Hybrid peptide dendrimer systems and extrahepatic delivery

Abstract

The present invention relates to compositions that can deliver therapeutic molecules, such as nucleic acids, to mammalian cells and to human and animal bodies, and methods of making and using the same. The compositions of the invention are useful in the medical field, such as in the treatment of cancer and autoimmune diseases.

Description

Technical Field

[0001] The present invention relates to compositions that can deliver therapeutic molecules (such as nucleic acids) to mammalian cells and to the human and animal body. Background Art

[0002] Nucleic acid therapeutics are expected to become the next generation of precision medicines that will transform healthcare. However, key challenges remain. One such challenge is the effective and safe delivery of nucleic acid therapeutics to patients. Current viral and non-viral vector platforms often fail in clinical translation due to off-target effects, immune activation, and difficulty in scalable production of the vectors.

[0003] In the context of in vivo delivery of nucleic acids, viral-derived vectors have been extensively studied, and some are clinically advanced, including adeno-associated virus (AAV) (Sheridan et al., 2011 and Wang et al., 2019). However, the use of these systems is limited to the delivery of DNA <5 kb and cannot transfer RNA or larger DNA. Despite the progress made in these delivery systems, there is still the potential for random insertions and immunotoxicity with the use of these systems. In particular, AAV systems can be highly immunogenic when targeting non-liver tissues that require the use of higher doses. This tendency to produce immune responses to AAV systems also limits the usefulness of the system for repeated dosing, as patients often develop immunity to AAV delivery systems. Finally, AAV delivery systems are known to be expensive and difficult to produce at the scale and good manufacturing practice (GMP) levels required for therapeutic use.

[0004] Non-viral vector systems for delivering nucleic acids to cells and tissues in vivo are also under study. These systems include systems designed to deliver smaller nucleic acids such as siRNA and antisense oligonucleotides (ASOs). One approach that has been explored is a bioconjugated oligonucleotide delivery system in which siRNA or ASO is conjugated to an antibody or ligand (Benizri et al., 2019). However, these methods are limited to gene silencing or exon skipping and cannot be used to express genes in target tissues. Bioconjugate-based delivery systems are challenging to apply to large genetic payloads such as plasmid DNA and mRNA because each of them is a large negatively charged molecule that cannot easily pass through the negatively charged plasma membrane of the cell.

[0005] To address the difficulty of plasmid DNA and mRNA passing through the plasma membrane, it may be useful to encapsulate nucleic acids and neutralize the charge for effective delivery. Lipid nanoparticles (LNPs) have been used to encapsulate and deliver mRNA, such as in COVID-19 vaccines (Qui et al., 2021), because they contain non-viral delivery vehicles. These systems can be used for vaccine delivery because only a small amount of muscle and immune cells local to the delivery site need to be transfected to train the immune system to fight infection. Therefore, RNA-based COVID-19 vaccines are administered intramuscularly. However, for many diseases, in order to effectively treat the disease, a large number of target cells will need to be transfected. Although the LNPs currently used clinically may be very suitable for targeting the liver when administered intravenously, they are not very suitable for targeting other tissues, so they are not very suitable for diseases related to 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-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (Braum, 2019; Ren et al., 2000).

[0007] To overcome the various shortcomings of the above-mentioned delivery systems, the addition of peptides to lipid-based carriers has been studied. In these hybrid systems, peptides and lipids are associated with nucleic acids to form nanoparticles that can be internalized by cells. The peptide elements can be linear (Kwok et al., 2016) or branched, such as peptide dendrimers (Kwok et al., 2013). These peptide / lipid systems were initially used to study the delivery of plasmid DNA and siRNA under serum-free in vitro conditions that are very different from in vivo conditions. However, it is not clear whether these systems can be used to deliver long nucleic acids in vivo. The only reports show that the use of peptide dendrimer / lipid nanoparticles for in vivo liver delivery of ASOs can only achieve a low increase of 20-30% compared to the ASO 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 inventors recently demonstrated that in vivo delivery of long nucleic acids, such as mRNA, can be achieved using a peptide dendrimer / lipid hybrid system. The inventors developed a framework for nucleic acid delivery using peptide dendrimers as disclosed in WO2022162200, which is incorporated by reference in its entirety. The dendrimers disclosed in WO2022162200 are branched peptides that display one, two, three or four amino acid residues between the "branch residues" that serve as branching units within the dendrimer molecule. WO2022162200 shows that these dendrimer / lipid compositions are unexpectedly effective in delivering larger nucleic acids (e.g., larger than antisense oligonucleotides ASO) to, for example, extrahepatic tissues. Although some smaller nucleic acids such as ASOs are easily absorbed into cells even without a carrier system, the composition of WO2022162200 allows for the effective delivery of larger nucleic acids, such as mRNA.

[0009] Despite the progress made 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 therapies.

[0010] The present invention is designed in view of the above considerations. Summary of the invention

[0011] The development of nucleic acid therapy depends on effective and targeted nucleic acid delivery. The inventors have found that the peptide dendrimer / lipid-based nucleic acid delivery system can be modified to improve the delivery of nucleic acids to specific cells and tissues. The inventors have found that one way to achieve cell and / or tissue-specific nucleic acid delivery is by changing the ratio of peptide dendrimers (also referred to herein as "dendrimers") to nucleic acids in nanoparticles and / or changing the ratio of lipids to nucleic acids. Alternatively, targeted delivery can be achieved by including cell or tissue-specific targeting motifs in nanoparticles. There is no particular limitation on the position of the targeting motif, and it can be, for example, covalently bound to a peptide dendrimer, covalently bound to a second peptide (which may be additionally included in the nanoparticle), or covalently bound to a polymer (which may be additionally included in the nanoparticle).

[0012] The inventors have also found that peptide dendrimer / lipid nanoparticles comprising a combination of two different dendrimers are even more effective in achieving in vitro and in vivo nucleic acid delivery than nanoparticles comprising a single peptide dendrimer. For example, combining a "first generation" peptide dendrimer with a "second" or "third generation" peptide dendrimer in a single composition can allow for more effective delivery of nucleic acids in vitro and in vivo than compositions comprising only a single first, second or third generation peptide dendrimer. This can be achieved, for example, by varying the number of charged and hydrophobic residues in each peptide dendrimer that can form non-covalent 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 the ability to form a monodisperse population of nanoparticles. For example, a nanoparticle can comprise a dendrimer that cannot form a monodisperse population of dendrimer / lipid nanoparticles when used alone (indicating a relatively high dendrimer-nucleic acid dissociation rate) with a second dendrimer that can form a monodisperse population of dendrimer / lipid nanoparticles alone (indicating a relatively low dendrimer-nucleic acid dissociation rate). Peptide dendrimers can therefore be selected based on the polydispersity index of each single dendrimer / lipid nanoparticle.In addition, the nanoparticles may comprise advantageous combinations of lipids as described herein, which may improve transfection efficiency.

[0013] These novel findings further expand the field of nucleic acid therapeutics and offer promising solutions to the outstanding challenges of providing efficient, safe, and targeted vectors for in vivo nucleic acid delivery.

[0014] The present invention can also be used for ex vivo transfection of cells / tissues derived from patients. For example, the nanoparticles of the present invention can be adapted to more effectively deliver nucleic acids to ex vivo myeloid or lymphoid cells. This discovery will be particularly useful in the field of therapeutic drugs based on chimeric antigen receptors (CARs), such as CAR-T and CAR-M for the treatment of various cancers. Embodiments in which, for example, favorable lipid combinations exhibit enhanced transfection efficiency can be applied to in vitro applications.

[0015] Based on the unexpected discovery in WO2022162200 that peptide dendrimer / lipid nanoparticles comprising a single peptide dendrimer can effectively deliver nucleic acid cargo to cells in vivo, it is expected that the current mixed peptide dendrimer / lipid nanoparticles will also effectively deliver nucleic acid cargo to cells in vivo. In addition, based on the improved efficiency of cargo delivery achieved in vitro with mixed dendrimer nanoparticles compared to single dendrimer nanoparticles, it is also expected that the efficiency of delivering nucleic acid cargo to cells in vivo will also be higher than that shown by single dendrimer / lipid nanoparticles. Therefore, taking into account the inventors' findings in WO2022162200, mixed dendrimer nanoparticles can effectively deliver nucleic acids, such as DNA and RNA, to various tissues, particularly to the lungs and immune cell-rich tissues (including spleen, lymph nodes and bone marrow). This enables the development of improved DNA- and RNA-based therapies for extrahepatic tissues.

[0016] The present invention can effectively and specifically deliver nucleic acids, including DNA and mRNA, to various cells and tissues, particularly to myeloid and lymphoid cells and to muscle and lung tissue and cancer cells. Nanoparticles can also be adapted to target other organs and tissues. The ability to deliver nucleic acids (and particularly mRNA) to immune cells and to a greater extent to myeloid cells (including monocytes, macrophages, neutrophils and dendritic cells) enables this technology to be developed for the treatment of all cancer types (including solid tumors and blood cancers, such as myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML), autoimmune diseases (such as diabetes including type I diabetes, rheumatoid arthritis, Crohn's disease, uveitis, inflammatory bowel disease) and other immune cell-related disorders, such as graft-versus-host disease, allogeneic transplant rejection, acute rejection after transplantation, chronic rejection after transplantation, primary graft dysfunction Functional disorders, chronic granulomatous disease (CGD) or Gaucher disease. The present invention also allows the delivery of nucleic acids including DNA and mRNA to the lungs and / or immune cells, making it possible to treat lung-related diseases such as cystic fibrosis and chronic obstructive pulmonary disease. In addition, the present invention allows the delivery of nucleic acids including DNA and mRNA to muscle tissue and muscle cells to treat myopathy, including muscular dystrophy (e.g., Duchenne muscular dystrophy, myotonic dystrophy, facioscapulohumeral muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, oculopharyngeal muscular dystrophy, Emory-Dreyfus muscular dystrophy, hereditary muscular dystrophy, congenital muscular dystrophy and distal muscular dystrophy) and muscle wasting diseases.

[0017] Thus, in one aspect, the present invention provides nanoparticles comprising peptide dendrimers, nucleic acids and lipids. It will be appreciated by the skilled artisan that more than one peptide dendrimer and / or more than one lipid may be included. The peptide dendrimer comprises at least: a core peptide sequence, a first branch residue and two first peptide motifs. The branch residues may be lysine, 2,4-diaminobutyric acid, ornithine or diaminopropionic acid. The nanoparticles may be targeted to specific cell types or tissues, such as myeloid cells, lymphoid cells, muscle cells, lung cells and / or cancer cells, and / or myeloid tissue, lymphoid tissue, muscle tissue, lung tissue and / or tumor tissue. The nanoparticles may be targeted to bone marrow, pancreas, neuronal tissue, kidney tissue, heart tissue, liver tissue, eyes, joints or prostate, or to stem cells, pancreatic cells, neuronal cells, kidney cells, heart cells, liver cells, eye cells, synovial cells or prostate cells. As used herein, myeloid tissue includes tissues of cells of the bone marrow and myeloid cell lineage. As used herein, lymphoid tissue includes any organ of the lymphatic system, including spleen, thymus, lymph nodes and bone marrow.Nanoparticles have in vivo, ex vivo and in vitro uses.Nanoparticles can be transfected with target cells or tissues 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%, such as myeloid cells, lymphoid cells, muscle cells, lung cells or cancer cells.Transfection efficiency can be determined by any standard method in the art, such as by fluorescence microscopy, real-time PCR (qPCR), plasmid reporter system, reporter gene assay, flow cytometry, protein blotting or immunofluorescence staining.This type of method is 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 the absorption of nanoparticles. Preferably, the transfection efficiency is measured after a cell population of 600 μL volume is transfected in a serum-containing medium in a 24-well plate for four hours at 37°C. The nanoparticle population is preferably added to the wells at a dose of 1.5 μg 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 reporter genes. Flow cytometry is used to determine reporter gene expression. In this case, reporter gene expression is measured after a cell population of 600 μL volume is transfected in a serum-containing medium in a 24-well plate for 24 hours at 37°C. The nanoparticle population is preferably added to the wells at a dose of 1.5 μg nucleic acid. Some modifications may be made to the test protocol, such as using a higher or lower cell volume, for example, in the range of 300 to 1,200 μL.In some embodiments, 12-well plates, 48-well plates, or 96-well plates can be used instead of 24-well plates. For example, 120,000 macrophages can be used in 24-well plates, or 240,000 macrophages can be used in 12-well plates. 60,000 C2c12 cells can be used in 24-well plates. The transfection time can vary, such as 1 or 2 hours of incubation with the nanoparticle population. Longer incubation times, such as 16 or 24 hours, can be used, particularly when flow cytometry is used to determine reporter gene expression. A slightly larger amount of nanoparticles, such as 3 μg, can also be used.

[0018] In another aspect, the present invention provides nanoparticles comprising peptide dendrimers, nucleic acids and lipids for use in medicine. The peptide dendrimers comprise at least: a core peptide sequence, a first branch residue and two first peptide motifs. The branch residues may be lysine, 2,4-diaminobutyric acid, ornithine or diaminopropionic acid. The nanoparticles target myeloid cells, lymphoid cells, muscle cells, lung cells and / or cancer cells, and / or myeloid tissue, lymphoid tissue, muscle tissue, lung tissue and / or tumors. The nanoparticles are capable of transfecting target cells or tissues, such as myeloid cells, lymphoid cells, muscle cells, lung cells 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%, at least 85%.

[0019] In a further aspect, the present invention provides nanoparticles comprising peptide dendrimers, nucleic acids and lipids for treating cancer, autoimmune diseases, lung diseases and / or myopathy. The peptide dendrimer comprises at least: a core peptide sequence, a first branch residue and two first peptide motifs. The branch residues can be lysine, 2,4-diaminobutyric acid, ornithine or diaminopropionic acid. The nanoparticles can target myeloid cells, lymphoid cells, muscle cells and / or lung cells, and / or myeloid tissue, lymphoid tissue, muscle tissue, lung tissue and / or tumors. The nanoparticles can be targeted to bone marrow, pancreas, neuronal tissue, kidney tissue, heart tissue, liver tissue, eyes, joints or prostate, or targeted to stem cells, pancreatic cells, neuronal cells, kidney cells, heart cells, liver cells, eye cells, synovial cells or prostate cells. The nanoparticles are capable of transfecting target cells or tissues, such as myeloid cells, lymphoid cells, muscle cells, lung cells 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%, at least 85%.

[0020] In a further aspect, the present invention provides a method for treating cancer, autoimmune diseases, lung diseases and / or myopathy, the method 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 branch residue and two first peptide motifs. The branch residues may be lysine, 2,4-diaminobutyric acid, ornithine or diaminopropionic acid. The nanoparticles may be targeted to myeloid cells, lymphoid cells, muscle cells, lung cells and / or cancer cells, and / or myeloid tissue, lymphoid tissue, muscle tissue, lung tissue and / or tumors. The nanoparticles may be targeted to bone marrow, pancreas, neuronal tissue, kidney tissue, heart tissue, liver tissue, eyes, joints or prostate, or to stem cells, pancreatic cells, neuronal cells, kidney cells, heart cells, liver cells, eye cells, synovial cells or prostate cells. The nanoparticles are capable of transfecting target cells or tissues, such as myeloid cells, lymphoid cells, muscle cells, lung cells 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%, at least 85%.

[0021] In a further aspect, the present invention provides nanoparticles comprising a peptide dendrimer selected from Table 1, a nucleic acid and a lipid. For example, the nanoparticles of the present invention may comprise a peptide dendrimer having the structure (RHL)4(KRHL)2KGSC-NH2, (HL)8(KRF)4(KRF)2KGSC-NH2, (LR)4(KRL)2KRHA-NH2, (LR)8(KRL)4(KRL)2KGSC-NH2, (LR)4(KRL)2KRHC-NH2, (LR)4(KRL)2KRHCR-(Acp)-RR-(β-A)-RR-(Acp)-RR-(β-A) -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-(Acp)-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)2KL Dendrimers of RL-NH2, (LR)8(KRL)4(KRL)2KGSCGAASSLNIA(Acp)-NH2, (LR)8(KRL)4(KRL)2KGSCHHHHHHGAASSLNIA(Acp)-NH2, (LR)4(KRL)2KGSGGSGGSGGSC[(SS)-α-D-thiomannose], (Ac-EEEE)2KGSGGSGGSC[(SS)-α-D-thiomannose].In some embodiments, the nanoparticles may include nanoparticles having the structures (RHL)4(KRHL)2KGSC-NH2, (HL)8(KRF)4(KRF)2KGSC-NH2, (LR)4(KRL)2KRHA-NH2, (LR)4(KRL)2KRHCR-(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 Dendrimers of (p)-RR-(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 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 branch residue and two first peptide motifs. The branch 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, wherein the composition comprises 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 branch residue and two first peptide motifs. The branch 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 of treating cancer and / or an autoimmune disease. The first and second peptide dendrimers comprise at least: a core peptide sequence, a first branch residue and two first peptide motifs. The branch residues may be lysine, 2,4-diaminobutyric acid, ornithine or diaminopropionic acid.

[0025] In another aspect, the 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 branch residue and two first peptide motifs. The branch residues may be lysine, 2,4-diaminobutyric acid, ornithine or diaminopropionic acid. The method may include treating a patient suffering from cancer and / or an autoimmune disease.

[0026] The skilled person will appreciate that these compositions comprising a first peptide dendrimer, a second peptide dendrimer may include more than two dendrimers, such as three or four or more dendrimers.

[0027] Relatedly, the present invention provides compositions comprising nanoparticles of the present invention. The present invention further provides pharmaceutical compositions comprising nanoparticles of the present invention and a pharmaceutically acceptable excipient. The pharmaceutical compositions can be used for medical treatment. The pharmaceutical compositions can be used to treat cancer, autoimmune diseases, lung diseases and / or myopathy. A method for treating cancer, autoimmune diseases, lung diseases and / or myopathy is also provided, wherein the method comprises administering the pharmaceutical composition to a patient or subject. In some embodiments, the composition or pharmaceutical composition is contained in a liquid. In other embodiments, the composition or pharmaceutical composition is provided as a dry composition, such as a dry powder. Freeze-drying and / or freeze-drying techniques can be used to prepare the dry composition.

[0028] The nanoparticles of the present invention may also comprise a myeloid cell, lymphoid cell, muscle cell, lung cell or cancer cell targeting motif. For example, the muscle cell targeting motif may comprise the peptide motif ASSLNIA (SEQ ID NO: 1), PYDQLRH (SEQ ID NO: 2) or KAMHQMQ (SEQ ID NO: 3). 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, relative to the peptide motif ASSLNIA (SEQ ID NO: 1), PYDQLRH (SEQ ID NO: 2) or KAMHQMQ (SEQ ID NO: 3), the variant muscle cell targeting motif may comprise one, two or three amino acid substitutions, deletions or additions, provided that the variant retains the ability to target the nanoparticle 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, relative to the peptide sequence CGFECVRQCPERC (SEQ ID NO: 4), the variant lung cell targeting motif may comprise one, two or three amino acid substitutions, deletions or additions, 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, relative to the peptide sequence CGFECVRQCPERC (SEQ ID NO: 5), the variant cancer cell targeting motif may comprise one, two or three amino acid substitutions, deletions or additions, provided that the variant retains the ability to target nanoparticles to cancer cells. In another example, the cancer cell targeting motif may be maltotriose. Maltotriose can be used because maltotriose 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 mannose 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, mannose glycosylation, and / or maltotriose may be used to target nanoparticles to macrophages with an M2 phenotype. For example, the targeting motif may comprise mannose or maltotriose, each of which may bind to the CD206 receptor expressed on M2 phenotype macrophages. Other sugars may be selected to be conjugated to 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, such as T cells. The cell targeting motif can be a CD3, CD4 or CD8 binding agent, such as an antibody that specifically binds to one of these markers. In some embodiments, an anti-CD3 antibody is used. This can be conjugated to a negatively charged polymer such as PGA or a glutamic acid-containing peptide.

[0033] Nanoparticles can be delivered to lung tissue by targeting. Targeted lung cells include alveolar macrophages, ciliated cells, epithelial cells, basal cells, secretory cells, rod cells, alveolar cells, fibroblasts and / or endothelial cells. In some embodiments, targeting to tissues such as lungs can be achieved without the need for other ligands or peptide motifs. For example, as described herein, regulating lipid: nucleic acid ratio can significantly enhance lung targeting. In addition, as described herein, regulating the surface charge of nanoparticles can also affect tissue targeting specificity.

[0034] The nanoparticles of the present invention may also include bone marrow, pancreas, neuronal tissue, kidney tissue, heart tissue, liver tissue, eyes, joints or prostate or stem cells, pancreatic cells, neuronal cells, kidney cells, heart cells, liver cells, eye cells, synovial cells or prostate cell targeting motifs. For example, the targeting motif may include a motif targeting bone marrow or stem cells, such as an antibody specifically binding to CD34 and / or a lipid comprising a bisphosphonate (BP) group (Xue et al., 2022, which is hereby incorporated by reference in its entirety). It will be appreciated by the technician that such lipids comprising BP can be conjugated to any component of the nanoparticle (e.g., a peptide and / or dendrimer component).

[0035] In some embodiments, the targeting motif is a pancreas targeting peptide, such as a glucagon-like peptide-1 (GLP-1) homolog. Jones et al., 2018 (hereby incorporated 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 and variants and binding fragments thereof can be used as pancreas targeting peptides of the present invention. Preferably, for example, the variant has at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% amino acid identity with SEQ ID NO: 9 over at least 20 consecutive amino acids, and the binding fragment preferably has 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, which is hereby incorporated by reference in its entirety). KKEEE is SEQ ID NO:10.

[0037] In some embodiments, the targeting motif is a neuronal tissue / cell targeting peptide, such as YTIWMPENPRPGTPCDIFTNSRGKRASNGGGG (SEQ ID NO: 11) or KSVRTWNEIIPSKGCLRVGGRCHPHVNGGG (SEQ ID NO: 12) (Wang et al., 2021, which is hereby incorporated by reference in its entirety). In some embodiments, the neuronal targeting peptide comprises a Phe-Arg-Trp (FRW) motif (Tang et al., 2019, which is hereby incorporated 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, which is hereby incorporated 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 binding agent is an N-acetylgalactosamine (Gal-NAc) sugar moiety (Cui et al., 2021, which is hereby incorporated by reference in its entirety). It will be appreciated by the skilled artisan that such sugar moieties can be conjugated to any component of the nanoparticle (e.g., a peptide and / or dendrimer component). In other embodiments, the liver targeting or hepatocyte targeting moiety is vitamin A (Senoo et al., 2010, which is hereby incorporated by reference in its entirety).

