrimer. Alternatively, or in addition, the cell-penetrating peptide, the endosomal escape peptide, the nuclear localization motif, and / or the fatty acid may be conjugated to the N-terminus of the first and / or second peptide dendrimer.

[0165] Relatedly, the present invention provides compositions comprising the nanoparticles of the present invention. The present invention further provides pharmaceutical compositions comprising the nanoparticles of the present invention and a pharmaceutically acceptable excipient. The pharmaceutical compositions may be used in medicine. The pharmaceutical compositions may be for use in treating cancer, autoimmune diseases, pulmonary diseases, and / or myopathies. Methods of treating cancer, autoimmune diseases, pulmonary diseases, and / or myopathies are also provided, comprising administering the pharmaceutical composition to a patient or subject. In some embodiments, the composition or pharmaceutical composition is contained within a liquid. In other embodiments, the composition or pharmaceutical composition is provided as a dry composition, e.g., a dry powder. The dry composition may be prepared using lyophilization and / or freeze-drying techniques.

[0166] In a further aspect, the present invention provides a method for producing coated nanoparticles capable of transfecting target cells by mixing a solution of a peptide with a solution of preformed nanoparticles to form coated nanoparticles. In some cases, the preformed nanoparticles have a positive surface charge and the peptide has a negative net charge. In some embodiments, the peptide is a dendrimer, PGA, or glutamic acid-containing peptide. The dendrimer may be first-, second-, or third-generation, as defined herein, and may have one, two, three, four, or more amino acids in each generation and in its core sequence. Without being bound by theory, surface charge and hydrophobicity may alter the binding of serum components to the nanocarrier (corona), which can affect tissue distribution. This can be achieved, for example, by coating positive nanoparticles with linear or branched PGA (as defined herein), since PGA is a negatively charged peptide. In some embodiments, the glutamic acid-containing peptide comprises a glutamic acid-rich domain comprising a total of at least 4, at least 6, or at least 8 amino acid residues, at least 2 of which are glutamic acid, and at least 20% of the amino acid residues in the glutamic acid-rich domain are glutamic acid. In some embodiments, a peptide dendrimer may comprise a glutamic acid-containing peptide. In some cases, the preformed nanoparticles have a negative surface charge and the dendrimer has a positive net charge. In some cases, the surface of the preformed nanoparticles is uncharged and the dendrimer comprises a hydrophobic region.

[0167] In some embodiments, the peptide dendrimer, PGA, or glutamic acid-containing peptide comprises a cell targeting motif, such as a bone marrow, lymphoid, muscle, lung, CD206+ cell, or tumor cell targeting motif. In some embodiments, the muscle cell targeting motif comprises an ASSLNIA (SEQ ID NO: 1), PYDQLRH (SEQ ID NO: 2), or KAMHQMQ (SEQ ID NO: 3) peptide motif. In some embodiments, the cell targeting motif comprises an integrin targeting motif, optionally comprising an RGD or ACDCRGDCFCG (SEQ ID NO: 5) peptide motif. In some embodiments, the lung targeting motif comprises the peptide sequence CGFECVRQCPERC (SEQ ID NO: 4). In still further embodiments, the cell targeting motif is a mannose receptor targeting motif, such as a mannose sugar or maltotriose. In some embodiments, the cell targeting motif targets lymphocytes, such as T cells. In further embodiments, the cell targeting motif may be a CD3, CD4, or CD8 binder, such as an antibody that specifically binds to one of these markers. In some embodiments, an anti-CD3 antibody is used.

[0168] In a related aspect, the invention provides coated nanoparticles capable of transfecting target cells, the coated nanoparticles comprising a peptide on their surface. In some embodiments, the nanoparticles are nanoparticles according to any one of the aspects described herein. In some embodiments, the peptide on the surface of the coated nanoparticles can be a linear PGA, e.g., a short PGA containing 2-100 glutamic acid residues, or the peptide can be a branched PGA. In some embodiments, the peptide is a glutamic acid-containing peptide comprising a glutamic acid-rich domain containing a total of at least 4, at least 6, or at least 8 amino acid residues, at least 2 of which are glutamic acid, and at least 20% of the amino acid residues in the glutamic acid-rich domain are glutamic acid. In some embodiments, the nanoparticles are coated with a dendrimer selected from Table 1 or Table 1B, or a peptide selected from Table 1A (wherein the peptide is not E100). In some embodiments, the nanoparticles are nanoparticles according to any one of the aspects described herein that are coated with a linear PGA, a branched PGA, a glutamic acid-containing peptide, a dendrimer selected from Table 1 or Table 1B, or a peptide selected from Table 1A, as defined herein.

[0169] In some embodiments, the linear or branched PGA or glutamic acid-containing peptide may further comprise a cell targeting motif. For example, the cell targeting motif may comprise a bone marrow, lymphoid, muscle, lung, CD206+ cell, or tumor cell targeting motif. In some embodiments, the muscle cell targeting motif comprises an ASSLNIA (SEQ ID NO: 1), PYDQLRH (SEQ ID NO: 2), or KAMHQMQ (SEQ ID NO: 3) peptide motif. In some embodiments, the cell targeting motif comprises an integrin targeting motif, optionally comprising an RGD or ACDCRGDCFCG (SEQ ID NO: 5) peptide motif. In some embodiments, the lung targeting motif comprises the peptide sequence CGFECVRQCPERC (SEQ ID NO: 4). In even further embodiments, the cell targeting motif is a mannose receptor targeting motif, e.g., a mannose sugar or maltotriose. In some embodiments, the cell targeting motif targets lymphocytes, e.g., T cells. The cell targeting motif can be a CD3, CD4 or CD8 conjugate, for example, an antibody that specifically binds to one of these markers. In some embodiments, an anti-CD3 antibody is used. It can be conjugated to a negatively charged polymer such as PGA or a glutamic acid-containing peptide, or conjugated to a negatively charged lipid.

[0170] In a related aspect, the present invention provides coated nanoparticles capable of transfecting target cells, the coated nanoparticles having a peptide on their surface. The nanoparticles of this aspect may be as defined according to the preceding aspects of the invention. The peptide preferably comprises a peptide dendrimer. In some cases, the peptide is a PGA, as defined herein.

[0171] The present invention includes combinations of the described embodiments and preferred features except where such combinations are clearly impermissible or explicitly avoided.

[0172] Embodiments and experiments illustrating the principles of the present invention will now be discussed with reference to the accompanying figures. [Brief explanation of the drawings]

[0173] [Figure 1] A third-generation dendrimer is illustrated (N-terminus on the left and C-terminus on the right). The circle represents 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 corresponding second-generation dendrimer would lack the third layer of eight peptide motifs. The first-generation dendrimer would lack the third layer of eight peptide motifs and the second layer of four peptide motifs, plus the layer of branching residues between the third and second layers.

[0174] [Figure 2] eGFP expression in mouse macrophages after transfection with peptide dendrimer / lipid nanoparticles, naked mRNA, or DOTMA / DOPE-mRNA nanoparticles (LPX-mRNA). Cells were transfected with 1) eGFP mRNA alone, 2) DOTMA / DOPE and eGFP mRNA (lipid:mRNA ratio of 3.2:1 w / w), 3) GSCG1,2-RHL, DOTMA / DOPE, and eGFP mRNA (N:P = 0.6:1), 4) GSCG1,2-RL,3-LR, DOTMA / DOPE, and eGFP mRNA (N:P = 0.16:1), or 5) RHCG1,2-RL,2-LR, DOTMA / DOPE, and eGFP mRNA (N:P = 8:1). D / D indicates DOTMA:DOPE (w / w = 10:1 w / w relative to mRNA).

[0175] [Figure 3]eGFP expression in human macrophages after transfection with peptide dendrimer / lipid nanoparticles, naked mRNA, or DOTMA / DOPE-mRNA nanoparticles (LPX-mRNA). Cells were 1) not transfected, or 2) eGFP mRNA alone, 3) DOTMA / DOPE and eGFP mRNA (w / w = 3.2:1 lipid:mRNA), 4) GSCG1,2-RL, 3-LR, DOTMA / DOPE and eGFP mRNA (N:P = 0.16:1), 5) GSCG1,2-RHL, DOTMA / DOPE and eGFP mRNA (N:P = 0.6:1), 6) GSCG1-LRLR, DOTMA / DOPE and eGFP mRNA (N:P = 0.6:1), 7) GSCG1,2-RHL, DOTMA / DOPE and eGFP mRNA (N:P = 5:1), 8) GSCG1,2-RF, 3HL, DOTMA / DOPE and eGFP mRNA (N:P = 0.6:1), or 9) RHCG1-R, DOTMA / DOPE and eGFP LPX-RNA formulations contained DOTMA:DOPE at a lipid:mRNA ratio of 3.2:1.

[0176] [Figure 4] eGFP expression in mouse macrophages after transfection with peptide dendrimer / lipid nanoparticles, naked mRNA, or DOTMA / DOPE-mRNA nanoparticles (LPX-mRNA). Cells were either 1) untransfected or transfected with 2) eGFP mRNA alone, 3) GSCG1,2-RHL, DOTMA / DOPE, and eGFP mRNA (N:P = 8:1), or 4) GSCG1,2-RHL, DOTMA / DOPE, and eGFP mRNA (N:P = 8:1) nanoparticles containing a mannose-G1-EEEE coating. The mannose-G1-EEEE dendrimer was used at 1 eq (1 equivalent) of mRNA in the nanoparticles. D / D indicates a DOTMA:DOPE (w / w ratio of 2.5:1 relative to mRNA).

[0177] [Figure 5] eGFP expression in mouse macrophages after transfection with peptide dendrimer / lipid nanoparticles, naked mRNA, or DOTMA / DOPE-mRNA nanoparticles (LPX-mRNA). Cells were either 1) untransfected or transfected with 2) eGFP mRNA alone, 3) DOTMA / DOPE and eGFP mRNA (lipid:mRNA ratio 3.2:1 w / w), 4) GSCG1,2-RHL, DOTMA / DOPE, and eGFP mRNA (N:P = 8:1), or 5) GSCG1,2-RHL, DOTMA / DOPE, and eGFP mRNA (N:P = 8:1) nanoparticles containing a mannose-G1-EEEE coating. The mannose-G1-EEEE dendrimer was used at 0.5 times the mass (0.5 eq) of mRNA in the nanoparticles. D / D indicates DOTMA:DOPE (5:1 w / w relative to mRNA). The LPX-RNA formulation contained DOTMA:DOPE at a lipid:mRNA ratio of 3.2:1 w / w.

[0178] [Figure 6] eGFP expression in mouse macrophages after transfection with peptide dendrimer / lipid nanoparticles, naked mRNA, or DOTMA / DOPE-mRNA nanoparticles (LPX-mRNA). Cells were either 1) untransfected or transfected with 2) eGFP mRNA alone, 3) DOTMA / DOPE and eGFP mRNA (lipid:mRNA ratio 3.2:1 w / w), 4) GSCG1,2-RHL, DOTMA / DOPE, and eGFP mRNA (N:P = 8:1), or 5) GSCG1,2-RHL, DOTMA / DOPE, and eGFP mRNA (N:P = 8:1) nanoparticles containing a mannose-G1-EEEE coating. Mannose-G1-EEEE dendrimer was used at 1 eq (1 equivalent) of mRNA in the nanoparticles. D / D indicates DOTMA:DOPE (10:1 w / w relative to mRNA). The LPX-RNA formulation contained DOTMA:DOPE at a lipid:mRNA ratio of 3.2:1 w / w.

[0179] [Figure 7] eGFP expression in mouse macrophages after transfection with peptide dendrimer / lipid nanoparticles, naked mRNA, or DOTMA / DOPE-mRNA nanoparticles (LPX-mRNA). Cells were either 1) untransfected or transfected with 2) eGFP mRNA alone, 3) DOTMA / DOPE and eGFP mRNA (lipid:mRNA ratio of 3.2:1 w / w), 4) GSCG1,2-RHL, DOTMA / DOPE, and eGFP mRNA (N:P = 0.6:1), or 5) GSCG1,2-RHL, DOTMA / DOPE, and eGFP mRNA (N:P = 0.16:1) nanoparticles containing a mannose-G1-EEEE coating. The mannose-G1-EEEE dendrimer was used at three times the mass (3 eq) of mRNA in the nanoparticles. D / D indicates DOTMA:DOPE (10:1 w / w relative to mRNA). The LPX-RNA formulation contained DOTMA:DOPE at a lipid:mRNA ratio of 3.2:1 w / w.

[0180] [Figure 8]eGFP expression in mouse macrophages after transfection with peptide dendrimer / lipid nanoparticles, naked mRNA, or DOTMA / DOPE-mRNA nanoparticles (mRNA-LPX). Cells were either 1) untransfected (HEPES) or transfected with 2) eGFP mRNA alone, 3) DOTMA / DOPE and eGFP mRNA (lipid:mRNA ratio of 3.2:1 w / w), 4) GSCG 1,2-RL, 3-LR, DOTMA / DOPE, and eGFP mRNA (N:P ratio of 8:1), or 5) GSCG 1,2-RL, 3-LR, DOTMA / DOPE, and eGFP mRNA (N:P ratio of 8:1) nanoparticles containing a mannose-G1-EEEE coating. Mannose-G1-EEEE dendrimers were used at 1 eq (1 equivalent) of mRNA in the nanoparticles. D / D indicates DOTMA:DOPE (10:1 w / w ratio relative to mRNA). The LPX-RNA formulation contained DOTMA:DOPE at a lipid:mRNA ratio of 3.2:1 w / w.

[0181] [Figure 9] mRNA delivery to muscle cells. Mouse muscle cells (C2c12) were transfected with mRNA expressing eGFP. Cells were transfected with mRNA alone, mRNA with GSCG1,2-RL, 3-LR and DOTMA:DOPE (N:P = 0.6:1), mRNA with NTX2 and DOTMA:DOPE (N:P = 0.6:1), or mRNA with NTX3 and DOTMA:DOPE (N:P = 0.6:1). Cells were harvested 24 hours after transfection. Relative fluorescence units (RFU) were measured and normalized to cellular protein content to obtain RFU / mg. NTX2 is GSCG1,2-RL, 3-LR linked to a myocyte-targeting peptide, while NTX3 is GSCG1,2-RL, 3-LR linked to a myocyte-targeting peptide containing six histidines. D / D indicates DOTMA:DOPE (w / w=10:1 relative to mRNA).

[0182] [Figure 10]mRNA delivery to muscle cells. Mouse muscle cells (C2c12) expressing eGFP after transfection with eGFP mRNA. Cells were transfected with mRNA alone, mRNA containing RHCG1-RL, 2-RL, and DOTMA:DOPE (N:P = 0.6:1), or mRNA containing NTX5 and DOTMA:DOPE (N:P = 0.6:1). Cells were harvested 24 hours after transfection. Relative fluorescence units (RFU) were measured and normalized to the cellular protein content to obtain RFU / mg. NTX5 is RHCG1-RL, 2-LR linked to a muscle cell targeting peptide. D / D indicates DOTMA:DOPE (w / w = 10:1 relative to mRNA).

[0183] [Figure 11A] Luciferase expression in mouse tissues after intravenous administration of compositions containing GSCG1-LRLR with DOTMA / DOPE and mRNA (w / w = 23:1, lipid to mRNA), DOTMA / DOPE and mRNA (w / w = 23:1, lipid to mRNA), or luciferase-encoding mRNA alone. The dendrimer compositions were injected at an N:P ratio of 0.6:1. Mice were injected with the compositions, and tissues were harvested 6 hours later to measure luciferase signal in the lungs, spleen, liver, heart, kidneys, muscle (gastrocnemius), and brain.

[0184] [Figure 11B] Luciferase expression in lung tissue of mice after intravenous administration of a composition containing GSCG1,2-RHL (N:P=0.6) with DOTMA / DOPE and mRNA (w / w=23:1, lipid to mRNA), DOTMA / DOPE and mRNA (w / w=23:1, lipid to mRNA), or luciferase-encoding mRNA alone. The dendrimer composition was injected at an N:P ratio of 0.6:1. Mice were injected with the composition, and luciferase signal in the lung region was measured 6 hours later by in vivo IVIS imaging.

[0185] [Figure 11C]Luciferase expression in mouse tissues after intravenous administration of a composition containing GSCG1,2-RHL with DOTMA / DOPE and mRNA (w / w = 23:1, lipid to mRNA) and luciferase-encoding mRNA alone. The dendrimer composition was injected at an N:P ratio of 0.6:1. Mice were injected with the composition, and tissues were harvested 6 hours later to measure luciferase signal in the lungs, spleen, liver, heart, kidneys, muscle (gastrocnemius), and brain.

[0186] [Figure 12A] Luciferase expression in mouse tissues after intravenous administration of a composition containing DOTMA / DOPE (w / w = 10:1 relative to mRNA) and luciferase-expressing mRNAs GSCG1,2-RL, GSCG3-LR (N:P = 0.16:1 relative to mRNA), GSCG1,2-RHL (N:P = 0.6:1 relative to mRNA), and GSCG1-LRLR (N:P = 0.6:1 relative to mRNA). Treatment with mRNA alone served as a control. 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, and brain.

[0187] [Figure 12B] Luciferase expression in mouse tissues after intravenous administration of a composition containing LPX with luciferase-expressing mRNA. LPX-RNA represents DOTMA:DOPE at a w / w ratio of 3.2:1 relative to the mRNA. BALB / c 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 (fat), and brain.

[0188] [Figure 12C]Luciferase expression in mouse tissues after intravenous administration of a composition containing DOTMA / DOPE (w / w = 10:1 relative to mRNA) and luciferase-expressing mRNAs containing GSCG1,2-RL, 3-LR (N:P = 0.16:1 relative to mRNA), GSCG1,2-RHL (N:P = 0.6:1 relative to mRNA), and GSCG1-LRLR (N:P = 0.6:1 relative to mRNA). Treatment with mRNA alone was used as a control. LPX-RNA represents DOTMA:DOPE at a w / w ratio of 3.2:1 relative to mRNA. CD-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 (adipose), and brain.

[0189] [Figure 12D] Luciferase expression in mouse tissues after intravenous administration of a composition containing LPX with luciferase-expressing mRNA. LPX-RNA represents DOTMA:DOPE at a w / w ratio of 3.2:1 relative to the mRNA. CD-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 (fat), and brain.

[0190] [Figure 13]In vivo mRNA delivery to lung and bone marrow cells in cancer models. MC38-bearing mice were treated with Alexafluor488-tagged mRNA (at a dose of 2.25 mg / kg (mRNA vs. body weight)) with LPX, Alexafluor488-tagged mRNA (at a dose of 2.25 mg / kg (mRNA vs. body weight)) with GSCG1,2-RL,3-LR, and DOTMA:DOPE (N:P=0.16:1), GSCG1,2-RL,3-LR, DOTMA:DOPE (N:P=0.16:1), and mannose-G Either Alexafluor488-tagged mRNA with a l-EEEE ((Ac-EEEE)2KGSGGSGGSC[(SS)-α-D-thiomannose]) coating (at a dose of 2.25 mg / kg (mRNA to body weight)) or Alexafluor488-tagged mRNA with GSCG1,2-RHL and DOTMA:DOPE (N:P = 0.6:1) (at a dose of 2.25 mg / kg (mRNA to body weight)) was injected. Mannose-G1-EEEE dendrimer was used at three times the mass (3 eq) of mRNA in the nanoparticles. D / D indicates DOTMA:DOPE (w / w = 10:1 relative to mRNA). LPX-RNA indicates DOTMA:DOPE (w / w = 3.2:1 relative to mRNA). Four hours after injection of the formulations, cells were isolated for flow cytometry analysis. (A) % of total live cells in the lung that have taken up Alexafluor488-tagged mRNA, (B) % of total live CD206+ (mannose receptor+) cells in the lung that have taken up Alexafluor488-tagged mRNA, (C) % of total live bone marrow cells in the lung that have taken up Alexafluor488-tagged mRNA, and (D) % of total live M2 macrophages (CD206+ (mannose receptor+) expressing cells) in the lung that have taken up Alexafluor488-tagged mRNA.

[0191] [Figure 14]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, GSCG1,2-RL, 3-LR (NP = 0.16:1), DOTMA / DOPE (w / w = 10:1 relative to mRNA) and luciferase mRNA, or Lipofectamine 2000™ and luciferase mRNA (upper left panel), or 2) eGFP mRNA alone, GSCG1,2-RL, 3-LR (NP = 0.16:1), DOTMA / DOPE (w / w = 10:1 relative to mRNA) and eGFP mRNA, or DLin-MC3-DMA:cholesterol:DSPC:DMG-PEG lipid nanoparticles and eGFP mRNA (upper right panel). C2c12 cells were transfected with luciferase mRNA alone, a composition containing GSCG1,2-RL,3-LR (NP = 8:1), DOTMA / DOPE (w / w = 10:1 relative to mRNA) and luciferase mRNA, Lipofectamine 2000™ and luciferase mRNA, or a composition containing polyethyleneimine and luciferase mRNA (bottom panel). Luciferase and eGFP expression were measured 24 hours after transfection. Peptide dendrimers are more efficient at transfecting HeLa and C2c12 cells in vitro than commercially available transfection reagents such as Lipofectamine 2000 and LNP.

[0192] [Figure 15]Comparison of the transfection efficiency of G1,2,3-RL with other commercially available transfection reagents for DNA delivery, including DOTMA / DOPE, polyethyleneimine, and Lipofectamine 2000. (A) Transfection of HeLa cells in the absence of serum, (B) Transfection of HeLa cells in the presence of serum, (C) Transfection of Neuro2A cells in the absence of serum, and (D) Transfection of Neuro2A cells in the presence of serum. Cells transfected with a luciferase expression plasmid (pCI-Luc) in the presence of GSCG1,2,3-RL with D / D (measured 24 h posttransfection). Luminescence values were normalized by dividing them by the analogous values from cells treated with D / D DNA complexes to obtain the percentage of transfection. Conditions: 1 x 104 cells were transfected with 0.25 μg of pCI-Luc for 4 h in the absence of serum or in the presence of 10% serum. Error bars represent the mean ± SEM of experiments performed in triplicate. GSCG1,2,3-RL-mediated transfection was significantly more potent than other reagents in HeLa and Neuro2A cells. * indicates p<0.05, *** indicates p<0.001, and **** indicates p<0.0001. D / D is 1:1 w / w DOTMA / DOPE.

[0193] [Figure 16]Comparison of transfection efficiencies of different generations (G1, G2, and G3) of dendrimers with different N / P ratios and cationic residues (e.g., KL vs. RL) for DNA delivery. (A) Transfection efficiency in HeLa cells; (B) Transfection efficiency in Neuro2A cells. Cells were transfected with a luciferase expression plasmid (pCI-Luc) in the presence of peptide dendrimers with D / D (measured 24 h posttransfection). Luminescence values were normalized by dividing them by the analogous value for cells treated with D / D DNA complexes (1:1 w / w, 0.25 μg) to obtain the percentage of transfection. Condition: 1 × 104 cells transfected with 0.25 μg pCI-Luc. Error bars represent the mean ± SEM of triplicate experiments. * indicates p<0.05. D / D is DOTMA / DOPE 1:1 w / w. The second and third generation dendrimers perform better than the first generation dendrimers in transfecting both HeLa and Neuro2A cells.

[0194] [Figure 17]Comparison of transfection efficiency between single and hybrid dendrimer compositions. HeLa cells were transfected with A) a 1:2 mixture of GSCG1,2-RL, 3-LR or GSCG1,2-RL, 3-LR and GSCG1-LRLR, B) a 2:1 mixture of RHCG1-RL, 2-LR, RHCG1-RL, 2-LR and RHCG1-R, or a 1:2 mixture of RHCG1-RL, 2-LR and RHCG1-R, C) a 1:2 mixture of RHCG1-RL, 2-LR or RHCG1-RL, 2-LR and RHCG1,2-R. A) a 1:1 mixture of RHCG1-RL and 2-LR or RHCG1-RL, 2-LR, and RHCG1-RLR, or a 1:2 mixture of RHCG1-RL, 2-LR, and RHCG1-RLR, or a 1:1 mixture of RHCG1-RL, 2-LR, and RHCG1-RLR, or a 1:2 mixture of RHCG1-RL, 2-LR, and RHCG1-RLR, or a 1:1 mixture of RHCG1-RL, 2-LR, or RHCG1-RL, 2-LR, and GSCG1-LRLR, or a 1:2 mixture of RHCG1-RL, 2-LR, and GSCG1-LRLR. The final N:P ratio for all compositions was 8:1. DOTMA:DOPE was added to the mRNA complex at a w / w ratio of 10:1. The ratio between dendrimers is the molar ratio of N contributed by each dendrimer. The percentage of transfection was calculated by multiplying the transfection value of the mRNA complex formed with one dendrimer by 100 (%), normalizing it to the transfection value of the mRNA complex formed with two dendrimers. Hybrid dendrimer systems are superior to single dendrimer systems with respect to in vitro transfection.

[0195] [Figure 18]Demonstration of transfection efficiency in C2c12 cells. Labeled mRNA delivery into cells. Mouse muscle cells (C2c12) took up mRNA labeled with Alexa488 fluorophore after transfection. Cells were transfected for 4 hours with labeled mRNA alone, NTX3 (N:P = 0.6:1), and labeled mRNA with DOTMA:DOPE, and mRNA uptake was measured by flow cytometry. NTX3 is GSCG1,2-RL,3-LR linked to a muscle cell targeting peptide with six histidines. DOTMA:DOPE was used at a w / w ratio of 10:1 relative to the mRNA.

[0196] [Figure 19] Demonstration of transfection efficiency in macrophages. Delivery of labeled mRNA to cells. Macrophages (J774 cells) took up mRNA labeled with Alexa488 fluorophore after transfection. Cells were transfected with labeled mRNA containing GSCG1,2-RHL (N:P=0.6:1) and DOTMA:DOPE for 4 hours, and mRNA uptake was measured by flow cytometry. Cells were transfected with different amounts of mRNA, ranging from 0.0015 μg to 1.5 μg. DOTMA:DOPE was used at a w / w ratio of 10:1 relative to mRNA.