[0040] In some embodiments, the targeting motif is an eye targeting or eye cell targeting moiety, such as CARSKNKDC (SEQ ID NO: 14) ( et al., 2021, which is hereby incorporated 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 and / or a hyaluronic acid saccharide that specifically binds to CD44 (Gorantla et al., 2021, which is hereby incorporated 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, which is hereby incorporated 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 a peptide sequence XRXRRBRRX RRBRXB (SEQ ID NO: 6), wherein X is 6-aminohexanoic acid, and B is β-alanine. The cell penetrating peptide may comprise a variant of the peptide sequence XRXRRBRRXRRBRXB (SEQ ID NO: 6). For example, relative to the peptide sequence XRXRRBRRXRRBRXB (SEQ ID NO: 6), the variant may comprise one, two or three amino acid substitutions, deletions or additions, provided that the variant retains biological activity.

[0044] In some embodiments, the myeloid cell, lymphoid cell, muscle cell or lung cell targeting motif can be an antibody. By "antibody" we include fragments or derivatives thereof, or synthetic antibodies or synthetic antibody fragments. Antibody fragments, such as Fab and Fab2 fragments, can also be used, as can genetically engineered antibodies and antibody fragments. Single-chain Fv (scFv) antibodies can also be used. The variable heavy chain (V H ) and variable light chain (V L ) domains are involved in antigen recognition, a fact first recognized by early protease digestion experiments. This was further confirmed by the "humanization" of rodent antibodies. The variable domains of rodent origin can be fused with the constant domains of human origin so that the resulting antibody retains the antigenic specificity of the rodent parent antibody (Morrison et al., (1984) Proc. Natl. Acad. Sd. USA 81, 6851-6855). What we call "ScFv molecules" means those in which V H and V L The chaperone domain is a molecule that is covalently linked (e.g., by a flexible oligopeptide). In some embodiments, the antibody can be a T cell-specific antibody, such as an anti-CD3 antibody (e.g., clone OKT3, BioXCell; catalog number BE0001-2). In some embodiments, the antibody can 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 can be covalently bound to a polymer or lipid. In some embodiments, the cell and / or tissue targeting motif can be covalently bound to a polymer or lipid included in the nanoparticle. The polymer or lipid can be positively charged. Alternatively, the polymer or lipid can be a negatively charged or electrically neutral polymer. The polymer or lipid can be selected from polyglutamic acid (PGA), poly (acrylic acid), alginic acid, polyethylene glycol (PEG), cholesterol hemisuccinate / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, electrically neutral zwitterionic polymers or lipids or peptides containing glutamic acid. PGA, poly (acrylic acid), alginic acid, PEG, cholesterol hemisuccinate / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, electrically neutral zwitterionic polymers or lipids or peptides containing glutamic acid may include further targeting domains as described herein, such as antibodies or their target binding fragments. Cell and / or tissue targeting motifs (e.g., antibodies) can be conjugated to one or more glutamic acid residues. PGA is a peptide comprising a region consisting mainly of glutamic acid residues, and preferably comprises at least two continuous glutamic acid residues. (Other amino acid residues may be present in the PGA molecule). In some embodiments, PGA is a peptide comprising a region (EXXEXX...) having a glutamic acid residue at every third position, and comprises a total of 2 to 50, 2 to 40, 2 to 30, 2 to 20 or 2 to 10 amino acid residues. PGA can be a linear or dendritic (branched) PGA. PGA can be derivatized at its N and / or C-terminus. For example, the C-terminus can be derivatized with an amine group (NH2). Linear PGA can be a short PGA. Short PGA is any PGA molecule with less than 100, less than 75, less than 50 or less than 20 glutamic acid residues. In embodiments where PGA is linear, nanoparticles preferably do not include PBAE polymers. For example, PGA can comprise 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, PGA can be a dendritic PGA. Dendritic PGA is a peptide dendritic polymer comprising two or more branches, each branch having at least two consecutive glutamic acid residues. Dendritic PGA can be a first generation, second generation or third generation peptide dendritic polymer. In some embodiments, the dendritic PGA is a short dendritic PGA. Short dendritic PGA may 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. It can be proved that short dendritic PGA is beneficial because it can provide better binding / coating of nanoparticles compared to linear PGA because short dendritic PGA can provide better flexibility to be "wrapped" by nanocarriers. PGA is a peptide polymer. Therefore, in embodiments comprising PGA molecules, PGA can 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 initially 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 comprising 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, the E residues account for at least 10%, at least 15%, at least 20%, at least 25% or at least 30% of the amino acid residues of this glutamic acid-rich domain. In general, the glutamic acid-rich domain may include at least 4 amino acids (e.g., splitting across two dipeptide motifs in a given generation of dendritic polymers) or at least 6, at least 8, at least 10 or at least 12 amino acid residues. Preferably, the E residues are present in the glutamic acid-containing domain at a frequency of every two, every three or every four amino acid residues (EXEX ...), (EXXEXX ...) or (EXXXEXXX ...). Most preferably, the E residues are present in a glutamic acid-rich domain spanning at least 10 amino acid residues at a frequency of every three amino acid residues. Preferably, the peptide containing glutamic acid comprises a total of 2 to 50, 2 to 40, 2 to 30, 2 to 20 or 2 to 10 amino acid residues. The peptide containing glutamic acid can be a first generation, second generation or third generation peptide dendrimer, or it can be a linear sequence. Cell and / or tissue targeting motifs (such as antibodies) can be conjugated with one or more glutamic acid residues. In some embodiments of the present invention, this type of peptide containing glutamic acid comprising several glutamic acid residues is referred to as "PGA". In the embodiment comprising the peptide containing glutamic acid, the peptide containing glutamic acid can be referred to as "polymer" or "second peptide". In other embodiments, the peptide containing glutamic acid contained in the nanoparticle is the only peptide. In these embodiments, the nanoparticle can be initially formed with only lipids and nucleic acids, and then coated with the peptide containing glutamic acid.

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

[0048] In some embodiments, the cell and / or tissue targeting motif is mannose covalently linked to a polymer. In embodiments where the polymer is a peptide, mannose can be linked to the C and / or N termini. In other embodiments where the polymer is a peptide, mannose can be linked to other positions along the peptide. In some embodiments, mannose is covalently linked to a PGA molecule. In some embodiments, mannose is covalently linked to a PGA molecule comprising 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. PGA molecules derived from mannose are particularly preferably used for targeting cells expressing CD206, such as specific lymphocytes 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 to 100, 2 to 75, 2 to 50, 2 to 40, 2 to 30, 2 to 20 or 2 to 10 glutamic acid residues. The antibody can be, for example, an anti-CD3 antibody.

[0050] In some embodiments, the cell and / or tissue targeting motif can be covalently bound to a linear peptide. For example, a linear peptide is not part of a peptide dendrimer. In the case where the targeting motif comprises a peptide sequence, the peptide sequence can be continuous 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 myocyte 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, relative to the peptide motif ASSLNIA (SEQ ID NO: 1), PYDQLRH (SEQ ID NO: 2) or KAMHQMQ (SEQ ID NO: 3), the variant myocyte targeting motif may comprise one, two or three amino acid substitutions, deletions or additions, provided that the variant retains the ability to target nanoparticles to myocytes. 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 CG FECVRQCPERC (SEQ ID NO: 5). For example, relative to the peptide sequence CGF ECVRQCPERC (SEQ ID NO: 5), the variant cancer cell targeting motif may comprise one, two or three amino acid substitutions, deletions or additions, provided that the variant retains the ability to target nanoparticles to cancer cells. In some embodiments, the linear peptide comprises a CGFECVRQCPER C (SEQ ID NO: 4) lung targeting motif. The lung cell targeting motif may comprise a variant of the peptide sequence CGFE CVRQCPERC (SEQ ID NO: 4). For example, relative to the peptide sequence CGFEC VRQCPERC (SEQ ID NO: 4), the variant lung cell targeting motif may comprise one, two or three amino acid substitutions, deletions or additions, provided that the variant retains the ability to target nanoparticles to lung cells. The linear peptide may comprise one or more units of the targeting motif. For example, the linear peptide may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more targeting motifs. In some embodiments, the linear peptide consists of a muscle targeting motif of ASSLNIA (SEQ ID NO: 1), PYDQLRH (SEQ ID NO: 2) or KAMHQMQ (SEQ ID NO: 3), an integrin targeting peptide motif of RGD or ACDCRGDCFCG (SEQ ID NO: 5), or a lung targeting peptide motif of CGFECVRQCPERC (SEQ ID NO: 4).In some embodiments, the linear peptide consists of the lung targeting peptide sequence CGFECVRQCP ERC (SEQ ID NO: 4). Where the targeting motif is a sugar, the linear peptide may comprise one or more glycosylation. For example, the linear peptide may comprise one or more mannose glycosylation.

[0051] In some embodiments, the cell and / or tissue targeting motif can be covalently bound to the peptide dendrimer of the nanoparticle. The cell and / or tissue targeting motif can be covalently bound to the C-terminus and / or N-terminus of the peptide dendrimer. The peptide dendrimer core can include the cell and / or tissue targeting motif. The cell and / or tissue targeting motif can be bound to the outermost layer of the peptide motif. In the case where the targeting motif is a sugar, the peptide dendrimer can include one or more glycosylations. For example, the peptide dendrimer can include one or more mannose glycosylations. For example, the peptide dendrimer can have the structure mannose-G1-RL,2-LR. In some embodiments, the peptide dendrimer can have the structure mannose-G1-EEEE. For example, the peptide dendrimer can have the structure mannose-G1-RL,2-LR. In some embodiments, the peptide dendrimer can 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 nanoparticle comprises 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 where the second peptide is a dendritic peptide, it may be referred to herein as a "second peptide dendrimer". Thus, in some embodiments, the nanoparticle comprises a second peptide dendrimer covalently bound to a cell and / or tissue targeting motif. The cell and / or tissue targeting motif may be covalently bound 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 bound to the outermost layer of the peptide motif of the second peptide dendrimer. In the case where 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] ("Ac" represents acetylation of 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, targeting of nanoparticles to specific tissues and / or cell types is achieved by selecting the NP ratio of dendrimer:nucleic acid and / or the w / w ratio of lipid:nucleic acid. For example, a higher NP ratio favors spleen targeting, and some lung targeting is also observed at a lipid:nucleic acid w / w ratio of about 10:1. Lung targeting can be enhanced by increasing the lipid:nucleic acid w / w ratio, for example to about 23:1.

[0054] The dendrimers used in the present invention are first, second or third generation peptide dendrimers, which means that they have up to three "layers" of peptide motifs interspersed between "branching" residues such as lysine. First generation dendrimers have the following structure shown in N-terminal to C-terminal orientation, with Lys as the branching unit:

[0055] (N-terminal-Pep1)2-Lys-(core)-(C-terminal)

[0056] The second generation dendrimers have the following structure shown in N-terminal to C-terminal orientation, with Lys as the branching unit:

[0057] (N-terminal-Pep2)4-Lys2-(Pep1)2-Lys-(core)-(C-terminal)

[0058] The third generation dendrimer has the following structure shown in N-terminal to C-terminal orientation, with Lys as the branching unit:

[0059] (N-terminal-Pep3)8-Lys4-(Pep2)4-Lys2-(Pep1)2-Lys-(core)-(C-terminal)

[0060] Figure 1 The diagram in FIG. 5 shows a third generation dendrimer (N-terminus on the left and C-terminus on the right).

[0061] exist Figure 1 In , the circles represent the core sequence. Each triangle represents a branching residue, such as lysine. Each rectangle represents a peptide motif. There are two peptide motifs in the first layer of the third generation dendrimer, four peptide motifs in the second layer, and eight peptide motifs in the third layer. As discussed herein, the N-terminus and C-terminus can be derivatized with further chemical motifs. For example, while in underivatized embodiments, the C-terminus is a carboxylic acid, in other embodiments, as a result of the chemical route used to synthesize the dendrimer, the C-terminus is derivatized, for example to include a primary amide group CONH2 (rather than COOH). Functionally important derivatizations are also contemplated, such as targeting moieties (e.g., antibodies, peptide groups, sugar groups, and / or lipid chains), which can be attached to the N- and / or C-termini or attached at other positions along the dendrimer. The N-terminus of the peptide dendrimers disclosed herein can be derivatized, for example, acetylated.

[0062] As described herein, the dendrimer can be of the first, second or third generation. This can be structurally defined as follows: The first generation dendrimer comprises a core peptide sequence, a first branch residue and two first peptide motifs each connected to the first branch residue. The two first peptide motifs are independently composed of a single amino acid, dipeptide, tripeptide or tetrapeptide motif. The second generation dendrimer further comprises two second branch residues (e.g., lysine) and four second peptide motifs, wherein one of the second branch residues is covalently bound to one of the first peptide motifs, and another second branch residue is covalently bound to another first peptide motif, and wherein each second branch residue is covalently bound to two second peptide motifs. The four second peptide motifs are independently composed of a single amino acid, dipeptide, tripeptide or tetrapeptide motif. The third generation dendrimer further comprises four third branch residues (e.g., lysine) and eight third peptide motifs, 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 wherein each third branch residue is covalently bound to two third peptide motifs. The eight third peptide motifs are independently composed of a single amino acid, a dipeptide, a tripeptide or a tetrapeptide motif. Each of the first, second and third peptide motifs, if present, may include (1) an amino acid with a basic side chain, such as but not limited to lysine (K) or arginine (R) or histidine (H), (2) an amino acid with an acidic side chain, such as but not limited to aspartic acid (D) and glutamic acid (E), (3) an amino acid with a non-polar side chain, such as but not limited to glycine (G), alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), β-alanine (B), tryptophan (W), proline (P), aminocaproic acid (X) and cysteine ​​(C), and (4) an amino acid with an uncharged polar side chain, such as but not limited to asparagine (N), glutamine (Q), serine (S), threonine (T) and tyrosine (Y).

[0063] The core peptide motif of the dendrimer is a single amino acid residue or a short peptide motif, such as a dipeptide or tripeptide motif. The core sequence may comprise any amino acid (L- and / or D-isomer), such as glycine (G), serine (S), cysteine ​​(C), alanine (A), lysine (K), leucine (L), valine (V), isoleucine (I), phenylalanine (F), methionine (M), tyrosine (Y), tryptophan (W), proline (P), threonine (T), asparagine (N), glutamine (Q), aspartic acid (D), glutamic acid (E), arginine (R) and / or histidine (H). The core sequence may also comprise non-naturally occurring amino acids (L- and / or D-isomers), such as β-alanine (B) and / or aminocaproic acid (X). In the case where the core is a tripeptide motif, it may comprise glycine (G), serine (S) and cysteine ​​(C) or alanine (A). Preferably, the core comprises ionizable residues, such as histidine (H). The core sequence may comprise arginine (R), histidine (H) and cysteine ​​(C). The core sequence may comprise arginine (R) or glycine (G), histidine (H) or serine (S) and cysteine ​​(C) or alanine (A). For example, the core sequence may be GSC or RHC. The tripeptide motif may comprise alanine (A), lysine (K) and leucine (L). For example, the core sequence may be KLA. The core peptide may be covalently bound to further moieties, such as cell-specific targeting peptides, or may be derivatized with lipid molecules. One, some or all of the amino acids of a dendrimer (e.g., a core peptide motif) may be covalently bound to further moieties, such as antibodies, cell-specific targeting peptides, sugar ligands such as glucose, mannose, galactose and GalNAc (or polysaccharides comprising them) and / or lipid substituents. The technician is easily able to select further moieties that will not adversely affect solubility or nucleic acid binding properties.

[0064] Preferred dendrimers are given in Table 1 below. Certain examples are discussed in particular. For example, in a dendrimer where each peptide motif is an Arg-His-Leu (RHL) tripeptide, this structure can be represented as G1-RHL, G1,2-RHL and G1,2,3-RHL. In a dendrimer where the peptide motifs are not identical in all generations, such as in a third generation dendrimer where the two first peptide motifs and the four second peptide motifs are Arg-Leu (RL) dipeptides and the eight third peptide motifs are Leu-Arg (LR) dipeptides, this structure can be represented as G1,2-RL,3-LR. In a dendrimer where the peptide motifs are not identical in a second generation dendrimer, such as in a dendrimer where the two first peptide motifs are Arg-Leu (RL) and the four second peptide motifs are Leu-Arg (LR) dipeptides, this structure can be represented as G1-RL,2-LR. In a dendrimer where each peptide motif is an Arg-Leu (RL) dipeptide, this structure can be represented as G1-RL, G1,2-RL and G1,2,3-RL. In a dendrimer where each peptide motif is a Lys-Leu (KL) dipeptide, this structure is represented as G1-KL, G1,2-KL and G1,2,3-KL. In a dendrimer where each peptide motif is a Leu-Arg (LR) dipeptide, this structure is represented as G1-LR, G1,2-LR and G1,2,3-LR.

[0065] "G1", "G2" and "G3" refer to "1st generation", "2nd generation" and "3rd generation" peptide motifs of the first, second and third layers, respectively. Each amino acid residue can be an L-amino acid or a D-amino acid. D-amino acids can be designated using lower case letters in the single letter code. Alternatively, a dendrimer in which each amino acid is of the D-subtype can be written with a preceding "D-" before the short form representation of the dendrimer.

[0066] Also discussed herein are peptide dendrimers having a specified core. For example, in a dendrimer where a defined peptide core is contemplated (e.g., an Arg-His-Cys peptide core), this structure may be represented as RHCG1,2-RL. It will be appreciated that, in accordance with the nomenclature of the peptide dendrimers disclosed herein, in the preceding examples, the "G" refers to a "generation" of the peptide motif, rather than a glycine residue. In contrast, for a peptide dendrimer whose structure is represented as GSCG1,2-RL,3-LR, it will be appreciated that the first "G" in this context refers to a glycine residue, while the second "G" refers to a "generation" of the peptide motif. In other examples where a defined core is contemplated, the core sequence will be underlined, leaving the "G" representing the generation ununderlined (e.g. GSC G1,2-RL,3-LR).

[0067] In some embodiments, the peptide dendrimer further comprises an alkyl chain, an alkenyl chain, an antibody or fragment thereof, a sugar and / or a fatty acid. The alkyl or alkenyl chain may be conjugated to the core peptide sequence, for example at the C-terminus of the peptide dendrimer. Alternatively or in addition, the alkyl or alkenyl chain may be conjugated to the N-terminus of the peptide dendrimer.

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

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

[0070] Preferably, the N / P ratio of the amount of peptide (measured by the number of nitrogen atoms with a 1+ charge on the peptide, N) to the amount of nucleic acid (measured by the number of phosphate groups with a 1-charge in the backbone, P) is greater than 0.05:1, for example, greater than 0.1:1. (The N / P ratio terminology may be expressed as "N / P", "N:P" or "NP"). In some embodiments, the N / P ratio is at or about or at least 0.15:1. In some embodiments, the N / P ratio is at or about or at least 0.16:1. In some embodiments, the N / P ratio is at or about or at least 0.6:1. In some embodiments, the N / P ratio is at least or greater than 1: 1, such as about 2: 1 or greater, about 2.5: 1 or greater, about 3: 1 or greater, about 4: 1 or greater, about 5: 1 or greater, about 10: 1, or up to 20: 1. In some embodiments, the N / P ratio is about 5: 1, about 8: 1, about 10: 1, or about 20: 1. In some embodiments, the N / P ratio is in the range of about 0.01: 1 and 100: 1, about 2: 1 to about 20: 1, or about 2.5: 1 to about 10: 1.

[0071] 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 G1-R.

[0072] 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, wherein the N:P ratio is between 0.05:1 and 20:1, for example, for delivery of nanoparticles to myeloid cells, the N:P ratio can be 0.16:1 or 0.6:1. In some embodiments, the peptide dendrimer has the structure GSC G1,2-RL,3-LR; RHC G1-RL,2-LR; GSC G1,2-RH L; GSC G1-LRLR; GSC G1,2-RF,3-HL; GSC G1-R, wherein the N:P ratio is between 0.05:1 and 20:1, for example, for delivery of nanoparticles to macrophages, the N:P ratio may be 0.16:1 or 0.6:1. In some embodiments, the peptide dendrimer has the structure GSC G1,2-RL,3-LR, wherein for delivery of nanoparticles to macrophages, the N:P ratio is 0.16:1. In some embodiments, the peptide dendrimer has the structure RHC G1-RL,2-LR, wherein for delivery of nanoparticles to macrophages, the N:P ratio is 8:1. In some embodiments, the peptide dendrimer has the structure GSC G1,2-RHL, where the N:P ratio is between 5:1 for nanoparticle delivery to macrophages.

[0073] Typically, when the nanoparticles have a dendrimer:nucleic acid NP ratio greater than about 2: 1 (e.g., N: P is about 8: 1), a composition comprising nanoparticles including first generation peptide dendrimers, lipids, and nucleic acids does not form a monodisperse population of nanoparticles. However, the use of a higher NP ratio enables control of certain properties of the nanoparticles. In addition, the ability to form a monodisperse population is important for the development of pharmaceutical compositions because it will limit batch-to-batch variability, allowing for more defined nanoparticle characterization and consistent results in vitro and in vivo. The distribution of the sizes of the nanoparticles in the population can be represented by a population polydispersity index (PDI). A PDI of 1 represents a completely polydisperse population of nanoparticles, while a PDI of 0 represents a completely monodisperse population of nanoparticles. Therefore, the PDI can be used as a measure of the uniformity of a population of nanoparticles in a composition. A PDI of 0.35 or less is considered to provide a suitable monodisperse population of nanoparticles, for example, for the development of a suitable pharmaceutical composition. 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 equal to or less than 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35 or 0.3. For simplicity and clarity, as used herein, where a PDI value is given for a particular peptide dendrimer or mixture of peptide dendrimers, this PDI value is for a population of nanoparticles comprising a peptide dendrimer, a nucleic acid and a lipid. For example, where a PDI of 0.566 is mentioned for the peptide dendrimer RHCG1-R, this PDI value is associated with nanoparticles comprising RHCG1-R, a nucleic acid and a lipid. The PDI of a particular nanoparticle formulation may be measured by any method standard in the art, for example using a Zetasizer Advance Series-Pro according to the manufacturer's instructions and the conditions disclosed in Example 3.

[0074] A non-uniform or non-monodisperse population of nanoparticles (e.g., where the PDI of the nanoparticles is greater than 0.35) may indicate that the binding of the peptide dendrimer to the nucleic acid in the nanoparticle is unstable (i.e., the peptide dendrimer and the nucleic acid have low binding affinity and are easily dissociated). In contrast, a uniform or monodisperse population of nanoparticles may indicate that the binding of the peptide dendrimer to the nucleic acid in the nanoparticle is stable (i.e., the peptide dendrimer and the nucleic acid are bound with high affinity and are not easily dissociated).

[0075] Without wishing to be bound by any particular theory, it is believed that nanoparticles comprising first generation peptide dendrimers tend to form a less uniform population of nanoparticles (and therefore have a "high" PDI greater than 0.35) than a population of nanoparticles comprising second or third generation peptide dendrimers because first generation dendrimers have fewer cationic groups per peptide dendrimer. Due to fewer cationic groups to interact with anionic groups on nucleic acids, first generation peptide dendrimers / nucleic acids can form unstable complexes that can dissociate at a relatively high rate compared to second / third generation dendrimers / nucleic acid complexes, thereby producing a heterogeneous mixture of nanoparticles in solution. By including a second or third generation peptide dendrimer in combination with a first generation peptide dendrimer, it is believed that this can balance the relatively stable association required to form uniform particles (provided by the second or third generation peptide dendrimer) with the relatively unstable association required to effectively release nucleic acids once delivered to cells (provided by the first generation peptide dendrimer).

[0076] The present inventors have also discovered that the transfection efficiency can be improved by using nanoparticles comprising two peptide dendrimers that can form a suitably uniform population of nanoparticles when used alone in nanoparticles comprising nucleic acid and lipid. That is, even if each peptide dendrimer alone forms a monodisperse population of nanoparticles in solution, an improvement in transfection efficiency can be achieved. For example, the first and second peptide dendrimers can be independently selected from second or third generation peptide dendrimers that form a monodisperse population of nanoparticles when used alone.

[0077] Thus, in some embodiments, a first peptide dendrimer can 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 can 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, the first peptide dendrimer has a PDI when used in a first reference nanoparticle consisting of the first peptide dendrimer, a nucleic acid, and a lipid that is higher than the PDI of a second reference nanoparticle consisting of the second peptide dendrimer, a nucleic acid, and a lipid, wherein the reference nanoparticle is prepared at an NP ratio of 8: 1.

[0078] In some embodiments, the PDI of the nanoparticles comprising the first peptide dendrimer, the second peptide dendrimer, the nucleic acid, and the lipid is less than or equal to 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. In some embodiments, the PDI of the nanoparticles comprising the first peptide dendrimer, the second peptide dendrimer, the nucleic acid, and the lipid is less than or equal to 0.35.

[0079] In some embodiments, the first dendritic polymer has a PDI 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, about 0.40. In some embodiments, the second dendrimer has a PDI of 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, about 0.20. In some embodiments, the first peptide dendrimer has a PDI greater than about 0.25, about 0.26, about 0.27, about 0.28, about 0.29, or about 0.30, and the second peptide dendrimer has a PDI less than about 0.25, about 0.24, about 0.23, about 0.22, about 0.21, 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 PDI of the first peptide dendrimer is greater than about 0.10 and the PDI of the second peptide dendrimer is less than about 0.20.

[0080] In some embodiments, the PDI of the first peptide dendrimer 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, about 0.9 greater than the PDI of the second peptide dendrimer. In some embodiments, the PDI of the first peptide dendrimer is between 0.05 and 0.9, between 0.06 and 0.8, between 0.07 and 0.6, between 0.08 and 0.5, between 0.09 and 0.4, about 0.1 and 0.3, between 0.12 and 0.2, between 0.13 and 0.15 greater than the PDI of the second peptide dendrimer.

[0081] In some embodiments, the first peptide dendrimer, when used in a first reference nanoparticle consisting of the first peptide dendrimer, a nucleic acid, and a lipid, has a PDI that is between 0.05 and 0.9, between 0.06 and 0.8, between 0.07 and 0.6, between 0.08 and 0.5, between 0.09 and 0.4, between 0.1 and 0.3, between 0.12 and 0.2, or between 0.13 and 0.15 greater than the PDI of a second reference nanoparticle consisting of the second peptide dendrimer, the nucleic acid, and the lipid.

[0082] The transfection efficiency and PDI of nanoparticles comprising two peptide dendrimers can be optimized by varying the relative levels of each peptide dendrimer in the nanoparticle. For example, the molar ratio relative to the nitrogen contributed by each of the first peptide dendrimer and the second peptide dendrimer in the nanoparticle can be selected from between 1:4 and 4:1, between 1:3 and 3:1, between 1:2 and 2:1. In some examples, 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, 2:1, 3:1 or 4:1.

[0083] 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.

[0084] 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 bound to one of the first peptide motifs, and another second branching residue is covalently bound to another first peptide motif. Each of the second branching residues is covalently bound to two second peptide motifs. Each of the first branching unit and the two second branching units can be independently selected from lysine, 2,4-diaminobutyric acid, ornithine or diaminopropionic acid.

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

[0086] The first peptide dendrimer may be a first generation peptide dendrimer comprising a core peptide sequence, a first branch unit and two first peptide motifs. Alternatively, the first peptide dendrimer is a second generation peptide dendrimer comprising a core peptide sequence, a first branch unit and two first peptide motifs, at least two second branch units and four second peptide motifs. One of the second branch residues is covalently bound to one of the first peptide motifs, and another second branch residue is covalently bound to another first peptide motif. Each of the second branch residues is covalently bound to two second peptide motifs. Each of the first branch unit and the two second branch units may be independently selected from lysine, 2,4-diaminobutyric acid, ornithine or diaminopropionic acid.

[0087] 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 branch residues is covalently bound to one of the first peptide motifs, and another second branch residue is covalently bound to another first peptide motif. Each of the second branch residues is covalently bound to two second peptide motifs. Each of the first branch unit and the two second branch units can be independently selected from lysine, 2,4-diaminobutyric acid, ornithine or diaminopropionic acid.

[0088] In some embodiments, the second peptide dendrimer is a third generation peptide dendrimer comprising a core peptide sequence, a first branch unit and two first peptide motifs, at least two second branch units and four second peptide motifs, and at least four third branch residues and eight third peptide motifs. One of the second branch residues is covalently bound to one of the first peptide motifs, and another second branch residue is covalently bound to another first peptide motif. Each of the second branch residues is covalently bound to two second peptide motifs. Each second peptide motif is covalently bound to one of the third branch residues, respectively, so that each third branch residue is covalently bound to one second peptide motif. Each third branch residue is covalently bound to two third peptide motifs. Each of the first branch unit, the two second branch units, and the four third branch units can be independently selected from lysine, 2,4-diaminobutyric acid, ornithine, or diaminopropionic acid.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] In some embodiments, the peptide motifs of the first and second peptide dendrimers are independently selected from a single amino acid, dipeptide, tripeptide or tetrapeptide motif. 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 a single amino acid, dipeptide, tripeptide or tetrapeptide motif.

[0094] 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, such as β-alanine (B) or aminocaproic 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, such as β-alanine (B) or aminocaproic acid (X or Acp).

[0095] In some embodiments, the first, second and / or third peptide motif (where present) of the first and / or second peptide dendrimer comprises amino acids with basic side chains.

[0096] In some embodiments, the core sequence of the first and / or second peptide dendrimer comprises amino acid residues having ionizable groups such as histidine.

[0097] In some embodiments, the first, second and / or third peptide motif (where present) of the first and / or second peptide dendrimer comprises amino acids having non-polar side chains.

[0098] In some embodiments, the first, second and / or third peptide motif (where present) of the first and / or second peptide dendrimer comprises amino acids having acidic side chains.

[0099] In some embodiments, the first, second and / or third peptide motif (where present) of the first and / or second peptide dendrimer comprises amino acids with uncharged polar side chains.

[0100] In some embodiments, each of the first, second and third peptide motifs, if present, may comprise (1) amino acids with basic side chains, such as but not limited to lysine (K) or arginine (R) or histidine (H), (2) amino acids with acidic side chains, such as but not limited to aspartic acid (D) and glutamic acid (E), (3) amino acids with non-polar side chains, such as but not limited to glycine (G), alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), β-alanine (B), tryptophan (W), proline (P), aminocaproic acid (X) and cysteine ​​(C), and (4) amino acids with uncharged polar side chains, such as but not limited to asparagine (N), glutamine (Q), serine (S), threonine (T) and tyrosine (Y).

[0101] Preferably, at least one of the first, second and third peptide motifs (where 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 (where present) may comprise leucine (L), arginine (R) and / or histidine (H). In some embodiments, all first, second and third peptide motifs (where present) of the first and / or second peptide dendrimer comprise leucine (L), arginine (R) and / or histidine (H).

[0102] 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, the first, second and third peptide motifs all comprise leucine (L).

[0103] In some embodiments, the core peptide sequence of the first and / or second peptide dendrimer comprises the amino acid sequence RHC, GSA or GSC.

[0104] In some embodiments, the peptide dendrimer, nucleic acid, and lipid form positively charged particles.

[0105] In other embodiments, the peptide dendrimers, nucleic acids, and lipids form negatively charged particles or particles that have neutral charge.

[0106] The lipid component of the nanoparticle may comprise a mixture of lipids, including cationic lipids. For example, the lipid component may comprise dioleoylphosphatidylethanolamine (DOPE) and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). For a given application, the DOPE:DOTMA w / w ratio can be easily determined to obtain the best properties, but will generally be in the range of 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 only lipid or in combination with DOPE.

[0107] In other embodiments, the lipid component of the composition may include other lipids, such as DODAP, DOTAP and / or DORI (N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(oleyloxy)propane-1-ammonium bromide) in addition to (or in place of) DOPE and DOTMA. Exemplary lipid components are given below:

[0108] Cationic lipids:

[0109] N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)

[0110] 1,2-Dioleoyl-3-trimethylammonium-propane chloride (DOTAP)

[0111] N-(2-Hydroxyethyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane-1-ammonium bromide (DORI)

[0112] 2,3-Dioleyl-N-(2[spermine-carboxamido]ethyl)-N,N-dimethyl-1-propylammonium trifluoroacetate (DOSPA)

[0113] 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-chol)

[0114] Neutral lipids:

[0115] 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)

[0116] 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC)

[0117] cholesterol

[0118] Anionic lipids:

[0119] 1,2-Dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG)

[0120] Ionizable lipids:

[0121] 1,2-Dioleoyloxy-3-(dimethylamino)propane (DODAP)

[0122] DLin-DMA

[0123] DLin-KC2-DMA

[0124] DLin-MC3-DMA

[0125] SM-102

[0126] ALC-0315

[0127] Other possible lipids include 4-(2-aminoethyl)-morpholino-cholesterol-hemisuccinate (MoChol), cholesterol hemisuccinate (CHEMS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DP PC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), cholesterol-(3-imidazol-1-ylpropyl) carbamate (CHIM), dimethyldioctadecyl ammonium bromide (DDAB), dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylglycerol (DOPG), and cholesterol sulfate (chol-SO4).

[0128] It is contemplated that any of the above lipids may be used alone or in combination with one another in the compositions of the present invention. Additionally, the lipids may be derivatized via attachment to a PEG group such as PEG2000.

[0129] In some embodiments, the lipids of the nanoparticles include cationic lipids, neutral lipids, anionic lipids, and / or ionizable lipids.

[0130] In some embodiments, the lipids of the nanoparticles comprise saturated fatty acids. Additionally or alternatively, the lipids of the composition may comprise unsaturated fatty acids.

[0131] 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.

[0132] In some embodiments, the lipids include dioleoylphosphatidylethanolamine (DOPE) and / or N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). In some embodiments, the lipids include dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylglycerol (DOPG).

[0133] The lipid component of the nanoparticles may comprise DOTMA, DOPE, DOPC and / or DOPG.

[0134] The amount of lipid component can be expressed as a weight:weight ratio ("w / w" or "w:w"), which can be in the range of 1:50 to 50:1 relative to the amount of nucleic acid in the nanoparticle. More preferably, the amount of lipid (by weight) relative to the amount of nucleic acid (by weight) 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 lipids. As described herein, the nanoparticles can contain lipids including more than one lipid component, such as a mixture of two, three or four lipids. The weight of the lipid component is the total (combined weight) of these lipid components. Preferably, each lipid component is mixed in approximately equal proportions.

[0135] In some embodiments, lipid: nucleic acid w / w can be selected to target nanoparticles to lung tissue. For example, the nanoparticle can include lipid: nucleic acid w / w between 2: 1 and 40: 1, so that the nanoparticle is targeted to lung and / or spleen. In a further example, the nanoparticle can include lipid: nucleic acid w / w of 10: 1 or 23: 1. When lipid: nucleic acid w / w is 10: 1, spleen and lung are targeted, and spleen is targeted more strongly than lung. When lipid: nucleic acid w / w is 23: 1, spleen and lung are targeted, and lung is targeted more strongly than spleen.

[0136] 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.

[0137] By including a dendrimer with one or more charged, hydrophobic and / or ionizable amino acids (e.g., arginine, aspartic acid, cysteine, glutamic acid, histidine, lysine, leucine, and tyrosine), transfection efficiency can be increased following targeted delivery of the nanoparticles to cells or tissues 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.

[0138] In some embodiments, the core peptide sequence comprises amino acids such as arginine, aspartic acid, cysteine, glutamic acid, histidine, lysine, and tyrosine. In some embodiments, the core peptide sequence comprises the ionizable amino acid histidine.

[0139] For example, inclusion of histidine in the core and / or peptide motif of one or both peptide dendrimers can confer extracellular stability to the nanoparticles but facilitate intracellular release of nucleic acids from the nanoparticles. For example, the five-membered imidazole ring of histidine contains two nitrogen atoms that can form hydrogen bonds, thereby providing stability to the nanoparticles. However, when exposed to the acidic endosomal environment, protonation of histidine can lead to endosomal swelling, cleavage and release of nucleic acids contained within the nanoparticles.

[0140] In some embodiments, the nucleic acid is RNA. For example, RNA can be selected from mRNA, circular RNA (circRNA), ssRNA, dsRNA, sgRNA, crRNA, tracrRNA, IncRNA, siRNA, saRNA and / or self-amplifying RNA.

[0141] In some embodiments, the nucleic acid is DNA. For example, the DNA can include ssDNA, dsDNA, plasmids and / or cDNA.

[0142] For the avoidance of any doubt, a nanoparticle may comprise more than one nucleic acid (eg more than one type of RNA molecule). Similarly, a composition may comprise more than one lipid.

[0143] In some embodiments, the nanoparticle comprises an RNA nucleic acid and a DNA nucleic acid. The RNA nucleic acid and the DNA nucleic acid can be part of a single nucleic acid molecule.

[0144] In some embodiments, the nucleic acid includes a modified nucleic acid.Exemplary nucleic acid modifications are described herein.

[0145] The nucleic acid can encode a transgene and can express the transgene in a target cell. The transgene can be a protein or a peptide. Additionally or alternatively, the nucleic acid can regulate the expression or activity of an endogenous gene. The regulation can be an increase in the expression of a gene and / or exogenous expression of more copies of a gene, or the regulation can be a decrease in the expression of a gene.

[0146] In some embodiments, the nucleic acid expresses a target transgene identified in Table A, and the nanoparticle composition is used to treat a corresponding disease identified in Table A.

[0147]

[0148] Table A. Exemplary target diseases and corresponding target transgenes.

[0149] In some embodiments, the endogenous gene that is regulated is a gene that expresses a protein or peptide.

[0150] 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.

[0151] In some embodiments, the nucleic acid encodes a CAR that specifically binds to carcinoembryonic antigen (CEA) or CEA cell adhesion molecule 7 (CEACAM7).

[0152] 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).

[0153] 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 κB kinase subunit β (IKK2), or CCAAT enhancer binding protein alpha (CEBPA).

[0154] 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 nucleic acid therapy is used to treat cancer.

[0155] In some embodiments, the transgene comprises a tumor antigen, a viral protein, a bacterial protein, or a protein of a microorganism that parasitizes the mammal.

[0156] In some embodiments, the nucleic acid comprises or encodes a self-amplifying RNA.

[0157] In some embodiments, the use comprises treating a genetic disorder in a subject.

[0158] In some embodiments, the nucleic acid expresses a functional form of a gene that is non-functional, down-regulated, inactivated, or impaired in the subject.

[0159] In some embodiments, nucleic acid encodes and / or comprises one or more components of a system for editing a genome or a system for changing gene expression. For example, a system for editing a genome or a system for changing gene expression can be a CRISPR / Cas system. Nucleic acid can encode Cas protein or peptide, and / or comprise sgRNA, crRNA and / or tracrRNA. Nucleic acid can comprise mRNA encoding Cas protein or peptide and RNA sequence comprising sgRNA. Compositions may comprise mRNA encoding Cas protein or peptide and another RNA (as a separate molecule) comprising sgRNA. In some embodiments, one or more of sgRNA, crRNA, tracrRNA and nucleic acid encoding Cas protein is part of a single nucleic acid when present. In some embodiments, one or more of sgRNA, crRNA, tracrRNA and nucleic acid encoding Cas protein is present on two or more nucleic acids when present.

[0160] It is contemplated that the present invention can be used to deliver nucleic acid therapeutics to treat myopathies. It is also contemplated that the present invention can be used to deliver nucleic acid therapeutics to treat muscular dystrophies, such as Duchenne muscular dystrophy, myotonic dystrophy, facioscapulohumeral muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, oculopharyngeal muscular dystrophy, Emory-Dreyfus muscular dystrophy, hereditary muscular dystrophy, congenital muscular dystrophy, and distal muscular dystrophy.

[0161] Nucleic acid therapy can be used to treat patients with muscle atrophy, such as cachexia. Nucleic acid therapy can be used to treat other muscle disorders, such as hereditary muscle disorders, such as myotonia congenita or familial periodic paralysis. Nucleic acid therapy can be used to treat motor neuron diseases, such as ALS (amyotrophic lateral sclerosis), spinal bulbar muscular atrophy (SBMA) or spinal muscular atrophy (SMA). Nucleic acid therapy can be used to treat mitochondrial diseases, such as Friedreich's ataxia (FA), or mitochondrial myopathy, such as Kearns-Sell 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 broken red fibers (MERRF), neuropathy, ataxia and retinitis pigmentosa (NARP), Pearson syndrome or progressive external ophthalmoplegia (PEO). Nucleic acid therapy can be used to treat congenital myopathies such as capillary myopathy, centronuclear myopathy, congenital myopathy with imbalanced fiber types, core myopathy, central core disease, multinuclear myopathy, myosin storage myopathy, myotubular myopathy, or nemaline myopathy. Nucleic acid therapy can be used to treat distal myopathies such as GNE myopathy / Nonaka myopathy / hereditary inclusion body myopathy (HIBM), Laing distal myopathy, Markesbery-Griggs distal myopathy late, Miyoshi myopathy, Udd myopathy / tibial muscular dystrophy, VCP myopathy / IBMPFD, vocal cord and pharyngeal distal myopathy, or Welander distal myopathy. Nucleic acid therapy can be used to treat endocrine myopathies such as hyperthyroid myopathy or hypothyroid myopathy. Nucleic acid therapy can be used to treat inflammatory myopathies such as dermatomyositis, inclusion body myositis, or polymyositis. Nucleic acid therapy can be used to treat metabolic myopathies such as acid maltase deficiency (AMD, Pompe disease), carnitine deficiency, carnitine palmitoyltransferase deficiency, debranching enzyme deficiency (Corey disease, Forbes disease), lactate dehydrogenase deficiency, myoadenylate deaminase deficiency, phosphofructokinase deficiency (Tarui disease), phosphoglycerate kinase deficiency, phosphoglycerate mutase deficiency, or phosphorylase deficiency (McArdle disease). Nucleic acid therapy can be used to treat myofibrillar myopathy or scapulohumeral myopathy. Nucleic acid therapy can be used to treat neuromuscular junction diseases such as congenital myasthenic syndrome (CMS), Lambert-Eaton myasthenic syndrome (LEMS), or myasthenia gravis (MG). Nucleic acid therapy can be used to treat peripheral nerve diseases such as Charcot-Marie-Tooth disease (CMT) or giant axonal neuropathy (GAN).Nucleic acid therapy can be used to treat cardiovascular diseases such as thromboangiitis obliterans / Burger's disease, diabetic peripheral neuropathy (also tested in ALS, critical limb ischemia, and foot ulcers), peripheral arterial disease, limb ischemia, critical limb ischemia (also called chronic limb-threatening ischemia and diabetic limb ischemia), severe peripheral arterial occlusive disease (PAOD), or intermittent claudication / atherosclerosis. Nucleic acid therapy can be used to treat cancers 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. Nucleic acid therapy can be used to treat allergies, such as peanut allergies. Nucleic acid therapy can be used to treat multiple sclerosis (MS). Nucleic acid therapy can be used to treat myelodysplastic syndrome (MDS).

[0162] Pompe disease is caused by a defect in human acid alpha-glucosidase (GAA), a lysosomal enzyme that cleaves terminal alpha 1-4 and alpha 1-6 glucose from glycogen. The compositions of the present invention can be used to treat Pompe disease. The compositions of the present invention comprising nucleic acids encoding GAA can be administered to a subject suffering from Pompe disease so that the nucleic acids are delivered to the target tissues of the subject, thereby expressing GAA in the target tissues described herein, particularly the liver and skeletal muscle. The enzyme can be secreted from the tissue into the circulation.

[0163] Follistatin is an inhibitor of TGF-β superfamily ligands, which inhibits skeletal muscle growth and promotes muscle atrophy. A composition comprising a nucleic acid encoding follistatin of the present invention can be administered to a subject suffering from a muscle atrophy disorder so that the nucleic acid is delivered to the target tissue of the subject, thereby expressing follistatin in the target tissue described herein (particularly skeletal muscle). The protein can be secreted from the tissue into the circulation.

[0164] Accordingly, the present invention provides methods of treating such disorders and compositions for use in such treatment methods.

[0165] In some embodiments, the first peptide dendrimer comprises the structure given 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 RHC In some embodiments, the first peptide dendrimer comprises the structure G1-RLR. In some embodiments, the first peptide dendrimer comprises the structure RHC In some embodiments, the first peptide dendrimer comprises the structure G1,2-R. In some embodiments, the first peptide dendrimer comprises the structure RHCG1,2-R. In 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.