[0197] [Figure 20] Demonstration of transfection efficiency in T cells. Delivery of labeled mRNA into cells. Human T cells (Jurkat cells) took up mRNA labeled with Alexa488 fluorophore after transfection. Cells were transfected with labeled mRNA containing GSCG1,2-RHL (N:P=0.6:1) and DOTMA:DOPE for 4 hours, and mRNA uptake was measured by flow cytometry. Cells were transfected with different amounts of mRNA, ranging from 0.0015µg to 1.5µg. DOTMA:DOPE was used at a w / w ratio of 10:1 relative to mRNA.

[0198] [Figure 21]Further demonstration of transfection efficiency in T cells. Labeled mRNA delivery to cells. HeLa cells took up mRNA labeled with Alexa488 fluorophore after transfection. Cells were transfected with labeled mRNA containing GSCG1,2-RHL (N:P=0.6:1 relative to mRNA) and DOTMA:DOPE for 2 hours, and mRNA uptake was measured by flow cytometry. Cells were transfected with different amounts of mRNA, ranging from 0.1875 μg to 1.5 μg. DOTMA:DOPE was used at a w / w ratio of 10:1 relative to mRNA.

[0199] [Figure 22] Transfection of HeLa cells using nanocarriers formulated with different lipid components. Nanocarriers containing the three lipid systems show higher transfection efficacy than nanocarriers containing DOTMA:DOPE.

[0200] [Figure 23] Transfection of HeLa cells using nanocarriers formulated with different lipid components. Nanocarriers containing the three lipid systems show higher transfection efficacy than nanocarriers containing DOTMA:DOPE.

[0201] [Figure 24] Transfection of A549 cells using nanocarriers formulated with different lipid components. Nanocarriers containing the three lipid systems show higher transfection efficacy than nanocarriers containing DOTMA:DOPE.

[0202] [Figure 25]Dendritic PGA coating enhances delivery to T cells. Human T cells were transfected with formulations expressing eGFP (alone) or containing mRNA with GSCG1,2-RHL and coated with either linear or dendritic PGA. Cellular eGFP expression was quantified by flow cytometry and normalized to the level achieved by formulations coated with linear PGA. Panel A (top): Equimolar amounts of linear and dendritic PGA. Panel B (bottom): Equimolar charges of linear and dendritic PGA.

[0203] [Figure 26] Enhanced targeting of undifferentiated muscle cells using particles containing mRNA expressing eGFP. Nanocarriers were coated with molar equivalents of dendritic PGA with or without a muscle-targeting domain. Particles coated with dendrimers containing the muscle-targeting domain achieved a dramatic increase in eGFP expression compared to particles coated with dendrimers without the muscle-targeting domain.

[0204] [Figure 27] Enhanced targeting of differentiated muscle cells using particles containing mRNA expressing eGFP. Nanocarriers were coated with molar equivalents of dendritic PGA with or without a muscle-targeting domain. Particles coated with dendrimers containing the muscle-targeting domain achieved a dramatic increase in eGFP expression compared to particles coated with dendrimers without the muscle-targeting domain.

[0205] [Figure 28] Enhanced targeting of tumor cells using particles containing mRNA expressing eGFP. Nanocarriers were coated with molar equivalents of dendritic PGA with or without an integrin targeting domain. Particles coated with dendrimers containing the integrin targeting domain achieved a dramatic increase in eGFP expression compared to particles coated with dendrimers without the integrin targeting domain.

[0206] [Figure 29] Enhanced targeting of human T cells using particles containing mRNA expressing eGFP. Nanocarriers were coated with 1x or 3x equivalents of dendritic PGA with or without anti-CD3 antibody (T cell targeting domain). Particles coated with dendrimers containing anti-CD3 antibody achieved a dramatic increase in eGFP expression compared to particles coated with dendrimers without anti-CD3 antibody. D / D indicates DOTMA:DOPE (w / w = 10:1 relative to mRNA). eq refers to equivalent, hCD3 refers to human CD3, Ab refers to antibody, ctr refers to control, and ITC refers to isotype control. eGFP-expressing mRNA was used.

[0207] [Figure 30] Functional delivery of two mRNA molecules to cancer cells in vitro using the nanocarriers of the present invention. HeLa cells were transfected with nanocarriers containing a mixture of two mRNAs or with a formulation of the two mRNAs without the nanocarriers of the present invention. When transfected with nanocarriers containing eGFP and / or mCherry, respectively, approximately 100% of the cells showed functional expression of eGFP and / or mCherry.

[0208] [Figure 31] Functional in vivo delivery of mRNA molecules to a subject using a nanocarrier of the present invention containing two nucleic acids. Six hours after intravenous administration of a nanocarrier containing luciferase mRNA and a CpG-containing nucleic acid to mice, high luciferase levels were observed in the lungs and spleen.

[0209] [Figure 32]Transfection of primary leukocytes with nanocarriers formulated with multiple nucleic acids. Primary mouse monocyte-derived dendritic cells (moDCs) were transfected with nanocarriers containing CpG molecules and mRNA expressing eGFP. Functional expression of eGFP is observed after 2 hours (panel A), 4 hours (panel B), and 22 hours (panel C). D / D indicates DOTMA:DOPE (w / w = 10:1 relative to mRNA).

[0210] [Figure 33] Induction of M1 macrophages using the nanocarriers of the present invention. Activated IRF5 was delivered to primary M2 macrophages using nanocarriers. Panel A is a volcano plot showing significantly upregulated M1 genes and downregulated M2 genes. Panel B plots the results of gene set enrichment analysis (GSEA), which shows the number of differentially expressed genes in cells delivered with modified IRF5 via the nanocarriers of the present invention.

[0211] [Figure 34] Cytokine secretion by M1 macrophages induced using the nanocarrier of the present invention. Modified IRF5 was delivered to M2 macrophages using the nanocarrier of the present invention, which polarized them into M1 macrophages. Control: M2 macrophages delivered with luciferase via the nanocarrier of the present invention. Cytokine secretion was measured 24 hours later. Panels A and B show the secretion of IL12p70 and p40, respectively, after repolarization of mouse macrophages. Panels C and D show the secretion of IL12p70 and TNFα, respectively, after repolarization of human macrophages.

[0212] [Figure 35] Delivery of therapeutic mRNA using the nanocarriers of the present invention significantly suppresses tumor growth. The therapeutic mRNA expresses modified IRF5, which induces M1 macrophages. Significantly suppressed the growth of MC38 carcinoma.

[0213] [Figure 36] Jurkat cells were transfected with nanocarriers formulated with different lipid components. The "mRNA alone" control and the control nanocarriers containing DOTMA:DOPE contained a 1.5 μg "dose" of eGFP-expressing mRNA (in a well of a 12-well plate). In contrast, nanocarriers with test lipids contained 20% of the mRNA used in the control transfection, e.g., 0.3 μg for each test nanocarrier formulation.

[0214] [Figure 37] HeLa cells were transfected with nanocarriers formulated with different lipid components. The "mRNA alone" control and the control nanocarriers containing DOTMA:DOPE contained a 0.25 μg "dose" of eGFP-expressing mRNA (in a well of a 96-well plate). In contrast, the nanocarriers with test lipids contained 67% of the mRNA used in the control transfection, e.g., 0.17 μg for each test nanocarrier formulation.

[0215] [Figure 38] A549 cells were transfected with nanocarriers formulated with different lipid components. The "mRNA alone" control and the control nanocarriers containing DOTMA:DOPE contained a 0.25 μg "dose" of eGFP-expressing mRNA (in a well of a 96-well plate). In contrast, the nanocarriers with test lipids contained 67% of the mRNA used in the control transfection, e.g., 0.17 μg for each test nanocarrier formulation.

[0216] [Figure 39]In vivo mRNA delivery to bone marrow cells within tumors. MC38 tumor-bearing mice were treated with Alexafluor488-tagged mRNA (at a dose of 2.25 mg / kg (mRNA vs. body weight)) containing LPX, GSCG1,2-RL,3-LR, and DOTMA:DOPE (N:P=0.16:1), and Alexafluor488-tagged mRNA (at a dose of 2.25 mg / kg (mRNA vs. body weight)) containing GSCG1,2-RL,3-LR, DOTMA:DOPE (N:P=0.16:1), and mannose- The mice were injected with either Alexafluor488-tagged mRNA with G1-EEEE ((Ac-EEEE)2KGSGGSGGSC[(SS)-α-D-thiomannose]) coating (at a dose of 2.25 mg / kg (mRNA to body weight)) or Alexafluor488-tagged mRNA with GSCG1,2-RHL and DOTMA:DOPE (N:P = 0.6:1) (at a dose of 2.25 mg / kg (mRNA to body weight)). Mannose-G1-EEEE dendrimers were used at three times the mass (3 eq) of mRNA in the nanoparticles. D / D indicates DOTMA:DOPE (w / w = 10:1 relative to mRNA). LPX-RNA indicates DOTMA:DOPE (w / w = 3.2:1 relative to mRNA). Four hours after injection of the formulations, cells were isolated for flow cytometry analysis. (A) Percentage of total live bone marrow cells that have taken up Alexafluor488-tagged mRNA, (B) Percentage of total live M2 macrophages (CD206+ (mannose receptor+) expressing cells) in the tumor that have taken up Alexafluor488-tagged mRNA, and (C) Percentage of total live myeloid-derived suppressor cells (MDSC) in the tumor that have taken up Alexafluor488-tagged mRNA.

[0217] [Figure 40]Intratumoral luciferase expression in MC38 tumor-bearing mice after intravenous administration of a composition containing DOTMA / DOPE (w / w ratio relative to mRNA: 10:1) and GSCG1,2-RHL with luciferase-expressing mRNA (N:P ratio relative to mRNA: 0.6:1). Treatment with mRNA alone and LPX-RNA were used as controls. LPX-RNA represents DOTMA:DOPE at a w / w ratio of 3.2:1 relative to mRNA. Mice were injected with the composition, and luciferase signals in the tumor area were measured 6 hours later by in vivo IVIS imaging.

[0218] [Figure 41A] Primary mouse bone marrow-derived macrophages were polarized to the M2 phenotype. The cells were then transfected for 24 hours with mRNA expressing a modified form of IRF5 protein, which can polarize M2 cells to M1 cells. Control: Cells were transfected with mRNA expressing luciferase (control mRNA). The modified IRF5 protein contains a mutation that functions as an activated form of WT IRF5. The formulations used included DOTMA / DOPE (w / w = 10:1 relative to mRNA) and GSCG1,2-RHL with mRNA (N:P = 0.6:1 relative to mRNA). WT IRF5 and modified IRF5 protein expression levels were detected by flow cytometry and expressed as % IRF5-expressing cells.

[0219] [Figure 41B]Primary mouse bone marrow-derived macrophages were polarized to the M2 phenotype. Then, for 24 hours, the cells were transfected with mRNA expressing a modified form of the IRF5 protein, which can polarize M2 cells to M1 cells. Control: Cells were transfected with mRNA expressing luciferase (control mRNA). The modified IRF5 protein has a mutation that functions as an activated form of WT IRF5. The formulations used included DOTMA / DOPE (w / w = 10:1 relative to mRNA) and GSCG1,2-RHL with mRNA (N:P = 0.6:1 relative to mRNA). M2 cells transfected with the modified IRF5 mRNA showed increased expression of the M1 marker (CD80), indicating M1 polarization.

[0220] [Figure 42] Nanocarriers coated with human CD3-targeting antibodies improve human T cell-targeted mRNA delivery. Jurkat cells were transfected with (1) GSCG1,2-RHL and DOTMA / DOPE carrying mRNA (N:P = 0.6 relative to mRNA), (2) GSCG1,2-RHL and DOTMA / DOPE carrying mRNA coated with 3 equivalents of anti-CD3 antibody-conjugated dendrimer (N:P = 0.6 relative to mRNA), (3) GSCG1,2-RHL and DOTMA / DOPE carrying mRNA coated with dendrimer alone (N:P = 0.6 relative to mRNA) (control for (2)), (4) GSCG1,2-RHL and DOTMA / DOPE carrying mRNA coated with 1 equivalent of anti-CD3 antibody-conjugated dendrimer (N:P = 0.6 relative to mRNA), and (5) GSCG1,2-RHL and DOTMA / DOPE coated with dendrimer alone (N:P = 0.6 relative to mRNA) (control for (4)). D / D indicates DOTMA:DOPE (w / w = 10:1 relative to mRNA). Transfections were analyzed by flow cytometry 24 hours after transfection of the formulations. eq refers to equivalent amount, hCD3 refers to human CD3, Ab refers to antibody, and ctr refers to control. eGFP-expressing mRNA was used. DETAILED DESCRIPTION OF THE INVENTION

[0221] Aspects and embodiments of the present invention will be described below with reference to the accompanying drawings and technical definitions that follow. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated herein by reference.

[0222] Current immunotherapy response rates are approximately 15–20%, and there is an urgent need to improve treatment outcomes. One strategy is to deliver mRNA to express proteins (e.g., CEBPA, IRF5, IRF8, cGAS-STING, SOCS1, and / or SOCS3) to reverse the immunosuppressive phenotype of myeloid cells within the tumor microenvironment, thereby providing a more favorable environment for immunotherapy response. Another strategy is to introduce mRNA into immune cells within the tumor to express cytokines (e.g., IL-2, IL-7, IL-12, IL-15, IL-21, and / or interferons), activating the immune cells to fight cancer cells. It is also possible to deliver mRNA to macrophages to express chimeric antigen receptors, which can activate macrophages to kill tumor cells. Delivering mRNA to express tumor antigens within antigen-presenting cells can help activate the immune system to attack cancer cells. These strategies can be applied to treat all tumors, especially non-small cell lung cancer or small cell lung cancer (SCLC), advanced melanoma, prostate cancer, ovarian cancer, breast cancer, lung cancer, bile duct cancer (cholangiocarcinoma), gallbladder cancer, neuroendocrine tumors, hepatocellular carcinoma, colorectal cancer, pancreatic cancer, liver cancer, prostate cancer, thyroid cancer, pancreatic cancer such as pancreatic ductal adenocarcinoma (PDAC), acute myeloid lymphoma (AML), myelodysplastic syndrome (MDS), colorectal cancer such as WT KRAS CRC, KRAS mutant metastatic tumors, hematological tumors, esophageal cancer, breast cancer, prostate cancer, bladder cancer, tumors of the GI tract, head and neck squamous cell carcinoma (HNSCC), kidney cancer, myelofibrosis, CD206+ cancer, melanoma, prostate cancer or anal cancer, and solid tumors.

[0223] The compositions of the present invention can be used to treat pulmonary diseases, such as pulmonary fibrosis, including cystic fibrosis, asthma, tuberculosis (TB), acute lung injury (ALI), idiopathic pulmonary fibrosis, allergic airway disease, chronic obstructive pulmonary disease (COPD), alpha-1 antitrypsin deficiency (AATD), pulmonary arterial hypertension, fibrotic lung disease, chronic lung disease, or respiratory tract infections. The compositions of the present invention can be used to treat muscle diseases, such as muscular dystrophy or muscle wasting diseases. The compositions of the present invention can be used to treat kidney diseases. The compositions of the present invention can be used to treat rare genetic diseases (in hematology, neurology, amyloidosis, pulmonology, endocrinology, and nephrology). The compositions of the present invention can be used to deliver anti-infectives (e.g., to target macrophages for bacterial killing / to deliver antibiotic payloads).

[0224] The nanoparticles and compositions of the present invention can be used to deliver the nucleic acids described herein to specific tissues in the human or animal body, such as skeletal muscle, liver, lung, heart, white and / or brown adipose tissue, brain, spleen, bone marrow, joints, kidneys, gastrointestinal tract, eyes, thymus, skin, lymph nodes, pancreas, adrenal glands, testes, prostate, ovaries, uterus, bladder, diaphragm, and tumors. In some embodiments, the present invention can be used to deliver nucleic acids to skeletal muscle, lung, spleen, bone marrow, thymus, lymph nodes, and tumors. In particular, the nanoparticles of the present invention are particularly effective in targeting the delivery of nucleic acid cargo to bone marrow, lymph, muscle, and lung cells.

[0225] With respect to myeloid cells, the nanoparticles of the invention can efficiently deliver nucleic acid cargo to CD206-expressing cells, such as macrophages, neutrophils, dendritic cells, endothelial cells and lymphoid cells, including, in particular, M2 phenotype macrophages.

[0226] With respect to lung cells, the nanoparticles of the present invention may target delivery of cargo to alveolar macrophages, ciliated cells, epithelial cells, basal cells, secretory cells, club cells, alveolar cells, fibroblasts, and / or endothelial cells. Improved delivery of nucleic acids to lung tissue using the nanoparticles of the present invention will lead to new and improved treatment options for lung diseases such as cystic fibrosis.

[0227] Cell and tissue targeting

[0228] The present inventors have found that the nanoparticles of the present invention can be highly efficiently and specifically targeted to cells within specific tissues by altering the physical properties of the nanoparticles. This can be achieved in a number of ways.

[0229] For example, the nanoparticle may include a tissue- or cell-specific targeting motif. In this regard, the tissue- or cell-specific targeting motif may be a cell surface receptor ligand. The receptor ligand may be a protein or a fragment of a protein ligand, such as a peptide. The protein or peptide ligand may form part of a peptide dendrimer. For example, the protein or peptide ligand may be present in the peptide dendrimer core or one or more peptide motifs. Alternatively, or in addition, the protein or peptide ligand may be present in a linear peptide contained within the nanoparticle.

[0230] The receptor ligand may also be a sugar. For example, the sugar may be selected from mannose, galactose, or glucose sugars. Preferably, the sugar is mannose. The sugar may be covalently bound to a component of the nanoparticle, for example, the sugar may be covalently bound to a peptide dendrimer or a linear peptide in the nanoparticle. The sugar may also be covalently bound to a polymer (e.g., PGA) or lipid.

[0231] Regardless of the type of ligand forming the cell and / or tissue targeting motif, the ligand will preferably bind to a receptor with limited tissue distribution. For example, the receptor may be expressed only on the target cell or within the target tissue. Alternatively, the receptor may be predominantly expressed within the target cell type or tissue. For example, CD206 on CD206+ cells. In one example, by including a mannose ligand within the nanoparticle, M2 phenotype macrophages can be targeted for nucleic acid delivery. Because M2 macrophages exhibit high levels of mannose receptor expression, the mannose ligand can target nucleic acid delivery to M2 macrophages.

[0232] The present inventors have also found that delivery of nucleic acids to specific cell and tissue types can be achieved by varying the lipid:nucleic acid weight / weight ratio. In particular, specific delivery of nucleic acids to the lung can be achieved using lipid:nucleic acid w / w ratios of 2:1 to 40:1, preferably 23:1 or 10:1.

[0233] The efficiency of macrophage transfection using nanoparticles containing one of three peptide dendrimers with varying N:P ratios was tested by transfecting human and mouse macrophages in vitro in full growth medium conditions. Each peptide dendrimer was shown to be able to transfect human and mouse macrophages with high efficiency (Figures 2 and 3). In particular, in the case of mouse macrophages, the peptide dendrimer / lipid nanoparticles were shown to be superior to lipid-based delivery vectors containing only DOTMA / DOPE (Figure 2).

[0234] Certain macrophage populations, i.e., M2 phenotype macrophages, are enriched in the expression of the mannose receptor CD206. Thus, in some examples, dendrimers used in the nanoparticles of the present invention include mannose sugars for binding to the CD206 receptor. Certain dendrimers of the present invention that are particularly preferred for mRNA delivery to myeloid cells include G1,2-RHL, G1,2-RL, 3-LR, G1-RL, 2LR, G1-RL, 2-LR, and especially GSC G1,2-RL, 3-LR, RHC G1-RL, 2-LR, GSC G1,2-RHL, GSC G1-LRLR, GSC G1,2-RF, 3-HL, or GSC G1-R. These dendrimers may be derivatized to include mannose glycosylation.

[0235] Certain dendrimers of the invention that are particularly preferred for mRNA delivery to lymphoid cells include dendrimers conjugated or covalently attached to anti-CD3 antibodies, or anti-CD3 antibody fragments.

[0236] Certain dendrimers of the invention that are particularly preferred for mRNA delivery to muscle cells include G1,2-RL, 3-LR and G1-RL, 2-LR, particularly those containing the ASSLNIA (SEQ ID NO: 1) peptide motif. GSC G1,2-RL, 3-LR and GSC G1-RL, 2-LR (e.g., NTX2, NTX3, and NTX5). The NP ratio may be 0.6:1.

[0237] Certain dendrimers of the invention that are particularly preferred for mRNA delivery to lung cells include G1-LRLR, especially GSC G1-LRLR. The NP ratio may be 0.6:1.

[0238] Coated nanoparticles

[0239] In embodiments providing nanoparticles coated with peptides and / or dendrimers, this means that the peptides / dendrimers are present on the surface of the coated nanoparticles. This can be easily achieved by first preparing nanoparticles (preferably containing one or more peptide dendrimers along with nucleic acids and lipids) and then mixing the nanoparticle formulation with the peptides and / or dendrimers to be coated on the surface of the nanoparticles. For nanoparticles with positive or negative surface charges, the success of the coating can be easily determined by monitoring the zeta potential of the nanoparticles. The zeta potential of positively charged nanoparticles becomes substantially non-positive after coating the nanoparticles with negatively charged peptides / dendrimers. Conversely, the zeta potential of negatively charged nanoparticles becomes substantially non-negative after coating the nanoparticles with negatively charged peptides / dendrimers. In some embodiments, the zeta potential of the nanoparticles changes from positive to negative or negative to positive after coating with peptides / dendrimers. (In contrast, incorporating such peptides / dendrimers throughout the nanoparticle does not substantially affect its zeta potential, which measures the charge on the surface of the nanoparticle.) Other analytical methods, such as determining particle size before and after coating, can be used. Such methods can be used, for example, to confirm the successful coating of uncharged nanoparticles with hydrophobic peptides / dendrimers.

[0240] mRNA transfection

[0241] The effect of using a composition containing two separate peptide dendrimers for mRNA delivery was tested by transfecting HeLa cells in full growth medium conditions. It was shown that including two separate peptide dendrimers in a transfection composition can significantly increase transfection efficiency (Figure 17). Specifically, it was shown that including a first-generation ("G1") dendrimer in combination with a second-generation ("G2") dendrimer can result in transfection efficiencies up to three times higher than those seen when using the G2 dendrimer alone (Figure 5B). Transfection efficiency can also be adjusted within a composition containing two separate peptide dendrimers by varying the ratio of G2:G1 peptide dendrimers in the composition. For example, the G2 dendrimer RHC G1-RL, 2-LR and G1 dendrimers RHC In compositions containing G1-R, a G2:G1 ratio of 2:1 has approximately 66% transfection efficiency compared to a G2:G1 ratio of 1:2 (FIG. 17B).

[0242] We also tested the effect of using two different G2 dendrimers on transfection efficiency, e.g., peptide dendrimers. RHC G1-RL, 2-LR and RHC The composition comprising G1,2-R is RHC We found that G1-RL and 2-LR improved transfection efficiency compared to G1-RL and 2-LR alone (Fig. 17C).

[0243] messenger RNA (mRNA)

[0244] Messenger RNA (mRNA) is a single-stranded RNA molecule containing the coding sequence of a gene, which is translated by ribosomes into the corresponding amino acid sequence. mRNA is produced during the transcription process, when an enzyme (RNA polymerase) converts the gene into the primary transcript mRNA (also known as pre-mRNA). This pre-mRNA typically still contains introns, which are regions that do not encode the final amino acid sequence. These are removed during the RNA splicing process, leaving only the exons, which encode the protein. This exon sequence constitutes the mature mRNA. The mature mRNA is then read by ribosomes to produce the encoded protein. The present invention can be used to deliver mRNA molecules to target cells and tissues as a means of inducing the expression of a desired protein or peptide. Induction of peptide / protein expression via mRNA delivery is particularly useful when transient expression is desired. To improve mRNA expression, synonymous codons within the mRNA can be altered based on the codon bias of the organism, i.e., the mRNA can be codon-optimized. For example, the mRNA can be codon-optimized for expression in the organism type to which the composition of the present invention is administered. For example, the mRNA may be codon-optimized for expression in a mammal, such as a human.

[0245] In particular, delivery of mRNA encoding chimeric antigen receptors (CARs) and transcription factors is envisioned.

[0246] mRNA and lncRNA (described below) are typically large molecules with negatively charged and hydrophobic faces. Therefore, mRNA and lncRNA require a balance of hydrophobic and hydrophilic interactions to be encapsulated and delivered to target tissues and cells. This balance of hydrophobic and hydrophilic interactions will be different from, for example, double-stranded nucleic acids with charges on both sides, such as pDNA and siRNA. Because mRNA and lncRNA are significantly larger than, for example, ASOs, the requirements for encapsulation and delivery may also be different. Therefore, the optimal dendrimer NP ratio and DOTMA / DOPE w / w ratio for mRNA and lncRNA delivery will be different compared to ASO delivery.

[0247] modified nucleic acid

[0248] Modified nucleotide bases may be used in addition to the naturally occurring bases and may impart advantageous properties to nucleic acids containing them.

[0249] For example, modified bases may increase the stability of a nucleic acid molecule, thereby reducing the amount required. The provision of modified bases may also provide a nucleic acid molecule that is more or less stable than an unmodified nucleic acid.

[0250] 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 3' hydroxyl group and other than a 5' phosphate group. 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.

[0251] 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-2-thiouracil, -D-methyl These include mannosylqueosine, 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.

[0252] RNA interference

[0253] The present invention facilitates therapeutic downregulation of target gene expression through the delivery of nucleic acids, including RNA interference (RNAi). Small RNA molecules can be used to control gene expression.

[0254] These include targeted degradation of mRNA by small interfering RNA (siRNA), post-transcriptional gene silencing (PTG), developmentally regulated sequence-specific translational repression of mRNA by microRNA (miRNA) and targeted transcriptional gene silencing.

[0255] 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 rapidly target and silence specific genes with homology. It 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 but short enough to evade host responses. The reduction in expression of target gene products can be widespread, with 90% silencing induced by a few siRNA molecules.

[0256] 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 either inducing mRNA elimination (RNAi) or preventing mRNA translation into protein. siRNAs are derived by processing long double-stranded RNAs and, when found in nature, are usually of exogenous origin. Microinterfering RNAs (miRNAs) are endogenously encoded small non-coding RNAs that are derived by processing short hairpins. Both siRNAs and miRNAs can inhibit the translation of mRNAs with partially complementary target sequences and degrade mRNAs with fully complementary sequences without cleaving the RNA.

[0257] Therefore, the present invention provides the use of these sequences in the composition of the present invention for downregulating the expression of target genes.For example, it is envisioned that the nanoparticles of the present invention can be used to deliver RNAi-based therapy for use in treating diabetes, for example, type I or type II diabetes.

[0258] siRNA ligands are typically double-stranded, and to optimize the effectiveness of RNA-mediated downregulation of target gene function, the length of the siRNA molecule is preferably selected to ensure correct recognition of the siRNA by the RISC complex, which mediates siRNA recognition of the mRNA target, and so that the siRNA is sufficiently short to reduce the host response.

[0259] 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 not translated into protein. The DNA sequence encoding 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-pair to form a partially double-stranded RNA segment. The design of microRNA sequences is described in John et al., 2004.

[0260] Typically, RNA ligands intended to mimic the effects 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 present invention using double-stranded siRNA, the molecules may have, for example, symmetric 3' overhangs of one or two (ribo)nucleotides, typically a UU dTdT 3' overhang. Based on the disclosure provided herein, those skilled in the art can easily design suitable siRNA and miRNA sequences using resources such as Ambion's online siRNA finder. siRNA and miRNA sequences can be synthetically produced and exogenously added to cause gene downregulation, or they can be produced using expression systems (e.g., vectors). In a preferred embodiment, the siRNA is synthetically synthesized.

[0261] Longer double-stranded RNAs may be processed intracellularly to produce siRNAs (see, e.g., Myers et al. (2003)). Longer dsRNA molecules may have, for example, symmetric 3' or 5' overhangs of one or two (ribo)nucleotides, or may have blunt ends. Longer dsRNA molecules may be 25 nucleotides or longer. Preferably, longer dsRNA molecules are 25 to 30 nucleotides in length. More preferably, longer dsRNA molecules are 25 to 27 nucleotides in length. Most preferably, longer dsRNA molecules are 27 nucleotides in length.

[0262] In one embodiment, siRNA, longer dsRNA or miRNA is produced endogenously (intracellularly) by transcription from vector.Vector can be introduced into cell by any method known in the art.Optionally, the expression of RNA sequence can be controlled using tissue-specific promoter.In another embodiment, siRNA, longer dsRNA or miRNA is produced exogenously (in vitro) by transcription from vector.

[0263] Alternatively, siRNA molecules can be synthesized using standard solid-phase or solution-phase synthesis techniques known in the art. The linkage between nucleotides can be a phosphodiester bond or alternative, such as a linking 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, where R is H (or salt) or alkyl (1-12C), and R6 is alkyl (1-9C), and is linked to adjacent nucleotides via -O- or -S-.

[0264] Long non-coding RNA

[0265] Mammalian genomes are extensively transcribed, generating a vast number of transcripts, including thousands of long non-coding RNA molecules (lncRNAs). It has been shown that lncRNAs can regulate chromatin state, transcription, RNA stability, and translation of certain genes. The lncRNAs can be delivered to target cells or tissues using the nanoparticles of the present invention.

[0266] RNA activation (RNAa)

[0267] RNA activation (RNAa) is an RNA-mediated process that enhances 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 dsRNAs with two-nucleotide overhangs at both ends. Despite the fact that saRNAs mediate gene activation in a sequence-specific manner, they share the same structure and chemical components as siRNAs. To activate gene expression, the guide strand of the saRNA is loaded onto AGO2, and the complex is then transported to the nucleus. Once inside the nucleus, the guide strand-AGO2 complex binds directly to the gene promoter or associated transcript and recruits key components, including RNA polymerase II, to initiate gene activation (Kwok et al. 2019).

[0268] Antisense oligonucleotides (ASOs)

[0269] Antisense oligonucleotide (ASO) is a single-stranded DNA or RNA complementary to target sequence.ASO hybridizes with target nucleic acid.For example, ASO can be used to target intracellular coding RNA molecules or non-coding RNA molecules.After target binding, ASO / target complex can be enzymatically degraded, for example, by RNase H.

[0270] circular RNA

[0271] The present invention contemplates the use of circular RNA (circRNA) as a nucleic acid component. CircRNA is a type of single-stranded RNA that forms a continuous closed loop via a covalent bond formed between the 5' and 3' ends of the RNA molecule. The closed-loop structure of circRNA and the lack of a poly(A) tail are predicted to confer exonuclease resistance, thereby increasing circRNA stability. As a result, circRNAs have an increased half-life compared to equivalent non-circular RNAs. For example, circRNAs arising from protein-coding genes as alternatively spliced forms are more stable than the corresponding linear mRNA of the same protein-coding gene. Numerous functions have been attributed to circRNAs, including scaffolding for protein complexes, regulating parent genes, RNA-protein interactions, and microRNA sponges. Recently, circRNAs have been recognized as potentially useful for various therapeutic approaches. For example, circRNAs may be used as microRNA "sponges" to capture microRNAs. CircRNAs may also be used as sources of protein translation, which can persist in cells longer than standard linear mRNAs. CircRNAs may also be used to regulate protein activity by acting as aptamers.

[0272] modified nucleic acid

[0273] Modified nucleotide bases may be used in addition to the naturally occurring bases and may impart advantageous properties to nucleic acids containing them.

[0274] For example, modified bases may increase the stability of a nucleic acid molecule, thereby reducing the amount required. The provision of modified bases may also provide a nucleic acid molecule that is more or less stable than an unmodified nucleic acid.

[0275] 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 3' hydroxyl group and other than a 5' phosphate group. 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.

[0276] 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-2-thiouracil, -D-methyl These include mannosylqueosine, 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.

[0277] medical therapy

[0278] The present invention contemplates use in gene therapy regimens. The use of gene therapy regimens using either DNA or RNA, particularly mRNA, is contemplated for use in the present invention. The nucleic acid can be present in a composition that, when introduced into target cells, results in expression of a therapeutic gene product, e.g., a transgene. Target cells include muscle cells, spleen cells, liver cells, astrocytes, 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. In some embodiments, target cells include muscle cells, spleen cells, lung cells, cardiomyocytes, stem cells, monocytes, macrophages, dendritic cells, neutrophils, B cells, T cells, myeloid-derived suppressor cells, tumor-associated macrophages, tumor-associated neutrophils, or tumor cells.

[0279] To make gene therapy practical, it is desirable to use a DNA / RNA delivery system that (1) delivers therapeutic sequences to target cells, (2) mediates uptake of therapeutic nucleic acids into a proportion of the target cell population, and (3) is suitable for in vivo and / or ex vivo use for therapeutic applications.

[0280] The compositions of the present invention are particularly well suited to mediating the uptake of therapeutic nucleic acids into a relatively high proportion of a target cell population, as shown, for example, in Example 9 and FIGS.

[0281] The nucleic acid encoding the transgene can express the transgene in the target cell. The transgene can be a protein or a peptide. Additionally, or alternatively, the nucleic acid can modulate the expression or activity of an endogenous gene. Modulation can be an increase in expression of the gene and / or exogenous expression of additional copies of the gene, or modulation can be a decrease in gene expression.

[0282] The transgene may be a viral protein, a bacterial protein, or a protein of a microorganism parasitic to a mammal. A composition expressing a viral protein, a bacterial protein, or a 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 subject's skeletal muscle in order to stimulate an immune response to the viral, bacterial, or parasitic protein. Thus, the present invention provides methods of vaccinating a subject and compositions for use in vaccinating a subject. In such an example, the transgene may be expressed in lymph node tissue cells, such as immune cells described herein, e.g., leukocytes, e.g., B lymphocytes, T lymphocytes, monocytes, neutrophils, dendritic cells, macrophages, or monocytes.

[0283] The transgene can be an immune molecule, such as 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 immune complex, or a tumor antigen.

[0284] The transgene may express a therapeutic protein for use in gene therapy. The gene therapy may be for treating cancer and / or genetic disorders, such as autoimmune disorders, e.g., type I diabetes (also known as juvenile onset diabetes), in a patient. The transgene may be a functional form of a gene that is non-functional, down-regulated, inactive, or impaired in the subject.

[0285] The genetic disorder may be a monogenic disease in the patient, e.g., muscular dystrophy. In embodiments where the monogenic disease 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(s) into the circulation.

[0286] It is contemplated that the present invention may be used to deliver nucleic acid therapies to treat myopathies, and it is also contemplated that the present invention may be used to deliver nucleic acid therapies 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, hereditary muscular dystrophies, congenital muscular dystrophies, and distal muscular dystrophy.

[0287] The nucleic acid therapy may be for treating muscle-wasting conditions such as cachexia. The nucleic acid therapy may be for treating other muscle disorders, such as genetic muscle disorders such as myotonia congenita or familial periodic paralysis. The nucleic acid therapy may be for treating motor neuron diseases 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 syndrome (subacute necrotizing encephalomyopathies), mitochondrial DNA depletion syndrome, mitochondrial encephalomyopathies, lactic acidosis, and stroke-like episodes (MELAS), mitochondrial neurogastrointestinal encephalomyopathies (MNGIE), myoclonic epilepsy with ragged-red fibers (MERRF), neuropathy, ataxia, and retinitis pigmentosa (NARP), Pearson syndrome, or progressive external ophthalmoplegia (PEO). The nucleic acid therapy may be for treating a congenital myopathy, such as cap myopathy, centronuclear myopathy, congenital fiber inequality, core myopathy, central core disease, multiminicore disease, myosin storage myopathy, myotubular myopathy, or nemaline myopathy. The nucleic acid therapy may be for treating a distal myopathy, such as GNE myopathy / Nonaka myopathy / hereditary inclusion body myopathy (HIBM), Laing distal myopathy, Markesbery-Griggs late-onset distal myopathy, Miyoshi myopathy, Udd myopathy / tibial muscular dystrophy, VCP myopathy / IBMPFD, distal vocal cord and pharyngeal myopathy, or Welander distal myopathy. The nucleic acid therapy may be for treating an endocrine myopathy, such as hyperthyroid myopathy or hypothyroid myopathy. The nucleic acid therapy may be for treating an inflammatory myopathy, 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, debranching 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 myopathy or scapuloperoneal myopathy. The nucleic acid therapy may be for treating neuromuscular junction disorders, 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 disease, such as Charcot-Marie-Tooth disease (CMT) or giant axonal neuropathy (GAN).

[0288] The nucleic acid therapy may be for treating cardiovascular diseases such as thromboangiitis obliterans / Buerger's disease, diabetic peripheral neuropathy (ALS, also referred to as critical limb ischemia and foot ulcers), peripheral arterial disease, lower limb ischemia, critical limb ischemia (also known as comprehensive severe chronic lower limb ischemia and diabetic lower limb ischemia), severe peripheral arterial occlusive disease (PAOD), or intermittent claudication / arteriosclerosis. For example, the nucleic acid may encode one or more of the following transgenes: hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and / or fibroblast growth factor (FGF). In particular, the transgenes hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and / or fibroblast growth factor (FGF) may be useful for treating lower limb ischemia, such as diabetic lower limb ischemia, in a subject.

[0289] The nucleic acid therapy may be for treating an infectious disease 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 a cancer 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 an allergy 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).

[0290] Pompe disease is caused by a deficiency of human acid α-glucosidase (GAA), a lysosomal enzyme that cleaves terminal α1-4 and α1-6 glucose from glycogen. The compositions of the present invention may be used to treat Pompe disease. The compositions of the present invention, which contain a nucleic acid encoding GAA, may 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 the liver and skeletal muscle. The enzyme may be secreted from the tissue into the circulation.

[0291] Follistatin is an inhibitor of TGF-β superfamily ligands that suppress skeletal muscle growth and promote muscle wasting. The compositions of the present invention containing 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.

[0292] Accordingly, the present invention provides methods for treating such disorders and compositions for use in such treatment.

[0293] The nucleic acid-containing nanoparticles of the present invention can be stored and administered in a pharmaceutically acceptable sterile carrier. Various sterile solutions can be used for administering the composition, including water, PBS, TRIS buffer, HEPES buffer, ethanol, lipids, etc. The concentration of DNA / RNA is sufficient to provide a therapeutic dose, which will depend on the efficiency of intracellular delivery.

[0294] The actual delivery of the compositions of the invention to a patient can be carried out by a variety of techniques, including by direct injection, instillation of lung and other epithelial surfaces, or intravenous injection. Administration can be by needle, trocar, cannula, catheter, etc., as a bolus, multiple doses, or prolonged infusion, etc. It is also envisioned that the nucleic acid cargo may be delivered to patient cells or donor cells ex vivo prior to perfusion of the patient or donor cells into a subject.

[0295] Chimeric antigen receptor (CAR)

[0296] The present invention contemplates transfecting target cells with DNA / RNA encoding chimeric antigen receptors (CARs). Target cells for CAR transfection include, but are not limited to, T cells, including γδ-T cells, macrophages, and natural killer (NK) cells. Examples of CAR constructs contemplated by the present invention include anti-carcinoembryonic antigen (CEA) CARs, anti-CEA cell adhesion molecule 7 (CEACAM7) CARs, and anti-CEACAM5 CARs.

[0297] CARs are a class of recombinant proteins that typically contain an antigen-recognition domain, typically a single-chain variable fragment (scFv), a hinge region or ectodomain, a transmembrane domain, and an intracellular signaling / activation domain. The scFv domain is a chimeric peptide containing the variable light (VL) and variable heavy (VH) domains of an immunoglobulin linked to each other so that the scFv can interact with the target antigen. Other antigen-recognition domains, such as TNF receptors, innate immune receptors, cytokines, structural proteins, and growth factors, may be used instead of the scFv domain.

[0298] The hinge region, also known as the ectodomain or spacer, is located between the antigen-recognition domain and the transmembrane domain. Ideally, the hinge region will lack FcγR binding activity. The hinge may be derived from IgG; for example, the hinge may comprise CH2 and CH3 of IgG. Alternatively, the hinge region may be derived from CD28 or CD8α, which naturally lack FcγR binding activity.

[0299] The transmembrane domain is located between the hinge region and the intracellular signaling domain.Any suitable transmembrane domain can be used for CAR.Typically, the transmembrane domain is derived from CD3-ξ, CD4, CD8 or CD28.

[0300] The intracellular signaling domain of the CAR typically comprises the CD3-zeta cytoplasmic domain as the main intracellular signaling domain. In addition, the CAR will usually also comprise one or more costimulatory domains. The costimulatory domains may be derived from CD27, CD28, CD134, and CD137.

[0301] Binding of the antigen recognition domain of the CAR to its target antigen triggers clustering of the CAR, which results in the transmission of an activation signal to the intracellular T cell signaling domain, thereby activating an intracellular signaling pathway to stimulate a desired biological response.

[0302] The CAR platform was first described in T cell-based immunotherapy (CAR-T) and has shown success in treating a number of hematological cancers. More recently, the CAR platform has been expanded to other white blood cells, such as CAR-expressing NK (CAR-NK) and CAR-expressing γδ-T cells (CAR-γδT). The CAR platform has also been expanded to myeloid cells, including CAR-expressing macrophages (CAR-M), which are particularly useful for targeting and treating solid tumors.

[0303] CAR-macrophages, polarized macrophages

[0304] Cancer macrophages often adopt an anti-inflammatory or "alternatively activated" phenotype (referred to herein as the M2 phenotype). M2 macrophages mediate tissue repair and can secrete immunomodulatory cytokines such as IL-4, IL-10, IL-13, and TGF-β. Within the tumor microenvironment, chemotactic factors such as CCL2 are often secreted, which recruit monocytes, which then progress to differentiation into M2-like macrophages that secrete the immunomodulatory cytokines IL-4, IL-10, IL-13, and TGF-β. M2-like macrophages within the tumor microenvironment favor regulatory T cell function over effector T cell function, promoting angiogenesis and tumorigenesis. Therefore, enrichment of the tumor microenvironment with M2-like macrophages often correlates with poor prognosis (Sloas, C., et al., 2021). Therefore, it is an object of the present invention to reprogram M2 phenotype macrophages, which are enriched in the tumor microenvironment and have tumor-promoting properties, into M1 phenotype macrophages, which have anti-tumor properties.

[0305] Thus, the present invention contemplates transfecting M2 phenotype macrophages, which are often enriched within the solid tumor microenvironment and have tumor-promoting properties, in situ or ex vivo with one or more transgenes suitable for reprogramming M2 phenotype macrophages to M1 phenotype macrophages. Examples of suitable transgenes include, but are not limited to, interferon regulatory factor 5 (IRF5), activated IRF5, inhibitor of nuclear factor kappa B kinase subunit beta (IKK2), or CCAAT enhancer-binding protein alpha (CEBPA).

[0306] In a further example, the present invention contemplates co-transfecting M2 phenotype macrophages in situ or ex vivo with one or more transgenes and a CAR construct suitable for reprogramming M2 phenotype macrophages to M1 phenotype macrophages. Suitable transgenes for reprogramming M2 phenotype macrophages include, for example, interferon regulatory factor 5 (IRF5), activated IRF5, inhibitor of nuclear factor kappa B kinase subunit beta (IKK2), or CCAAT enhancer-binding protein alpha (CEBPA). Suitable CAR constructs include, for example, an anti-CEA CAR, an anti-CEACAM7 CAR, and an anti-CEACAM5 CAR. In some examples, M2 phenotype macrophages are transfected with an anti-CEA CAR and activated IRF5. In some examples, M2 phenotype macrophages are transfected with an anti-CEA CAR and activated IRF5. In some examples, M2 phenotype macrophages are transfected with an anti-CEACAM5 CAR and activated IRF5.

[0307] It is also contemplated that the nanoparticles of the present invention may be used to transfect M2 and / or M1 phenotype macrophages in vivo or ex vivo with nanoparticles of the present invention comprising a nucleic acid encoding a CAR. Exemplary CARs include anti-CEA CARs, anti-CEACAM7 CARs, and anti-CEACAM5 CARs. The macrophages may be derived from a patient or from a donor blood sample.

[0308] Gene editing

[0309] 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.

[0310] 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 functions to guide the Cas nuclease to a target site within the genome, and the tracrRNA functions 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-stranded guide RNA (sgRNA) that guides the Cas nuclease to a target site within the target gene. The sgRNA comprises a target-specific crRNA fused to the scaffold tracrRNA in a single nucleic acid.

[0311] 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, crRNA, and tracrRNA. In some embodiments, the nucleic acid comprises DNA or mRNA encoding a Cas protein or peptide and an sgRNA.

[0312] The CRISPR / Cas system can also be used to induce the repair or modification of a target gene. For example, the CRISPR / Cas system can include a nucleic acid template for promoting DNA repair or for introducing an exogenous nucleic acid sequence into a target gene, for example, by promoting homologous recombination repair. The CRISPR / Cas system can also be used to introduce targeted modifications into target genomic DNA, for example, using base editing techniques. This can be achieved using a Cas protein fused to a base editor, such as cytidine deaminase, as disclosed, for example, in WO2017070633A2 (incorporated by reference). In another example, the CRISPR / Cas system can be used to "rewrite" a nucleic acid sequence within a genome. For example, the CRISPR / Cas system can be a primed editing system. In such a primed editing system, a fusion protein can be used. For example, the fusion protein can include a catalytically impaired Cas domain (e.g., a "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 synthesis of edited genetic information.

[0313] In some embodiments, the CRISPR / Cas gene editing system may comprise a nucleic acid template that directs repair of a target gene of interest. In other embodiments, the Cas protein or peptide may comprise a base editor. In yet further embodiments, the CRISPR / Cas system may be a primed editing system.

[0314] CRISPR / Cas gene silencing and gene activation

[0315] CRISPR / Cas systems are suitable for use in gene silencing and activation. Such systems are contemplated for use with the present invention. For example, in some embodiments, a 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 dead. The fusion protein may be guided to a site of interest within the genome by either an sgRNA or crRNA. Upon binding of the fusion protein to the site of interest, the transcriptional repressor or activator may control expression of the gene of interest.

[0316] Nucleic acid-based vaccines

[0317] DNA vaccines, as defined by the World Health Organization (WHO), and RNA vaccines involve the direct introduction of plasmids containing DNA sequences or RNA encoding the antigen(s) into the appropriate tissue (of the vaccinated subject) where an immune response to the antigen(s) is desired and rely on in situ production of the target antigen. These approaches offer numerous potential advantages over conventional approaches, including stimulation of both B and T cell responses, improved vaccine stability, the absence of any infectious agents, and relative ease of large-scale production. As proof of principle for DNA vaccination, immune responses in animals have been obtained using genes derived from various 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 has also been obtained in animals. However, the value and benefits of DNA vaccines must be evaluated on a case-by-case basis, and their applicability will depend on the nature of the agent being immunized against, the nature of the antigen, and the type of immune response required for protection.

[0318] The field of DNA and RNA vaccination is rapidly evolving. Vaccines currently in development not only use DNA but also include adjuvants that may assist the DNA in entering cells, target the DNA to specific cells, or act as adjuvants in stimulating or inducing an immune response. As of 2020, the WHO noted that the first nucleic acid vaccines licensed for sale likely used plasmid DNA derived from bacterial cells, but that in the future, others may use RNA or complexes of nucleic acid molecules with other entities. However, with the outbreak of the COVID-19 pandemic in 2020, a concerted effort was made to commercialize the first RNA-based COVID-19 vaccines, which were approved for use in mid- to late 2020. Since their approval, these RNA-based vaccines have been successfully deployed worldwide to immunize populations against COVID-19.

[0319] Intramuscular delivery of DNA vaccines, as with other vaccine technologies, is a common approach (Lim et al., 2020). The low replication rate of myocytes (muscle cells) in skeletal muscle makes them an attractive target for DNA vaccination, as stable expression does not depend on genomic integration.

[0320] Commercially available RNA vaccines currently use antigen-encoding mRNA as a 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 features of mRNA and may include a 5' cap, a 3' polyA tail, and 5' and 3' untranslated regions (UTRs). In addition to encoding the antigen of interest, the self-amplifying RNA will also contain 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. Upon translation of the RDRA by the subject's translational machinery, the RDRA can bind to the self-amplifying RNA and replicate the RNA. The inclusion of a system for self-amplification reduces the minimum RNA required for the vaccine, thereby reducing the likelihood of a subject experiencing side effects.

[0321] Combination therapy

[0322] The compounds of the present invention or compounds identified by the methods of the present invention may be used to treat tumors and cancer in subjects in need thereof. The compounds may be administered alone or in combination with other anti-cancer agents.

[0323] "Anticancer agent" refers to any agent useful in treating a neoplastic condition. One class of anticancer 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.

[0324] Cytotoxic chemotherapeutic agents cause cell death through mechanisms or means that are not receptor-mediated. They cause cell death by interfering with cell division, metabolism, or functions necessary for cell survival. Because of this mechanism of action, rapidly proliferating (meaning growth or division) or metabolically active cells are preferentially killed over non-proliferating cells. The mitotic or energy-utilizing state of different cells in the body (which is the metabolic activity that supports cell function) determines the dose of chemotherapeutic agent that will cause cell death. Cytotoxic chemotherapeutic agents include, but are not limited 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 irradiation, cytarabine, etoposide, melphalan, pentostatin, and radiation. Ibrutinib (a BTK inhibitor) is another anti-cancer drug that can be used in combination with the medical use of the present invention. BTK inhibitors enhance TAM repolarization to the M1 phenotype. This combination therapy may be particularly useful for treating solid tumors, particularly "cold" tumors such as PDAC.

[0325] Anticancer drugs also include protein kinase inhibitors, which can be used to treat various cancers, including blood cancers and lung cancers.Protein kinases typically promote cell proliferation, survival and migration, and are often constitutively overexpressed or active in cancers.Therefore, protein kinase inhibitors are common drug targets in cancer treatment.Examples of kinase inhibitors for clinical use include crizotinib, ceritinib, aleitinib, brigatinib, bosutinib, dasatinib, imatinib, nilotinib, ponatinib, vemurafenib, dabrafenib, ibrutinib, palbociclib, sorafenib, and ribociclib.

[0326] Anti-cancer drugs also include agents used in immunotherapy, including antibodies. Immunotherapy can induce, amplify, reduce, or suppress immune responses depending on the specific disease condition. For example, tumor cells expressing PDL1 ligands suppress the subject's normal immune response 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 clinically 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.

[0327] Pharmaceutical Composition

[0328] The nanoparticles of the present invention may be formulated as pharmaceutical compositions or formulations. The pharmaceutical compositions provided herein may contain one or more pharmaceutically acceptable excipients or carriers, such as solvents, solubility enhancers, suspending agents, buffers, isotonicity agents, antioxidants, antimicrobial preservatives, diluents, binders, lubricants, and disintegrants. "Pharmaceutically acceptable" refers to molecular entities and compositions that are "generally regarded as safe," e.g., physiologically tolerated and typically do not cause allergic or similar adverse reactions, such as gastric irritation, when administered to humans. In some embodiments, the term refers to molecular entities and compositions that have been approved by U.S. federal or state regulatory authorities as GRAS listed under Sections 204(s) and 409 of the Federal Food, Drug, and Cosmetic Act, and are subject to premarket review and approval by the FDA or a similar listing, the U.S. Pharmacopeia, or another generally recognized pharmacopeia, for use in animals, and more specifically, for use in humans.