[0166] In some embodiments, the second peptide dendrimer comprises the structures given in Table 8. In some embodiments, the second peptide dendrimer comprises the structures G1,2-RL, G3-LR. In some embodiments, the second peptide dendrimer comprises the structures GSC In some embodiments, the second peptide dendrimer comprises the structures G1-RL, G2-LR. In some embodiments, the second peptide dendrimer comprises the structures RHC In some embodiments, the second peptide dendrimer comprises the structure G1,2-KL. In some embodiments, the second peptide dendrimer comprises the structure 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 RHC 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-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 GSC G1-RHL, 2-LHR.

[0167] 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 comprises the structure GSCG1,2-RL,3-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 G1-RL,2-LR. 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 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. In some embodiments, the first peptide dendrimer comprises the structure RHC G1-RLR, and the second peptide dendrimer comprises the structure RHC 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 comprises the structure RHC G1-RL,2-LR.

[0168] 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.

[0169] In some embodiments, delivery of a 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%, 300% compared to delivery of the same nucleic acid to the same tissue or cell type using a composition comprising a lipid, a nucleic acid and only the first or second peptide dendrimer. The cell or tissue is a cell type or organ / tissue as defined herein. For example, the tissue can be spleen, lymphoid organs, skeletal muscle, brain and adipose tissue as well as lung, tumor tissue, heart, skeletal muscle, adipose tissue, brain, liver and kidney.

[0170] In some embodiments, the first and / or second peptide dendrimer 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: 1), wherein X is 6-aminohexanoic acid and B is β-alanine.

[0171] In some embodiments, the first and / or second peptide dendrimers further comprise 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.

[0172] 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.

[0173] In some embodiments, the first and / or second 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.

[0174] In some embodiments, the lipids of the composition include cationic lipids, neutral lipids, anionic lipids, and / or ionizable lipids.

[0175] In some embodiments, the lipids of the composition comprise saturated fatty acids. Additionally or alternatively, the lipids of the composition may comprise unsaturated fatty acids.

[0176] 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.

[0177] In some embodiments, the lipid comprises dioleoylphosphatidylethanolamine (DOPE) and / or N-[1-(2,3-dioleoyloxy)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-ammonium bromide (DORI).

[0178] In some embodiments, the peptide dendrimer, nucleic acid, and lipid form positively charged particles.

[0179] In other embodiments, the peptide dendrimers, nucleic acids, and lipids form negatively charged particles or particles that have neutral charge.

[0180] The lipid component of the composition may comprise DOTMA, DOPE, DOPC and / or DOPG.

[0181] The lipid-based nucleic acid delivery system can be DOTMA / DOPE.

[0182] The amount of lipid component can be expressed as a weight:weight ratio ("w / w" or "w:w") relative to the amount of nucleic acid in the composition, and can be in the range of 1:50 to 50:1. More preferably, the amount of lipid (by weight) relative to the amount of nucleic acid (by weight) 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 about 10:1 to 25:1. These ratios refer to the weight of total lipids. As described herein, the composition can include lipids including more than one lipid component, such as a mixture of two, three or four lipids. The weight of the lipid component is the total (combined weight) of these lipid components. Preferably, each lipid component is mixed in approximately equal proportions.

[0183] Preferably, the N / P ratio of the amount of peptide (measured by the number of nitrogen atoms with a 1+ charge on the peptide, N) to the amount of nucleic acid (measured by the number of phosphate groups with a 1-charge in the backbone, P) is greater than 0.05:1, for example, greater than 0.1:1. (The N / P ratio terminology may be expressed as "N / P", "N:P" or "NP"). In some embodiments, the N / P ratio is at or about 0.15:1 or at least 0.15:1. In some embodiments, the N / P ratio is at 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, for example, 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 and 100:1, about 2:1 to about 20:1, or about 2.5:1 to about 10:1. The first and / or second peptide dendrimers 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 a fatty acid may be conjugated to the C-terminus of the first and / or second peptide dendrimers. Alternatively or in addition, the cell penetrating peptide, the endosomal escape peptide, the nuclear localization motif, and / or a fatty acid may be conjugated to the N-terminus of the first and / or second peptide dendrimers.

[0184] Relatedly, the present invention provides compositions comprising nanoparticles of the present invention. The present invention further provides pharmaceutical compositions comprising nanoparticles of the present invention and a pharmaceutically acceptable excipient. The pharmaceutical compositions can be used for medical treatment. The pharmaceutical compositions can be used to treat cancer, autoimmune diseases, lung diseases and / or myopathy. A method for treating cancer, autoimmune diseases, lung diseases and / or myopathy is also provided, wherein the method comprises administering the pharmaceutical composition to a patient or subject. In some embodiments, the composition or pharmaceutical composition is contained in a liquid. In other embodiments, the composition or pharmaceutical composition is provided as a dry composition, such as a dry powder. Freeze-drying and / or freeze-drying techniques can be used to prepare the dry composition.

[0185] In a further aspect, the present invention provides a method for producing coated nanoparticles that can transfect 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 a peptide containing glutamic acid. The dendrimer can be of the first, second, or third generation and can have one, two, three, four, or more amino acids in each generation and its core sequence, as defined herein. Without being bound by theory, surface charge and hydrophobicity can change the binding of serum components to the nanocarrier (protein corona), which can affect tissue distribution. This can be achieved, for example, by coating positive nanoparticles with linear or branched PGA (as defined herein) because PGA is a negatively charged peptide. In some embodiments, the glutamic acid-containing peptide comprises a glutamic acid-rich domain, the 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 wherein at least 20% of the amino acid residues of the glutamic acid-rich domain are glutamic acid. In some embodiments, the peptide dendrimer may comprise a glutamic acid-containing peptide. In some cases, the preformed nanoparticle has a negative surface charge and the dendrimer has a positive net charge. In some cases, the surface of the preformed nanoparticle is uncharged and the dendrimer comprises a hydrophobic region.

[0186] In some embodiments, the peptide dendrimer, PGA or glutamic acid-containing peptide comprises a cell targeting motif, such as a myeloid cell, lymphoid cell, muscle cell, lung cell, 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 mannose or maltotriose. In some embodiments, the cell targeting motif targets lymphocytes, such as T cells. In further embodiments, the cell targeting motif can be a CD3, CD4 or CD8 binding agent, such as an antibody that specifically binds to one of these markers. In some embodiments, an anti-CD3 antibody is used.

[0187] In a related aspect, the present invention provides coated nanoparticles capable of transfecting target cells, wherein the coated nanoparticles comprise peptides on their surface. In some embodiments, the nanoparticles are nanoparticles according to any aspect described herein. In some embodiments, the peptide on the surface of the coated nanoparticles may be a linear PGA, such as a short PGA comprising 2 to 100 glutamic acid residues, or the peptide may be a branched PGA. In some embodiments, the peptide is a glutamic acid-containing peptide comprising a glutamic acid-rich domain, the 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 of the glutamic acid-rich domain are glutamic acid. In some embodiments, the nanoparticles are coated with a dendritic polymer 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 aspect described herein, coated with linear PGA, branched PGA, glutamic acid-containing peptides defined herein, dendritic polymers selected from Table 1 or Table 1B, or a peptide selected from Table 1A.

[0188] 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 myeloid cell, a lymphoid cell, a muscle cell, a lung cell, a CD206+ cell, or a tumor cell targeting motif. In some embodiments, the muscle cell targeting motif comprises an ASSLNIA (SEQ ID NO: 1), a PYDQLRH (SEQ ID NO: 2), or a 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 mannose or maltotriose. In some embodiments, the cell targeting motif targets lymphocytes, such as T cells. The cell targeting motif may be a CD3, CD4, or CD8 binding agent, such as an antibody that specifically binds to one of these markers. In some embodiments, an anti-CD3 antibody is used. This can be conjugated to a negatively charged polymer such as PGA or a glutamic acid-containing peptide, or to a negatively charged lipid.

[0189] In a related aspect, the present invention provides coated nanoparticles capable of transfecting target cells, wherein the coated nanoparticles have peptides on their surface. The nanoparticles in this regard may be defined as in accordance with the aforementioned aspects of the present invention. The peptides preferably comprise peptide dendrimers. In some cases, the peptides are PGA as defined herein.

[0190] The present invention includes any combination of the described aspects and preferred features unless such a combination is expressly impermissible or explicitly avoided. BRIEF DESCRIPTION OF THE DRAWINGS

[0191] Embodiments and experiments illustrating the principles of the present invention will now be discussed with reference to the accompanying drawings, in which:

[0192] Figure 1 .A diagram shows a third generation dendrimer (N-terminus on the left and C-terminus on the right). The circles represent the core sequence. Each triangle represents a branch residue, such as lysine. Each rectangle represents a peptide motif. A third generation dendrimer has two peptide motifs in the first layer, four peptide motifs in the second layer, and eight peptide motifs in the third layer. A corresponding second generation dendrimer would lack the third layer of eight peptide motifs. A first generation dendrimer would lack the third layer of eight peptide motifs and the second layer of four peptide motifs as well as the layer of branch residues between the third and second layers.

[0193] 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 (w / w = 3.2:1 lipid:mRNA); 3) GSC G1,2-RH L, DOTMA / DOPE and eGFP mRNA (N:P=0.6:1); 4) GSC G1, 2-RL, 3-LR, DOTMA / DOPE and eGFP mRNA (N:P=0.16:1); or 5) RHC G1, RL, 2-LR, DOTMA / DOPE and eGFP mRNA (N:P=8:1). D / D indicates DOTMA:DOPE (w / w=10:1 for mRNA).

[0194] 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 either 1) untransfected or transfected with: 2) eGFP mRNA alone; 3) DOTMA / DOPE and eGFP mRNA (w / w=3.2:1 lipid:mRNA); 4) GSC G1,2-RL,3-LR, DOTMA / DOPE and eGFP mRNA (N:P=0.16:1); 5) GSCG1,2-RHL, DOTMA / DOPE and eGFPmRNA (N:P=0.6:1); 6) GSC G1-LRLR, DOTMA / DOPE and eGFP mRNA (N:P=0.6:1); 7) GSC G1,2-RHL, DOTMA / DOPE and eGFP mRNA (N:P=5:1); 8) GSC G1, 2-RF, 3HL, DOTMA / DOPE and eGFP mRNA (N:P=0.6:1); or 9) RHC G1-R, DOTMA / DOPE and eGFP mRNA (N:P=0.6:1). D / D indicates DOTMA:DOPE (w / w=10:1 for mRNA). LPX-RNA formulations included DOTMA:DOPE at w / w=3.2:1 lipid:mRNA.

[0195] Figure 4 eGFP expression in mouse macrophages following 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) GSC G1,2-RHL, DOTMA / DOPE and eGFP mRNA (N:P=8:1); or 4) containing mannose-G1-EEEE coating GSC G1,2-RHL, DOTMA / DOPE and eGFP mRNA (N:P=8:1) nanoparticles. Mannosyl-G1-EEEE dendrimer was used at 1 times the mass of mRNA in the nanoparticles (1 equivalent). D / D represents DOTMA:DOPE (w / w=2.5:1 for mRNA).

[0196] 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 (w / w = 3.2:1 lipid:mRNA); 4) GSC G1,2-RHL, DOTMA / DOPE and eGFP mRNA (N:P=8:1); or 5) containing mannose-G1-EEEE coating GSCG1,2-RHL, DOTMA / DOPE and eGFP mRNA (N:P=8:1) nanoparticles. Mannosyl-G1-EEEE dendrimer was used at 0.5 times the mass of mRNA in the nanoparticles (0.5 equivalents). D / D represents DOTMA:DOPE (w / w=5:1 for mRNA). LPX-RNA formulations contained DOTMA:DOPE w / w=3.2:1 lipid:mRNA.

[0197] 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 (w / w = 3.2:1 lipid:mRNA); 4) GSC G1,2-RHL, DOTMA / DOPE and eGFP mRNA (N:P=8:1); or 5) containing mannose-G1-EEEE coating GSC G1,2-RHL, DOTMA / DOPE and eGFP mRNA (N:P=8:1) nanoparticles. Mannosyl-G1-EEEE dendrimer was used at 1 times the mass of mRNA in the nanoparticles (1 equivalent). D / D represents DOTMA:DOPE (w / w=10:1 for mRNA). LPX-RNA formulations included DOTMA:DOPE at w / w=3.2:1 lipid:mRNA.

[0198] 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 (w / w = 3.2:1 lipid:mRNA); 4) GSC G1,2-RHL, DOTMA / DOPE and eGFP mRNA (N:P=0.6:1); or 5) containing mannose-G1-EEEE coating GSCG1,2-RHL, DOTMA / DOPE and eGFP mRNA (N:P=0.16:1) nanoparticles. Mannosyl-G1-EEEE dendrimer was used at 3 times the mass of mRNA in the nanoparticles (3 equivalents). D / D represents DOTMA:DOPE (w / w=10:1 for mRNA). LPX-RNA formulations included DOTMA:DOPE at w / w=3.2:1 lipid:mRNA.

[0199] 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 (w / w = 3.2:1 lipid:mRNA); 4) GSC G1,2-RL,3-LR, DOTMA / DOPE and eGFP mRNA (N:P=8:1); or 5) containing mannose-G1-EEEE coating GSC G1,2-RL,3

[0200] -LR, DOTMA / DOPE and eGFP mRNA (N:P=8:1) nanoparticles. Mannosyl-G1-EEEE dendrimer was used at 1 times the mass of mRNA in the nanoparticles (1 equivalent). D / D represents DOTMA:DOPE (w / w=10:1 for mRNA). LPX-RNA formulations contained DOTMA:DOPE at w / w=3.2:1 lipid:mRNA.

[0201] Fig. 9 . Delivery of mRNA to muscle cells. Mouse muscle cells (C2c12) transfected with mRNA expressing eGFP. Cells were transfected with: mRNA alone, with GSC mRNA with G1,2-R,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 the protein content of the cells to give RFU / mg. NTX2 was linked to a myocyte targeting peptide GSC G1,2-RL,3-LR; NTX3 is linked to a myocyte targeting peptide with 6 histidines GSCG1, 2-RL, 3-LR. D / D indicates DOT MA:DOPE (w / w = 10:1 for mRNA).

[0202] Fig.10 . Delivery of mRNA to muscle cells. Mouse muscle cells (C2c12) expressing eGFP after transfection with eGFP mRNA. Cells were transfected with: mRNA alone, with RHC mRNAs of G1-RL, 2-RL and DOTMA:DOPE (N:P=0.6:1), mRNAs of 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 protein content of the cells to obtain RFU / mg. NTX5 was linked to a myocyte targeting peptide RHC G1-RL, 2-LR. D / D indicates DOTMA:DOPE (w / w=10:1 for mRNA).

[0203] Fig.11 .A: Luciferase expression in mouse tissues following intravenous administration of a composition comprising: GSC G1-LRLR with DOTMA / DOPE and mRNA (lipid to mRNA ratio w / w=23:1), DOTMA / DOPE and mRNA (lipid to mRNA ratio w / w=23:1), or mRNA encoding luciferase alone. 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 signals in lung, spleen, liver, heart, kidney, muscle (gastrocnemius), and brain. B: Luciferase expression in lung of mouse tissues after intravenous administration of a composition comprising: GSC G1,2-RHL (N:P = 0.6) with DOTMA / DOPE and mRNA (lipid to mRNA ratio w / w = 23:1), DOTMA / DOPE and mRNA (lipid to mRNA ratio w / w = 23:1), or mRNA encoding luciferase alone. Dendrimer compositions were injected at an N:P ratio of 0.6:1. Mice were injected with the compositions, and luciferase signals were measured in the lung region by in vivo IVIS imaging 6 hours later. C: Luciferase expression in mouse tissues after intravenous administration of a composition comprising: GSCG1,2-RHL with DOTMA / DOPE and mRNA (lipid to mRNA ratio w / w=23:1) and mRNA encoding luciferase alone. 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 signals in lung, spleen, liver, heart, kidney, muscle (gastrocnemius) and brain.

[0204] Fig.12 .A: Luciferase expression in mouse tissues following intravenous administration of a composition comprising: GSC G1,2-RL,3-LR (N:P 0.16:1 for mRNA), G SC G1,2-RHL (N:P=0.6:1 for mRNA), GSCG1-LRLR (N:P=0.6:1 for mRNA) with DOTMA / DOPE (w / w=10:1 for mRNA) and mRNA expressing luciferase. mRNA treatment alone was used 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. B. Luciferase expression in mouse tissues after intravenous administration of a composition comprising LPX and mRNA expressing luciferase. LP X-RNA represents DOTMA:DOPE at w / w=3.2:1 for 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. C. Luciferase expression in mouse tissues after intravenous administration of a composition comprising GSCG1,2-RL,3-LR (N:P 0.16:1 for mRNA), GSCG1,2-RHL (N:P = 0.6:1 for mRNA), GSCG1-LRLR (N:P = 0.6:1 for mRNA) with DOTMA / DOPE (w / w = 10:1 for mRNA) and mRNA expressing luciferase. mRNA treatment alone was used as a control. LPX-RNA represents DOTMA:DOPE at w / w = 3.2:1 for 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. D. Luciferase expression in mouse tissues after intravenous administration of a composition comprising LPX with mRNA expressing luciferase. LPX-RNA represents DOTMA:DOPE w / w=3.2:1 against mRNA. CD-1 mice were injected with the composition and tissues were harvested 6 hours later to measure luciferase signal in muscle (gastrocnemius), liver, lung, heart, spleen, kidney, adipose tissue (fat) and brain.

[0205] Fig.13 In vivo mRNA delivery to lung and myeloid cells in cancer models. MC38-bearing mice were injected with Alexafluor488-labeled mRNA and LPX (dose 2.25 mg / kg, mRNA to body weight ratio), Alexafluor488-labeled mRNA and GSC G1,2-RL,3-LR and DOTMA:DOPE (N:P=0.16:1) (dose 2.25mg / kg, mRNA to body weight ratio), Alexafluor488 labeled mRNA and GSCG1,2-RL,3-LR, DOTMA:DOPE (N:P=0.16:1) and mannose-G1-EEEE ((Ac-EEEE)2KGSGGSGGSC[(SS)-α-D-thiomannose]) coating (dose 2.25 mg / kg, mRNA to body weight ratio) and Alexafluor488 labeled mRNA and GSC G1,2-RHL and DOTMA:DOPE (N:P = 0.6:1) (dose 2.25 mg / kg, mRNA to body weight ratio). Manno-G1-EEEE dendrimer was used at 3 times the mass of mRNA in the nanoparticles (3 equivalents). D / D represents DOTMA:DOPE (w / w = 10:1 for mRNA). LPX-RNA represents DOTMA:DOPE w / w = 3.2:1 for mRNA. Cells were isolated 4 hours after injection of the formulation for flow cytometry analysis. (A) % of all viable cells uptake of Alexafluor488-labeled mRNA in the lung, (B) % of all viable CD206+ (mannose receptor+) cells uptake of Alexafluor488-labeled mRNA in the lung, (C) % of all viable myeloid cells uptake of Alexafluor488-labeled mRNA in the lung, and (D) % of all viable M2 macrophages (CD206+, mannose receptor+ expressing cells) uptake of Alexafluor488-labeled mRNA in the lung.

[0206] Fig.14 . Luciferase and eGFP expression in HeLa or C2c12 cells after transfection with dendrimer compositions or commercially available transfection reagents. HeLa cells were transfected with: 1) luciferase mRNA alone, GSC A composition of G1,2-RL,3-LR (NP=0.16:1), DOTMA / DOPE (w / w=10:1 for mRNA) and luciferase mRNA or Lipofectamine 2000 TM and luciferase mRNA (upper left); or 2) eGFP mRNA alone, comprising GSC G1,2-RL,3-LR (NP = 0.16:1), DOTMA / DOPE (w / w = 10:1 for mRNA) and eGFP mRNA or a composition comprising DLin-MC3-DMA: cholesterol: DSPC: DMG-PEG lipid nanoparticles and eGF P mRNA (upper right). C2c12 cells were transfected with the following substances: luciferase mRNA alone, containing GSCComposition of G1,2-RL,3-LR (NP=8:1), DOTMA / DOPE (w / w=10:1 for mRNA) and luciferase mRNA, including Lipofectamine 2000 TM and luciferase mRNA or a combination of polyethyleneimine and luciferase mRNA (bottom). Luciferase and eGFP expression were measured 24 hours after transfection. Compared with commercially available transfection reagents such as Lipofectamine 2000 and LNP, peptide dendrimers were more efficient in transfecting HeLa cells and C2c12 cells in vitro.

[0207] Fig.15 The transfection efficiency of G1,2,3-RL was compared with other commercial transfection reagents for DNA delivery, including DOTMA / DOPE, polyethyleneimine, and Lipofectamine 2000. (A) Transfection in HeLa cells in the absence of serum, (B) transfection in HeLa cells in the presence of serum, (C) transfection in Neuro2A cells in the absence of serum, and (D) transfection in Neuro2A cells in the presence of serum. GSC Cells transfected with luciferase expression plasmid (pCI-Luc) in the presence of G1,2,3-RL and D / D (measured 24 h after transfection). Luminescence values ​​were normalized by dividing them by similar values ​​for cells treated with D / D DNA complexes to generate % transfection. Conditions: 1×10 cells were transfected with 0.25 μg pCI-Luc in the absence of serum or in the presence of 10% serum. 4 The error bars are the mean ± SEM of three replicates. In HeLa and Neuro2A cells, GSC G1,2,3-RL mediated transfection was significantly more potent than the other agents. * indicates p<0.05, *** indicates p<0.001, and **** indicates p<0.0001. D / D is DOTMA / DOPE at 1:1 w / w.

[0208] Fig.16Comparison of transfection efficiency of dendrimers of different generations (G1, G2 and G3) 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 transfected with luciferase expression plasmid (pCI-Luc) in the presence of peptide dendrimers with D / D (measured 24 h after transfection). Luminescence values ​​were normalized by dividing them by similar values ​​for cells treated with D / D DNA complexes (w / w 1:1, 0.25 μg) to generate % transfection. Conditions: 1×10 4 Cells were transfected with 0.25 μg pCI-Luc. Error bars are mean ± SEM of triplicate experiments. * indicates p < 0.05. D / D is DOTMA / DOPE at 1:1 w / w. Both 2nd and 3rd generation dendrimers performed better in transfecting HeLa and Neuro2A cells compared to 1st generation dendrimers.