[0329] When used, the excipients of the composition will not adversely affect the stability, bioavailability, safety, and / or efficacy of the active ingredient. Therefore, those skilled in the art will understand that a composition that is not incompatible with any of the formulation ingredients is provided. The excipients may be selected from the group consisting of buffers, tonicity agents, chelating agents, antioxidants, antibacterial agents, and preservatives.

[0330] The nucleic acid-containing compositions of the present invention can be stored and administered in a pharmaceutically acceptable sterile carrier. Various sterile solutions can be used for administering the compositions, including water, PBS, ethanol, lipids, etc. The concentration of DNA / RNA is sufficient to provide a therapeutic dose, which will depend on the efficiency of intracellular delivery.

[0331] Therapeutic product development

[0332] The nucleic acid delivered by the composition of the present invention may exhibit a therapeutic effect (e.g., by directly acting to down- or up-regulate a target gene), or it may express a gene product (which may be a therapeutic protein or therapeutic nucleic acid) via an expression cassette comprising 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 regulatory nucleotide sequence are covalently linked in such a way that expression of the coding sequence is under the influence or control of the regulatory sequence. Thus, a regulatory sequence is operably linked to a selected nucleotide sequence if the regulatory sequence is capable of effecting 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.

[0333] Administration route

[0334] Compositions according to aspects of the present invention may be formulated for administration by a number of routes, including, but not limited to, intravenous, parenteral, intraarterial, intramuscular, intratumoral, subcutaneous, oral, and intranasal.

[0335] The actual delivery of the compositions of the invention to a subject (human or animal) can be accomplished by a variety of techniques, including direct injection, inhalation, instillation of lungs and other epithelial surfaces, or by intravenous, parenteral, intraarterial, intramuscular, intratumoral, or subcutaneous injection. Administration can be by needle, trocar, cannula, catheter, etc., as a bolus, multiple doses, or prolonged infusion, etc.

[0336] subject

[0337] The subject to be treated may be any animal or human. The subject is preferably a mammal, more preferably a human. The subject may also be a non-human mammal, but is more preferably a human. The subject may be male or female. The subject may be a patient. Therapeutic use may be performed on humans or animals (veterinary use).

[0338] The features disclosed in the foregoing description, or the following claims, or the accompanying drawings, and presented in a particular form or in terms of means for performing a disclosed function, or a method or process for obtaining a disclosed result, may be used separately or in any combination of such features as appropriate to realize the invention in various forms thereof.

[0339] While the present invention has been described in conjunction with the above exemplary embodiments, many equivalent modifications and variations will become apparent to those skilled in the art given this disclosure. Accordingly, the above exemplary embodiments of the invention are considered to be illustrative and not limiting. Various changes may be made to the described embodiments without departing from the spirit and scope of the invention.

[0340] For the avoidance of doubt, the 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.

[0341] Any section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0342] Throughout this specification, including the claims that follow, unless the context clearly indicates otherwise, the words "comprise" and "include" and variations thereof (such as "comprises," "comprising," and "including") are understood to mean the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of other integers or steps or groups of integers or steps.

[0343] 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 connection with numerical values is arbitrary and means, for example, + / - 10%. [Example]

[0344] Example 1 - Methods and Materials for In Vitro Transfection of Cells and Tissues

[0345] The in vitro macrophage transfection efficiency of eGFP mRNA was assessed using certain dendrimers from the table below and can be described in the Examples below. Tables 1, 1A and 1B below present dendrimers and peptides that may be used in various aspects and embodiments of the present invention.

[0346] [Table 2] TIFF2025525843000004.tif178151TIFF2025525843000005.tif215157

[0347] [Table 3]

[0348] [Table 4]

[0349] method

[0350] Murine BMDM (bone marrow-derived monocyte) polarization

[0351] BMDM cells were isolated from mouse bone marrow cell suspensions using the StemCell Tech EasySep Mouse Monocyte Isolation Kit. Cells were resuspended in complete medium (DMEM + 1% glutamax + 10% heat-inactivated FBS + 1% Pen / Strep) and concentrated to 1E+6 cells / ml with 50 ng / ml mouse M-CSF (1:1000 of a 50 μg / ml stock solution). Two million cells (i.e., 2 mL) were seeded into each well of a 6-well plate(s). Plates were incubated at 37°C for 4 days. A half-medium exchange was performed using complete medium enriched with 50 μg / ml mouse M-CSF and 40 ng / ml mouse IL-4. The following day (day 5), cells were washed with PBS and incubated with 5 mM EDTA (diluted in PBS) for 10 minutes at 37°C, followed by tapping the plate to resuspend the cells. The cells were resuspended in complete medium containing 50 μg / ml of mouse M-CSF and 20 ng / ml of mouse IL-4 (activation medium) and seeded into each well of a 24-well plate. The cells were stained with F4 / 80 and CD11b to confirm their macrophage phenotype.

[0352] Mouse BMDM transfection

[0353] The medium covering the polarized BMDM cells was aspirated and replaced with 480 μl / well of activation medium. 120 μl / well of each mRNA formulation was added to the cells. Cells were transfected at 37°C for 24 hours.

[0354] J774.2 subculture and macrophage polarization

[0355] J774.2 cells were subcultured in DMEM supplemented with 2 mM glutamax and 10% heat-inactivated FBS (complete medium). Twice weekly (approximately 70-80% confluent, every 3-4 days), cells were collected by scraping into Falcon tubes and pelleted at 400 g for 5 minutes. Cells were resuspended in complete medium, counted, and seeded into 10 cm dishes at a density of 1.7-2.5E+4 cells / cm². For M2 polarization, cells were treated for 48 hours with complete medium supplemented with 20 ng / ml recombinant mouse IL-4 and 20 ng / ml recombinant mouse IL-13 (activation medium). After 24 hours in IL-4- and IL-13-containing medium, cells were harvested by scraping as described above. Cells were seeded in 24-well plates at a density of 1.2E+5 cells / well in 0.5 ml / well of complete medium containing 20 ng / ml of IL-4 and IL-13.

[0356] Transfection

[0357] The medium covering the polarized J774.2 cells was aspirated and replaced with 480 μl / well of activation medium. 120 μl / well of each test condition (total volume was 600 μl / well) was added (test conditions were prepared with 25 mM HEPES solution). Control conditions were prepared consisting of 25 mM pure HEPES and an mRNA control. The plate was shaken back and forth between additions of each test condition to ensure mixing. The plate was placed in the incubator overnight.

[0358] Human THP-1 subculture and macrophage polarization

[0359] THP-1 cells (a human leukemia-monocyte cell line) were subcultured at 3E+5 cells / ml in RPMI (containing glutamax) + 10% inactivated FBS (complete medium) and passaged every 3–4 days. For macrophage polarization, 7.5E+6 cells were seeded in a T75 flask in a total of 20 ml of complete medium containing 5 ng / ml PMA (1:1000 of a 5 μg / ml stock solution). The cells were transferred to a 37°C incubator overnight. After 24 h, THP-1 cells were washed with complete medium, and fresh flask complete medium (without PMA) was overlaid on top of the cells. After an additional 72 h, the complete medium was replaced with fresh complete medium containing 20 ng / ml rhIL-4 (1:500 of a 10 μg / ml stock solution). After 24 h, the cells were harvested by first washing with PBS and then treating with 5 mM EDTA (diluted in PBS) for 5 min at 37°C. The cells were spun down at 300g for 5 minutes and resuspended at 2.4E+5 cells / ml in complete medium containing 20ng / ml rhIL-4. 500ul of cells were seeded into each well of a 24-well plate and placed in a 37°C incubator overnight.

[0360] THP-1 transfection

[0361] The medium covering the polarized THP-1 cells was aspirated and replaced with 480 μl / well of complete medium containing 20 ng / ml rhIL-4. 120 μl / well of each mRNA formulation was added (formulations were prepared in 25 mM HEPES solution). Control conditions consisted of 25 mM pure HEPES and an mRNA control. Cells were transfected for 24 hours at 37°C.

[0362] Staining and fixation of polarized macrophages

[0363] Staining and fixation of both human and mouse macrophages were performed as follows. After 24 hours, the medium covering each test condition was transferred to the corresponding 1.5 ml Eppendorf tube (to preserve cells that may have died during transfection). The cells were then washed with 300 μl / well of PBS and transferred to the corresponding tube. 500 μl / well of 5 mM EDTA was added to each well, and the plate(s) were transferred to a 37°C incubator for 5-10 minutes until the cells began to detach. The cells were then transferred from the plate to the corresponding 1.5 ml tube. The cells were pelleted at 300 g for 5 minutes and resuspended in 100 μl / sample of fixable Live / Dead Aqua stain. For compensation, ArC beads were stained with 3 μl of pure Live / Dead Aqua. The cells were stained on ice for 30 minutes. The tubes were then spun down at 300 g for 5 minutes. The supernatant was discarded, and the cells were resuspended in 500 μl / sample of PBS. Negative ArC beads were added as compensation controls. GFP compensation beads were dispensed into a separate tube as a GFP compensation control. Samples were spun down at 300g for 5 minutes. Samples were then resuspended in 100ul / sample of Reagent A from the Invitrogen Fix / Perm kit and left at room temperature for 15 minutes. Samples were spun down at 400g for 5 minutes, and the supernatant was discarded. Samples were washed once more with PBS and spun down at 300g for 5 minutes. Finally, cells were resuspended in 400ul / sample of FACS buffer (PBS + 2mM EDTA + 0.5% w / v BSA) for flow analysis. Samples were stored in the refrigerator before flow analysis, which was performed within 24 hours of fixation.

[0364] Surface marker staining of BMDM and J774.2 cells (after live / dead staining and before fixation)

[0365] Cells were resuspended in 50 μl / sample of 2x FcX block diluted in FACS buffer (1:50, final concentration 1:100) and incubated in the refrigerator for 5 minutes. 50 μl / sample of 2x surface antigen-antibody mix (final concentrations: F4 / 80-PE 1:100, CD206-BV605 1:25, CD86-PECy7 1:25, and CD11b 1:200) was added on top of the FACS buffer (equal volume to the block). For each antibody used, one drop of UltraComp beads and 3 μl of each antibody were added on top (antibody compensation controls). Cells were incubated in the refrigerator for 20 minutes and spun down at 400 g for 5 minutes. After a PBS wash, cells were fixed and proceeded to subsequent steps as detailed in the section above.

[0366] C2c12 muscle cell culture and transfection

[0367] Cell lines, transfection reagents, and mRNA. C2c12 cells were maintained in DMEM medium containing 10% (v / v) FCS and 1% (v / v) L-glutamine in a humidified atmosphere of 5% CO2 and 37°C. Alexa Fluor 488-tagged mRNA expressing eGFP was purchased from RiboPro. mRNA expressing either eGFP or firefly luciferase was purchased from Trilink. DOTMA:DOPE, 1:1 (w / w), was obtained from Invitrogen or Encapsula NanoSciences LLC.

[0368] Transfection procedure. 24 h prior to transfection, C2c12 cells were seeded into 96-well plates to reach 70% confluence. mRNA transfection complexes were formed by mixing mRNA with dendrimer in 25 mM HEPES buffer, followed by DOTMA:DOPE in 25 mM HEPES buffer at 25 °C. The transfection complexes were then layered onto cells in complete growth medium. Cells were harvested 4 h post-transfection for FACS analysis or 24 h post-transfection for reporter gene assays.

[0369] Cell harvesting and staining.

[0370] The cell culture medium and the PBS used to wash each well were collected from each treatment, followed by the addition of 5 μM EDTA in PBS. After a 10-minute incubation at 37°C, cells were gently detached from the plate and pelleted at 400 g for 8 minutes at 4°C. Cells were stained using LIVE / DEAD fixable aqua fluorescent reactive dye (Invitrogen) according to the manufacturer's instructions. For compensation, ArC beads were stained with 3 μl of pure Live / Dead Aqua. After staining, cells were pelleted at 400 g for 8 minutes at 4°C and resuspended in a 500 μl PBS wash per sample before repeating the centrifugation. Negative ArC beads were added as compensation controls. GFP compensation beads were aliquoted into a separate tube as GFP compensation controls. All samples containing beads were fixed with 4% paraformaldehyde in PBS for 15 minutes at room temperature. Samples were then spun down again at 400g for 8 minutes at 4°C and resuspended in FACS buffer (PBS + 2mM EDTA + 0.5% w / v BSA). Samples were stored at 4°C and flow analysis was performed within 24 hours of fixation.

[0371] Flow cytometry.

[0372] Data were collected using a BD LSRFortessa I analyzer running FACSDIVA software (Beckton Dickinson). All collected data were analyzed using FlowJo 10.0 software.

[0373] Nanoparticle Formulation Procedure

[0374] Tube A: The peptide dendrimer stock solution was added to a 0.5 ml sterile polypropylene tube containing 100 mM HEPES buffer (1.00 μl) and sterile water (to obtain a final volume of 4.0 μl). The tube was gently shaken and then spun down using a minicentrifuge for 10-15 seconds to ensure that all the liquid was at the bottom of the tube. The concentration and volume of the dendrimer stock solution and water were varied depending on the molecular weight and charge of the desired dendrimer, as well as the N / P ratio. The final concentration of HEPES buffer in Tube A was 25 mM.

[0375] Tube B (800.0 μg / ml mRNA in 25 mM HEPES buffer) (excess): 42.00 μl of 1 mg / ml mRNA stock solution was added to a 0.5 ml sterile polypropylene tube containing 3.94 μl of sterile water and 6.56 μl of 200 mM HEPES buffer. The tube was gently shaken to mix, then spun down for 10-15 seconds using a minicentrifuge to ensure all the liquid was at the bottom of the tube.

[0376] Tube C (3076.9 μg / ml DOTMA / DOPE liposomes in 25 mM HEPES buffer) (excess): 11.88 μl of 25 mM (17.68 mg / ml) DOTMA / DOPE liposome solution was added to 47.84 μl of sterile water and 8.53 μl of 200 mM HEPES buffer in a 0.5 ml sterile polypropylene falcon tube. The tube was gently shaken to mix, then spun down for 3-5 seconds in a minicentrifuge to ensure all the liquid was at the bottom of the tube.

[0377] Mixing: 800 μg / ml mRNA solution (10 μl, 8.0 μg, 2.42 × 10-5 mmol phosphate) was mixed in Tube A by rapidly pipetting up and down 10-15 times. This was allowed to stand for 2-5 minutes. 3076.9 μg / ml DOTMA / DOPE liposome solution (26.0 μl, 80.0 μg) was mixed in Tube A by rapidly pipetting up and down 10-15 times.

[0378] Formulation Example 1: 40 μl formulation of (RHL) 4 (KRHL) 2 KGSC-NH 2 , N / P ratio 0.6 (4912 g / mol, 10 charges per dendrimer).

[0379] Tube A (dendrimer): To a 0.5 ml sterile polypropylene tube was added 5 mg / ml dendrimer stock solution (1.430 μl, 7.14 μg, 1.45 × 10 mmol dendrimer, 1.45 × 10 mmol N), sterile water (1.570 μl), and 100 mM HEPES buffer (1.00 μl).

[0380] Formulation Example 2: 30 ul formulation of (RHL)4(KRHL)2KGSC-NH2, N / P ratio 8 (4912 g / mol, 10 charges per dendrimer).

[0381] Tube A (dendrimer): To a 0.5 ml sterile polypropylene tube was added 50 mg / ml dendrimer stock solution (1.430 μl, 71.4 μg, 1.45 × 10 mmol dendrimer, 1.45 × 10 mmol N), sterile water (0.821 μl), and 100 mM HEPES buffer (0.75 μl).

[0382] Various liposome / mRNA ratios

[0383] To change the mass ratio of liposomes to mRNA in the formulation, the concentration of liposomes in tube C must be changed so that the same volume of liposome solution is still added (i.e., 26 µl for formulations with a final volume of 40 µl).

[0384] Application of mannose-dendrimer coating

[0385] The amount of coating applied is defined as the mass ratio of coated dendrimer to mRNA in the sample. The mass ratio of applied mRNA:coating is typically 1:0.5, 1:1, or 1:3.

[0386] Twelve microliters of nanoparticle formulation was dispensed into a 0.5 ml sterile polypropylene tube. 4 μl of a 0.3 mg / ml, 0.6 mg / ml, or 1.8 mg / ml solution of (Ac-EEEE)2KGSGGSGGSC[(SS)-α-D-thiomannose] (for mRNA:coating mass ratios of 1:0.5, 1:1, or 1:3, respectively) was added and mixed by rapidly pipetting up and down 10–15 times and allowed to incubate for 5 min.

[0387] In vivo delivery of peptide dendrimers / nanoparticles

[0388] All animal procedures were performed in accordance with the Animal Research Reporting of In Vivo Experiments and were approved by the UK Home Office for conducting regulatory procedures with permission. For the data underlying Figure 11, 4-week-old Balb / c mice were purchased from Jackson's Laboratory and allowed to acclimate to their new housing environment for 1 week upon arrival. All mice were weighed and warmed to 37°C in a heating chamber for 15 minutes. Formulations were delivered intravenously (5 mL / kg) via the tail vein using a 30G insulin syringe (BD biosciences).

[0389] Bioluminescence Imaging (BLI)

[0390] 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 (GoldBio) at a dose of 10 μL / g. Mice were anesthetized with 2.5% isoflurane in a chamber and then placed on a heated imaging platform while maintaining 2.5% isoflurane. For the data underlying Figure 11, mice were imaged 10 minutes after D-luciferin administration with an exposure time set at 45 seconds to ensure the acquired signal was within the effective detection range (medium binning). Bioluminescence signals were quantified by measuring photon flux (photons / second) 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. Each tissue was then placed on a dish containing D-luciferin in PBS during collection. The organ 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 tissue was placed in a storage vial and flash-frozen in liquid nitrogen. Blood was collected in an EDTA-K2 tube and spun at 10,000 rpm for 15 minutes. Plasma was transferred to a new tube and frozen.

[0391] Example 2 - Mouse and human macrophage transfection using peptide dendrimers

[0392] Mouse macrophage transfection

[0393] The transfection efficiency of macrophage-polarized primary bone marrow-derived monocytes using various peptide dendrimer / lipid nanoparticles was evaluated and compared to lipid-only nanoparticles (LPX-RNA). Each nanoparticle, with or without dendrimers, contained eGFP mRNA, allowing analysis of the absolute number of transfected cells to be assessed by flow cytometry. As can be seen in Figure 2, the transfection efficiency of all peptide dendrimer / lipid-based nucleic acid delivery systems tested was superior to that of either naked mRNA or mRNA contained in lipid-only nanoparticles (DOTMA / DOPE lipid-based nanoparticles). While lipid-based nanoparticles were only able to achieve approximately 10-15% transfection efficiency, GSC G1,2-RHL (NP=0.6:1), GSC G1,2-RL, 3-LR (NP=0.16:1) or GSC The transfection efficiencies of G1,RL, and 2-LR (NP=8:1) were approximately 35%, 40%, and 50%, respectively (FIG. 2).

[0394] Human macrophage transfection

[0395] Next, to test whether peptide dendrimer / lipid nanoparticles were useful for transfecting human cells in general, and human macrophages in particular, a panel of peptide dendrimer / lipid eGFP mRNA-containing nanoparticles was tested for transfection efficiency in human THP-1 cells. As seen with primary mouse macrophage cells, each of the dendrimers tested was able to transfect THP-1 cells with high efficiency, in each case exceeding the efficiency demonstrated by lipid-only nanoparticles (Figure 3). GSC G1,2-RL, 3-LR (NP=0.16:1), GSC G1-LRLR (NP = 0.6); GSC G1,2-RHL (NP=5:1) and GSCG1,2-RF and G1,2-RHL nanoparticles (NP = 0.6:1) each achieve transfection efficiencies of 50% to 60%, whereas using lipid-only nanoparticles, only approximately 20% of cells express eGFP after transfection. Interestingly, G1,2-RHL nanoparticles with a lower NP ratio (NP = 0.6:1 compared to NP = 5:1) can further increase the transfection efficiency of these nanoparticles by approximately 10%. RHC G1-R (NP = 0.6:1) also outperforms lipid-only nanoparticles, achieving a transfection efficiency of approximately 30–35%.

[0396] Considering the primary macrophage mouse transfection experiments and human macrophage transfection experiments, it is clear that peptide dendrimer / lipid nanoparticles provide an improved mechanism for achieving transfection of macrophages.

[0397] Example 3 - Mannose-conjugated peptide dendrimers increase transfection efficiency

[0398] M2 phenotype macrophages are highly enriched in many solid tumors and are known to be anti-inflammatory, promote regulatory T cell function, and promote angiogenesis and tumorigenesis. Patients with high intratumoral M2 phenotype macrophage content typically have a poor prognosis.

[0399] With the above in mind, we next investigated whether macrophage transfection efficiency could be further improved by targeting peptide dendrimer / lipid nanoparticles to bind to specific receptors on macrophages. Macrophages, particularly M2 phenotype macrophages, are known to have abundant surface expression of the mannose-liganded receptor CD206. Therefore, we coated various peptide dendrimer / lipid nanoparticles with a second peptide dendrimer conjugated to mannose sugars to investigate whether this could improve delivery of mRNA cargo to macrophages.

[0400] GSC The performance of G1,2-RHL / lipid nanoparticles (NP = 8:1, lipid:mRNA w / w ratio 2.5:1) was poor, with very low levels of macrophage transfection of approximately 2-3% (Figure 4). This poor performance can be attributed to the lipid:mRNA w / w ratio of 2.5:1 in this formulation, which is generally GSC G1,2-RHL / lipid nanoparticles transfect macrophages with high efficiency when the lipid:mRNA ratio is increased (see, for example, Figures 2, 3, and 5-7). However, even when using suboptimal lipid:mRNA ratios, coating nanoparticles with (Ac-EEEE)2KGSGGSGGSC[(SS)-α-D-thiomannose] at a 1:1 w / w ratio (1 eq) of coating:mRNA can achieve an increase in transfection efficiency of approximately 2-3% to approximately 25% (Figure 4). Therefore, even "poorly performing" peptide dendrimer / lipid nanoparticles may demonstrate improved mRNA delivery to macrophages by coating the nanoparticles with mannose sugars.

[0401] Further optimized GSC Using the G1,2-RHL / lipid nanoparticle composition (NP = 8:1, lipid:mRNA w / w ratio of 5:1), GSC G1,2-RHL-containing nanoparticles can transfect macrophages with higher efficiency than lipid-only nanoparticles (Figure 5, approximately 60% vs. approximately 10%). Transfection efficiency can be further improved by applying the same dendrimer-mannose coating to the nanoparticles at a 0.5:1 w / w ratio (0.5 eq) of coating:mRNA. Using this dendrimer-mannose coating, transfection efficiency further increases from approximately 60% to approximately 75% (Figure 5). This further demonstrates that coating dendrimer / lipid nanoparticles with ligands for receptors expressed on target cells can increase transfection efficiency.

[0402] Next, when the w / w ratio of DOTMA / DOPE:mRNA was further increased from 5:1 (Figure 5) to 10:1 (Figure 6), GSC We investigated whether the transfection efficiency of G1,2-RHL / lipid nanoparticles could be further improved. Using a 10:1 DOTMA / DOPE:mRNA w / w ratio, we achieved a transfection rate of approximately 60%, as seen when using a 5:1 DOTMA / DOPE:mRNA w / w ratio (Figure 5). GSC It does not appear to further increase the transfection efficiency of nanoparticles containing G1,2-RHL (Figure 6). Importantly, however, we note that increasing the lipid:mRNA ratio does not adversely affect transfection efficiency, further demonstrating the versatility of the peptide dendrimer / lipid nanoparticle nucleic acid delivery system.

[0403] Again, we observed that when the dendrimer-mannose coating was applied at a 1:1 w / w ratio (1 eq) of coating:mRNA, the transfection efficiency was again increased by approximately 10% compared to uncoated nanoparticles ( Figure 6 ).

[0404] Next, the effect of NP ratio on transfection efficiency was investigated. NP with lipid:mRNA w / w ratios of 0.6:1 and 10:1 was used. GSC Using nanoparticles containing G1,2-RHL, transfection efficiencies of greater than 70% can be achieved (Figure 7). This data again further demonstrates the versatility of these delivery systems, providing a wide working range of NP ratios and lipid:mRNA w / w ratios.

[0405] Similar to the other tested nanoparticle formulations, nanoparticles were coated with mannose-conjugated dendrimers, in this case at a 3:1 w / w ratio (3 eq) of coating:mRNA, with a lipid:mRNA w / w ratio of NP = 0.6:1 and 10:1. GSC It is possible to further increase the transfection efficiency of G1,2-RHL / lipid nanoparticles (Figure 7).

[0406] To sum up, GSC The experiments performed above with G1,2-RHL demonstrate that efficient transfection of macrophages can be achieved using these dendrimer / lipid nanoparticles, and that improved transfection can be achieved by optimizing the NP ratio (dendrimer:mRNA) and / or w / w ratio (lipid:mRNA). Furthermore, the increased transfection efficiency observed when the same nanoparticles were coated with mannose-dendrimer suggests that targeted delivery of nanoparticles to CD206-expressing macrophages may be achieved.

[0407] To further explore the general applicability of the mannose coating technique to improve delivery of nucleic acid cargo to macrophages, we performed mannose-coated macrophages with and without the coating. GSC A comparison of nanoparticles containing G1,2-RL and 3-LR / lipid was performed. As can be seen in Figure 8, GSC G1,2-RL,3-LR / lipid shows moderate transfection efficiency (approximately 20%) of macrophages in vitro. Transfection efficiency can be increased by approximately 30% to approximately 50% by including coating the nanoparticles with (Ac-EEEE)2KGSGGSGGSC[(SS)-α-D-thiomannose] at a 3:1 w / w ratio (3 eq) of coating:mRNA. These data support the use of mannose-conjugated dendrimers to improve the transfection efficiency of macrophages using peptide dendrimer / lipid nanoparticles.

[0408] Example 4 - Peptide motifs can be used to increase muscle cell transfection

[0409] As shown in Example 3, peptide dendrimer / lipid nanoparticles can be targeted to macrophages by including a targeting motif in the nanoparticle (e.g., a mannose-conjugated dendrimer). To further explore the ability of these new nucleic acid delivery systems to target specific cell types, we next generated various peptide dendrimers containing the muscle cell targeting peptide motif (ASSLNIA (SEQ ID NO: 1)) to determine whether nucleic acid delivery and translation could be increased within muscle cells.

[0410] Peptide dendrimers containing the ASSLNIA (SEQ ID NO: 1) muscle-targeting peptide GSC Two variants (NTX2 and NTX3) of G1,2-RL,3-LR((LR)8(KRL)4(KRL)2KGSC-NH2) were generated. GSC Muscle cell transfection with G1,2-RL,3-LR NTX2 ((LR)8(KRL)4(KRL)2KGSCGAASSLNIA(Acp)-NH2) or NTX3 ((LR)8(KRL)4(KRL)2KGSCHHHHHHGAASSLNIA(Acp)-NH2) was performed using eGFP mRNA, and transfection efficiency was assayed using either flow cytometry or transgene expression assays. Transfection with eGFP mRNA allows for determination of mRNA internalization and levels of translated protein from transfected mRNA. We expect that nanocarriers with a cell targeting domain (i.e., a muscle targeting domain in this case) will result in higher transfection efficacy compared to nanocarriers without a targeting domain.

[0411] Both NTX2 and NTX3 GSCCompared with G1,2-RL and G3-LR, we transfected myocytes with eGFP mRNA with higher efficiency. Transfection using NTX2 nanoparticles increased eGFP expression by twofold, while transfection using NTX3 nanoparticles increased expression by threefold (Figure 9). NTX3 contains six additional histidines in its core sequence, which may act as a proton sponge to facilitate endosomal escape. This may explain why NTX3 mediates higher transfection efficacy than NTX2.

[0412] RHC Similar results were obtained when G1-RL, 2-LR ((LR)4(KRL)2KRHC-NH2) dendrimer / lipid nanoparticles were compared with a similar peptide dendrimer containing the ASSLNIA (SEQ ID NO: 1) peptide motif (NTX5,(LR)4(KRL)2KRHCGAASSLNIA-(Acp)-NH2). As seen in Figure 10, the inclusion of the ASSLNIA muscle-targeting motif increased mRNA delivery to muscle cells (Figure 10) and also increased eGFP expression by approximately two-fold. Taken together, Figures 9 and 10 suggest that the inclusion of the muscle-targeting motif ASSLNIA (SEQ ID NO: 1) within the peptide dendrimer increased mRNA delivery to muscle cells and transgene expression within muscle cells.

[0413] Example 5 - Peptide dendrimers can enhance tissue-specific mRNA expression

[0414] We wished to investigate whether the inclusion of peptide dendrimers in lipid-based nanoparticles could improve tissue-specific nucleic acid cargo delivery. Using a DOTMA / DOPE lipid delivery system (w / w = 23:1 relative to mRNA), luciferase mRNA was efficiently delivered to the lung and spleen, as shown by the bioluminescence in Figure 11. Delivery of mRNA cargo to the lung and spleen was GSC G1-LRLR (NP=0.6:1) or GSC Further improvement can be achieved by including either G1,2-RHL (NP=0.6:1).

[0415] The above examples highlight that peptide dendrimer / lipid nanoparticles are highly versatile tools for delivering nucleic acid cargo to a variety of target cells and tissues, including bone marrow cells such as macrophages, particularly M2 phenotype macrophages, muscle cells, lung, and spleen.

[0416] Example 6 - In vivo tissue distribution

[0417] The present inventors investigated the tissue distribution of nucleic acid expression achieved by the nanoparticles of the present invention.

[0418] All animal procedures in our study were performed in compliance with UK law and included ethical approval. For the data underlying Figure 12, 6-8 week old female BALB / C and CD-1 mice were acclimated for 1 week upon their arrival. All mice were weighed and formulations were delivered intravenously (100 μl) via the tail vein using a 30G insulin syringe (BD biosciences).

[0419] Bioluminescence Imaging (BLI)

[0420] 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. Six minutes after D-luciferin administration, mice were anesthetized with 5% isoflurane in a chamber and then placed on a heated imaging platform while maintaining 2.5% isoflurane. For the data underlying Figure 12, mice were imaged 10 minutes after D-luciferin administration, using an exposure time set to ensure that the acquired signal was within the effective detection range (open filter, binning 8, aperture 1). Bioluminescence signals were quantified by measuring photon flux (photons / second) within 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.

[0421] Each tissue was placed in 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. The results are presented in Figure 12.

[0422] Example 7 - Nanoparticle size, PDI and surface charge characterization

[0423] Dynamic Light Scattering

[0424] Hydrodynamic size was measured using the dynamic light scattering (DLS) technique. DLS is a highly sensitive, non-invasive method for measuring the size and size distribution of nanoparticles in liquids. The Brownian motion of nanoparticles in suspension results in laser light being scattered at different intensities. By analyzing these intensity fluctuations, the speed of Brownian motion can be calculated. The size of the nanoparticles can be determined by using the Stokes-Einstein relation. Latest techniques can measure nanoparticles smaller than 1 nm.

[0425] Particle size can also be measured by: Nanoparticle tracking analysis Atomic force microscopy Electron microscopy Disc centrifugation Adjustable resistive pulse sensing Particle scattering and diffusion measurement method

[0426] Polydispersity Index (PDI)

[0427] The PDI is used to estimate the average homogeneity of a particle solution. The cumulant method is a standard technique for analyzing DLS data for sample polydispersity. The PDI is a number calculated from a two-parameter fit (cumulant analysis) to the correlation data. Cumulant analysis is used to evaluate the autocorrelation function generated by a DLS experiment. The calculation is defined in ISO 13321 and ISO 22412. A PDI value greater than 0.7 indicates that the sample has a very broad size distribution and is not suitable for DLS techniques. The calculation of these parameters is defined in ISO Standards 13321:1996E and ISO 22412:2008.

[0428] Zeta potential

[0429] Zeta potential is a measure of the magnitude of electrostatic or charge repulsion / attraction between particles. It can be determined by analyzing the mobility and charge (zeta potential) of particles using electrophoretic light scattering (ELS) techniques.

[0430] The nanoparticle size, PDI and surface charge properties of nanoparticles of the invention comprising only second generation dendrimers are shown in Table 2, along with the properties of nanoparticles of the invention comprising second generation dendrimers together with additional PGA dendrimers derivatized with mannose:

[0431] [Table 5]

[0432] Example 8 - In vivo delivery of mRNA using nanoparticles with or without mannose coating

[0433] The present inventors investigated the in vivo distribution of nucleic acids by the nanoparticles of the present invention with and without coating with Mannose-G1-EEEE dendrimers.

[0434] method

[0435] Six-week-old female C57Bl / 6 mice were supplied by Envigo (UK). Mice were acclimated in the animal facility for at least 7 days before use. MC38 cells were added at a concentration of 1 × 10 per mouse. 7 Cells were implanted into the left flank. Tumors were measured by caliper three times weekly as soon as they were palpable. When tumors reached 0.15 cm3 (caliper measurement), mice were randomized into four groups (n=3) and administered IV at 2.25 mg / kg or 3 mg / kg.

[0436] Four hours after administration of the formulation, the animals were culled and tissues were harvested. Terminal blood was collected into EDTA tubes and treated with erythrocyte lysis buffer HYBRI-MAX (Merck) before antibody staining. Spleens were dissociated by passing them through a 70 μM cell strainer using a syringe plunger. The cell suspension was then treated with erythrocyte lysis buffer HYBRI-MAX (Merck) before antibody staining. Lung and tumor samples were cut into small pieces and subjected to enzymatic digestion using a Lung and Tumor Dissociation Kit (mouse) and a gentleMACS™ Octo Dissociator with Heaters (Miltenyi) according to the manufacturer's instructions.

[0437] Prior to antibody staining, the final cell suspension was blocked with mouse Fc block. Cells were incubated with the antibody master mix for 20 minutes at 4°C and then fixed with 4% PFA for 30 minutes at room temperature. They were analyzed within 24 hours of fixation using an Attune NxT flow cytometer (Thermo Fisher Scientific). Data were analyzed using FlowJo_v10.8.1 software. Compensation was performed using Attune compensation beads.

[0438] result

[0439] [Table 6]

[0440] conclusion

[0441] As shown in Figure 13, nanocarriers coated with mannose-G1-EEEE mediated higher uptake in lung cells compared to uncoated nanocarriers and LPX-mRNA alone. Without being bound by theory, this may be due to the fact that the dendritic PGA coating allows for interaction with serum components, which may more easily guide the nanocarriers to lung tissue.

[0442] All CD206+ cells internalized nanocarriers coated with mannose-G1-EEEE more effectively than uncoated nanocarriers and LPX-mRNA alone, which may suggest that mannose targeting is specific to CD206+-expressing cells, including M2 macrophages.

[0443] Table 3 shows that mRNA with G1,2-RL, 3-LR (N:P 0.16:1) and DOTMA:DOPE (w / w 10:1 relative to mRNA) mediated efficient uptake of myeloid cells, such as macrophages, neutrophils, and dendritic cells, within the tumor.

[0444] As shown in Figure 39, CD206+ M2 macrophage cells internalized nanocarriers coated with mannose-G1-EEEE more effectively than uncoated nanocarriers and LPX-mRNA alone, suggesting that mannose targeting is specific to CD206+ expressing cells, including M2 macrophages.

[0445] The above examples highlight that peptide dendrimer / lipid nanoparticles are highly effective in delivering nucleic acids to myeloid cells within tissues, and in some cases to non-myeloid cells (e.g., non-immune cells in the lung). Cell specificity can be further improved by adding targeting motifs to the nanocarrier (e.g., mannose for CD206 targeting).

[0446] Example 9 - Comparison of third generation dendrimer transfection efficiency of mRNA and DNA compared to commercially available transfection reagents

[0447] In vitro transfection efficiency for mRNA and DNA delivery was evaluated using certain dendrimers in Table 1 (shown in Example 1 above) and described in the following examples:

[0448] Cationic lipid-based nucleic acid delivery systems are one of the most studied and efficient non-viral vector platforms described to date, and the rational design and development of peptide vectors using natural amino acids is particularly attractive for therapeutic applications due to the non-toxic nature of amino acids. We developed a structural framework for nucleic acid delivery using peptide dendrimers. The structural framework comprises layers of peptide (or dipeptide) motifs linked to lysine residues. We found that the distribution of cationic amino acid residues (Lys or Arg) in each generation (layer) resulted in peptide dendrimers transfecting more efficiently than dendrimers with charges localized only on the surface (Kwok et al., 2013). Using a solid-phase peptide dendrimer synthesis procedure, we were able to precisely manipulate the position of every amino acid residue incorporated within the dendritic scaffold. This allows for greater control over the structure and function of the dendrimer, which was typically not 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 vector showed high transfection efficiency, good reproducibility of results, and low toxicity.

[0449] The present inventors compared the in vitro transfection efficiency of these novel peptide dendrimer systems with other known transfection reagents. GSC The cells were transfected with mRNA encoding luciferase using either a composition containing G1,2-RL, 3-LR (NP to mRNA = 0.16:1) and DOTMA / DOPE (w / w 10:1 to mRNA) or the commercially available Lipofectamine™ 2000. As shown in the upper left panel of Figure 14, GSC A composition containing G1,2-RL, 3-LR (NP to mRNA = 0.16:1) and DOTMA / DOPE increases in vitro transfection efficiency by approximately one order of magnitude compared to the commercially available transfection reagent Lipofectamine™ 2000. GSCHeLa cells transfected with mRNA encoding eGFP using G1,2-RL, 3-LR (NP to mRNA = 0.16:1) and DOTMA / DOPE increased eGFP expression approximately 4-fold compared to mRNA transfected using the DLin-MC3-DMA:cholesterol:DSPC:DMG-PEG lipid nanoparticle delivery system (Figure 14, upper right panel).

[0450] To further validate the peptide-dendrimer system, C2C12 cells were cultured in 1) mRNA-only medium; 2) mRNA-containing medium; GSC C2C12 cells were transfected for 24 hours with either G1,2-RL, 3-LR (NP to mRNA = 8:1) and DOTMA / DOPE (w / w = 10:1 to mRNA), 3) mRNA with DOTMA / DOPE (w / w = 10:1 to mRNA), 4) mRNA with polyethyleneimine, or 5) mRNA with Lipofectamine 2000. As shown in the bottom panel of Figure 14, the peptide dendrimer formulation significantly improved mRNA delivery into C2C12 cells compared to commercially available lipid-based transfection reagents.

[0451] The present inventors have also demonstrated the ability of third-generation peptide dendrimers GSC Using G1,2,3-RL, we compared the transfection efficiency of DNA using this dendrimer with that of commercially available transfection reagents. HeLA cells (Figure 15, upper panel) or Neuro2A cells (Figure 15, lower panel) were transfected with 1) GSC The cells were transfected with DNA using 1) G1,2,3-RL and DOTMA / DOPE, 2) DOTMA / DOPE alone, 3) polyethyleneimine, or 4) Lipofectamine 2000. The dendrimers were transfected under both serum-free (Figure 15, left panel) and serum-free (Figure 15, right panel) conditions. GSCG1,2,3-RL outperformed all commercially available transfection reagents tested: The third-generation dendrimer transfected HeLa and Neuro2A cells 2-600 times better than several widely used commercially available reagents, such as polyethyleneimine (PEI), Lipofectin (also known as DOTMA / DOPE), and Lipofectamine 2000 (Figure 15).

[0452] In general, the G3 dendrimers tested efficiently transfect mRNA and DNA in HeLa cells, at least mRNA in C2C12 cells, and DNA in Neuro2A cells.

[0453] Example 10 - Comparison of transfection efficiency of first, second and third generation dendrimers

[0454] We investigated the effect of the number of generations, from G1 to G2-G3, based on the KL repeat unit on delivery efficiency. We also replaced the KL unit with the RL repeat unit to compare the effect of protonated basic groups with different pKa. Interestingly, we observed a relationship between the generation and transfection in cells (Figure 16). The generation dependence on transfection was observed in first-generation dendrimers. GSC G1-KL or GSC We demonstrate that G1-RL does not transfect HeLa or Neuro2A cells that have lysine or arginine as the charged residue, which may be due to the fact that the G1 peptide is not physically large enough to completely encapsulate the plasmid DNA and form stable nanoparticles, as shown by complex stability assays (data not shown).

[0455] However, in the second generation, arginine-containing GSC G1,2-RL is GSCCompared to G1,2-KL, it shows higher transfection efficiency (N / P ratio of 10:1 or 20:1 for mRNA in HeLa cells and N / P ratio of 20:1 for mRNA in Neuro2A cells) (Figure 16). GSC For G1,2-KL GSC Such transfection advantages of G1,2-RL include DNA GSC Forms more stable transfection complexes than G1,2-KL GSC This is consistent with the ability of G1,2-RL (data not shown).

[0456] Example 11 - Comparison of single and hybrid dendrimer systems

[0457] Hybrid dendrimer systems were investigated to determine whether transfection efficiency could be further improved compared to single dendrimer systems.

[0458] Materials and Methods

[0459] Cell lines, transfection reagents, and mRNA. HeLa cells were maintained in RPMI medium containing 10% (v / v) FCS and 1% (v / v) P / S in a humidified atmosphere at 5% CO and 37°C. eGFP mRNA was purchased from Trilink (CleanCap® EGFP mRNA (5 moU)-(L-7201)). DOTMA:DOPE, 1:1 (w / w), was obtained from Invitrogen (Lipofectin™ Transfection Reagent) (Fisher-18292037) or Encapsula NanoSciences LLC.

[0460] Nanoparticle Formulation Procedure: To formulate hybrid dendrimer nanoparticles, peptide dendrimer 1 was mixed with peptide dendrimer 2 in a molar ratio relative to the resulting N, with the final N / P ratio typically being 8 overall. For example, a 10 mg / ml peptide dendrimer 2 solution (1.886 μl, 18.9 μg, 7.50 × 10 mmol dendrimer, 5.25 × 10 mmol N, N / P) in sterile water (2.90 μl) and 200 mM HEPES buffer (0.814 μl) was prepared. 5.33) was added to Tube A, which contained peptide dendrimer 1 (10 mg / ml peptide dendrimer 1 solution (0.904 μl, 9.0 μg, 2.33 × 10-7 mmol dendrimer, 2.56 × 10-6 mmol N, N / P = 2.67). The tube was gently shaken and then spun down using a minicentrifuge for 10-15 seconds to ensure all the liquid was at the bottom of the tube. 16.25 μl of mRNA (25 mM HEPES A 200.0 μg / ml solution of mRNA (3.25 μg, 9.85 × 10-6 mmol phosphate) in buffer was added to the tube and mixed by rapidly pipetting up and down 10-15 times to form a complex at N / P = 8. This was allowed to stand for 2-5 minutes. A 796.2 μg / ml DOTMA / DOPE liposome solution (42.2 μl, 32.5 μg) was added to the tube by rapidly pipetting up and down 10-15 times.

[0461] Transfection Procedure. 24 hours prior to transfection, HeLa cells were seeded in 96-well plates to reach 70% confluence. mRNA transfection complexes were formed by mixing mRNA with dendrimer in 25 mM HEPES buffer, followed by DOTMA:DOPE in 25 mM HEPES buffer at 25°C. The entire transfection mixture was formulated with a final dendrimer:mRNA NP ratio of 8:1. DOTMA:DOPE was added to the mRNA complex at a w / w ratio of 10:1. The transfection complexes were then overlaid onto the cells in complete growth medium. 24 hours after transfection, cells were harvested for reporter gene assays.

[0462] Transgene expression assay: Cells were washed twice with PBS and incubated with 50 μl of 1x M-PER lysis buffer (Thermo 11874111). Cells were protected from light and gently agitated at room temperature for 15 minutes to aid cell lysis. 40 μl of lysate from each well was transferred to a black 96-well plate, and eGFP relative fluorescence units (RFU), absorbance at 535 nm, were quantified using a Molecular Devices SpectraMax iD5.

[0463] Protein content determination: The protein content of each cell lysate was determined by mixing 25 μL of lysate with a Pierce™ BCA Protein Assay Kit (200 μl, Thermo Scientific). After 30 minutes of incubation at 37°C in the dark, absorbance was measured at 562 nm using a Molecular Devices SpectraMax iD5 and converted to protein concentration using a BSA standard curve. RFU per mg of protein represented eGFP expression. Values shown in the transfection figures are expressed after normalization to a control transfection experiment using DOTMA / DOPE and are shown as a percentage.

[0464] Size, Zeta Potential, and Polydispersity Index (PDI) Measurements: Hydrodynamic size was measured using dynamic light scattering (DLS) technology using a Zetasizer Advance Series-Pro (Malvern Panalytical Ltd, Malvern, UK) according to the manufacturer's instructions. DLS is a highly sensitive, non-invasive method for measuring the size and size distribution of nanoparticles in liquids. Brownian motion of nanoparticles in suspension results in laser light being scattered at different intensities. By analyzing these intensity fluctuations, the rate of Brownian motion can be calculated. Nanoparticle size can be determined using the Stokes-Einstein relationship. Current technology allows the measurement of nanoparticles smaller than 1 nm.

[0465] Data acquired from DLS measurements can also be used to calculate the PDI of particles in solution. The PDI is used to estimate the average homogeneity of a particle solution. The cumulant method is a standard technique for analyzing DLS data for sample polydispersity. The PDI is a number calculated from a two-parameter fit (cumulant analysis) to the correlation data. Cumulant analysis is used to evaluate the autocorrelation function generated by a DLS experiment. Calculations are defined in ISO 13321 and ISO 22412. A PDI value greater than 0.7 indicates that the sample has a very broad size distribution and is not suitable for DLS techniques. Calculations of these parameters are defined in ISO 13321:1996E and ISO 22412:2008.

[0466] To measure size and PDI, samples were diluted 16-fold in 25 mM HEPES buffer (5 μl sample + 75 μl buffer). Parameters: Reference material: polystyrene latex, dispersant: water, 25° C. PDI measurements were performed on nanoparticles containing an 8:1 dendrimer:nucleic acid NP ratio.

[0467] Zeta potential is a measure of the magnitude of electrostatic or charge repulsion / attraction between particles. It can be determined by analyzing particle mobility and charge (zeta potential) using electrophoretic light scattering (ELS) techniques. To measure zeta potential, samples were diluted 150-fold in 25 mM HEPES buffer (10 μl sample + 690 μl buffer) and added to sterile DTS1070 cells. Parameters: Reference material: polystyrene latex, dispersant: water, 25°C. Zeta potential measurements were performed using a Zetasizer Advance Series-Pro (Malvern Panalytical Ltd, Malvern, UK) according to the manufacturer's instructions.

[0468] result

[0469] The PDI of nanoparticles containing mRNA, lipids, and single peptide dendrimers was investigated. The polydispersity index (PDI) can be used to measure the ability of a particular composition, including a particular peptide dendrimer, to form a monodisperse nanoparticle population. A higher PDI is associated with a decreased formation of a monodisperse population of nanoparticles. As seen in Table 5, for example, nanoparticles containing RHCG1-R, RHCG1-RLR, or G1-LRLR have a PDI greater than 0.35, indicating that these dendrimers are unable to form sufficiently monodisperse mRNA nanoparticles at an 8:1 NP ratio. This inability to form a monodisperse population indicates that dendrimer-mRNA complexes in nanoparticles with a PDI greater than 0.35 are relatively unstable compared to those in nanoparticles with a PDI of 0.35 or less.

[0470] [Table 7] TIFF2025525843000011.tif79162

[0471] With the above in mind, we investigated whether the inclusion of dendrimers with a PDI greater than 0.35 in compositions containing stable dendrimer-mRNA complexes (i.e., PDIs of 0.35 or less) could increase transfection efficiency by increasing the dissociation rate of the mRNA after entry into cells.

[0472] GSC G1,2-RL, 3-LR alone or GSC G1,2-RL, 3-LR and GSC A first comparison was made between HeLa cells transfected with either a mixture containing G1-LRLR in a 1:2 ratio. GSC G1 dendrimer in a composition containing G1, 2-RL and 3-LR GSC With G1-LRLR included, GSCThe transfection efficiency of mRNA was significantly increased compared to the single dendrimer composition containing G1,2-RL,3-LR alone (Figure 17A). The transfection efficiency increased by approximately 100% in the hybrid dendrimer composition compared to the single dendrimer composition.

[0473] A second G1 dendrimer was investigated for use in hybrid dendrimer systems. GSC G1,2-RL, 3-LR alone or in a 2:1 or 1:2 ratio GSC G1,2-RL, 3-LR and RHC Comparisons were made between transfection mixtures containing either G1-R in a 2:1 ratio. GSC G1,2-RL, 3-LR: RHC G1-R resulted in an increase in transfection efficiency of approximately 100% (Figure 17B). By changing the G2:G1 ratio to 1:2, a further increase in transfection efficiency was obtained, resulting in a transfection efficiency of approximately 300% compared to the G2 dendrimer mixture alone.

[0474] next, RHC G1-RL, 2-LR-containing dendrimer transfection mixture, RHC G1,2-R, RHC G1-RLR, or GSC G1-RLR were investigated (Figures 17C-E, respectively). Using a 1:1 G2:G1 ratio appears to result in a slight increase in transfection efficiency of approximately 20-50% compared to the G2 dendrimer alone (Figures 17D and E, compare the first and second columns). However, in line with previous results, using a 1:2 ratio results in an additional increase in transfection efficiency of approximately 60-100% (Figures 17D and E, compare the first and third columns).

[0475] Considering the results presented in Figures 17A-B and 17D-E, the addition of the G1 dendrimer to form a hybrid dendrimer system with either the G2 or G3 dendrimer appears to improve mRNA transfection compared to mRNA complexes formed with a single dendrimer alone. For example, Figure 17A shows the effect of the G1 dendrimer on the mRNA transfection of the G2 or G3 dendrimer. GSC G1-RLRL is a G3 dendrimer GSC Combining G1,2-RL and G2-LR improves transfection by 200%. Figures 17B, D-E also show that adding G1 dendrimers to G2 dendrimers can enhance transfection by up to 300%.

[0476] Surprisingly, the two G2 dendrimers RHC G1-RL, 2-LR and RHC It was also observed that the hybrid dendrimer mixture containing RHCG1,2-R could also increase mRNA transfection efficiency (Figure 17C). Although it remains unclear what causes this increase in transfection efficiency, one hypothesis is that the inclusion of the relatively less stable RHCG1,2-R in the nanoparticles increases the overall instability of the nanoparticles, which promotes mRNA release once the nanoparticles enter cells.

[0477] As can be seen in Table 5, the PDI of RHCG1,2-R nanoparticles is relatively higher than that of RHCG1-RL,2-LR (0.252 vs. 0.109). RHC This indicates that nanoparticles containing G1,2-R form monodisperse populations that are acceptable. RHCThe increased relative instability compared to the highly stable nanoparticles formed using RHCG1-RL,2-LR may aid in the dissociation of mRNA from nanoparticles containing both dendrimers. This is supported by the results of PDI assays of mixed dendrimer compositions (Table 6, discussed further below), which demonstrate that the PDI of nanoparticles containing a 1:1 ratio of RHCG1-RL,2-LR and RHCG1,2-R is higher than that of nanoparticles containing only RHCG1-RL,2-LR (0.155 vs. 0.109, respectively).

[0478] Example 12 - PDI assay of mixed dendrimer systems

[0479] While improved transfection efficiency is essential for the development of improved nucleic acid therapeutics, it is also crucial that any new formulations have pharmaceutically acceptable properties. For example, it is important that new formulations have a sufficiently monodisperse nanoparticle population so that any in vivo results are predictable. Therefore, the PDI of the above "hybrid" nanoparticles was analyzed. Table 6 demonstrates that the majority of the nanoparticles tested had a sufficiently monodisperse nanoparticle population suitable for use in pharmaceutical compositions, i.e., all but one of the combinations tested had a PDI of less than 0.35.