[0209] Fig.17 Comparison of transfection efficacy between single dendrimer compositions and hybrid dendrimer compositions. HeLa cells were transfected with: A) GSC G1,2-RL,3-LR or GSC G1,2-RL,3-LR and GSC A 1:2 mixture of G1-LRLR; B) RHC G1-RL,2-LR, RHC G1-RL, 2-LR and RHC A 2:1 mixture of G1-R or RHC G1-RL, 2-LR and RHC 1:2 mixture of G1-R; C) RHC G1-RL,2-LR or R HC G1-RL, 2-LR and RHC 1:1 mixture of G1,2-R; D) RHC G1-RL,2-LR or RHC G1-RL, 2-LR and RHC 1:1 mixture of G1-RLR or RHC G1-RL, 2-LR and RHC 1:2 mixture of G1-RLR; E) RHC G1-RL,2-LR or RHC G 1-RL, 2-LR and GSC 1:1 mixture of G1-LRLR or RHC G1-RL, 2-LR and G SC1:2 mixture of G1-LRLR. Final N:P ratio of all compositions was 8:1. DOTMA:DOPE was added to mRNA complexes at w / w=10:1. Ratio between dendrimers is the molar ratio of N contributed by each dendrimer. % transfection was calculated by normalizing the transfection value of mRNA complexes formed with 1 dendrimer to the transfection value of mRNA complexes formed with 2 dendrimers multiplied by 100% For in vitro transfection, hybrid dendrimer systems performed better than single dendrimer systems.

[0210] Fig.18 . Demonstration of transfection efficiency in C2c12 cells. Labeled mRNA was delivered to cells. After transfection, mouse muscle cells (C2c12) took up mRNA labeled with Alexa488 fluorophore. Cells were transfected for 4 hours with: labeled mRNA alone, labeled mRNA with NTX3 (N:P=0.6:1) and DOTMA:DOPE, and the uptake of mRNA was measured by flow cytometry. NTX3 was linked to a muscle cell targeting peptide with 6 histidines. GSC G1, 2-RL, 3-LR. DOTMA:DOPE was used at a w / w ratio of 10:1 for mRNA.

[0211] Fig.19 . Demonstration of transfection efficiency in macrophages. Labeled mRNA was delivered to cells. After transfection, macrophages (J774 cells) took up mRNA labeled with Alexa488 fluorophore. Cells were incubated with labeled mRNA and GSC G1,2-RHL (N:P = 0.6:1) and DOT MA:DOPE were transfected for 4 hours and the uptake of mRNA was measured by flow cytometry. Cells were transfected with different amounts of mRNA from 0.0015ug to 1.5ug. DOTMA:DOPE was used at w / w = 10:1 for mRNA.

[0212] Fig. 20 . Demonstration of transfection efficiency in T cells. Delivery of labeled mRNA to cells. After transfection, human T cells (Jurkat cells) took up mRNA labeled with Alexa488 fluorophore. Cells were incubated with labeled mRNA and GSC G1,2-RHL (N:P = 0.6:1) and DOTMA:DOPE were transfected for 4 hours and the uptake of mRNA was measured by flow cytometry. Cells were transfected with different amounts of mRNA from 0.0015ug to 1.5ug. DOTMA:DOPE was used at w / w = 10:1 for mRNA.

[0213] Fig.21. Further demonstrate transfection efficiency in T cells. Delivery of labeled mRNA to cells. After transfection, HeLa cells take up mRNA labeled with Alexa488 fluorophore. Cells are incubated with labeled mRNA and GSC G1,2-RHL (N:P=0.6:1 for mRNA) and DOTMA:DOPE were transfected for 2 hours, and the uptake of mRNA was measured by flow cytometry. Cells were transfected with different amounts of mRNA from 0.1875ug to 1.5ug. DOTMA:DOPE was used at w / w=10:1 for mRNA.

[0214] Fig. 22 HeLa cells were transfected with nanocarriers formulated with different lipid compositions. The nanocarriers containing the three lipid systems showed higher transfection efficacy than the nanocarriers containing DOTMA:DOPE.

[0215] Fig.23 HeLa cells were transfected with nanocarriers formulated with different lipid compositions. The nanocarriers containing the three lipid systems showed higher transfection efficacy than the nanocarriers containing DOTMA:DOPE.

[0216] Fig.24 A549 cells were transfected with nanocarriers formulated with different lipid compositions. The nanocarriers containing the three lipid systems showed higher transfection efficacy than the nanocarriers containing DOTMA:DOPE.

[0217] Fig.25 Dendritic PGA coating enhances delivery to T cells. Human T cells were treated with a formulation containing mRNA expressing eGFP (alone) or with GSC G1,2-RHL was transfected and coated with either linear PGA or dendritic PGA. The eGFP expression of the cells was quantified by flow cytometry and normalized to the level achieved with the preparation coated with linear PGA. Panel A (top): Equivalent molar amounts of linear and dendritic PGA. Panel B (bottom): Equivalent molar charge of linear and dendritic PGA.

[0218] Fig.26 Enhanced targeting of undifferentiated muscle cells with 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 a muscle targeting domain achieved a substantial increase in eGFP expression compared to particles coated with dendrimers without a muscle targeting domain.

[0219] Fig. 27Enhanced targeting of differentiated muscle cells with 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 a muscle targeting domain achieved a substantial increase in eGFP expression compared to particles coated with dendrimers without a muscle targeting domain.

[0220] Fig.28 Enhanced targeting of tumor cells with particles containing mRNA expressing eGFP. Nanocarriers were coated with molar equivalents of dendritic PGA with or without integrin targeting domains. Particles coated with dendrimers containing integrin targeting domains achieved a substantial increase in eGFP expression compared to particles coated with dendrimers without integrin targeting domains.

[0221] Fig.29 .Enhanced targeting of human T cells with 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 antibodies achieved a substantial increase in eGFP expression compared to particles coated with dendrimers without anti-CD3 antibodies. D / D denotes DOTMA:DOPE (w / w=10:1 for mRNA). eq refers to equivalents; hCD3 refers to human CD3; Ab refers to antibody; and ctr refers to control; ITC refers to isotype control. mRNA expressing eGFP was used.

[0222] Fig.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 mixed with two mRNAs or with a preparation of 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.

[0223] Fig.31 Functional delivery of mRNA molecules into a subject using nanocarriers of the invention comprising two nucleic acids. High luciferase levels were observed in the lungs and spleen six hours after intravenous administration of nanocarriers comprising luciferase mRNA and CpG-containing nucleic acids to mice.

[0224] Fig.32. Transfection of primary leukocytes with nanocarriers formulated with various nucleic acids. Primary mouse monocyte-derived dendritic cells (moDCs) were transfected with nanocarriers containing CpG molecules and mRNA expressing eGFP. Functional expression of eGFP was observed after 2 hours of transfection (Fig. A), 4 hours of transfection (Fig. B), and 22 hours of transfection (Fig. C). D / D indicates DOTMA:DOPE (w / w = 10:1 for mRNA).

[0225] Fig.33 . Inducing M1 macrophages using the nanocarriers of the present invention. Activated IRF5 was delivered to primary M2 macrophages using nanocarriers. Figure A is a volcano plot showing significant upregulation of M1 genes and downregulation of M2 genes. Figure B depicts the results of gene set enrichment analysis (GSEA), showing the number of differentially expressed genes in cells to which modified IRF5 was delivered via the nanocarriers of the present invention.

[0226] Fig.34 . Inducing cytokine secretion by M1 macrophages using the nanocarriers of the present invention. Modified IRF5 was delivered to M2 macrophages using the nanocarriers of the present invention to polarize them to M1 macrophages. Control: M2 macrophages to which luciferase was delivered via the nanocarriers of the present invention. Cytokine secretion was measured after 24 hours. Figures A and B show IL12 p70 and p40 secretion after repolarization of mouse macrophages, respectively. Figures C and D show IL12 p70 and TNFα secretion after repolarization of human macrophages, respectively.

[0227] Fig.35 .Delivery of therapeutic mRNA using the nanocarrier of the present invention significantly inhibited tumor growth. Therapeutic mRNA expression induced modified IRF5 in M1 macrophages. MC38 cancer growth was significantly inhibited.

[0228] Fig.36 Jurkat cells were transfected with nanocarriers formulated with different lipid compositions. The "mRNA alone" control and the control nanocarriers containing DOTMA:DOPE contained a 1.5 ug "dose" of mRNA expressing eGFP (in a well of a 12-well plate). In contrast, the nanocarriers with the test lipids contained 20% of the mRNA used in the control transfection, e.g., 0.3 ug per test nanocarrier formulation.

[0229] Fig.37HeLa cells were transfected with nanocarriers formulated with different lipid compositions. The "mRNA alone" control and the control nanocarriers containing DOTMA:DOPE contained a 0.25ug "dose" of mRNA expressing eGFP (in a well of a 96-well plate). In contrast, the nanocarriers with the test lipids contained 67% of the mRNA used in the control transfection, e.g., 0.17ug per test nanocarrier formulation.

[0230] Fig.38 A549 cells were transfected with nanocarriers formulated with different lipid compositions. The "mRNA alone" control and the control nanocarriers containing DOTMA:DOPE contained a 0.25ug "dose" of mRNA expressing eGFP (in a well of a 96-well plate). In contrast, the nanocarriers with the test lipids contained 67% of the mRNA used in the control transfection, e.g., 0.17ug per test nanocarrier formulation.

[0231] Fig.39 . In vivo delivery of mRNA to myeloid cells in tumors. Mice bearing MC38 tumors were injected with Alexafluor488-labeled mRNA and LPX (dose of 2.25 mg / kg, mRNA to body weight ratio), Alexafluor488-labeled mRNA and GSC G1,2-RL,3-LR and DOTMA:DOPE (N:P=0.16:1) (dose of 2.25 mg / kg, ratio of mRNA to body weight), Alexafluor488-labeled mRNA and GSC G1,2-RL,3-LR, DOTMA:DOPE (N:P=0.16:1) and mannose-G1-EEEE ((Ac-EEEE)2K GSGGSGGSC[(SS)-α-D-thiomannose]) coating (dose 2.25 mg / kg, mRNA to body weight ratio) and Alexafluor488-labeled mRNA GSCG1,2-RHL and DOTMA:DOPE (N:P=0.6:1) (dose 2.25 mg / kg, mRNA to body weight ratio). Mannosyl-G1-EEEE dendrimer was used at 3 times the mass of mRNA in the nanoparticles (3 equivalents). D / D represents DOTMA:DOPE (w / w=10:1 for mRNA). LPX-RNA represents DOTMA:DOPE at w / w=3.2:1 for mRNA. Cells were isolated 4 hours after injection of the formulation for flow cytometry analysis. (A) % of all viable myeloid cells uptake of Alexafluor488-labeled mRNA, (B) % of all viable M2 macrophages (CD206+, mannose receptor+ expressing cells) uptake of Alexafluor488-labeled mRNA in tumors, and (C) % of all viable myeloid-derived suppressor cells (MDSC) uptake of Alexafluor488-labeled mRNA in tumors.

[0232] Fig.40 .Intravenous administration includes GSC Luciferase expression in tumors of mice bearing MC38 tumors following treatment with a composition of G1,2-RHL (N:P=0.6:1 for mRNA) with DOTMA / DOPE (w / w=10:1 for mRNA) and mRNA expressing luciferase. mRNA treatment alone and LPX-RNA were used as controls. LPX-RNA represents DOTMA:DOPE at w / w=3.2:1 for mRNA. Mice were injected with the composition and luciferase signal in the tumor area was measured by in vivo IVIS imaging 6 hours later.

[0233] Fig.41 .A: Primary mouse bone marrow-derived macrophages were polarized to an M2 phenotype. The cells were then transfected with mRNA expressing a modified form of the IRF5 protein for 24 hours, which can polarize the M2 cells to M1 cells. Control: Cells were transfected with mRNA expressing luciferase (control mRNA). The modified IRF5 is a protein with a mutation that acts as an activated form of WT IRF5. The formulation used contained GSCG1,2-RHL (N:P=0.6:1 for mRNA) with DOTMA / DOPE (w / w=10:1 for mRNA) and mRNA. WT IRF5 and modified IRF5 protein expression levels were detected by flow cytometry and expressed as % of IRF5 expressing cells. B: Primary mouse bone marrow-derived macrophages were polarized to an M2 phenotype. The cells were then transfected with mRNA expressing a modified form of the IRF5 protein for 24 hours, which can polarize M2 cells to M1 cells. Control: Cells were transfected with mRNA expressing luciferase (control mRNA). Modified IRF5 is a protein with a mutation that acts as an activated form of WT IRF5. The formulation used contained GSC G1,2-RHL (N:P=0.6:1 for mRNA) with DOTMA / DOPE (w / w=10:1 for mRNA) and mRNA. M2 cells transfected with modified IRF5 mRNA resulted in increased expression of M1 markers (CD80), indicating M1 polarization.

[0234] Fig.42 .Nanocarriers coated with human CD3-targeting antibodies improve human T cell-targeted mRNA delivery. Jurkat cells were transfected with: (1) GSC G1,2-RHL and DOTMA / DOPE (N:P=0.6 for mRNA) and mRNA, (2) GSC G1,2-RHL and DOTMA / DOPE (N:P=0.6 for mRNA) and mRNA, coated with 3 equivalents of anti-CD3 antibody-conjugated dendrimer, (3) GSC G1,2-RHL and DOTMA / DOPE (N:P=0.6 for mRNA) with mRNA, dendrimer alone (control for (2)), (4) GSC G1,2-RHL and DOTMA / DOPE (N:P=0.6 for mRNA) with mRNA, coated with 1 equivalent of anti-CD3 antibody-conjugated dendrimer, and (5) GSC G1,2-RHL and DOTMA / DOPE (N:P = 0.6 for mRNA) with mRNA, dendrimer alone coated (control of (4)). D / D indicates DOTMA:DOPE (w / w = 10:1 for mRNA). Transfection was analyzed by flow cytometry 24 hours after formulation. eq means equivalent; hCD3 means human CD3; Ab means antibody, and ctr means control. mRNA expressing eGFP was used. DETAILED DESCRIPTION

[0235] Various aspects and embodiments of the present invention will now be discussed with reference to the accompanying drawings and the following technical definitions. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated herein by reference.

[0236] The current immunotherapy response rate is around 15-20%, and there is an urgent need to improve the treatment effect. One strategy is to deliver mRNA to express proteins (such as CEBPA, IRF5, IRF8, cGAS–STING, SOCS1 and / or SOCS3) to restore the immunosuppressive phenotype of myeloid cells in the tumor microenvironment, which will provide a more favorable response environment for immunotherapy. Another strategy may be to transfer mRNA to immune cells in the tumor to express cytokines (such as IL-2, IL-7, IL-12, IL-15, IL-21 and / or interferon), thereby activating immune cells to fight cancer cells. mRNA can also be delivered to macrophages to express chimeric antigen receptors so that macrophages can be activated to kill tumor cells. Delivery of mRNA to express tumor antigens in antigen presenting cells will help activate the immune system to attack cancer cells. These strategies can be used 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, biliary tract 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, GI tract tumors, head and neck squamous cell carcinoma (HNSCC), renal cancer, myelofibrosis, CD206+ cancers, melanoma, prostate cancer or anal cancer and solid tumors.

[0237] The compositions of the present invention can be used to treat lung diseases such as 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 hypertension, fibrotic lung disease, chronic lung disease or respiratory tract infection. The compositions of the present invention can be used to treat muscle diseases such as muscular dystrophy or muscular dystrophy. The compositions of the present invention can be used to treat kidney disease. The compositions of the present invention can be used to treat rare genetic diseases (hematology, neurology, amyloidosis, pulmonology, endocrinology and nephrology). The compositions of the present invention can be used to deliver anti-infective drugs (e.g., to target macrophages to kill bacteria / deliver antibiotic payloads).

[0238] Nano-particle of the present invention and composition can be used for delivering nucleic acid as described herein to certain tissues of human or animal body.For example, it is possible to deliver to skeletal muscle, liver, lung, heart, white and / or brown adipose tissue, brain, spleen, bone marrow, joint, kidney, gastrointestinal tract, eyes, thymus, skin, lymph node, pancreas, adrenal gland, testis, prostate, ovary, uterus, bladder, diaphragm and tumor.In some embodiments, the present invention can be used for delivering nucleic acid to skeletal muscle, lung, spleen, bone marrow, thymus, lymph node and tumor.Especially, nano-particle of the present invention is particularly effective when delivering nucleic acid goods to myeloid cell, lymphoid cell, myocyte and pneumonocyte targeting.

[0239] With regard to myeloid cells, the nanoparticles of the present invention can effectively deliver nucleic acid cargo to cells expressing CD206, such as macrophages, neutrophils, dendritic cells, endothelial cells, and lymphocytes, particularly M2 phenotype macrophages.

[0240] About lung cells, the nanoparticles of the present invention can target the delivery of cargo to alveolar macrophages, ciliated cells, epithelial cells, basal cells, secretory cells, club cells, alveolar cells, fibroblasts and / or endothelial cells. Improving the 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.

[0241] Cell and tissue targeting

[0242] The inventors have found that by changing the physical properties of the nanoparticles, the nanoparticles of the invention can be specifically targeted to cells within a particular tissue with greater efficiency. This can be achieved in a variety of ways.

[0243] For example, the nanoparticle may comprise a tissue or cell specific targeting motif. In this context, the tissue or cell specific targeting motif may be a cell surface receptor ligand. The receptor ligand may be a fragment of a protein or 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 in one or more peptide motifs. Alternatively or in addition, the protein or peptide ligand may also be present in a linear peptide included in the nanoparticle.

[0244] The receptor ligand may also be a sugar. For example, the sugar may be selected from mannose, galactose or glucose. Preferably, the sugar is mannose. The sugar may be covalently bound to a component of the nanoparticle, for example, it may be covalently bound to a peptide dendrimer, or it may be covalently bound to a linear peptide in the nanoparticle. The sugar may also be covalently bound to a polymer (e.g., PGA) or a lipid.

[0245] Regardless of the type of ligand forming the cell and / or tissue targeting motif, the ligand will preferentially 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 primarily expressed in the target cell type or tissue. For example, CD206 on CD206+ cells. In an example, M2 phenotype macrophages may be targeted for nucleic acid delivery by including mannose ligands in nanoparticles. Mannose ligands may target nucleic acid delivery to M2 macrophages because M2 macrophages exhibit high levels of mannose receptor expression.

[0246] The inventors have also found that by changing the lipid:nucleic acid weight / weight ratio, the delivery of nucleic acid to specific cells and tissue types can be achieved. In particular, the specific delivery of nucleic acid to the lungs can be achieved by using a lipid:nucleic acid w / w ratio between 2:1 and 40:1, preferably 23:1 or 10:1.

[0247] The efficiency of macrophage transfection using nanoparticles containing one of the three peptide dendrimers with different N:P ratios was studied by transfecting human and mouse macrophages in vitro under complete growth medium conditions. The results showed that each peptide dendrimer was able to transfect human and mouse macrophages with high efficiency ( Figure 2 and 3 In particular, the results showed that for mouse macrophages, peptide dendrimer / lipid nanoparticles outperformed lipid-based delivery vehicles containing only DOTMA / DOPE ( Figure 2 ).

[0248] Certain macrophage populations, namely M2 phenotype macrophages, have enriched expression of the mannose receptor CD206. Thus, in some instances, the dendrimers used in the nanoparticles of the present invention comprise mannose for binding to the CD206 receptor. Certain dendrimers of the present invention that are particularly preferred for delivering mRNA to myeloid cells include G1,2-RHL, G1,2-RL,3-LR, G1-RL,2LR, G1-RL,2-LR, and particularly 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 can be derivatized to include mannose glycosylation.

[0249] Certain dendrimers of the invention that are particularly preferred for delivering mRNA to lymphoid cells include dendrimers conjugated or covalently bound to anti-CD3 antibodies or anti-CD3 antibody fragments.

[0250] Certain dendrimers of the invention that are particularly preferred for delivering mRNA to muscle cells include G1,2-RL,3-LR and G1-RL,2-LR, and in particular those containing the ASSLNIA (SEQ ID NO: 1) peptide motif (e.g., NTX2, NTX3, and NTX5). GSC G1,2-RL,3-LR and GSC G1-RL, 2-LR. NP ratio can be 0.6:1.

[0251] Certain dendrimers of the invention that are particularly preferred for delivering mRNA to lung cells include G1-LRLR, and in particular GSC G1-LRLR.NP ratio can be 0.6:1.

[0252] Coated Nanoparticles

[0253] In the aspect of providing nanoparticles coated with peptides and / or dendrimers, this means that the peptide / dendrimer is present on the surface of the coated nanoparticles. This can be achieved simply by first preparing nanoparticles (which preferably contain one or more peptide dendrimers and nucleic acids and lipids), and then mixing the nanoparticle preparation with the peptide and / or dendrimer to be coated on the surface of the nanoparticles. For nanoparticles with positive or negative surface charges, successful coating can be easily determined by monitoring the zeta potential of the nanoparticles. After a positively charged nanoparticle has been coated with a negatively charged peptide / dendrimer, its zeta potential becomes substantially less positive. Conversely, after a negatively charged nanoparticle has been coated with a negatively charged peptide / dendrimer, its zeta potential becomes substantially less negative. In some embodiments, after coating with the peptide / dendrimer, the zeta potential of the nanoparticle changes from positive to negative, or from negative to positive. (In contrast, the inclusion of 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 may be used, such as measuring particle size before and after coating. Such methods can be used to confirm whether uncharged nanoparticles have been successfully coated with, for example, a hydrophobic peptide / dendrimer.

[0254] mRNA transfection

[0255] The effect of delivering mRNA using a composition comprising two different peptide dendrimers was investigated by transfecting HeLa cells under complete growth medium conditions. The results showed that by including two different peptide dendrimers in the transfection composition, the transfection efficiency could be significantly improved ( Fig.17Specifically, the results showed that by including a generation 1 ("G1") dendrimer in combination with a generation 2 ("G2") dendrimer, this could result in transfection efficiencies up to 3 times greater than when using the G2 dendrimer alone ( Figure 5 B) Transfection efficiency in compositions comprising two different peptide dendrimers can also be modulated by varying the ratio of G2:G1 peptide dendrimers in the composition. RHC G1-RL, 2-LR and G1 dendrimers RHC In the composition of G1-R, the G2:G1 ratio of 2:1 had a transfection efficiency of approximately 66% compared to the G2:G1 ratio of 1:2 ( Fig.17 B).