[0480] The PDI of nanoparticles containing mixed dendrimer populations can be tuned by varying the ratio of dendrimers in the nanoparticles. For example, a combination of dendrimers in a 1:2 ratio versus a 2:1 ratio can be used. RHC G1-RL, 2-LR and RHC The PDIs of G1-R are 0.521 and 0.108, respectively. Similarly, the dendrimer combinations in a 1:1 ratio vs. a 1:2 ratio RHC G1-RL, 2-LR and RHC The PDIs of G1-RLR are 0.0819 and 0.248, respectively. Finally, the combination of dendrimers in a 1:1 ratio versus a 1:2 ratio RHC G1-RL, 2-LR and GSCThe PDIs of G1-LRLR are 0.141 and 0.289, respectively. Taken together, these results indicate that the PDI of mixed dendrimer nanoparticles can be increased by increasing the proportion of dendrimers that have a higher PDI when used alone.

[0481] [Table 8]

[0482] Increasing the proportion of dendrimers with higher PDI in nanoparticles containing two peptide dendrimers generally appears to increase the transfection efficiency of the nanoparticles. However, as the proportion of dendrimers with higher PDI increases, the PDI also increases. This observation may allow for the optimization of the ratio of peptide dendrimers in the nanoparticles to give the highest transfection efficiency while maintaining the PDI within acceptable boundaries (e.g., 0.35 or less).

[0483] Example 13 - Exemplary dendrimers for use in mixed peptide dendrimer / lipid nanoparticles

[0484] [Table 9]

[0485] [Table 10]

[0486] Example 14 - Determination of transfection efficiency

[0487] Transfection of C2c12 cells:

[0488] C2c12 cells were maintained in DMEM medium containing 10% (v / v) FCS and 1% (v / v) L-glutamine in a humidified atmosphere at 5% CO2 and 37°C. Alexa Fluor 488-tagged mRNA expressing eGFP was purchased from RiboPro. 24 hours prior to transfection, C2c12 cells were seeded into 96-well plates to reach 70% confluence. mRNA transfection complexes were formed by mixing mRNA with dendrimer in 25 mM HEPES buffer, followed by DOTMA:DOPE (1:1 w / w) in 25 mM HEPES buffer at 25°C. The transfection complex ("NTX3") was then overlaid onto the cells in complete growth medium. Four hours after transfection, cells were harvested for FACS analysis. The cell culture medium and the PBS used to wash each well were collected from each treatment, and 5 μM EDTA in PBS was added. After a 10-minute incubation at 37°C, cells were gently detached from the plate and pelleted at 400g for 8 minutes at 4°C. Cells were stained using LIVE / DEAD fixable aqua fluorescent reactive dye (Invitrogen) according to the manufacturer's instructions. For compensation, ArC beads were stained with 3uL of pure Live / Dead Aqua. After staining, cells were pelleted at 400g for 8 minutes at 4°C and resuspended in a 500uL / sample PBS wash before repeating the centrifugation. Negative ArC beads were added as compensation controls. GFP compensation beads were aliquoted into a separate tube as GFP compensation controls. All samples containing beads were fixed with 4% paraformaldehyde in PBS for 15 minutes at room temperature. Samples were then spun again at 400g for 8 minutes at 4°C and resuspended in FACS buffer (PBS + 2mM EDTA + 0.5% w / v BSA). Samples were stored at 4°C and flow analysis was performed within 24 hours of fixation. Data were collected using a BD LSRFortessa I analyzer running FACSDIVA software (Beckton Dickinson). Collected data were analyzed using FlowJo 10.0 software. Figure 18 shows that approximately 100% of C2c12 myocytes were successfully transfected.

[0489] Transfection of J774 cells:

[0490] The day before transfection, J774 cells cultured in 10 cm dishes were washed with PBS, scraped using a cell scraper, and collected in a 50 ml Falcon tube. The cells were spun at 400 g for 5 minutes and resuspended in 10 ml of J774 complete medium (DMEM containing 10% heat-inactivated FBS and 2 mM GlutaMAX supplement) for counting. The cells were seeded into 24-well plates. The cells were transferred to an incubator overnight. For transfection, the medium covering the J774 cells was replaced with 480 μl / well of fresh J774 complete medium. The cells were transfected with 120 μl / well of 25 mM HEPES or formulations (various concentrations of encapsulated Alexa Fluor 488-eGFP mRNA) in an incubator for 4 hours (i.e., the total volume during transfection was 600 μl). After 4 hours, J774 cells were washed with PBS and harvested using a cell scraper, with 5 mM EDTA incubated with the cells for 15 minutes at 37°C. The J774 cells were then spun down. The supernatant was discarded, and the cells were resuspended in 100 μl / sample of LIVE / DEAD fixable red diluted in PBS (1:250) and left in the refrigerator for 30 minutes. The cells were then spun down at 400 g for 5 minutes and washed with PBS. The cells were spun down again, resuspended in 100 μl / sample of 4% PFA (diluted in PBS), and fixed for 10 minutes at room temperature. After fixation, the cells were spun down at 500 g for 5 minutes and washed with PBS. The cells were spun down again, resuspended in 400 μl / sample of FACS buffer (PBS containing 0.5% w / v BSA and 2 mM EDTA), and passed through a 70 μm filter before flow analysis. Data were collected using a BD LSRFortessa I analyzer running FACSDIVA software (Beckton Dickinson). Collected data were analyzed using FlowJo 10.0 software.

[0491] Figure 19 shows that approximately 100% of J774 macrophages were successfully transfected when 1.5 μg of nucleic acid was applied using the compositions of the present invention, and approximately 50% of J774 macrophages were successfully transfected when 0.015 μg of nucleic acid was applied using the compositions of the present invention.

[0492] Transfection of Jurkat cells:

[0493] On the day of transfection, Jurkat cells were harvested, spun down at 300g for 5 minutes, and resuspended in 10ml of Jurkat complete medium (DMEM containing 10% heat-inactivated FBS and 2mM GlutaMAX supplement) for counting. Cells were diluted to 4.17E+5 cells / ml and seeded into a 24-well format at 480µl / well (i.e., 2E+5 cells / well). Cells were left for 30 minutes before transfection. Cells were transfected with 120µl / well of 25mM HEPES or formulations (various concentrations of encapsulated Alexa Fluor 488-eGFP mRNA) in an incubator for 4 hours (i.e., the total volume during transfection was 600µl). After 4 hours of transfection, cells were collected in a 1.5ml Eppendorf tube and spun down at 300g for 5 minutes. Cells were then washed once with 750µl / sample of PBS and then spun down. The supernatant was discarded, and the cells were resuspended in 100 μl / sample of LIVE / DEAD fixable red diluted in PBS (1:250) and left in the refrigerator for 30 minutes. The cells were then spun down at 400 g for 5 minutes and washed with PBS. The cells were spun down again, resuspended in 100 μl / sample of 4% PFA (diluted in PBS), and fixed for 10 minutes at room temperature. After fixation, the cells were spun down at 500 g for 5 minutes and washed with PBS. The cells were spun down again, resuspended in 400 μl / sample of FACS buffer (PBS containing 0.5% w / v BSA and 2 mM EDTA), and passed through a 70 μm filter before flow analysis. Data were collected using a BD LSR Fortessa I analyzer running FACSDIVA software (Beckton Dickinson). The collected data were analyzed using FlowJo 10.0 software.

[0494] Figure 20 shows that approximately 100% of Jurkat T cells were successfully transfected when 1.5 ug of nucleic acid was applied using the compositions of the present invention, and approximately 50% of Jurkat T cells were successfully transfected when 0.15 ug of nucleic acid was applied using the compositions of the present invention.

[0495] Transfection of HeLa cells:

[0496] HeLa cells were seeded in 96-well plates to reach 70% confluence. mRNA transfection complexes were formed by mixing mRNA with dendrimer in 25 mM HEPES buffer, followed by DOTMA:DOPE in 25 mM HEPES buffer at 25°C. The mRNA expressed eGFP and was tagged with Alexa Fluor 488. The transfection complexes were layered on the cells in complete growth medium. Cells were harvested 2 hours post-transfection as follows: Cells were washed and incubated at 37°C for 10 minutes. Cells were then gently detached from the plate and pelleted. Cells were stained with LIVE / DEAD fixable aqua fluorescent reactive dye (Invitrogen) according to the manufacturer's instructions. For compensation, ArC beads were stained with 3 μL of pure Live / Dead Aqua. After staining, cells were pelleted at 4°C and resuspended in 500 μL / sample of PBS wash, followed by repeated centrifugation. Negative ArC beads were added for compensation controls. GFP compensation beads were dispensed into a separate tube as a GFP compensation control. All samples containing beads were fixed with 4% paraformaldehyde in PBS for 15 minutes at room temperature. Afterwards, samples were spun down again at 4°C and then resuspended in FACS buffer (PBS + 2 mM EDTA + 0.5% w / v BSA). Samples were stored at 4°C, and flow analysis was performed within 24 hours of fixation. Data were collected using a BD LSRFortessa I analyzer running FACSDIVA software (Beckton Dickinson). Collected data were analyzed using FlowJo 10.0 software.

[0497] Figure 21 shows that approximately 100% of HeLa cells were successfully transfected when 1.5 μg or 1.125 μg of nucleic acid was applied using the compositions of the present invention, and approximately 75% of HeLa cells were successfully transfected when 0.1875 μg of nucleic acid was applied using the compositions of the present invention.

[0498] Example 15 - Lipid Mixture (i)

[0499] To formulate nanocarriers with dendrimers and mRNA with different lipids, the individual lipids dissolved in a solvent were mixed in the desired molar ratio and combined with the dendrimer and mRNA.

[0500] To form an exemplary nanoparticle composition: GSC G1,2-RHL ((RHL)4(KRHL)2KGSC-NH2) and EGFP mRNA were prepared and mixed in an aqueous buffer solution. The liposome mixture described below was then added.

[0501] DODAP:DOTAP:DOPE 1:1:1. DODAP (1,2-dioleoyloxy-3-(dimethylamino)propane), DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), and DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) were combined in a 1:1:1 molar ratio in ethanol and mixed with the peptide and mRNA solution to obtain a turbid solution. DLS measurement (27-fold dilution in 25 mM HEPES buffer): 115.9 nm, PDI 0.11.

[0502] DODAP:DOTMA:DOPE 1:1:1. DODAP, DOTMA (1N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride), and DOPE were combined in a 1:1:1 molar ratio in ethanol solvent and mixed with the peptide and mRNA solution to obtain a turbid solution. DLS measurement (27-fold dilution in 25 mM HEPES buffer): 116.9 nm, PDI 0.10.

[0503] DODAP:DOTAP:DOPE 1:1:2. DODAP, DOTAP, and DOPE were combined in a molar ratio of 1:1:2 in ethanol solvent and mixed with the peptide and mRNA solution to obtain a turbid solution. DLS measurement (27-fold dilution in 25 mM HEPES buffer): 127.3 nm, PDI 0.09.

[0504] DODAP:DOTMA:DOPE 1:1:2. DODAP, DOTMA, and DOPE were combined in a molar ratio of 1:1:2 in ethanol solvent and mixed with the peptide and mRNA solution to obtain a turbid solution. DLS measurement (27-fold dilution in 25 mM HEPES buffer): 136.9 nm, PDI 0.08.

[0505] DODAP:DOTMA:DOPE 2:1:1. DODAP, DOTMA, and DOPE were combined in a molar ratio of 2:1:1 in ethanol solvent and mixed with the peptide and mRNA solution to obtain a turbid solution. DLS measurement (27-fold dilution in 25 mM HEPES buffer): 139.5 nm, PDI 0.07.

[0506] DODAP:DOTAP:DOPE 2:1:1. DODAP, DOTAP, and DOPE were combined in a molar ratio of 2:1:1 in ethanol solvent and mixed with the peptide and mRNA solution to obtain a turbid solution. DLS measurement (27-fold dilution in 25 mM HEPES buffer): 160.3 nm, PDI 0.06.

[0507] DODAP:DOTAP 1:1. DODAP and DOTAP were combined in a 1:1 molar ratio in ethanol solvent and mixed with the peptide and mRNA solution to obtain a turbid solution. DLS measurement (27-fold dilution in 25 mM HEPES buffer): 124.6 nm, PDI 0.12.

[0508] DODAP:DORI:DOPE 1:1:1. DODAP, DORI (N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium bromide), and DOPE were combined in a 1:1:1 molar ratio in ethanol and mixed with the peptide and mRNA solution to obtain a turbid solution. DLS measurement (27-fold dilution in 25 mM HEPES buffer): 148.9 nm, PDI 0.06.

[0509] DODAP:DORI:DOPE 1:1:2. DODAP, DORI, and DOPE were combined in a molar ratio of 1:1:1 in ethanol solvent and mixed with the peptide and mRNA solution to obtain a turbid solution. DLS measurement (27-fold dilution in 25 mM HEPES buffer): 160.3 nm, PDI 0.06.

[0510] DOPE has CAS number 4004-05-1 and formula: C 41 H 78 NO8P), and DOTAP has CAS number: 132172-61-3 and formula: C 42 H 80 NO4Cl), and DODAP has CAS number 127512-29-2 and formula: C 41 H 77 DORI has the CAS number 153312-59-5 and the formula: C 43 H 82 DOTMA has the CAS number 104162-48-3 and the formula: C 42 H 84 DOTMA can also be named 1,2-di-O-octadecenyl-3-trimethylammonium propane (chloride salt) (CAS number: 104872-42-6).

[0511] The transfection efficacy of nanocarriers containing each lipid mixture was evaluated in human cancer cell lines (HeLa and A549).

[0512] HeLa cells were transfected with nanocarriers formulated with different lipid components using the methods described herein. The nanocarriers used were: (1) DOTMA:DOPE (using a 1:1 ratio of moles of DOTMA to moles of DOPE); GSC G1,2-RL, 3-LR (N:P=0.16:1 relative to mRNA) (total lipid amount was w / w=10:1 relative to mRNA), (2) with DOTMA:DOPE (1:1 molar ratio of DOTMA to DOPE) GSC G1,2-RL, 3-LR (N:P=8:1 relative to mRNA) (total lipid amount was 10:1 w / w relative to mRNA), (3) DOTMA:DOPE (1:1 molar ratio of DOTMA to DOPE) GSC G1,2-RHL (N:P=0.6:1 relative to mRNA) (total lipid amount was w / w=10:1 relative to mRNA), (4) with DODAP:DOTAP (using 1:1 moles of DODAP to moles of DOTAP) GSC G1,2-RHL (N:P=0.6:1 relative to mRNA) (total lipid amount was w / w=10:1 relative to mRNA), (5) with DODAP:DOTAP:DOPE (using 1:1:1 moles of DODAP to moles of DOTAP to moles of DOPE) GSC G1,2-RHL (N:P=0.6:1 relative to mRNA) (total lipid amount was w / w=10:1 relative to mRNA), and (6) DODAP:DOTAP:DOPE (using 1:1:2 moles of DODAP to moles of DOTAP to moles of DOPE). GSC G1,2-RHL (N:P=0.6:1 relative to mRNA) (total lipid amount was 10:1 w / w relative to mRNA), (7) with DODAP:DOTMA:DOPE (1:1:1 moles of DODAP to moles of DOTMA to moles of DOPE) GSC G1,2-RHL (N:P=0.6:1 relative to mRNA) (total lipid amount was w / w=10:1 relative to mRNA), and (8) DODAP:DOTMA:DOPE (using 1:1:2 moles of DODAP to moles of DOTMA to moles of DOPE). GSC The transfections were carried out using either G1,2-RHL (N:P = 0.6:1 relative to mRNA) or G1,2-RHL (total lipid amount was 10:1 w / w relative to mRNA). The mRNA used was mRNA expressing eGFP, and cells were harvested 24 hours after transfection and analyzed for fluorescent protein expression using a plate reader. Control: Cells transfected with mRNA alone. Figures 22 and 23 show the increased transduction efficiency achieved by three lipid systems in nanoparticles containing a representative second-generation peptide dendrimer when transfecting HeLa cells.

[0513] A549 cells were transfected with nanocarriers formulated with different lipid components using the methods described herein. The nanocarriers used were: (1) DOTMA:DOPE (using a 1:1 ratio of moles of DOTMA to moles of DOPE); GSC G1,2-RL, 3-LR (N:P=0.16:1 relative to mRNA) (total lipid amount was w / w=10:1 relative to mRNA), (2) with DOTMA:DOPE (1:1 molar ratio of DOTMA to DOPE) GSC G1,2-RL, 3-LR (N:P=8:1 relative to mRNA) (total lipid amount was 10:1 w / w relative to mRNA), (3) DOTMA:DOPE (1:1 molar ratio of DOTMA to DOPE) GSC G1,2-RHL (N:P=0.6:1 relative to mRNA) (total lipid amount was w / w=10:1 relative to mRNA), (4) with DODAP:DOTAP:DOPE (2:1:1 moles of DODAP to moles of DOTAP to moles of DOPE) GSC G1,2-RHL (N:P=0.6:1 relative to mRNA) (total lipid amount was w / w=10:1 relative to mRNA), and (5) DODAP:DORI:DOPE (using 1:1:1 moles of DODAP to moles of DORI to moles of DOPE). GSCG1,2-RHL (N:P=0.6:1 relative to mRNA) (total lipid amount was w / w=10:1 relative to mRNA), (6) with DODAP:DORI:DOPE (1:1:2 moles of DODAP to moles of DORI to moles of DOPE) GSC G1,2-RHL (N:P=0.6:1 relative to mRNA) (total lipid amount was w / w=10:1 relative to mRNA), (7) with DODAP:DOTMA:DOPE (2:1:1 moles of DODAP to moles of DOTMA to moles of DOPE) GSC The transfections were carried out using either G1,2-RHL (N:P = 0.6:1 relative to mRNA) or G1,2-RHL (total lipid content was 10:1 w / w relative to mRNA). The mRNA used was eGFP-expressing mRNA. Cells were harvested 24 hours after transfection and analyzed for fluorescent protein expression using a plate reader. Formulations (4), (5), (6), and (7) contained only 67% of the eGFP mRNA content compared to formulations (1), (2), and (3). Control: cells transfected with mRNA alone. Figure 24 shows the increased transduction efficiency achieved by three lipid systems in nanoparticles containing a representative second-generation peptide dendrimer when transfecting A549 cells.

[0514] [Table 11]

[0515] Example 16 - Comparison of Linear and Dendritic PGA

[0516] Human T cells were transfected with naked mRNA expressing eGFP (enhanced GFP) or with nanocarriers containing mRNA expressing eGFP using the methods described herein. The nanocarriers were composed of DOTMA / DOPE (10:1 w / w ratio relative to mRNA) and mRNA. GSC It contained G1,2-RHL (N:P=0.6:1 relative to mRNA).

[0517] In the first experiment, nanocarriers were coated with equal numbers of molecules of either linear or dendritic PGA (Figure 25A). Linear PGA was a linear PGA with 100 glutamic acids, and dendritic PGA was GSEGSEGSEC(OH)G1-(Ac)ESGESGESG.

[0518] [Table 12]

[0519] [Table 13]

[0520] In a second experiment, nanocarriers were coated with equimolar charges of either linear or dendritic PGA (Figure 25B). Linear PGA was a linear PGA with 100 glutamic acids, and dendritic PGA was G1-EEEE.

[0521] [Table 14]

[0522] [Table 15]

[0523] Intracellular eGFP protein expression was quantified by flow cytometry. Figures 25A and 25B show expression levels normalized to the level achieved by the formulation coated with linear PGA ("normalized relative transfection"). Negative control cells were either not transfected or transfected with mRNA alone.

[0524] Example 17 - Muscle-targeted nanocarriers

[0525] The nanoparticles of the present invention were coated with dendrimers containing muscle-targeting peptides to improve targeting and transfection of muscle cells, and even differentiated muscle cells.

[0526] DOTMA / DOPE (w / w=10:1 relative to mRNA) and mRNA GSC Nanocarriers containing G1,2-RHL (N:P = 0.6:1 relative to mRNA) were first prepared as described. The nanoparticles were then coated with the muscle-targeting domain-containing dendrimer (muscle-G1-EEEE(Ac-EEEE)2KGSCGAASSLNIA-(Acp)-NH2) at different mRNA:coating molar ratios ranging from 0.5:1 to 5:1, preferably approximately 2.74:1. As a control, nanoparticles were coated with the muscle-targeting domain-free dendrimer (G1-EEEE) at different mRNA:coating molar ratios ranging from 0.5:1 to 5:1, preferably approximately 2.74:1.

[0527] In the first experiment, mouse muscle cells (C2c12 cells) were transfected with a formulation containing mRNA expressing eGFP. The formulation used was a DOTMA / DOPE (w / w = 10:1 relative to mRNA) and mRNA. GSC Dendrimers containing G1,2-RHL (N:P=0.6:1 relative to mRNA) were coated with either the same molar equivalent of dendritic PGA with or without a muscle-targeting domain. Cells were harvested 24 hours after transfection. Control: Cells were not transfected with mRNA. The dendrimer with the muscle-targeting domain was (Ac-EEEE)2KGSCGAASSLNIA-(Acp)-NH2 (designated "Muscle-G1-EEEE." ASSLNIA (SEQ ID NO: 1) is the muscle-targeting motif). The dendrimer without the muscle-targeting domain was (Ac-EEEE)2KGSGGSGGSC-NH2 ("GSGGSGGSCG1-EEEE"). Figure 26 shows a dramatic increase in eGFP expression in muscle cells transfected with nanocarriers containing the muscle-targeting domain.

[0528] In the second experiment, differentiated mouse muscle cells (C2c12 cells) were transfected with a formulation containing mRNA expressing eGFP. Muscle cells were differentiated as follows: C2c12 cells were seeded and grown in growth medium until confluent. Once the cells were confluent, the medium on the cells was replaced with differentiation medium for two days to allow the cells to express myotube characteristics. Thirty minutes prior to transfection, the differentiation medium was replaced with growth medium. Cells were transfected with the nanocarriers shown in Figure 27 for 24 hours, harvested, and assayed for eGFP reporter gene expression using a plate reader. The mRNA used expressed eGFP. Growth medium contained high-glucose DMEM (Gibco), 10% FBS (Gibco), and 2 mM L-glutamine (Gibco). The differentiation medium contained high glucose DMEM (Gibco), 2% horse serum (Gibco), 2 mM L-glutamine (Gibco), and 1 uM insulin.

[0529] As in the first experiment, the formulation used was DOTMA / DOPE (w / w=10:1 relative to mRNA) and mRNA. GSC Dendrimers containing G1,2-RHL (N:P=0.6:1 relative to mRNA) were coated with either the same molar equivalent of dendritic PGA with or without a muscle-targeting domain. Cells were harvested 24 hours after transfection. Control: Cells were not transfected with mRNA or were transfected with mRNA only. Dendrimers with a muscle-targeting domain were Muscle-G1-EEEE. Dendrimers without a muscle-targeting domain were GSGGSGGSCG1-EEEE. Figure 27 shows a dramatic increase in eGFP expression in differentiated muscle cells transfected with nanocarriers containing a muscle-targeting domain.

[0530] Nanoparticles coated with Muscle-G1-EEEE dendrimers at different molar ratios, such as 2.72:1 and 2.06:1 (dendrimer to mRNA), demonstrated improved mRNA transfection in differentiated C2c12 cells compared to nanoparticles coated with dendrimers lacking a targeting domain. Muscle-G1-EEEE coated at 2.72:1 had a PDI of 0.14, a zeta potential of -44.0 mV, and a size of 172 nm. Muscle-G1-EEEE coated at 2.06:1 had a PDI of 0.15, a zeta potential of -38.7 mV, and a size of 176 nm. GSGGSGGSCG1-EEEE coated at 2.72:1 had a PDI of 0.13, a zeta potential of -35.4 mV, and a size of 169 nm. GSGGSGGSCG1-EEEE coated at 2.06:1 had a PDI of 0.12, a zeta potential of -33.2 mV, and a size of 166 nm. Uncoated particles had a PDI of 0.21, a zeta potential of +37.1, and a size of 190 nm.

[0531] Example 18 - Tumor-targeted nanocarriers

[0532] A549 is a cancer cell line that highly expresses integrins (Guo et al., 2009). A549 cells were transfected with dendritic PGA-coated nanocarriers containing mRNA expressing eGFP, with or without the integrin targeting domain ACDCRGDCFCG (SEQ ID NO: 5). The nanocarriers also GSCThe dendrimer contained G1,2-RHL (N:P=0.6:1 relative to mRNA) and DOTMA / DOPE (w / w=10:1 relative to mRNA). Molar equivalents of dendritic PGA (with or without integrin targeting domain) were used. Cells were harvested 24 hours after transfection. Control: Cells were not transfected with mRNA or were transfected with mRNA only. The dendrimer with the integrin targeting domain was (Ac-EEEE)2KGSGGSGGSACDCRGDCFCG-NH2 (disulfide bridges: C1-C4, C2-C3). The dendrimer without the integrin targeting domain was (Ac-EEEE)2KGSGGSGGSC-NH2 ("GSGGSGGSCG1-EEEE").