[0256] The present inventors have also investigated the effect of using two different G2 dendrimers on transfection efficiency and found that peptide dendrimers comprising e.g. RHC G1-RL, 2-LR and RHC G1,2-R combination and single RHC G1-RL, 2-LR have improved transfection efficiency ( Fig.17 C).

[0257] Messenger RNA (mRNA)

[0258] Messenger RNA (mRNA) is a single-stranded molecule of RNA that translates the coding sequence of a gene into a corresponding amino acid sequence by ribosomes. mRNA is produced during the transcription process, where an enzyme (RNA polymerase) converts the gene into a primary transcript mRNA (also referred to as pre-mRNA). This pre-mRNA usually still contains introns, which are regions that will not continue to encode the final amino acid sequence. These are removed during RNA splicing, leaving only exons, which are regions that will encode proteins. This exon sequence constitutes mature mRNA. The mature mRNA is then read by ribosomes to produce encoded proteins. The present invention can be used to deliver mRNA molecules to target cells and tissues as a means of inducing the expression of desired proteins or peptides. When transient expression is required, it is particularly useful to deliver induced peptide / protein expression via mRNA. To improve mRNA expression, synonymous codons in mRNA can be changed based on the codon bias of the organism-that is, mRNA can be codon optimized. For example, mRNA can be codon optimized to be expressed in the organism type of the subject to whom the composition of the present invention is to be applied. For example, the mRNA can be codon optimized for expression in a mammal (eg, a human).

[0259] In particular, delivery of mRNA encoding a chimeric antigen receptor (CAR) and a transcription factor is contemplated.

[0260] mRNA and lncRNA (discussed below) are typically macromolecules with one negatively charged side and a hydrophobic side. Therefore, mRNA and lncRNA will need to strike a balance between hydrophobic and hydrophilic interactions to be encapsulated and delivered to target tissues and cells. This balance between hydrophobic and hydrophilic interactions will be different from, for example, double-stranded nucleic acids with charges on both sides, such as pDNA and siRNA. Since mRNA and lncRNA are much larger than, for example, ASOs, the requirements for encapsulation and delivery will also likely be different. Therefore, compared to ASO delivery, the optimal NP ratio of dendrimers for mRNA and lncRNA delivery and the w / w ratio of DOTMA / DO PE will be different.

[0261] Modified Nucleic Acids

[0262] In addition to naturally occurring bases, modified nucleotide bases can also be used and can impart advantageous properties to nucleic acids containing them.

[0263] For example, modified bases can increase the stability of nucleic acid molecules, thereby reducing the amount required. Providing modified bases can also provide nucleic acid molecules that are more stable or less stable than unmodified nucleic acids.

[0264] The term "modified nucleotide base" encompasses nucleotides with covalently modified bases and / or sugars. For example, modified nucleotides include nucleotides with sugars that are covalently linked to low molecular weight organic groups other than the hydroxyl group at the 3' position and the phosphate group at the 5' position. Thus, modified nucleotides may 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'-halogenated or azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose, furanose, and sedoheptose.

[0265] Modified nucleotides are known in the art and include alkylated purines and pyrimidines, acylated purines and pyrimidines, and other heterocyclic compounds. These classes of pyrimidines and purines are known in the art and include pseudoisocytosine, N4, N4-ethylenecytosine, 8-hydroxy-N6-methyladenine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)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-methoxy uracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-hydroxyacetic acid methyl ester, pseudouracil, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-hydroxy acid methyl ester, uracil 5-hydroxyacetic acid, quarkside, 2-thiocytosine, 5-propyluracil, 5-propylcytosine, 5-ethyluracil, 5-ethylcytosine, 5-butyluracil, 5-pentyluracil, 5-pentylcytosine and 2,6, diaminopurine, methyl pseudouracil, 1-methylguanine, 1-methylcytosine.

[0266] RNA interference

[0267] The present invention promotes therapeutic downregulation of target gene expression via delivery of nucleic acids. These include RNA interference (RNAi). Small RNA molecules can be used to regulate gene expression.

[0268] These include targeted degradation of mRNA by small interfering RNA (siRNA), posttranscriptional gene silencing (PTG), developmentally regulated sequence-specific translational inhibition of mRNA by microRNA (miRNA), and targeted transcriptional gene silencing.

[0269] The role of RNAi mechanisms and small RNAs in epigenetic gene silencing at targeted heterochromatin complexes and specific chromosomal loci has also been demonstrated. Double-stranded RNA (dsRNA)-dependent post-transcriptional silencing, also known as RNA interference (RNAi), is a phenomenon in which dsRNA complexes can target homologous specific genes for silencing in a short period of time. It acts as a signal to promote the degradation of mRNA with sequence identity. 21-nt siRNAs are generally long enough to induce gene-specific silencing, but short enough to escape host responses. The reduction in expression of targeted gene products can be extensive, with 90% of silencing induced by a few siRNA molecules.

[0270] In the art, these RNA sequences are referred to as "short or small interfering RNA" (siRNA) or "micro RNA" (miRNA), depending on their origin. Both types of sequences can be used to downregulate gene expression by binding to complementary RNA and triggering mRNA elimination (RNAi) or preventing mRNA translation into protein. siRNA is obtained by processing long double-stranded RNA and is usually of exogenous origin when found in nature. Microinterfering RNA (miRNA) is an endogenously encoded small non-coding RNA obtained by processing short hairpins. Both siRNA and miRNA can inhibit the translation of mRNA with partially complementary target sequences without RNA cleavage, and degrade mRNA with completely complementary sequences.

[0271] Therefore, the present invention provides the use of these sequences in the compositions of the present invention for down-regulating the expression of target genes.For example, it is envisioned that the nanoparticles of the present invention can be used to deliver RNAi-based therapies for the treatment of diabetes (eg, type I or type II diabetes).

[0272] siRNA ligands are typically double-stranded, and to optimize the effectiveness of RNA-mediated downregulation of target gene function, it is preferred to select the length of the siRNA molecule to ensure proper recognition of the siRNA by the RISC complex, which mediates recognition of the siRNA to the mRNA target, and to make the siRNA short enough to reduce host responses.

[0273] miRNA ligands are usually single-stranded and have partially complementary regions that enable the ligand to form a hairpin. miRNA is an RNA gene that is transcribed from DNA but not translated into protein. The DNA sequence encoding the miRNA gene is longer than the miRNA. This DNA sequence includes the miRNA sequence and an approximately reverse complementary sequence. When this DNA sequence is transcribed into a single-stranded RNA molecule, the miRNA sequence and its reverse complementary base pair to form a partial double-stranded RNA segment. John et al. discussed the design of microRNA sequences in 2004.

[0274] Typically, the RNA ligand intended to simulate the effect of siRNA or miRNA has between 10 and 40 ribonucleotides (or synthetic analogs thereof), more preferably between 17 and 30 ribonucleotides, more preferably between 19 and 25 ribonucleotides, and most preferably between 21 and 23 ribonucleotides. In some embodiments of the present invention using double-stranded siRNA, the molecule may have a symmetrical 3' overhang, such as a 3' overhang of one or two (ribose) nucleotides, typically the UU of dTdT3' overhang. Based on the disclosure provided herein, the technician can easily design suitable siRNA and miRNA sequences, such as using resources such as Ambion's online siRNA finder. siRNA and miRNA sequences can be synthesized and exogenously added to cause gene downregulation, or produced using an expression system (e.g., vector). In a preferred embodiment, siRNA is synthesized synthetically.

[0275] Longer double-stranded RNA can be processed in cells to produce siRNA (see, e.g., Myers et al., (2003)). Longer dsRNA molecules can have symmetrical 3' or 5' overhangs, e.g., 3' or 5' overhangs of one or two (ribo) nucleotides, or can have blunt ends. Longer dsRNA molecules can be 25 nucleotides or longer. Preferably, the longer dsRNA molecule length is between 25 and 30 nucleotides. More preferably, the longer dsRNA molecule length is between 25 and 27 nucleotides. Most preferably, the longer dsRNA molecule length is 27 nucleotides.

[0276] In one embodiment, siRNA, longer dsRNA or miRNA is produced endogenously (in the cell) by transcription from a vector. The vector can be introduced into the cell in any manner known in the art. Optionally, a tissue-specific promoter can be used to regulate the expression of the RNA sequence. In a further embodiment, siRNA, longer dsRNA or miRNA is produced exogenously (in vitro) by transcription from a vector.

[0277] 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 a surrogate bond, such as a linker of the formula P(O)S (thioester), P(S)S (dithioester), P(O)NR'2, P(O)R', P(O)OR6, CO or CONR'2, wherein R is H (or salt) or alkyl (1-12C), and R6 is alkyl (1-9C), connected to adjacent nucleotides via -O- or -S-.

[0278] Long noncoding RNA

[0279] The mammalian genome is universally transcribed, producing a large number of transcripts, including thousands of long non-coding RNA molecules (lncRNA). It has been shown that lncRNA can regulate chromatin state, transcription, RNA stability and translation of certain genes. The nanoparticles of the present invention can be used to deliver lncRNA to target cells or tissues.

[0280] RNA activation (RNAa )

[0281] RNA activation (RNAa) is a process mediated by RNA to enhance gene expression via a highly regulated and evolutionarily conserved pathway. RNAa can be induced by small activating RNA (saRNA), which is a class of non-coding RNA consisting of 21 nucleotide dsRNA with 2 nucleotide overhangs at both ends. SaRNA has the same structure and chemical components as siRNA, despite the fact that saRNA mediates gene activation in a sequence-specific manner. In order to activate gene expression, the guide strand of saRNA is loaded onto AGO2, and then the complex is transported to the nucleus. Once in the nucleus, the guide strand-AGO2 complex directly binds to the gene promoter or related transcript, recruiting key components including RNA polymerase II to initiate gene activation (Kwok et al., 2019).

[0282] Antisense Oligonucleotides (ASOs)

[0283] Antisense oligonucleotides (ASOs) are single strands of DNA or RNA that are complementary to a target sequence. ASOs hybridize with target nucleic acids. For example, ASOs can be used to target coding or non-coding RNA molecules in cells. After target binding, the ASO / target complex can be enzymatically degraded, for example by RNase H.

[0284] Circular RNA

[0285] 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 due to the formation of a covalent bond 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. Therefore, compared with comparable non-circular RNA, circRNA has an increased half-life. For example, circRNA produced by protein-coding genes as an alternative splicing form is more stable than the corresponding linear mRNA of the same protein-coding gene. Many functions are attributed to circRNA, including protein complex scaffolds, parental gene regulation, RNA-protein interactions, and microRNA sponges. Recently, it has been recognized that circRNA may be useful in a range of therapeutic methods. For example, circRNA can be used as a microRNA "sponge" to isolate microRNA. CircRNA can also be used as a source of protein translation, which can persist in cells longer than standard linear mRNA. CircRNA can also be used to control protein activity by acting as an aptamer.

[0286] Modified Nucleic Acids

[0287] In addition to naturally occurring bases, modified nucleotide bases can also be used and can impart advantageous properties to nucleic acids containing them.

[0288] For example, modified bases can increase the stability of nucleic acid molecules, thereby reducing the amount required. Providing modified bases can also provide nucleic acid molecules that are more stable or less stable than unmodified nucleic acids.

[0289] The term "modified nucleotide base" encompasses nucleotides with covalently modified bases and / or sugars. For example, modified nucleotides include nucleotides with sugars that are covalently linked to low molecular weight organic groups other than the hydroxyl group at the 3' position and the phosphate group at the 5' position. Thus, modified nucleotides may 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'-halogenated or azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose, furanose, and sedoheptose.

[0290] Modified nucleotides are known in the art and include alkylated purines and pyrimidines, acylated purines and pyrimidines, and other heterocyclic compounds. These classes of pyrimidines and purines are known in the art and include pseudoisocytosine, N4, N4-ethylenecytosine, 8-hydroxy-N6-methyladenine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)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-methoxy uracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-hydroxyacetic acid methyl ester, pseudouracil, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-hydroxy acid methyl ester, uracil 5-hydroxyacetic acid, quarkside, 2-thiocytosine, 5-propyluracil, 5-propylcytosine, 5-ethyluracil, 5-ethylcytosine, 5-butyluracil, 5-pentyluracil, 5-pentylcytosine and 2,6, diaminopurine, methyl pseudouracil, 1-methylguanine, 1-methylcytosine.

[0291] Medical treatment

[0292] The present invention contemplates the use in gene therapy schemes. Using DNA or RNA, and particularly mRNA, gene therapy schemes are considered for the present invention. Nucleic acid may be present in a composition, and the composition causes the expression of therapeutic gene products such as transgenes when introduced into target cells. Target cells include myocytes, splenocytes, hepatocytes, stellate cells, brain cells (neurons, astrocytes), splenocytes, lung cells, heart cells, kidney cells, adipocytes, stem cells, monocytes, macrophages, dendritic cells, neutrophils, B cells, T cells, myeloid-derived suppressor cells, tumor-associated macrophages, tumor-associated neutrophils or tumor cells. In some embodiments, target cells include myocytes, splenocytes, lung cells, heart cells, stem cells, monocytes, macrophages, dendritic cells, neutrophils, B cells, T cells, myeloid-derived suppressor cells, tumor-associated macrophages, tumor-associated neutrophils or tumor cells.

[0293] In order to make gene therapy practical, it is necessary to adopt a DNA / RNA transfer system that: (1) directs the therapeutic sequence into target cells, (2) mediates the uptake of the therapeutic nucleic acid into a certain proportion of the target cell population, and (3) is suitable for in vivo and / or ex vivo therapeutic applications.

[0294] The compositions of the present invention are particularly suitable for mediating the uptake of therapeutic nucleic acids into a relatively high proportion of the target cell population, such as Example 9 and Fig.14 and 15 as shown in .

[0295] 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 regulate the expression or activity of an endogenous gene. Regulation can be an increase in the expression of the gene and / or exogenous expression of more copies of the gene, or regulation can be a decrease in the expression of the gene.

[0296] Transgenic can be a protein of a viral protein, a bacterial protein or a microorganism parasitizing a mammal. The composition expressing a viral protein, a bacterial protein or a parasitic microorganism protein can be used as a vaccine. For example, an effective amount of the composition can be systemically delivered to a subject (e.g., intravenously) to achieve expression of a viral protein, a bacterial protein or a parasitic microorganism protein in the skeletal muscle of the subject, so as to induce an immune response to the virus, bacteria or parasitic protein. Therefore, the present invention provides a method for vaccinating a subject and a composition for vaccinating a subject. In such embodiments, transgenic can be expressed in immune cells as described herein, such as leukocytes, such as B lymphocytes, T lymphocytes, monocytes, neutrophils, dendritic cells, macrophages or monocytes; lymph node tissue cells.

[0297] 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 co-stimulatory receptor, a co-stimulatory ligand, a checkpoint inhibitor, an immunoconjugate, or a tumor antigen.

[0298] Transgenes can express therapeutic proteins for gene therapy. Gene therapy can be used to treat autoimmune disorders in patients, such as type I diabetes (also known as juvenile diabetes), cancer, and / or genetic disorders. Transgenes can be functional versions of genes that are non-functional, down-regulated, inactivated, or damaged in a subject.

[0299] The inherited disorder can be a monogenic disorder, such as muscular dystrophy in a patient. In embodiments where the monogenic disorder is muscular dystrophy, the transgene can be dystrophin. In embodiments where the disorder is ischemia, the transgene can be hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and fibroblast growth factor (FGF). In embodiments where the disorder is muscular atrophy, the transgene can be follistatin. In embodiments where the disorder is a neuromuscular disease, the transgene can be acid alpha-glucosidase (GAA). Although the transgene can be expressed in one or more tissues disclosed herein, the expressed protein can be secreted from the tissue into the circulation.

[0300] It is contemplated that the present invention may be used to deliver nucleic acid therapeutics to treat myopathies. It is also contemplated that the present invention may be used to deliver nucleic acid therapeutics to treat muscular dystrophies, such as Duchenne muscular dystrophy, myotonic dystrophy, facioscapulohumeral muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, oculopharyngeal muscular dystrophy, Emory-Dreyfus muscular dystrophy, hereditary muscular dystrophy, congenital muscular dystrophy, and distal muscular dystrophy.

[0301] Nucleic acid therapy can be used to treat patients with muscle atrophy, such as cachexia. Nucleic acid therapy can be used to treat other muscle disorders, such as hereditary muscle disorders, such as myotonia congenita or familial periodic paralysis. Nucleic acid therapy can be used to treat motor neuron diseases, such as ALS (amyotrophic lateral sclerosis), spinal bulbar muscular atrophy (SBMA) or spinal muscular atrophy (SMA). Nucleic acid therapy can be used to treat mitochondrial diseases, such as Friedreich's ataxia (FA), or mitochondrial myopathy, such as Kearns-Sell 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 broken red fibers (MERRF), neuropathy, ataxia and retinitis pigmentosa (NARP), Pearson syndrome or progressive external ophthalmoplegia (PEO). Nucleic acid therapy can be used to treat congenital myopathies such as capillary myopathy, centronuclear myopathy, congenital myopathy with imbalanced fiber types, core myopathy, central core disease, multinuclear myopathy, myosin storage myopathy, myotubular myopathy, or nemaline myopathy. Nucleic acid therapy can be used to treat distal myopathies such as GNE myopathy / Nonaka myopathy / hereditary inclusion body myopathy (HIBM), Laing distal myopathy, Markesbery-Griggs distal myopathy late, Miyoshi myopathy, Udd myopathy / tibial muscular dystrophy, VCP myopathy / IBMPFD, vocal cord and pharyngeal distal myopathy, or Welander distal myopathy. Nucleic acid therapy can be used to treat endocrine myopathies such as hyperthyroid myopathy or hypothyroid myopathy. Nucleic acid therapy can be used to treat inflammatory myopathies such as dermatomyositis, inclusion body myositis, or polymyositis. Nucleic acid therapy can be used to treat metabolic myopathies such as acid maltase deficiency (AMD, Pompe disease), carnitine deficiency, carnitine palmitoyltransferase deficiency, debranching enzyme deficiency (Corey disease, Forbes disease), lactate dehydrogenase deficiency, myoadenylate deaminase deficiency, phosphofructokinase deficiency (Tarui disease), phosphoglycerate kinase deficiency, phosphoglycerate mutase deficiency, or phosphorylase deficiency (McArdle disease). Nucleic acid therapy can be used to treat myofibrillar myopathy or scapulohumeral myopathy. Nucleic acid therapy can be used to treat neuromuscular junction diseases such as congenital myasthenic syndrome (CMS), Lambert-Eaton myasthenic syndrome (LEMS), or myasthenia gravis (MG). Nucleic acid therapy can be used to treat peripheral nerve diseases such as Charcot-Marie-Tooth disease (CMT) or giant axonal neuropathy (GAN).

[0302] Nucleic acid therapy can be used to treat cardiovascular diseases such as thromboangiitis obliterans / Burger's disease, diabetic peripheral neuropathy (also tested in ALS, critical limb ischemia, and foot ulcers), peripheral arterial disease, limb ischemia, critical limb ischemia (also known as chronic limb-threatening ischemia and diabetic limb ischemia), severe peripheral arterial occlusive disease (PAOD), or intermittent claudication / atherosclerosis. For example, the nucleic acid can encode one or more of a transgenic hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and / or fibroblast growth factor (FGF). In particular, a transgenic hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and / or fibroblast growth factor (FGF) can be used to treat limb ischemia, such as diabetic limb ischemia, in a subject.

[0303] Nucleic acid therapy can be used to treat infectious diseases such as COVID-19, HIV, HBV, HCV, Ebola and Marburg viruses, West Nile fever, SARS, avian influenza, HPV, cytomegalovirus, or malaria. Nucleic acid therapy can be used to treat cancers such as sarcoma, melanoma, breast cancer, lung cancer, pancreatic cancer, prostate cancer, liver cancer, acute myeloid leukemia, or B-cell lymphoma. Nucleic acid therapy can be used to treat allergies, such as peanut allergy. Nucleic acid therapy can be used to treat multiple sclerosis (MS). Nucleic acid therapy can be used to treat myelodysplastic syndrome (MDS).

[0304] Pompe disease is caused by a defect in human acid alpha-glucosidase (GAA), a lysosomal enzyme that cleaves terminal alpha 1-4 and alpha 1-6 glucose from glycogen. The compositions of the present invention can be used to treat Pompe disease. The compositions of the present invention comprising nucleic acids encoding GAA can be administered to a subject suffering from Pompe disease so that the nucleic acids are delivered to the target tissues of the subject, thereby expressing GAA in the target tissues described herein, particularly the liver and skeletal muscle. The enzyme can be secreted from the tissue into the circulation.

[0305] Follistatin is an inhibitor of TGF-β superfamily ligands, which inhibits skeletal muscle growth and promotes muscle atrophy. A composition comprising a nucleic acid encoding follistatin of the present invention can be administered to a subject suffering from a muscle atrophy disorder so that the nucleic acid is delivered to the target tissue of the subject, thereby expressing follistatin in the target tissues described herein (particularly liver and skeletal muscle). The protein can be secreted from the tissue into the circulation.

[0306] Accordingly, the present invention provides methods of treating such disorders and compositions for use in such treatments.

[0307] The nanoparticles containing nucleic acids of the present invention can be stored and administered in a sterile pharmaceutically acceptable carrier. Various sterile solutions can be used to administer the composition, including water, PBS, TRIS buffer, HEPES buffer, ethanol, lipids, etc. The concentration of DNA / RNA will be sufficient to provide a therapeutic dose, which will depend on the efficiency of transport into the cell.

[0308] The actual delivery of the compositions of the invention to the patient can be carried out by a variety of techniques, including direct injection, instillation into the lungs and other epithelial surfaces, or by intravenous injection. Administration can be carried out by means of a syringe needle, a cannula, a cannula, a catheter, etc., as a push injection, multiple doses, or long-term infusion. It is also contemplated that the nucleic acid cargo can be delivered ex vivo to the patient's cells or donor cells before the patient's or donor cells are infused into the subject.

[0309] Chimeric Antigen Receptor (CAR)

[0310] The present invention contemplates transfection of target cells with DNA / RNA encoding chimeric antigen receptors (CAR). CAR transfected target cells 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) CAR, anti-CEA cell adhesion molecule 7 (CEACAM7) CAR, and anti-CEACAM5 CAR.