[0533] Methods: First, an initial solution of GSCG1,2-RHL and DOTMA:DOPE nanoparticles in 25 mM HEPES buffer (150 μg / mL mRNA) was prepared as follows: In a sterile polypropylene tube, add 10 mg / mL of peptide (RHL)4(KRHL)2KGSC-NH2 (4912 μg / mol with TFA counterion, 10 N per peptide) (4.240 μL, 42.4 μg, 8.632 × 10 -6 millimole, 8.632 x 10 -5 In a second tube, 1 mg / mL CleanCap® EGFP mRNA (5 moU) (46.44 uL, 46.44 μg, 1.407 x 10) in 1 mM sodium citrate (pH 6.4) was added and mixed. -4 In a third tube, 25 mM (17.68 mg / mL) DOTMA:DOPE liposomes (1:1 molar ratio) in water (16.27 uL, 287.7 μg, 3.287 × 10 -4 43.20 uL of peptide mixture (7.847 x 10 mmol), sterile water (109.2 uL) and 200 mM HEPES buffer (19.34 uL) were added and mixed. -6millimole, 7.847 x 10 -5 100.8 uL of the mRNA mixture (1.309 x 10) was transferred to a new polypropylene tube. -4 144.0 uL of liposome mixture (3.057 x 10 mmol) was added to the tube containing the peptide and mixed quickly by pipetting up and down. The solution was allowed to incubate for 2 minutes. -4 100 millimolar N) was added to the tube containing the peptide and mRNA and mixed rapidly by pipetting up and down to obtain a turbid solution. DLS measurements (40-fold dilution in 25 mM HEPES buffer): 184 nm, PdI 0.13). Zeta potential measurements (3-fold dilution in 25 mM HEPES, then further diluted 60-fold in water): +33.5 mV.

[0534] Coating stock solution:

[0535] Int-GSGGSGGSCG1-EEEE(Ac-EEEE)2KGSGGSGGSACDCRGDCFCG (disulfide bridge between C1 and C4 and between C2 and C3, 3081 g / mol, 8 negative charges per peptide). 2.00 mg was dissolved in 133.3 μL of 50 mM ammonium carbonate solution, followed by 266.7 μL of 25 mM HEPES buffer to give a final concentration of 5.0 mg / mL. The tubes were centrifuged at 11,000 g for 3 minutes, aliquoted, and stored at -80°C.

[0536] GSGGSGGSCG1-EEEE ((Ac-EEEE)2KGSGGSGGSC-NH2, 1912 g / mol, 8 negative charges per peptide). 10 mg was dissolved in 156.2 uL of 50 mM ammonium carbonate solution, followed by 468.8 uL of 25 mM HEPES buffer to give a final concentration of 16.0 mg / mL. Tubes were centrifuged at 11,000 g for 3 minutes, aliquoted, and stored at -80°C.

[0537] Coated nanoparticles:

[0538] The base nanoparticles described above were diluted 2-fold in 25 mM HEPES to an mRNA concentration of 75 μg / mL. 134.0 μL (10.05 μg mRNA, 3.045 x 10 -5 To millimolar amounts of these nanoparticles (P), 67.0 uL of coating material dissolved in 25 mM HEPES was added and mixed quickly by pipetting up and down. The final mRNA concentration of the solution was 50 ug / mL. The coating breakdown is provided in the table below:

[0539] [Table 16]

[0540] FIG. 28 shows a dramatic increase in eGFP expression in A549 cells transfected with nanocarriers containing tumor-targeting integrin-binding domains.

[0541] Example 19 - Nanocarriers targeted via antibody-conjugated dendrimers

[0542] 10 mg / mL of GSEGSEGSEC(OH)G1-(Ac)ESGESGESG ([(Ac-ESGESGESG)2K]GSEGSEGSEC, 2793 g / mol, 9 negative charges per peptide) in water (180 μg, 6.446 × 10 -5 millimole, 5.801 x 10 -4 To a 500 uL polypropylene tube was added 2 mg / mL of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC.HCl, 191.7 g / mol) in water (6.80 uL, 13.59 uL, 7.090 x 10 -5 2 mg / mL N-hydroxysulfosuccinimide sodium salt (Sulfo-NHS, 217.1 g / mol) (8.47 uL, 16.94 uL, 7.799 x 10 mmol) in water was added and the solution was allowed to incubate at room temperature for 2 minutes. -5InVivoMAb anti-human CD3 (BioXCell BE0001-2, 150 kDa) at 7.19 mg / mL in PBS (pH 7.0) (134.5 uL, 967 ug, 6.446 x 10 -6 1000 mM PEG-1000 sucrose (0.5 mmol) was added and the solution was allowed to incubate at room temperature for 4 hours. 182.2 μL of PBS was added and 350 μL of the solution was dialyzed overnight at 4° C. in 500 mL of PBS (MWCO 50 kDa). The resulting conjugate had a theoretical concentration of 3.373 mg / mL in PBS (0.529 mg / mL of GSEGSEGSEC(OH)G1-(Ac)ESGESGESG peptide, 2.844 mg / mL of antibody).

[0543] An initial solution of lipid nanoparticles containing mRNA is prepared and then coated with antibody-conjugated dendrimers by mixing them in a volume ratio of, for example, 2:1. Increasing the coating material decreases the zeta potential, indicating coating of the nanoparticles.

[0544] Example 20 - T cell-targeted nanocarriers comprising CD3-binding antibodies

[0545] Nanocarriers coated with human CD3-targeting antibodies improve human T cell-targeted mRNA delivery. Jurkat cells were either untransfected or transfected with the following compositions:

[0546] (1) eGFP-expressing mRNA only, (2) with mRNA GSC G1,2-RHL (N:P=0.6 relative to mRNA) and DOTMA / DOPE (w / w=10 relative to mRNA) (control for (4) and (7)), (3) coated with 1 equivalent of isotype antibody control (ITC) conjugated dendrimer with mRNA. GSC G1,2-RHL (N:P=0.6 relative to mRNA) and DOTMA / DOPE (w / w=10 relative to mRNA) (control for (4)), (4) coated with 1 equivalent of anti-CD3 antibody-conjugated dendrimer and containing mRNA. GSCG1,2-RHL (N:P = 0.6 relative to mRNA) and DOTMA / DOPE (w / w = 10 relative to mRNA), (5) coated with dendrimer alone, carrying mRNA. GSC G1,2-RHL (N:P=0.6 relative to mRNA) and DOTMA / DOPE (w / w=10 relative to mRNA) (control for (7)), (6) coated with 3 equivalents of isotype antibody control (ITC) conjugated dendrimer with mRNA. GSC G1,2-RHL (N:P=0.6 relative to mRNA) and DOTMA / DOPE (w / w=10 relative to mRNA) (control for (7)), (7) coated with 3 equivalents of anti-CD3 antibody-conjugated dendrimer and carrying mRNA. GSC G1,2-RHL (N:P=0.6 relative to mRNA) and DOTMA / DOPE (w / w=10 relative to mRNA). D / D indicates DOTMA:DOPE (w / w=10:1 relative to mRNA).

[0547] T cells were transfected in 24-well plates with 4.5 μg of mRNA for 2 hours. eGFP expression was analyzed by cytometry 24 hours after transfection of the formulations. Isotype control (ITC): InVivoMAb mouse IgG2a isotype control (BioXCell BE0085, C1.18.4 clone).

[0548] Example 21 - In vitro delivery of multiple nucleic acids in a single formulation to cells

[0549] HeLa cells were transfected with a formulation containing two nucleic acids selected from the following: a first mRNA expressing eGFP, a second mRNA expressing mCherry, and a third mRNA expressing luciferase. The nanocarrier also contained DOTMA / DOPE (w / w = 10:1 relative to the mRNA). GSCG1,2-RHL (N:P = 0.6:1 relative to mRNA) was also included. The mRNA molecules were mRNA expressing eGFP and mRNA expressing mCherry or mRNA expressing luciferase (Fluc). 24 hours after transfection, cells were harvested and fluorescent protein expression was analyzed by flow cytometry. The eGFP+Fluc mRNA alone or with nanocarriers consisted of a 1:1 w / w ratio of eGFP mRNA to Fluc mRNA, the mCherry+Fluc mRNA alone or with nanocarriers consisted of a 1:1 w / w ratio of mCherry mRNA to Fluc mRNA, and the eGFP+mCherry mRNA alone or with nanocarriers consisted of a 1:1 w / w ratio of eGFP mRNA to mCherry mRNA. Figure 30 shows that nanocarriers can encapsulate and deliver more than one mRNA for functional protein expression.

[0550] [Table 17]

[0551] Example 22 - In vivo delivery of multiple nucleic acids to a subject in a single formulation

[0552] Has mRNA GSC Nanocarriers containing G1,2-RHL (N:P = 0.6:1 for mRNA), DOTMA:DOPE (10:1 for mRNA) were prepared as described. CpG, DOTMA:DOPE (10:1 for mRNA and CpG), and mRNA were prepared as described. GSC To prepare nanocarriers for G1,2-RHL (N:P=0.6:1 relative to mRNA), mRNA and CpG were first mixed at a ratio of 9:1 (w / w), mRNA to CpG. GSC G1,2-RHL (N:P = 0.6:1) was added, followed by the addition of DOTMA:DOPE (10:1 relative to mRNA and CpG). The CpG used was CpG ODN 2006 (CpG7909) purchased from InvivoGen.

[0553] Luciferase expression in mouse tissues after intravenous administration of a composition containing DOTMA / DOPE (w / w = 10:1 relative to mRNA), luciferase-expressing mRNA, and GSCG1,2-RHL (N:P = 0.6:1 relative to mRNA) with CpG molecules containing DOTMA / DOPE (w / w = 10:1 relative to nucleic acid). CpG oligonucleotides (ODNs) are synthetic ODNs containing unmethylated CpG dinucleotides (CpG motifs). Treatment with mRNA alone served as a control. 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 (fat), and brain.

[0554] FIG. 31 shows that high levels of luciferase were detected in the lungs and spleen 6 hours after administration of nanocarriers containing luciferase mRNA and CpG (white bars).

[0555] [Table 18]

[0556] Example 23 - Functional delivery to primary leukocytes using nanoparticles carrying two nucleic acids

[0557] Bone marrow cells were isolated from BALB / c mice and resuspended at 2–3E+5 cells / ml in complete DC medium (RPMI-1640 + 10% heat-inactivated FBS + 1% P / S + 40 ng / ml mouse GM-CSF (Peprotech)) and seeded onto 10 cm dishes (2–3E+6 cells / dish). Two to three days after seeding, an equal volume of complete DC medium was added. Half-medium changes were performed on days 5–6 and 8 after seeding.

[0558] Transfection and staining: 13-day cultured suspension cells (moDCs) were spun down and resuspended in complete DC medium at a concentration of 5.68E+5 cells / ml and seeded into a 24-well plate format at 2.5E+5 cells / well (i.e., 440 μl / well). The cells were then transfected with (1) DOTMA / DOPE (10:1 w / w ratio relative to mRNA) and eGFP-encoding mRNA. GSC G1,2-RHL (N:P = 0.6:1 relative to mRNA) or (2) DOTMA / DOPE (w / w = 10:1 relative to mRNA), carrying mRNA encoding eGFP and CpG nucleotides. GSC moDCs were transfected with G1,2-RHL (N:P = 0.6:1 relative to mRNA) for 2, 4, or 22 hours. Transfection efficiency (% eGFP+) of moDCs was measured using flow cytometry 22–24 hours after the start of transfection. moDCs were distinguished by the lack of surface expression of CD11c and F4 / 80 (macrophage marker). D / D refers to DOTMA:DOPE (w / w = 10:1 relative to mRNA).

[0559] The cells were then spun down, resuspended in 1 ml of fresh complete medium, and replated in 24-well plates. The next day, cells were harvested and stained with live / dead aqua (1:250 in PBS) for 30 minutes. After a PBS wash, cells were blocked for 5 minutes with 50 μl / sample of 2x TruStain human FcX block diluted in FACS buffer (1:20, final concentration 1:40). Cells were then stained for 20 minutes with 50 μl / sample of a 2x cocktail of surface marker antibodies diluted in FACS buffer. The final concentrations of these antibodies used were 1:100 CD11c-PerCP-Cy5.5 (Biolegend, 117328), 1:200 CD11b-APC (Biolegend, 101212), and 1:100 F4 / 80-PE (Biolegend, 123110). After washing with FACS buffer, cells were fixed with 4% PFA for 10 minutes. Cells were washed with PBS and resuspended in FACS buffer for flow cytometry analysis. Arc, eGFP, and UltraComp Plus beads were used for compensation. Figure 32 shows that all three transfections achieved similar levels of eGFP expression.

[0560] Example 24 - Functional repolarization of primary macrophages

[0561] The nanocarrier of the present invention was used to deliver modified IRF5, which polarized M2 macrophages to M1 macrophages. Modified IRF5 is a protein with a mutation that functions as an activated form of wild-type IRF5. The nanocarrier formulation used contained DOTMA / DOPE (w / w = 10:1 relative to mRNA) and mRNA expressing modified IRF5. GSC It contained G1,2-RHL (N:P=0.6:1 relative to mRNA).

[0562] Primary murine bone marrow-derived macrophages were polarized to the M2 phenotype, which were then transfected with the nanocarrier formulation for 24 hours.

[0563] The M1 characterization and activation induced by modified IRF5 mRNA was then confirmed as follows. RNA sequencing was performed to assess the underlying gene expression profile. The volcano plot shows the change in gene expression on the x-axis against the -Log10 P-value on the y-axis. P-values were adjusted using the Benjamini-Hochberg method. Fold changes were adjusted using the visualization method described by Love et al., 2014.

[0564] Figure 33A shows that many genes were significantly differentially expressed (DE) relative to the luciferase control. Genes upregulated in activated M1 macrophages (interleukins: Il27, Il12b, Il1b, and the nitric oxide synthesis gene Nos2) or M2 macrophages (Arg1, Cd163, Mrc1) were labeled, indicating significantly upregulated M1 and downregulated M2 genes. Figure 33B shows the results of gene set enrichment analysis (GSEA) performed on the RNA-sequencing dataset to discover a set of DE genes significantly enriched in cells transfected with modified IRF5 mRNA (FDR < 0.05). Cells were highly enriched in genes associated with cell death, cytokine activity, and interleukin production. GSEA was performed in WebGestalt using the Wald statistic ranking input.

[0565] IL12 secretion in primary mouse macrophages transfected with IRF5 using the nanocarrier of the present invention: Primary mouse bone marrow-derived macrophages were polarized to the M2 phenotype. The cells were then transfected for 24 hours with mRNA expressing a modified form of the IRF5 protein that can polarize M2 cells to M1 cells. Cytokine secretion was measured 24 hours after transfection. Control: Cells were transfected with mRNA expressing luciferase (control mRNA). Modified IRF5 is a protein with a mutation that functions as an activated form of WT IRF5. The formulation used was a mixture of DOTMA / DOPE (w / w = 10:1 relative to mRNA) and mRNA. GSCG1,2-RHL (N:P=0.6:1 relative to mRNA). Secretion of IL12p70 and IL12p40 was significantly increased in cells transfected with modified IRF5 mRNA (see Figures 34A and 34B).

[0566] IL12 and TNF secretion in primary human macrophages transfected with IRF5 using the nanocarrier of the present invention: Primary human macrophages were polarized to the M2 phenotype. The cells were then transfected for 24 hours with mRNA expressing a modified form of the IRF5 protein that can polarize M2 cells to M1 cells. Cytokine secretion was measured 24 hours after transfection. Control: Cells were transfected with mRNA expressing luciferase (control mRNA). Modified IRF5 is a protein with a mutation that functions as an activated form of WT IRF5. The formulation used was a mixture of DOTMA / DOPE (w / w = 10:1 relative to mRNA) and mRNA. GSC G1,2-RHL (N:P=0.6:1 relative to mRNA). Secretion of IL12p70 and TNF was significantly increased in cells transfected with modified IRF5 mRNA (see Figures 34C and 34D).

[0567] Methods for cytokine secretion experiments: Mouse (bone marrow-derived) and human (peripheral blood-derived) monocytes were differentiated by cytokine exposure using a standard 6-day protocol. Transfection was performed 6 days after seeding. Approximately 20–24 hours after transfection, the overlay medium was aspirated and replaced with 500 μl / well of complete BMDM medium (mouse) or ImmunoCult medium (human) containing cytokines (IL4 + M-CSF). 24 hours after medium addition, the medium was collected, spun for 10 minutes, and transferred to a series of 96-well plates stored in a -80°C freezer. Meanwhile, cells were washed with PBS and lysed with 200 μl / well of RIPA buffer on a shaker for 5 minutes. Lysates were spun for 10 minutes, and the supernatants were subsequently analyzed for cytokine content. Mouse cytokines were analyzed using the LEGENDplex Mouse Macrophage / Microglia Panel (Biolegend, 740845). Human cytokines were analyzed using the LEGENDplex Human Macrophage / Microglia Panel (Biolegend, 740503). Lysates were analyzed using the Pierce BCA assay (ThermoFisher, 23225) to determine the total protein content of each well.

[0568] Example 25 - In vivo cancer treatment

[0569] MC38 tumor-bearing mice were treated with vehicle control or nanocarriers of the invention delivering therapeutic mRNA, which expresses a modified (activated) IRF5 protein.

[0570] Mice were treated with 1.75 mg / kg of therapeutic mRNA nanocarriers three times a week, starting 9 days after tumor implantation.

[0571] As shown in Figure 35, significant inhibition of tumor growth was observed after treatment with nanocarriers delivering therapeutic mRNA (p=0.03, two-way ANOVA).

[0572] The median survival of mice treated with mRNA therapeutics (via the nanocarriers of the present invention) was significantly extended (time to endpoint, 1.5 cm 3 p=0.004, log-rank (Mantel-Cox) test was performed.

[0573] Example 26 - Lipid mixture (ii)

[0574] Jurkat, HeLa and A549 cells were transfected with nanocarriers of the present invention formulated with different lipid components (see Figures 36, 37 and 38, respectively).

[0575] Jurkat cells were transfected with the following formulations:

[0576] (1) DOTMA:DOPE (using a 1:1 ratio of moles of DOTMA to moles of DOPE) GSC G1,2-RHL (N:P=0.6:1 relative to mRNA) (total lipid amount was w / w=10:1 relative to mRNA), (2) LNPs containing ionizable MC3 lipids (LNPs with MC3), (3) LNPs containing ionizable SM-102 lipids (LNPs with SM-102), (4) LNPs with DODAP:DOTAP:DOPE (using 1:1:1 moles of DODAP to moles of DOTAP to moles of DOPE). GSC G1,2-RHL (N:P=0.6:1 relative to mRNA) (total lipid amount was w / w=10:1 relative to mRNA), (5) with DODAP:DOTAP:DOPE:DMG-PEG (using 1:1:1:0.046 moles of DODAP to moles of DOTAP to moles of DOPE to moles of DMG-PEG) GSC G1,2-RHL (N:P=0.6:1 relative to mRNA) (total lipid amount was 10:1 w / w relative to mRNA), (6) with DODAP:DOTMA:DOPE (1:1:1 moles of DODAP to moles of DOTMA to moles of DOPE) GS...

Claims

1. A nanoparticle comprising a peptide dendrimer, a nucleic acid, and a lipid, wherein the peptide dendrimer comprises at least a core peptide sequence, a first branched residue, and two first peptide motifs; the nanoparticles target target cells or tissues, such as bone marrow, lymphatic, muscle, lung cells, CD206+ cells and / or tumor cells, and / or bone marrow, lymphatic, muscle, lung tissue and / or tumor tissue; The nanoparticles are capable of transfecting the target cells in vitro with an efficiency of at least 10%, at least 12.5%, at least 15%, at least 17.5%, at least 20%, at least 25%, at least 30%, 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%, or at least 85%.

2. the nanoparticle comprises a bone marrow, lymph, muscle, lung, CD206+ cell, or tumor cell targeting motif; a. a muscle-targeting motif optionally comprising an ASSLNIA (SEQ ID NO: 1), a PYDQLRH (SEQ ID NO: 2), or a KAMHQMQ (SEQ ID NO: 3) peptide motif; b. an integrin targeting motif, optionally including an RGD or ACDCRGDCFCG (SEQ ID NO: 5) peptide motif; c. a lung targeting motif optionally comprising the peptide sequence CGFECVRQCPERC (SEQ ID NO: 4); d. a mannose receptor targeting motif, optionally a mannose sugar or maltotriose, or e. An antibody or target-binding fragment thereof (optionally, the antibody specifically binds to CD3) f. Lipids linked to bisphosphonate (BP) groups The nanoparticle of claim 1, wherein the nanoparticle is selected from the group consisting of:

3. 3. The nanoparticles of claim 1 or claim 2, wherein the lung cells are selected from alveolar macrophages, ciliated cells, epithelial cells, basal cells, secretory cells, club cells, alveolar cells, fibroblasts, CD206+ cells and / or endothelial cells.

4. 4. The nanoparticle of claim 2, wherein the bone marrow, lymphatic, muscle, lung, CD206+ cell and / or tumor cell targeting motif is attached to a negatively or neutrally charged polymer or lipid.

5. 5. The nanoparticle of claim 4, wherein the polymer or lipid is selected from polyglutamic acid (PGA), poly(acrylic acid), alginic acid, polyethylene glycol (PEG), or cholesteryl hemisuccinate / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, a neutrally charged zwitterionic polymer or lipid, or a glutamic acid-containing peptide.

6. 6. The nanoparticles of claim 4 or claim 5, wherein the polymer is a linear or dendritic PGA.

7. 6. The nanoparticle of claim 4 or claim 5, wherein the polymer is a dendritic glutamic acid-containing peptide.

8. 8. The nanoparticle of any one of claims 2 to 7, wherein the bone marrow, lymphatic, muscle, lung, CD206+ cell or tumor cell targeting motif is covalently attached to the peptide dendrimer, or the nanoparticle further comprises a second peptide comprising the bone marrow, lymphatic, muscle, lung or tumor cell targeting motif.

9. 9. The nanoparticle of claim 8, wherein the myeloid, lymphoid or lung cell targeting motif comprises a mannose sugar, and optionally the mannose sugar is covalently attached to the C-terminus and / or N-terminus of the peptide dendrimer and / or the second peptide.

10. 10. The nanoparticle of claim 9, wherein the mannose sugar is covalently attached to a second peptide comprising polyglutamic acid (PGA).

11. The second peptide comprises a linear PGA comprising 2 to 10, 2 to 25, 2 to 15 or 2 to 100 glutamic acid residues, or the second peptide comprises a branched PGA comprising 2 to 10, 2 to 25, 2 to 15 or 2 to 100 glutamic acid residues in each branch and / or in the core sequence, and optionally the second peptide has the structure (Ac-EEEE) 2 The nanoparticles of claim 10, comprising KGSGGSGGSC[(SS)-α-D-thiomannose].

12. 10. The nanoparticle of any one of the preceding claims, wherein the lipid:nucleic acid weight / weight ratio is from 0.5:1 to 40:1, 1:1 to 30:1, or 2:1 to 25:1, optionally wherein the lipid:nucleic acid weight / weight ratio is at least 10:1 and the nanoparticles are targeted to the spleen and lungs, or wherein the lipid:nucleic acid weight / weight ratio is about 23:1 and the nanoparticles are targeted to the lungs and spleen.

13. 10. The nanoparticle of claim 1, wherein the lipids comprise two lipids, three lipids, four lipids, five lipids, or more than five lipids, and optionally the lipids comprise DOPE, DOTMA, DODAP, DORI, DMG-PEG, and / or DOTAP.

14. The nanoparticle of claim 13 , wherein the lipid comprises DOTMA and DOPE.

15. 15. The nanoparticle of claim 13 or claim 14, wherein the lipid comprises three or four lipid components, each lipid component being selected from the group comprising DOPE, DOTMA, DODAP, DORI, DMG-PEG and DOTAP.

16. 16. The nanoparticle of claim 13 or claim 15, wherein the lipid comprises DODAP, DOTMA and DOPE, or the lipid comprises DODAP, DOTAP and DOPE, or the lipid comprises DODAP, DOTMA, DOPE and DMG-PEG, or the lipid comprises DODAP, DOTAP, DOPE and DMG-PEG.

17. the peptide dendrimer further comprises two second branched residues and four second peptide motifs; 10. The nanoparticle of claim 9, wherein one of the second branched residues is covalently bound to one of the first peptide motifs and the other second branched residue is covalently bound to the other first peptide motif, and each second branched residue is covalently bound to two second peptide motifs.

18. the peptide dendrimer further comprises at least four third branch residues and eight third peptide motifs; 18. The nanoparticle of claim 17, wherein each second peptide motif is 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.

19. 10. The nanoparticle of claim 1, wherein the peptide dendrimer is selected from Table 1 or Table 1B.

20. 10. The nanoparticle of any one of the preceding claims, wherein the peptide dendrimer has a structure selected from G1,2-RL, 3-LR; G1,2-R; G1-RL, 2-LR; G1,2-RHL; G1-LRLR; G1,2-RF, 3-HL; G1-R; GSCG1,2-RL, 3-LR; RHCG1,2-R; RHCG1-RL, 2-LR; GSCG1,2-RHL; GSCG1-LRLR; GSCG1,2-RF, 3-HL; or GSCG1-R.

21. The nucleic acid a. DNA, optionally antisense oligonucleotides, ssDNA, dsDNA, plasmids, cDNA and / or CpG molecules, or b. RNA, optionally mRNA, circRNA, ssRNA, dsRNA, sgRNA, crRNA, tracrRNA, lncRNA, siRNA, saRNA and / or self-amplifying RNA 10. The nanoparticle of any one of the preceding claims, wherein:

22. 10. The nanoparticle of any one of the preceding claims, wherein the nucleic acid encodes a chimeric antigen receptor (CAR) and / or a transcription factor.

23. 23. The nanoparticle of claim 22, wherein the CAR specifically binds to carcinoembryonic antigen (CEA) or CEA cell adhesion molecule 5 (CEACAM5) or CEA cell adhesion molecule 7 (CEACAM7).

24. 23. The nanoparticle of claim 22, wherein the transcription factor is selected from interferon regulatory factor 5 (IRF5), activated IRF5, inhibitor of nuclear factor kappa B kinase subunit beta (IKK2), and CCAAT enhancer binding protein alpha (CEBPA).

25. Nanoparticles according to any one of claims 1 to 24 for use in medicine.

26. Nanoparticles according to any one of claims 1 to 24 for use in a method for treating cancer, an autoimmune disease, an immune cell-related disease, a lung disease and / or a myopathy.

27. 27. The nanoparticle for use according to claim 26, wherein the cancer comprises a solid tumor.

28. 28. The nanoparticles for use according to claim 26 or claim 27, wherein the cancer is lung cancer, such as NSCLC or small cell lung cancer (SCLC), liver cancer, prostate cancer, thyroid cancer, pancreatic cancer such as pancreatic ductal adenocarcinoma (PDAC), acute myeloid lymphoma (AML), myelodysplastic syndrome (MDS), colorectal cancer such as WT KRAS CRC, KRAS mutant metastatic tumors, hematological tumors, esophageal cancer, breast cancer, prostate cancer, bladder cancer, tumors of the GI tract, head and neck squamous cell carcinoma (HNSCC), kidney cancer, myelofibrosis, CD206+ cancer, melanoma, prostate cancer or anal cancer.

29. 27. The nanoparticles for use according to claim 26, wherein the autoimmune disease is selected from multiple sclerosis (MS), type I or type II diabetes, rheumatoid arthritis, Crohn's disease, uveitis, inflammatory bowel disease or systemic lupus erythematosus, or the immune-related disease is Gaucher's disease, graft-versus-host disease, allograft rejection, acute rejection after transplantation, chronic rejection after transplantation, primary graft dysfunction or chronic granulomatous disease (CGD).

30. 27. The nanoparticles for use according to claim 26, wherein the lung disease is pulmonary fibrosis such as cystic fibrosis, asthma, tuberculosis (TB), acute lung injury (ALI), idiopathic pulmonary fibrosis, allergic airway disease, chronic obstructive pulmonary disease (COPD), alpha-1 antitrypsin deficiency (AATD), pulmonary arterial hypertension, pulmonary alveolar proteinosis, fibrotic lung disease, chronic lung disease or respiratory tract infection, or the muscle disease is muscular dystrophy or a muscle wasting disease.

31. Nanoparticles according to any one of claims 1 to 24 for use as a vaccine.

32. 1. A method for producing coated nanoparticles capable of transfecting target cells, comprising mixing a solution of peptides or peptide dendrimers with a solution of preformed nanoparticles to form said coated nanoparticles; a) the preformed nanoparticles have a positive surface charge and the peptide dendrimer has a negative net charge; or b) the preformed nanoparticles have a negative surface charge and the peptide or peptide dendrimer has a positive net charge; or c) the surface of the preformed nanoparticles is uncharged and the peptide or peptide dendrimer comprises a hydrophobic region; or d) the preformed nanoparticles have a positive surface charge and the peptide comprises more than 2 and less than 100 amino acid residues.

33. 32(a), wherein the peptide dendrimer comprises a PGA or a glutamic acid-containing peptide comprising a glutamic acid-rich domain comprising a total of at least 4, at least 6, or at least 8 amino acid residues, at least 2 of which are glutamic acid, and at least 20% of the amino acid residues in the glutamic acid-rich domain are glutamic acid; or 31(d), wherein the peptide comprises a linear PGA comprising more than 2 and less than 100 glutamic acid residues.

34. the peptide dendrimer comprises a myeloid, lymph, muscle, lung, CD206+ cell, tumor cell, bone marrow, pancreas, nervous tissue, kidney tissue, heart tissue, liver tissue, eye, joint or prostate, or stem cell, pancreatic cell, nervous cell, kidney cell, heart cell, liver cell, eye cell, synovial cell or prostate cell targeting motif; a. a muscle-targeting motif optionally comprising an ASSLNIA (SEQ ID NO: 1), a PYDQLRH (SEQ ID NO: 2), or a KAMHQMQ (SEQ ID NO: 3) peptide motif; b. an integrin targeting motif, optionally comprising an RGD or ACDCRGDCFCG (SEQ ID NO: 5) peptide motif; c. a lung targeting motif optionally comprising the peptide sequence CGFECVRQCPERC (SEQ ID NO: 4); d. a mannose receptor targeting motif, optionally a mannose sugar or maltotriose; e. an antibody or target-binding fragment thereof (optionally, the antibody specifically binds to CD3); f. A bone marrow or stem cell targeting motif, optionally comprising an antibody that specifically binds to CD34 and / or a lipid containing a bisphosphonate (BP) group; g. A pancreatic targeting motif optionally comprising a glucagon-like peptide-1 (GLP-1) homologue, such as exendin-4 (SEQ ID NO: 9), or a variant or binding fragment thereof; h.Optionally (KKEEE) 3 a kidney targeting motif containing K, i. a neural tissue or cell targeting motif, optionally including YTIWMPENPRPGTPCDIFTNSRGKRASNGGGG (SEQ ID NO: 11) or KSVRTWNEIIPSKGCLRVGGRCHPHVNGGG (SEQ ID NO: 12) or a neural targeting peptide containing a Phe-Arg-Trp (FRW) motif; j. a cardiac targeting or cardiac cell targeting motif, optionally including APWHLSSQYSRT (SEQ ID NO: 13); k. a liver-targeting or hepatocyte-targeting motif, optionally including an asialoglycoprotein receptor binder such as an N-acetylgalactosamine (Gal-NAc) sugar, or a vitamin A moiety; l. an eye or eye cell targeting motif, optionally including CARSKNKDC (SEQ ID NO: 14); m. a joint or synovial cell targeting motif, optionally including an antibody that specifically binds to CD44 and / or a hyaluronan glycoprotein; or n. A prostate targeting motif optionally comprising PKRGFQD (SEQ ID NO: 15) or SNTRVAP (SEQ ID NO: 16) 34. The method of claim 32 or 33, wherein the method is selected from the group consisting of:

35. Coated nanoparticles capable of transfecting target cells, said coated nanoparticles comprising a peptide on their surface; a) the nanoparticles are nanoparticles according to any one of claims 1 to 24, and / or b) the peptide is a linear PGA containing more than 2 and less than 100 glutamic acid residues, or the peptide is a branched PGA; and / or c) the peptide is a glutamic acid-containing peptide comprising a glutamic acid-rich domain comprising a total of at least 4, at least 6, or at least 8 amino acid residues, at least 2 of which are glutamic acid, and at least 20% of the amino acid residues in the glutamic acid-rich domain are glutamic acid; and / or d) the nanoparticles are coated with a dendrimer selected from Table 1 or Table 1B; or e) The coated nanoparticles, wherein the nanoparticles are coated with a peptide selected from Table 1A, wherein the peptide is not E100.

36. the PGA and / or glutamic acid-containing peptide comprises a bone marrow, lymph, muscle, lung, CD206+ cell, tumor cell, nervous tissue, kidney tissue, heart tissue, neuronal cell, kidney cell, or cardiac cell targeting motif; a. a muscle-targeting motif optionally comprising an ASSLNIA (SEQ ID NO: 1), a PYDQLRH (SEQ ID NO: 2), or a KAMHQMQ (SEQ ID NO: 3) peptide motif; b. an integrin targeting motif, optionally comprising an RGD or ACDCRGDCFCG (SEQ ID NO: 5) peptide motif; c. a lung targeting motif optionally comprising the peptide sequence CGFECVRQCPERC (SEQ ID NO: 4); d. a mannose receptor targeting motif, optionally a mannose sugar or maltotriose; e. an antibody or target-binding fragment thereof (optionally, the antibody specifically binds to CD3); f. A bone marrow or stem cell targeting motif, optionally comprising an antibody that specifically binds to CD34 and / or a lipid containing a bisphosphonate (BP) group; g. A pancreatic targeting motif optionally comprising a glucagon-like peptide-1 (GLP-1) homologue, such as exendin-4 (SEQ ID NO: 9), or a variant or binding fragment thereof; h.Optionally (KKEEE) 3 a kidney targeting motif containing K, i. a neural tissue or cell targeting motif, optionally including YTIWMPENPRPGTPCDIFTNSRGKRASNGGGG (SEQ ID NO: 11) or KSVRTWNEIIPSKGCLRVGGRCHPHVNGGG (SEQ ID NO: 12) or a neural targeting peptide containing a Phe-Arg-Trp (FRW) motif; j. a cardiac targeting or cardiac cell targeting motif, optionally including APWHLSSQYSRT (SEQ ID NO: 13); k. a liver-targeting or hepatocyte-targeting motif, optionally including an asialoglycoprotein receptor binder such as an N-acetylgalactosamine (Gal-NAc) sugar, or a vitamin A moiety; l. an eye or eye cell targeting motif, optionally including CARSKNKDC (SEQ ID NO: 14); m. a joint or synovial cell targeting motif, optionally including an antibody that specifically binds to CD44 and / or a hyaluronan glycoprotein; or n. A prostate targeting motif optionally comprising PKRGFQD (SEQ ID NO: 15) or SNTRVAP (SEQ ID NO: 16) 35(b), (c), (d) or (e) of the coated nanoparticle of claim 35, optionally selected from:

37. A composition comprising nanoparticles comprising a first peptide dendrimer, a second peptide dendrimer, a nucleic acid, and a lipid, wherein the first and second peptide dendrimers comprise at least a core peptide sequence, a first branching residue, and two first peptide motifs.

38. 38. The composition of claim 37, wherein the first peptide dendrimer is a first generation peptide dendrimer comprising a core peptide sequence, a first branched residue, and two first peptide motifs.

39. 39. The composition of claim 37 or claim 38, wherein the second peptide dendrimer further comprises at least two second branch residues and four second peptide motifs, one of the second branch residues covalently bonded to one of the first peptide motifs and another second branch residue covalently bonded to another first peptide motif, and each second branch residue covalently bonded to two second peptide motifs.

40. 40. The composition of any one of claims 37 to 39, wherein the second peptide dendrimer further comprises at least four third branch residues 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.

41. 41. The composition of any one of claims 37 to 40, wherein the branching residues of the first and second peptide dendrimers are independently selected from lysine, 2,4-diaminobutyric acid, ornithine, or diaminopropionic acid.

42. 42. The composition of any one of claims 37 to 41, wherein the peptide motifs of the first and second peptide dendrimers independently consist of a single amino acid, a dipeptide, a tripeptide, or a tetrapeptide motif.

43. 43. The composition of any one of claims 37-42, wherein the first and second peptide dendrimer peptide motifs independently comprise amino acids having basic side chains, non-polar side chains, acidic side chains, and / or uncharged polar side chains.

44. 44. The composition of any one of claims 37-43, wherein the first and second peptide dendrimer peptide motifs independently comprise leucine (L), arginine (R) and / or histidine (H) residues.

45. 45. The composition of any one of claims 37 to 44, wherein the core sequence of the first and / or second peptide dendrimer comprises at least two amino acids.

46. 46. The composition of any one of claims 37 to 45, wherein the core sequence of the first and / or second peptide dendrimer comprises a maximum of 30 amino acids.

47. 47. The composition of any one of claims 37 to 46, wherein the first and / or second peptide dendrimer core peptide sequence comprises the peptide sequence RHC, GSA, or GSC.

48. 48. The composition of any one of claims 37 to 47, wherein the core sequence of the first and / or second peptide dendrimer comprises an ionizable amino acid, such as histidine.

49. 49. The composition of any one of claims 37-48, wherein the nanoparticles have a polydispersity index of less than 0.6, 0.55, 0.5, 0.45, 0.4, 0.39, 0.38, 0.37, 0.36, 0.35, 0.34, 0.33, 0.32, 0.31, or 0.

30.

50. 50. The composition of any one of claims 37 to 49, wherein when the first peptide dendrimer is used in a first reference nanoparticle consisting of the first peptide dendrimer, a nucleic acid, and a lipid, the first reference nanoparticle has a PDI that is 0.05 to 0.9, 0.06 to 0.8, 0.07 to 0.6, 0.08 to 0.5, 0.09 to 0.4, 0.1 to 0.3, 0.12 to 0.2, or 0.13 to 0.15 higher than the PDI of a second reference nanoparticle consisting of the second peptide, a nucleic acid, and a lipid.

51. 51. The composition of any one of claims 37 to 50, wherein the molar ratio to nitrogen contributed by each of the first peptide dendrimer and the second peptide dendrimer is between 10:1 and 1:10, between 5:1 and 1:5, between 1:4 and 4:1, between 1:3 and 3:1, or between 1:2 and 2:

1.

52. 52. The composition of any one of claims 37 to 51, wherein the molar ratio of nitrogen contributed by each of the first peptide dendrimer and the second peptide dendrimer is 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, or 4:

1.

53. 53. The composition of any one of claims 37-52, wherein the first peptide dendrimer comprises a structure set forth in Table 7 and / or the second peptide dendrimer comprises a structure set forth in Table 8.

54. 54. The composition of any one of claims 37 to 53, wherein the first peptide dendrimer comprises the structure G1-LRLR; G1-R; G1,2-R; G1-RLR; GSCG1-LRLR; RHCG1-R; RHCG1,2-R; or RHCG1-RLR, and / or the second peptide dendrimer comprises the structure G1,2-RL,3-LR; G1-RL,2-LR; GSCG1,2-RL,3-LR; or RHCG1-RL,2-LR.

55. The nucleic acid a. DNA, optionally antisense oligonucleotides, ssDNA, dsDNA, plasmids, CpG molecules and / or cDNA, or b. RNA, optionally mRNA, circRNA, ssRNA, dsRNA, sgRNA, crRNA, tracrRNA, lncRNA, siRNA, saRNA and / or self-amplifying RNA The composition of any one of claims 37 to 54, selected from:

56. 56. The composition of any one of claims 37 to 55, wherein the nucleic acid encodes a protein or peptide, optionally wherein the protein or peptide comprises a chimeric antigen receptor (CAR), a transcription factor, 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.

57. 57. The composition of any one of claims 37-56, wherein delivery of said nucleic acid to a tissue or cell is increased by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 85%, 90%, 95%, or 100% compared to delivery of the same nucleic acid to the same tissue or cell type using a composition comprising only a lipid, a nucleic acid, and either the first or second peptide dendrimer.

58. A composition according to any one of claims 37 to 57 for use in medicine.

59. A composition according to any one of claims 37 to 57 for use as a vaccine.

60. 58. The composition of any one of claims 37 to 57 for use in a method for treating cancer, an autoimmune disease and / or a lung disease.

61. 61. The composition for use of claim 60, wherein the cancer is a solid tumor, lung cancer, such as NSCLC or small cell lung cancer (SCLC), liver cancer, prostate cancer, thyroid cancer, pancreatic cancer such as pancreatic ductal adenocarcinoma (PDAC), acute myeloid lymphoma (AML), myelodysplastic syndrome (MDS), colorectal cancer such as WT KRAS CRC, KRAS mutant metastatic tumors, blood tumors, esophageal cancer, breast cancer, prostate cancer, bladder cancer, tumors of the GI tract, head and neck squamous cell carcinoma (HNSCC), kidney cancer, CD206+ cancer, melanoma, prostate cancer or anal cancer.

62. 61. The composition for use of claim 60, wherein the autoimmune disease is selected from multiple sclerosis (MS), type I or type II diabetes, rheumatoid arthritis, Crohn's disease, uveitis, or inflammatory bowel disease or systemic lupus erythematosus, or the immune-related disease is Gaucher disease, graft-versus-host disease, allograft rejection, acute rejection after transplantation, chronic rejection after transplantation, primary graft dysfunction or chronic granulomatous disease (CGD), or the pulmonary disease is cystic fibrosis, asthma, tuberculosis (TB), acute lung injury (ALI), pulmonary fibrosis such as idiopathic pulmonary fibrosis, allergic airway disease, chronic obstructive pulmonary disease (COPD), alpha-1 antitrypsin deficiency (AATD), pulmonary arterial hypertension, pulmonary alveolar proteinosis, fibrotic lung disease, chronic lung disease or respiratory tract infection, or the muscle disease is muscular dystrophy or a muscle wasting disease.

63. A nanoparticle comprising a peptide dendrimer, a nucleic acid, and a lipid, wherein the peptide dendrimer comprises at least a core peptide sequence, a first branched residue, and two first peptide motifs; The nanoparticles target bone marrow, pancreas, nerve tissue, kidney tissue, heart tissue, liver tissue, eye, joint or prostate, or stem cells, pancreatic cells, nerve cells, kidney cells, heart cells, liver cells, eye cells, synovial cells or prostate cells.

64. The nanoparticles are a. a bone marrow or stem cell targeting motif, optionally comprising an antibody that specifically binds to CD34, and / or a lipid containing a bisphosphonate (BP) group; b. a pancreatic targeting motif optionally comprising a glucagon-like peptide-1 (GLP-1) homologue, such as exendin-4 (SEQ ID NO: 9), or a variant or binding fragment thereof; c. Arbitrarily (KKEEE) 3 a kidney targeting motif containing K, d. A neural tissue or cell targeting motif, optionally including YTIWMPENPRPGTPCDIFTNSRGKRASNGGGG (SEQ ID NO: 11) or KSVRTWNEIIPSKGCLRVGGRCHPHVNGGG (SEQ ID NO: 12), or a neural targeting peptide containing a Phe-Arg-Trp (FRW) motif; e. a cardiac targeting or cardiac cell targeting motif, optionally including APWHLSSQYSRT (SEQ ID NO: 13); f. a liver-targeting or hepatocyte-targeting motif, optionally including an asialoglycoprotein receptor binder such as an N-acetylgalactosamine (Gal-NAc) sugar, or a vitamin A moiety; g. An eye or eye cell targeting motif, optionally including CARSKNKDC (SEQ ID NO: 14); h. a joint or synovial cell targeting motif, optionally including an antibody that specifically binds to CD44 and / or a hyaluronan glycoprotein; or i. A prostate targeting motif optionally comprising PKRGFQD (SEQ ID NO: 15) or SNTRVAP (SEQ ID NO: 16) 64. The nanoparticle of claim 63, comprising a targeting motif selected from:

65. 65. The nanoparticle of claim 63 or claim 64, wherein the nanoparticle comprises a cell targeting motif that targets the stem cells, pancreatic cells, neural cells, kidney cells, cardiac cells, liver cells, eye cells, synovial cells or prostate cells.

66. 66. The nanoparticle of any one of claims 63 to 65, wherein the nanoparticle is capable of transfecting the target stem cells, pancreatic cells, neuronal cells, kidney cells, cardiac cells, liver cells, eye cells, synovial cells or prostate cells in vitro with an efficiency of at least 10%, at least 12.5%, at least 15%, at least 17.5%, at least 20%, at least 25%, at least 30%, 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%.

67. 67. The nanoparticle of any one of claims 63 to 66, wherein the bone marrow, pancreas, nerve tissue, kidney tissue, heart tissue, liver tissue, eye, joint or prostate, stem cell, pancreatic cell, nerve cell, kidney cell, heart cell, liver cell, eye cell, synovial cell or prostate cell targeting motif is attached to a negatively or neutrally charged polymer or lipid.

68. 68. The nanoparticle of claim 67, wherein the polymer or lipid is selected from polyglutamic acid (PGA), poly(acrylic acid), alginic acid, polyethylene glycol (PEG), or cholesteryl hemisuccinate / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, a neutrally charged zwitterionic polymer or lipid, or a glutamic acid-containing peptide.

69. 69. The nanoparticle of claim 67 or claim 68, wherein the polymer is linear or dendritic PGA.

70. 69. The nanoparticle of claim 67 or claim 68, wherein the polymer is a dendritic glutamic acid-containing peptide.

71. 71. The nanoparticle of any one of claims 63-70, wherein the bone marrow, pancreas, nervous tissue, kidney tissue, heart tissue, liver tissue, eye, joint or prostate, stem cell, pancreatic cell, nerve cell, kidney cell, heart cell, liver cell, eye cell, synovial cell or prostate cell targeting motif is covalently attached to the peptide dendrimer, or the nanoparticle further comprises a second peptide comprising the bone marrow, pancreas, nervous tissue, kidney tissue, heart tissue, liver tissue, eye, joint or prostate, stem cell, pancreatic cell, nerve cell, kidney cell, heart cell, liver cell, eye cell, synovial cell or prostate cell targeting motif.

72. The second peptide comprises a linear PGA comprising 2 to 10, 2 to 25, 2 to 15 or 2 to 100 glutamic acid residues, or the second peptide comprises a branched PGA comprising 2 to 10, 2 to 25, 2 to 15 or 2 to 100 glutamic acid residues in each branch and / or in the core sequence, and optionally the second peptide has the structure (Ac-EEEE) 2 72. The nanoparticle of claim 71, comprising KGSGGSGGSC[(SS)-α-D-thiomannose].

73. 73. The nanoparticle of any one of claims 63-72, wherein the lipid:nucleic acid weight / weight ratio is between 0.5:1 and 40:1, 1:1 and 30:1, or 2:1 and 25:1, optionally wherein the lipid:nucleic acid weight / weight ratio is at least 10:1 and the nanoparticle is targeted to the spleen and lung, or wherein the lipid:nucleic acid weight / weight ratio is about 23:1 and the nanoparticle is targeted to the lung and spleen.

74. 74. The nanoparticle of any one of claims 63 to 73, wherein the lipids comprise two lipids, three lipids, four lipids, five lipids, or more than five lipids, optionally wherein the lipids comprise DOPE, DOTMA, DODAP, DORI, DMG-PEG, and / or DOTAP.

75. 75. The nanoparticle of claim 74, wherein the lipid comprises DOTMA and DOPE.

76. 76. The nanoparticle of claim 74 or claim 75, wherein the lipid comprises three lipid components or four lipid components, each lipid component being selected from the group comprising DOPE, DOTMA, DODAP, DORI, DMG-PEG and DOTAP.

77. 77. The nanoparticle of claim 75 or claim 76, wherein the lipid comprises DODAP, DOTMA and DOPE, or the lipid comprises DODAP, DOTAP and DOPE, or the lipid comprises DODAP, DOTMA, DOPE and DMG-PEG, or the lipid comprises DODAP, DOTAP, DOPE and DMG-PEG.

78. the peptide dendrimer further comprises two second branched residues and four second peptide motifs; 78. The nanoparticle of any one of claims 63 to 77, wherein one of the second branched residues is covalently bound to one of the first peptide motifs and the other second branched residue is covalently bound to the other first peptide motif, and each second branched residue is covalently bound to two second peptide motifs.

79. the peptide dendrimer further comprises at least four third branch residues and eight third peptide motifs; 79. The nanoparticle of claim 78, wherein each second peptide motif is 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.

80. 10. The nanoparticle of any one of the preceding claims, wherein the peptide dendrimer is selected from Table 1 or Table 1B, or the nanoparticle is coated with a peptide selected from Table 1A.

81. 81. The nanoparticle of any one of claims 63 to 80, wherein the peptide dendrimer has a structure selected from G1,2-RL, 3-LR; G1,2-R; G1-RL, 2-LR; G1,2-RHL; G1-LRLR; G1,2-RF, 3-HL; G1-R; GSCG1,2-RL, 3-LR; RHCG1,2-R; RHCG1-RL, 2-LR; GSCG1,2-RHL; GSCG1-LRLR; GSCG1,2-RF, 3-HL; or GSCG1-R.

82. The nucleic acid a. DNA, optionally antisense oligonucleotides, ssDNA, dsDNA, plasmids, cDNA and / or CpG molecules, or b. RNA, optionally mRNA, circRNA, ssRNA, dsRNA, sgRNA, crRNA, tracrRNA, lncRNA, siRNA, saRNA and / or self-amplifying RNA The nanoparticles according to any one of claims 63 to 81, wherein

83. The nanoparticle of any one of claims 63 to 82, wherein the nucleic acid encodes a chimeric antigen receptor (CAR) and / or a transcription factor.

84. The nanoparticle of claim 83, wherein the CAR specifically binds to carcinoembryonic antigen (CEA) or CEA cell adhesion molecule 5 (CEACAM5) or CEA cell adhesion molecule 7 (CEACAM7).

85. 84. The nanoparticle of claim 83, wherein the transcription factor is selected from interferon regulatory factor 5 (IRF5), activated IRF5, inhibitor of nuclear factor kappa B kinase subunit beta (IKK2), and CCAAT enhancer binding protein alpha (CEBPA).

86. Nanoparticles according to any one of claims 63 to 85 for use in medicine.

87. Nanoparticles according to any one of claims 63 to 85 for use as a vaccine.

88. 86. Nanoparticles according to any one of claims 63 to 85 for use in a method for treating cancer, an autoimmune disease, an immune cell related disease, a lung disease and / or a myopathy.

89. 89. The nanoparticle for use according to claim 88, wherein the cancer comprises a solid tumor.

90. 90. The nanoparticles for use according to claim 88 or claim 89, wherein the cancer is lung cancer, such as NSCLC or small cell lung cancer (SCLC), liver cancer, prostate cancer, thyroid cancer, pancreatic cancer such as pancreatic ductal adenocarcinoma (PDAC), acute myeloid lymphoma (AML), myelodysplastic syndrome (MDS), colorectal cancer such as WT KRAS CRC, KRAS mutant metastatic tumors, hematological tumors, esophageal cancer, breast cancer, prostate cancer, bladder cancer, tumors of the GI tract, head and neck squamous cell carcinoma (HNSCC), kidney cancer, myelofibrosis, CD206+ cancer, melanoma, prostate cancer or anal cancer.

91. 89. The nanoparticles for use according to claim 88, wherein the autoimmune disease is selected from multiple sclerosis (MS), type I or type II diabetes, rheumatoid arthritis, Crohn's disease, uveitis, or inflammatory bowel disease, or the immune-related disease is Gaucher's disease, graft-versus-host disease, allograft rejection, acute rejection after transplantation, chronic rejection after transplantation, primary graft dysfunction, or chronic granulomatous disease (CGD).

92. 89. The nanoparticle for use according to claim 88, wherein the lung disease is cystic fibrosis, asthma, tuberculosis (TB), acute lung injury (ALI), pulmonary fibrosis such as idiopathic pulmonary fibrosis, allergic airway disease, chronic obstructive pulmonary disease (COPD), alpha-1 antitrypsin deficiency (AATD), pulmonary arterial hypertension, pulmonary alveolar proteinosis, fibrotic lung disease, chronic lung disease or respiratory tract infection, or the muscle disease is muscular dystrophy or a muscle wasting disease.

Images