[0311] CAR is a class of recombinant proteins, which generally include an antigen recognition domain, generally a single-chain variable fragment (scFv), a hinge region or an extracellular domain, a transmembrane domain, and an intracellular signal transduction / activation domain. The scFv domain is a chimeric peptide comprising a variable light chain (VL) domain and a variable heavy chain (VH) domain of an immunoglobulin, and the VL domain and the VH domain are connected together so that the scFv can interact with the target antigen. Other antigen recognition domains can be used to replace the scFv domain, such as TNF receptors, innate immune receptors, cytokines, structural proteins, and growth factors.

[0312] The hinge region is also called the extracellular domain or spacer and is present 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, CD8α, which naturally lack FcγR binding activity.

[0313] The transmembrane domain is present between the hinge region and the intracellular signaling domain. Any suitable transmembrane domain can be used in CAR. Typically, the transmembrane domain is derived from CD3-ξ, CD4, CD8 or CD28.

[0314] The intracellular signaling domain of CAR generally comprises a CD3-ζ cytoplasmic domain as the main intracellular signaling domain. In addition, CAR will generally also comprise one or more costimulatory domains. The costimulatory domain can be derived from CD27, CD28, CD134 and CD137.

[0315] Binding of the antigen recognition domain of the CAR to its target antigen results in aggregated CAR. This aggregation results in the transmission of activation signals from the intracellular T cell signaling domain, which in turn activates intracellular signaling pathways to stimulate the desired biological response.

[0316] The CAR platform was first described in T-cell-based immunotherapy (CAR-T) and has been shown to successfully treat a variety of blood cancers. More recently, the CAR platform has been extended to other white blood cells, such as CAR-expressing NK cells (CAR-NK) and γδ-T cells (CAR-γδT). The CAR platform has also been extended to myeloid cells, including CAR-expressing macrophages (CAR-M), which are particularly useful in targeting and treating solid tumors.

[0317] CAR-macrophages; polarized macrophages

[0318] Macrophages in cancer often adopt an anti-inflammatory or "alternative activation" phenotype - referred to herein as the M2 phenotype. M2 macrophages can mediate tissue repair and secrete immunoregulatory cytokines such as IL-4, IL-10, IL-13, and TGF-β. In the tumor microenvironment, chemoattractants such as CCL2 are often secreted, which recruit monocytes, which then continue to differentiate into M2-like macrophages, which secrete immunoregulatory cytokines IL-4, IL-10, IL-13, and TGF-β. M2-like macrophages in the tumor microenvironment will favor regulatory T cell functions rather than effector T cell functions, promote angiogenesis and tumorigenesis. Therefore, the enrichment of M2-like macrophages in the tumor microenvironment is generally associated with poor prognosis (Sloas, C. et al., 2021). Therefore, the purpose of the present invention is to reprogram M2 phenotype macrophages that are enriched in the tumor microenvironment and have pro-tumor properties into M1 phenotype macrophages with anti-tumor properties.

[0319] Therefore, the present invention contemplates transfecting M2 phenotype macrophages, which are usually enriched in the microenvironment of solid tumors and have tumor-promoting properties, with one or more transgenes suitable for reprogramming M2 phenotype macrophages into M1 phenotype macrophages in situ or ex vivo. 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).

[0320] In a further example, the present invention contemplates co-transfection of M2 phenotype macrophages in situ or in vitro with one or more transgenics and CAR constructs suitable for reprogramming M2 phenotype macrophages to M1 phenotype macrophages. Suitable transgenics for reprogramming M2 phenotype macrophages include, for example, interferon regulatory factor 5 (IRF5), activated IRF5, inhibitors of nuclear factor κB kinase subunit β (IKK2) or CCAAT enhancer binding protein α (CEBPA). Suitable CAR constructs include, for example, anti-CEACAR, anti-CEACAM7 CAR and anti-CEACAM5 CAR. In some instances, M2 phenotype macrophages are transfected with anti-CEA CAR and activated IRF5. In some instances, M2 phenotype macrophages are transfected with anti-CEACAM7 CAR and activated IRF5. In some instances, M2 phenotype macrophages are transfected with anti-CEACAM5 CAR and activated IRF5. In some instances, M2 phenotype macrophages are transfected with anti-CEACAM5 CAR and activated IRF5.

[0321] It is also envisioned that the nanoparticles of the present invention can be used to transfect M2 phenotype macrophages and / or M1 phenotype macrophages in vivo or ex vivo with nanoparticles of the present invention comprising nucleic acids encoding CAR. Examples of CAR include anti-CEA CAR, anti-CEACAM7 CAR, and anti-CEACAM5 CAR. Macrophages can be patient-derived or derived from donor blood samples.

[0322] Gene Editing

[0323] The present invention contemplates use in gene editing therapies, including gene editing therapies using techniques well known in the art such as CRISPR / Cas (e.g., CRISPR / Cas9 system), TALENS, and zinc finger nucleases.

[0324] 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, crRNA comprises a sequence complementary to the target DNA and is used to guide the Cas nuclease to the target site in the genome, and tracrRNA serves as a binding scaffold for the Cas nuclease required for Cas activity. In some embodiments, the CRISPR / Cas system comprises a Cas nuclease and a single guide RNA (sgRNA) to guide the Cas nuclease to the target site in the target gene. sgRNA comprises a target-specific crRNA fused to the scaffold tracrRNA in a single nucleic acid.

[0325] In some embodiments, the nucleic acid comprises a DNA or mRNA encoding a Cas protein or peptide (e.g., 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 a DNA or mRNA, a crRNA, and a tracrRNA encoding a Cas protein or peptide. In some embodiments, the nucleic acid comprises a DNA or mRNA and an sgRNA encoding a Cas protein or peptide.

[0326] The CRISPR / Cas system can also be used to guide the repair or modification of the target gene. For example, the CRISPR / Cas system may include a nucleic acid template to promote DNA repair, or introduce an exogenous nucleic acid sequence into the target gene by, for example, promoting homology-directed repair. The CRISPR / Cas system can also be used to introduce targeted modifications to the target genomic DNA, such as using base editing technology. This can be achieved using a Cas protein fused to a base editor (such as cytidine deaminase), such as disclosed in WO2017070633A2, which is incorporated by reference. In another example, the CRISPR / Cas system can be used to "rewrite" the nucleic acid sequence in the genome. For example, the CRISPR / Cas system can be a lead editing system. In this lead editing system, a fusion protein can be used. For example, a fusion protein may include a catalytically impaired Cas domain (such as a "nickase") and a reverse transcriptase. The catalytically impaired Cas domain may be able to cut a single strand of DNA to produce a nicked DNA duplex. The prime editing system may include a prime editing guide RNA (pegRNA), which includes an extended sgRNA containing a primer binding site and a reverse transcriptase template sequence. When the catalytically impaired Cas makes a nick on the DNA duplex, the primer binding site allows the 3' end of the nicked DNA strand to hybridize with the pegRNA, while the RT template serves as a template for synthesizing the edited genetic information.

[0327] In some embodiments, the CRISPR / Cas gene editing system may include a nucleic acid template to guide the repair of a target gene of interest. In other embodiments, the Cas protein or peptide may include a base editor. In further embodiments, the CRISPR / Cas system may be a lead editing system.

[0328] CRISPR / Cas gene silencing and gene activation

[0329] The CRISPR / Cas system has been adapted for use in gene silencing and activation. Such systems are envisioned for use in the present invention. For example, in some embodiments, the nucleic acid may encode a fusion protein comprising a Cas protein or peptide fused to a transcriptional repressor or activator. In some embodiments, the Cas protein is catalytically dead. The fusion protein can be directed to a site of interest in the genome by sgRNA or crRNA. When the fusion protein binds to the site of interest, the transcriptional repressor or activator can regulate the expression of the gene of interest.

[0330] Nucleic acid-based vaccines

[0331] DNA vaccines and RNA vaccines, as defined by the World Health Organization (WHO), involve the direct introduction of plasmids containing DNA sequences or RNA encoding antigens into appropriate tissues (of the subject to be vaccinated), against which immune responses are sought, and rely on the in situ production of target antigens. These methods offer many potential advantages over traditional methods, including stimulation of B cell and T cell responses, improved vaccine stability, the absence of any infectious agents, and relatively easy large-scale production. As proof of the principle of DNA vaccination, genes from a variety of infectious agents including influenza virus, hepatitis B virus, human immunodeficiency virus, rabies virus, lymphocytic choriomeningitis virus, malarial parasites, and mycoplasma have been used to obtain immune responses in animals. In some cases, disease protection for animals has also been obtained. However, the value and advantages of DNA vaccines must be evaluated on a case-by-case basis, and their applicability will depend on the nature of the infectious agent to which immunity is directed, the nature of the antigen, and the type of immune response required for protection.

[0332] The field of DNA and RNA vaccination is rapidly evolving. Vaccines currently in development use not only DNA, but also include adjuvants that assist in the entry of DNA into cells, target it to specific cells, or can act as an adjuvant in stimulating or directing an immune response. As of 2020, the WHO states that the first nucleic acid vaccines approved for marketing will likely use plasmid DNA derived from bacterial cells, but in the future, other vaccines may use RNA, or may use complexes of nucleic acid molecules and other entities. However, with the outbreak of COVID-19 in 2020, there was a concerted effort to bring the first RNA-based COVID-19 vaccines to market, and these vaccines were approved for use in mid-to-late 2020. Since their approval, these RNA-based vaccines have been successfully rolled out around the world, immunizing populations against COVID-19.

[0333] As with other vaccine technologies, intramuscular delivery of DNA vaccines 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 because stable expression does not rely on genomic integration.

[0334] RNA vaccines currently on the market use mRNA encoding antigens as payloads. One area that is now being explored to improve the effectiveness of RNA vaccines is the use of self-amplification RNA. Self-amplification RNA shares many structural features of mRNA, and may include a 5' cap, a 3' poly A tail, and 5' and 3' untranslated regions (UTRs). In addition to encoding the antigen of interest, self-amplification RNA will also include a system for self-amplification. For example, self-amplification RNA can also encode RNA-dependent RNA polymerase (RDRA), a promoter, and an antigen of interest. When the subject's translation mechanism translates RDRA, RDRA can engage self-amplification RNA and replicate RNA. Including a system for self-amplification reduces the minimum RNA required in the vaccine, and therefore will reduce the possibility of the subject experiencing side effects.

[0335] Combination therapy

[0336] The compounds of the present invention or compounds identified by the methods of the present invention can be used to treat tumors and cancers in a subject in need of treatment. The compounds can be administered alone or in combination with other anticancer agents.

[0337] "Anticancer" refers to any agent that can be used to treat a patient with a tumor. One class of antiancer includes chemotherapeutic agents. "Chemotherapy" means administering one or more chemotherapeutic drugs and / or other agents to a cancer patient by various methods including intravenous, oral, intramuscular, intraperitoneal, intravesical, subcutaneous, transdermal, buccal, or inhaled, or in the form of a suppository. Some chemotherapeutic agents are cytotoxic.

[0338] Cytotoxic chemotherapeutics trigger cell death via non-receptor mediated mechanisms or modes. Cytotoxic chemotherapeutics trigger cell death by interfering with the functions necessary for cell division, metabolism or cell survival. Due to this mechanism of action, cells that grow rapidly (which means proliferation or division) or are metabolically active will be killed preferentially than cells that are not such. The state of different cell divisions or energy consumption (i.e., metabolic activities that support cell functions) in the body determines the dosage of the chemotherapeutics that trigger cell death. Cytotoxic chemotherapeutics non-exclusively relate to alkylating agents, antimetabolites, plant alkaloids, topoisomerase inhibitors, antineoplastic agents and arsenic trioxide, carmustine, fludarabine, IDA ara-C, myalotang, GO, nitrogen mustard, cyclophosphamide, gemcitabine, bendamustine, whole body irradiation, cytarabine, etoposide, melphalan, pentostatin and radiation. Ibrutinib (BTK inhibitor) is another anticancer agent that can be used in combination with the medical application of the present invention. BTK inhibitors enhance TAM repolarization to M1 phenotype. This combination therapy may be particularly useful for treating solid tumors, particularly "cold" tumors such as PDAC.

[0339] Anticancer agents also include protein kinase inhibitors, which can be used to treat a wide variety of cancers, including blood cancers and lung cancers. Protein kinases generally promote cell proliferation, survival, and migration, and are generally constitutively overexpressed or active in cancer. Therefore, inhibitors of protein kinases are common drug targets in cancer therapy. Examples of kinase inhibitors used in clinical practice include crizotinib, ceritinib, alectinib, brigatinib, bosutinib, dasatinib, imatinib, nilotinib, ponatinib, vemurafenib, dabrafenib, ibrutinib, palbociclib, sorafenib, and ribociclib.

[0340] Anticancer agents also include agents for immunotherapy, including antibodies. Immunotherapy can trigger, amplify, reduce or inhibit immune responses, depending on the specific disease condition. For example, tumor cells expressing PDL1 ligands suppress normal immune responses in subjects by binding to PD-1 receptors 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 tumor cell killing. Other examples of immunotherapy include oncolytic virus therapy, T cell therapy, and cancer vaccines.

[0341] Pharmaceutical composition

[0342] The nanoparticles of the present invention can be formulated as pharmaceutical compositions or preparations. The pharmaceutical compositions provided herein may include one or more pharmaceutically acceptable excipients or carriers, such as solvents, solubility enhancers, suspending agents, buffers, isotonic agents, antioxidants, antimicrobial preservatives, diluents, binders, lubricants and disintegrants. "Pharmaceutically acceptable" refers to molecular entities and compositions that are "generally regarded as safe", such as when applied to humans, they are physiologically tolerable and generally do not produce allergic or similar adverse reactions, such as stomach discomfort, etc. In some embodiments, this term refers to molecular entities and compositions approved by regulatory agencies of the U.S. federal or state governments, such as the GRAS list (which has been reviewed before marketing and approved by the FDA) or similar lists in accordance with sections 204 (s) and 409 of the Federal Food, Drug, and Cosmetic Act, the U.S. Pharmacopeia or another generally recognized pharmacopoeia approved for use in animals, more specifically in humans.

[0343] When used, the excipients of the composition will not adversely affect the stability, bioavailability, safety and / or efficacy of the active ingredient. Thus, the skilled artisan will appreciate that there is no incompatibility between any of the components of the dosage form in the provided compositions. The excipients may be selected from the group consisting of buffers, tonicity agents, chelating agents, antioxidants, antimicrobials and preservatives.

[0344] The compositions containing nucleic acid of the present invention can be stored and applied in a sterile pharmaceutically acceptable carrier. Various sterile solutions can be used for applying the compositions, including water, PBS, ethanol, lipids, etc. The concentration of DNA / RNA will be sufficient to provide a therapeutic dose, which will depend on the transport efficiency into the cell.

[0345] Expression of therapeutic products

[0346] The nucleic acid delivered by the composition of the present invention can show a therapeutic effect (e.g., by directly acting to downregulate or upregulate a target gene), or it can express a gene product (which can be a therapeutic protein or a therapeutic nucleic acid) via an expression cassette comprising a coding sequence operably connected to a promoter. In this specification, the term "operably connected" may include a situation in which a selected nucleotide sequence and a regulatory nucleotide sequence are covalently linked in a manner that causes the expression of the coding sequence to be affected or controlled by the regulatory sequence. Therefore, if the regulatory sequence is able to affect the transcription of a portion or all of the coding sequence forming the selected nucleotide sequence, the regulatory sequence is operably connected to the selected nucleotide sequence. Where appropriate, the resulting transcript can then be translated into a desired protein or polypeptide.

[0347] Route of administration

[0348] Compositions according to various aspects of the invention may be formulated for administration by a variety of routes including, but not limited to, intravenous, parenteral, intraarterial, intramuscular, intratumoral, subcutaneous, oral, and nasal.

[0349] The actual delivery of the compositions of the present invention to a subject (human or animal) can be carried out by a variety of techniques, including direct injection, inhalation, instillation of the lungs and other epithelial surfaces, or by intravenous, parenteral, intraarterial, intramuscular, intratumoral or subcutaneous injection. Administration can be carried out by means of a syringe needle, a cannula, a cannula, a catheter, etc., in a push injection, multiple doses or long-term infusion.

[0350] Subjects

[0351] The subject to be treated may be any animal or human. The subject is preferably a mammal, more preferably a human. The subject may be a non-human mammal, but more preferably a human. The subject may be male or female. The subject may be a patient. The therapeutic use may be for humans or animals (veterinary use).

[0352] ***

[0353] The features disclosed in the above description or in the following claims or in the accompanying drawings, expressed in a specific form or in terms of a means for performing a disclosed function or a method or process for obtaining a disclosed result, may, where appropriate, be used alone or in any combination of such features to realize the invention in its various forms.

[0354] Although the present invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when this disclosure is given. Therefore, the exemplary embodiments of the present invention set forth above are considered to be illustrative rather than restrictive. Various changes may be made to the described embodiments without departing from the spirit and scope of the present invention.

[0355] For the avoidance of any doubt, any theoretical explanations provided herein are provided to enhance the reader's understanding. The inventors do not wish to be bound by any of these theoretical explanations.

[0356] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0357] Throughout the specification, including the appended claims, unless the context requires otherwise, the words "comprises" and "comprising" and variations will be understood to imply the inclusion of stated integers or steps or groups of integers or steps but not the exclusion of any other integers or steps or groups of integers or steps.

[0358] It must 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 indicates 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 a value is expressed as an approximation by using the antecedent "about", it will be understood that the particular value forms another embodiment. The term "about" in relation to a numerical value is optional and means, for example, + / - 10%.

[0359] Example

[0360] Example 1 - Methods and materials for in vitro transfection of cells and tissues

[0361] Certain dendrimers from the following table can be used to assess the in vitro macrophage transfection efficiency of eGFP mRNA and are discussed in the Examples below. Tables 1, 1A and 1B below set forth dendrimers and peptides that can be used in various aspects and embodiments of the present invention.

[0362]

[0363]

[0364]

[0365] Table 1. Exemplary dendrimers. The NH2 group present at the C-terminus of the core sequence is a result of the peptide synthesis method. X or Acp = 6-aminohexanoic acid, B or β-A = β-alanine, Dab = 2,4-diaminobutyric acid. Lowercase letters refer to D-form amino acids, while uppercase letters refer to L-form amino acids. l is the D-form of leucine. Glycine has no D or L form. Italics represent branching residues. Ac indicates acetylation at the N-terminus.

[0366] name Peptide structure MW PGA-100 (E)100 15000 Linear ESG Ac-ESGESGESGGSEGSEGSEC 1803 Linear E8 Ac-EEEEEEEEGSGGSGGSC 1742

[0367] Table 1A. Exemplary peptides. Ac indicates acetylation at the N-terminus.

[0368]

[0369]

[0370] Table 1B. Exemplary dendrimers. The NH2 group present at the C-terminus of the core sequence is a result of the peptide synthesis method. Lowercase letters refer to D-amino acids, while uppercase letters refer to L-amino acids. l is the D-form of leucine. Glycine does not have a D or L form. Italics represent branching residues. Ac indicates acetylation at the N-terminus.

[0371] method

[0372] Mouse BMDM (bone marrow-derived mononuclear cells) polarization

[0373] BMDM cells were isolated from mouse bone marrow cell suspension using StemCell Tech EasySep mouse monocyte isolation kit. Cells were resuspended in complete medium (DMEM+1% glutamax+10% heat-inactivated FBS+1% Pen / Strep) rich in 50ng / ml mouse M-CSF (1:1000 50ug / ml stock solution) at 1E+6 cells / ml. 2 million cells (i.e. 2mL) were inoculated in each well of a 6-well plate. The plate was incubated at 37°C for 4 days. Half of the culture medium was exchanged using complete medium rich in 50ug / ml mouse M-CSF and 40ng / ml mouse IL-4. Cells were then harvested on the second day (day 5) by washing the cells with PBS and incubating them with 5mM EDTA (diluted in PBS) for 10 minutes at 37°C, followed by tapping the plate and resuspending. The cells were resuspended in complete medium containing 50ug / ml murine M-CSF and 20ng / ml murine 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 the macrophage phenotype.

[0374] Mouse BMDM transfection

[0375] Aspirate the medium covering the polarized BMDM cells and exchange with 480ul / well of activation medium. Add 120ul / well of each mRNA preparation to the cells. Transfect the cells at 37°C for 24 hours.

[0376] J774.2 subculture and macrophage polarization

[0377] J774.2 cells are subcultured in DMEM (complete medium) supplemented with 2mM glutamax and 10% heat-inactivated FBS. Twice a week (confluence is about 70-80%; every 3-4 days) cells are scraped and collected in Falcon tubes, and precipitated at 400g for 5 minutes. Cells are resuspended in complete medium, counted, and inoculated into 10cm culture dishes at a density of 1.7-2.5E+4 cells / cm^2. For M2 polarization, cells are treated with complete medium (activation medium) supplemented with 20ng / ml recombinant mouse IL-4 and 20ng / ml recombinant mouse IL-13 over a period of 48 hours. After 24 hours in a medium containing IL-4 and IL-13, cells are scraped and collected as described above. Cells are inoculated into 24-well plates at a density of 1.2E+5 cells / well in 0.5ml / well complete medium containing 20ng / ml IL-4 and IL-13.

[0378] Transfection

[0379] Aspirate the culture medium covering polarized J774.2 cells and exchange with 480ul / well activation culture medium. Add 120ul / well (total volume is 600ul / well) of each test condition (prepare test conditions in 25mM HEPES solution). Prepare control conditions consisting of pure 25mM HEPES and mRNA control. Between the addition of each test condition, the plate is shaken back and forth to ensure mixing. The plate is placed in an incubator overnight.

[0380] Human THP-1 subculture and macrophage polarization

[0381] THP-1 cells (human leukemia monocytic cell line) were subcultured at 3E+5 cells / ml in RPMI (containing glutamax) + 10% heat-inactivated FBS (complete medium), and were subcultured every 3-4 days. For macrophage polarization, 7.5E+6 cells were seeded in a total of 20 ml complete medium containing 5ng / ml PMA (1:1000 of 5ug / ml stock solution) in a T75 flask. The cells were transferred to a 37°C incubator overnight. After 24 hours, the THP-1 cells were washed with complete medium, and fresh flask complete medium (without PMA) was covered on the cells. After another 72 hours, the complete medium was replaced with fresh complete medium containing 20ng / ml rhIL-4 (1:500 of 10μg / ml stock solution). Cells were harvested 24 hours later by first washing the cells with PBS, then treating with 5mM EDTA (diluted in PBS) for 5 minutes at 37°C. The cells were spun at 300 g for 5 minutes and resuspended in complete medium containing 20 ng / ml rhIL-4 at 2.4E+5 cells / ml. 500 ul of cells were seeded into each well of a 24-well plate and placed in a 37°C incubator overnight.

[0382] THP-1 transfection

[0383] Aspirate the medium covering the polarized THP-1 cells and exchange with 480ul / well of complete medium containing 20ng / ml rhIL-4. Add 120ul / well of each mRNA preparation (preparation prepared in 25mM HEPES solution). Prepare control conditions consisting of pure 25mM HEPES and mRNA control. Transfect cells at 37°C for 24 hours.

[0384] Staining and fixation of polarized macrophages

[0385] As follows, human and mouse derived macrophages were stained and fixed. After 24 hours, the culture medium covering each test condition was transferred to the corresponding 1.5ml Eppendorf tube (in order to preserve cells that may have died during transfection). The cells were then washed with 300ul / well PBS and transferred to the corresponding tube. 500ul / well 5mM EDTA was added to each well, and the plate was transferred to a 37°C incubator for 5-10 minutes until the cells began to break away. The cells were then transferred from the plate to the corresponding 1.5ml tube. The cells were precipitated at 300g for 5 minutes and then resuspended in 100ul / sample of the fixable Live / Dead Aqua dye. ArC beads were stained with 3ul pure Live / Dead Aqua for compensation. These cells were stained on ice for 30 minutes. After this, the tube was rotated at 300g for 5 minutes. The supernatant was discarded and the cells were resuspended in 500ul / sample PBS. Negative ArC beads were added to the compensation control. GFP compensation beads are distributed in separate tubes as GFP compensation controls. The samples are spun at 300g for 5 minutes. The samples are then resuspended in 100ul / sample of reagent A from Invitro gen Fix / Perm test kit and left at room temperature for 15 minutes. The samples are spun at 400g for 5 minutes and the supernatant is disposed of. The samples are washed with PBS again and spun at 300g for 5 minutes. Finally the cells are resuspended in 400ul / sample of FACS buffer (PBS+2mM EDTA+0.5%w / v BSA) for flow analysis. The samples are stored in a refrigerator before flow analysis and tested within 24 hours of fixation.

[0386] Surface marker staining of BMDM and J774.2 cells (after Live / Dead staining and before fixation)

[0387] The cells were resuspended in 2x FcX blocking solution (1:50; final concentration 1:100) of 50ul / sample diluted in FACS buffer and incubated in a refrigerator for 5 minutes. A mixture of surface antigen antibodies (final concentration: F4 / 80-PE1:100, CD206-BV605 1:25, CD86-PECy7 1:25 and CD11b 1:200) of 50ul / sample was added to the top of the FACS buffer (equal volume with blocking solution). For each antibody used, 1 drop of UltraComp beads and 3ul of each antibody (antibody compensation control) were added to the top. The cells were incubated in a refrigerator for 20 minutes and spun at 400g for 5 minutes. After PBS washing, the cells were fixed and the subsequent steps were performed as described in detail in the previous section.

[0388] C2c12 myocyte culture and transfection

[0389] 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-labeled mRNA expressing eGFP was purchased from RiboPro. mRNA expressing eGFP or firefly luciferase was purchased from Trilink. DOT MA:DOPE, 1:1 (w / w) was obtained from Invitrogen or Encapsula NanoSciences LLC.

[0390] Transfection procedure. C2c12 cells were seeded in 96-well plates 24 hours prior to transfection so as to reach 70% confluence. mRNA transfection complexes were formed by mixing mRNA with dendrimer in 25 mM HEPES buffer at 25°C and then with DOTMA:DOPE in 25 mM HEPES buffer. The transfection complexes were then overlaid onto the cells in complete growth medium. Cells were harvested 4 hours after transfection for FACS analysis or 24 hours after transfection for reporter gene assays.

[0391] Cell harvesting and staining.

[0392] Before adding 5uM EDTA in PBS, collect the cell culture medium from each treatment and the PBS for washing each well.After incubation at 37 ℃ for 10 minutes, make the cells gently detach from the plate and precipitate 8 minutes at 400g at 4 ℃.According to the manufacturer's instructions, cells are stained with LIVE / DEAD fixable aqueous fluorescent active dye (Invitrogen).ArC beads are stained with 3ul pure Live / Dead Aqua to compensate.After staining, cells are precipitated 8 minutes at 4 ℃ with 400g, and resuspended in the PBS washing solution of 500ul / sample before repeating centrifugation.Negative ArC beads are added to the compensation control.GFP compensation beads are distributed in a separate tube as GFP compensation control.All samples (including beads) are fixed with 4% paraformaldehyde in PBS for 15 minutes at room temperature.After this, the sample is once again spun at 4 ℃ with 400g for 8 minutes, and resuspended in FACS buffer (PBS+2mM EDTA+0.5%w / v BSA). Samples were stored at 4°C and subjected to flow analysis within 24 hours of fixation.

[0393] Flow cytometry.

[0394] Data were collected using a BD LSRFortessa I analyzer running FACSDIVA software (Beckton Dickinson). All collected data were analyzed using FlowJo 10.0 software.

[0395] Nanoparticle Formulation Procedure

[0396] Tube A: Add the peptide dendrimer stock solution to a sterile 0.5 ml polypropylene tube containing 100 mM HEPES buffer (1.00 μl), sterile water (to give a final volume of 4.0 μl). Gently shake the tube and then spin for 10-15 seconds using a microcentrifuge to ensure that all the liquid is at the bottom of the tube. The concentration and volume of the dendrimer stock solution and water vary depending on the molecular weight and charge of the dendrimer and the target N / P ratio. The final concentration of HEPES buffer in tube A is 25 mM.

[0397] Tube B (800.0 μg / ml mRNA in 25 mM HEPES buffer) (excess volume): Add 1 mg / ml mRNA stock solution (42.00 μl) to a sterile 0.5 ml polypropylene tube containing sterile water (3.94 μl) and 200 mM HEPES buffer (6.56 μl). Gently shake the tube to mix, then spin for 10-15 seconds using a microcentrifuge to ensure that all the liquid is at the bottom of the tube.

[0398] Tube C (3076.9 μg / ml DOTMA / DOPE liposomes in 25 mM HEPES buffer) (excess volume): Add 25 mM (17.68 mg / ml) DOTMA / DOPE liposome solution (11.88 μl) to a sterile 0.5 ml polypropylene falcon tube containing sterile water (47.84 μl) and 200 mM HEPES buffer (8.53 μl). Gently shake the tube to mix, then spin for 3-5 seconds using a microcentrifuge to ensure that all the liquid is at the bottom of the tube.

[0399] Mixing: Mix 800 μg / ml mRNA solution (10 μl, 8.0 μg, 2.42×10-5 mmolar phosphate) into tube A by pipetting up and down 10-15 times quickly. Let it stand for 2-5 minutes. Mix 3076.9 μg / ml DOTMA / DOPE liposome solution (26.0 μl, 80.0 μg) into tube A by pipetting up and down 10-15 times quickly.

[0400] Formulation Example 1: (RHL)4(KRHL)2KGSC-NH2, N / P ratio 0.6 (4912 g / mol, 10 charges / dendrimer) for 40 μl formulation.

[0401] Tube A (Dendrimer): To a sterile 0.5 ml polypropylene tube was added 5 mg / ml dendrimer stock solution (1.430 μl, 7.14 μg, 1.45×10-6 mmol dendrimer 1.45×10-5 mmol N), sterile water (1.570 μl) and 100 mM HEPES buffer (1.00 μl).

[0402] Formulation Example 2: (RHL)4(KRHL)2KGSC-NH2, N / P ratio 8 (4912 g / mol, 10 charges / dendrimer) for 30 ul formulation.

[0403] Tube A (Dendrimer): To a sterile 0.5 ml polypropylene tube was added 50 mg / ml dendrimer stock solution (1.430 μl, 71.4 μg, 1.45×10-5 mmol dendrimer 1.45×10-4 mmol N), sterile water (0.821 μl) and 100 mM HEPES buffer (0.75 μl).

[0404] Changing the lipid / mRNA ratio

[0405] To change the mass ratio of liposomes to mRNA in the formulation, the concentration of liposomes in tube C should be changed so that the same volume of liposome solution is still added (ie, 26 μl if the final volume of the formulation is 40 μl).

[0406] Application of mannose-dendrimer coating

[0407] The amount of coating applied is defined as the mass ratio of coated dendrimer relative to mRNA in the sample. The mRNA:coating mass ratio applied was typically 1:0.5, 1:1 or 1:3.

[0408] 12 μl of nanoparticle preparation was aliquoted into sterile 0.5 ml polypropylene tubes. 4 μl of 0.3 mg / ml, 0.6 mg / ml or 1.8 mg / ml solution of (Ac-EEEE)2KGSGGSGGSC[(SS)-α-D-thiomannose] (mRNA: coating mass ratio of 1:0.5, 1:1 or 1:3, respectively) was mixed by rapid pipetting up and down 10-15 times and allowed to incubate for 5 minutes.

[0409] In vivo delivery of peptide dendrimers / nanoparticles

[0410] All animal experimental procedures were performed in accordance with the Animal Research Report for In Vivo Experiments and were approved by the UK Home Office under the Permitted Regulatory Process. Fig.11The basic data of 4 week-old Balb / c mice were purchased from Jackon Laboratory, and they were adapted to 1 week in a new living environment after arrival. All mice were weighed and warmed in a 37°C heating chamber for 15 minutes. A 30G insulin syringe (BD biosciences) was used to deliver the preparation (5 mL / kg) intravenously via the tail vein.

[0411] Bioluminescence imaging (BLI)

[0412] All mice were imaged 6 hours after injection. BLI was performed using an IVIS Lumina II (Perkin Elmer) imaging system. Mice were administered a 10 uL / g dose of D-luciferin (GoldBio). Mice were anesthetized in a chamber containing 2.5% isoflurane and then placed on a heated imaging platform while maintaining 2.5% isoflurane. Fig.11 Based on the basic data of , mice were imaged after 10 minutes of D-luciferin administration, and the exposure time was set to 45 seconds to ensure that the obtained signal was within the effective detection range (bin culture medium). Using Living image software (Perkin Elmer), bioluminescent signals were quantified by measuring photon flux (photons / s). After whole-body imaging in vivo, mice were euthanized, heart blood was drawn, and tissues were extracted for ex vivo imaging. During the collection period, each tissue was placed on a culture dish containing D-luciferin in PBS. The organ was placed in the center of the imaging platform (Lumina II system) and the acquisition settings described in detail above were used to measure the signal. Finally, the tissue was placed in a storage vial and quickly frozen in liquid nitrogen. Blood was collected in EDTA-K2 tubes and rotated at 10,000rpm for 15 minutes. Plasma was transferred to a fresh tube and frozen.

[0413] Example 2 - Transfection of Mouse and Human Macrophages Using Peptide Dendrimers Transfection of Mouse Macrophages

[0414] Transfection efficiency of primary bone marrow-derived monocytes polarized to macrophages using various peptide dendrimer / lipid nanoparticles was evaluated and compared to lipid-only nanoparticles (LPX-RNA). Each nanoparticle, with or without dendrimer, included eGF P mRNA to allow the absolute number of transfected cells to be evaluated by flow cytometry analysis. Figure 2 As can be seen in Figure 2, all peptide dendrimer / lipid-based nucleic acid delivery systems tested outperformed naked mRNA or mRNA contained in lipid-only nanoparticles (DOT MA / DOPE lipid-based nanoparticles). Lipid-based nanoparticles were only able to achieve approximately 10-15% transfection efficiency, while the use of GSC G1,2-RHL (NP = 0.6:1), G SC G1,2-RL,3-LR(NP=0.16:1) or GSC G1, RL, 2-LR (NP = 8:1) showed approximately 35%, 40%, and 50% transfection efficiency, respectively ( Figure 2 ).

[0415] Human macrophage transfection

[0416] Next, to test whether the peptide dendrimer / lipid nanoparticles could be used to transfect human cells in general, and human macrophages in particular, a panel of peptide dendrimer / lipid eGFP mRNA-containing nanoparticles was tested for transfection efficiency on human THP-1 cells. As observed in primary mouse macrophages, each of the dendrimers tested was able to transfect THP-1 cells with high efficiency, and in each case exceeded the efficiency exhibited by the lipid-only nanoparticles ( Figure 3 ). GSC G1,2-RL,3-LR(NP=0.16:1), G SC G1-LRLR(NP=0.6), GSC G1,2-RHL (NP = 5:1) and GSC G1,2-RF,3HL (NP=0.6:1) can each achieve transfection efficiencies between 50% and 60%, while only about 20% of cells expressed eGFP after transfection using lipid-only nanoparticles. 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 about 10%. RHC G1-R (NP=0.6:1) also outperformed lipid-only nanoparticles, reaching a transfection efficiency of -30-35%.

[0417] From both primary macrophage mouse transfection experiments and human macrophage transfection experiments, it is clear that peptide dendrimer / lipid nanoparticles provide an improved mechanism to achieve macrophage transfection.

[0418] Example 3 - Mannose-conjugated peptide dendrimers improve transfection efficiency

[0419] 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. Generally, patients with high levels of M2 phenotype macrophages in their tumors have a poor prognosis.

[0420] In view of the above, the inventors next investigated whether macrophage transfection efficiency could be further improved by targeting peptide dendrimer / lipid nanoparticles to bind to specific receptors on macrophages. It is known that macrophages (and in particular M2 phenotype macrophages) have enriched surface expression of the receptor CD206, whose ligand is mannose. Therefore, various peptide dendrimer / lipid nanoparticles were coated with a second peptide dendrimer conjugated to mannose to investigate whether this could improve delivery of mRNA cargo to macrophages.

[0421] Observed GSC G1,2-RHL / lipid nanoparticles (NP=8:1, lipid:mRNA w / w ratio of 2.5:1) performed poorly and transfected macrophages at very low levels of ~2-3% ( Figure 4 This poor performance can be attributed to the lipid:mRNA w / w ratio of 2.5:1 in this formulation, as generally when the lipid:mRNA ratio is increased, GSC G1,2-RHL / lipid nanoparticles transfect macrophages with high efficiency (see e.g. Figure 2 , 3 and 5-7). However, even when using suboptimal lipid:mRNA ratios, coating the nanoparticles with (Ac-EEEE)2KGSGGSGGSC[(SS)-α-D-thiomannose] at a w / w ratio of 1:1 (1 equiv) relative to coating:mRNA can achieve an increase in transfection efficiency from ∼2-3% to ∼25% ( Figure 4 ). Thus, it appears that even “poorly performing” peptide dendrimer / lipid nanoparticles can improve mRNA delivery to macrophages by coating the nanoparticles with mannose.

[0422] Using further optimized GSC G1,2-RHL / lipid nanoparticle composition (NP=8:1, lipid:mRNA w / w ratio is 5:1), containing GSC Nanoparticles of G1,2-RHL can transfect macrophages with higher efficiency than when lipid-only nanoparticles are used ( Figure 5 ; ~60% vs. ~10%). The transfection efficiency can be further improved by applying the same dendrimer-mannose coating to the nanoparticles at a w / w ratio of 0.5:1 (0.5 equivalents) relative to coating:mRNA. Using this dendrimer-mannose coating, the transfection efficiency was further increased from ~60% to ~75% ( Figure 5 ). This further demonstrates that coating dendrimer / lipid nanoparticles with ligands for receptors expressed on target cells can increase transfection efficiency.

[0423] Next, we investigated the effect of increasing the DOTMA / DOPE:mRNA w / w ratio from 5:1 ( Figure 5 ) further increased to 10:1( Figure 6 ) can it be further improved GSC The transfection efficiency of G1,2-RHL / lipid nanoparticles was not further increased by using a 10:1 DOTMA / DOPE:mRNA w / w ratio. GSC The transfection efficiency of the nanoparticles of G1,2-RHL was ∼60%. Figure 6 ), as seen when a 5:1 DOTMA / DOPE:mRNA w / w ratio was used ( Figure 5 ). However, it is important to point out that increasing the lipid:mRNA ratio did not negatively affect transfection efficiency, further demonstrating the versatility of the peptide dendrimer / lipid nanoparticle nucleic acid delivery system.

[0424] Again, it was also observed that application of the dendrimer-mannose coating at a 1:1 (1 equivalent) w / w ratio of coating:mRNA again increased transfection efficiency by -10% compared to uncoated nanoparticles ( Figure 6 ).

[0425] Next, the effect of NP ratio on transfection efficiency was investigated. GSC G1,2-RHL nanoparticles can achieve a transfection efficiency of up to 70% ( Figure 7 ). This data again further demonstrates the versatility of these delivery systems, which provide a wide working range of NP ratios and lipid:mRNA w / w ratios.

[0426] As with the other nanoparticle formulations tested, it was possible to further increase the NPs of 0.6:1 and a lipid:mRNA w / w ratio of 10:1 by coating the nanoparticles with mannose-conjugated dendrimers. GSC Transfection efficiency of G1,2-RHL / lipid nanoparticles, in this case relative to a coating:mRNA w / w ratio of 3:1 (3 equivalents) ( Figure 7 ).

[0427] In summary, the above GSCExperiments performed by G1,2-RHL demonstrated 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). Further, the increased transfection efficiency observed when the same nanoparticles were coated with mannose-dendrimers suggests that targeted delivery of nanoparticles to CD206-expressing macrophages can be achieved.

[0428] To further investigate the general applicability of the mannose coating technique for improving the delivery of nucleic acid cargo to macrophages, GSC The nanoparticles of G1, 2-RL, and 3-LR / lipid were compared. Figure 8 It can be seen that GSC G1,2-RL,3-LR / lipid exhibited moderate macrophage transfection efficiency in vitro (~20%). By including coating the nanoparticles with (Ac-EEEE)2KGSGGSGGSC[(SS)-α-D-thiomannose] at a w / w ratio of 3:1 (3 equiv) relative to coating:mRNA, the transfection efficiency could be increased by ~30%, reaching ~50% efficiency. These data demonstrate that mannose-conjugated dendrimers can be used to increase transfection efficiency of macrophages using peptide dendrimer / lipid nanoparticles.

[0429] Example 4 - Peptide motifs can be used to increase myocyte transfection

[0430] As shown in Example 3, peptide dendrimer / lipid nanoparticles can be targeted to macrophages by including a targeting motif (e.g., mannose-conjugated dendrimer) in the nanoparticle. To further investigate the ability of these novel nucleic acid delivery systems to target specific cell types, the inventors next prepared various peptide dendrimers that included a myocyte targeting peptide motif (ASSLNIA (SEQ ID NO: 1)) to determine whether nucleic acid delivery and translation could be increased in myocytes.

[0431] Peptide dendrimers GSC Two variants (NTX2 and NTX3) of G1,2-RL,3-LR ((LR)8(KRL)4(KRL)2KGSC-NH2) included the ASSLNIA (SEQ ID NO:1) muscle targeting peptide. eGFP mRNA was used GSCMyocytes were transfected 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) and transfection efficiency was determined using flow cytometry or transgene expression assays. Transfection with eGFP mRNA allows determination of the levels of internalized mRNA and protein translated from the transfected mRNA. Nanocarriers with a cell targeting domain (in this case, a muscle targeting domain) are expected to yield higher transfection efficacy than nanocarriers without a targeting domain.

[0432] and GSC Both NTX2 and NTX3 transfected myocytes with eGFP mRNA at higher efficiencies than G1,2-RL,3-LR, with eGFP expression increasing twofold when transfected with NTX2 nanoparticles and threefold when transfected with NTX3 nanoparticles ( Fig. 9 NTX3 contains an additional 6 histidines in the core sequence, which can act as a proton sponge to promote endosomal escape. This may explain why NTX3 mediates higher transfection efficacy than NTX2.

[0433] When the general RHC Similar results were obtained when the G1-RL,2-LR ((LR)4(KRL)2KRHC-NH2) dendrimer / lipid nanoparticles were compared to a similar peptide dendrimer including the ASSLNIA (SEQ ID NO: 1) peptide motif (NTX5, (LR)4(KRL)2KRHCGAASSLNIA-(Acp)-NH2). Fig.10 As can be seen, including the ASSLNIA muscle targeting motif increased mRNA delivery to muscle cells ( Fig.10 ), and also increased eGFP expression by ~2-fold. Fig. 9 and 10 It was shown that inclusion of the muscle targeting motif ASSLNIA (SEQ ID NO: 1) in the peptide dendrimer increased mRNA delivery to muscle cells and increased transgene expression in muscle cells.

[0434] Example 5 - Peptide dendrimers can enhance tissue-specific mRNA expression

[0435] The present inventors wanted to investigate whether tissue-specific nucleic acid cargo delivery could be improved by including peptide dendrimers in lipid-based nanoparticles. Using the DOTMA / DO PE lipid delivery system (w / w = 23:1 for mRNA), luciferase mRNA was effectively delivered to the lungs and spleen, as shown by Fig.11The bioluminescence of GSC G1-LRLR (NP = 0.6:1) or GSC G1,2-RHL (NP = 0.6:1) can further improve the delivery of mRNA cargo to the lungs and spleen.

[0436] The above examples highlight that peptide dendrimer / lipid nanoparticles are very versatile tools for delivering nucleic acid cargo to a variety of target cells and tissues, including myeloid cells such as macrophages, particularly M2 phenotype macrophages, myocytes, lung, and spleen.

[0437] Example 6 - In vivo tissue distribution

[0438] The present inventors investigated the tissue distribution of nucleic acid expression achieved by the nanoparticles of the present invention.

[0439] All animal experimental procedures performed in our studies complied with UK legislation and ethical approval was obtained. Fig.12 Based on the basic data in , female BALB / C mice and CD-1 mice aged 6-8 weeks were allowed to acclimate for 1 week after arrival. All mice were weighed and the formulations (100 μl) were delivered intravenously via the tail vein using a 30G insulin syringe (BD biosciences).

[0440] Bioluminescence imaging (BLI)

[0441] All mice were imaged 6 hours after injection. BLI was performed using an IVIS Lumina II (Perkin Elmer) imaging system. Mice were administered a 150 mg / kg dose of D-luciferin (30 mg / mL, XenoLight, Perkin Elmer). Mice were anesthetized 6 minutes after receiving D-luciferin in a chamber containing 5% isoflurane and then placed on a heated imaging platform while maintaining 2.5% isoflurane. Fig.12 Based on the data of the experiment, mice were imaged 10 minutes after the administration of D-luciferin, and the exposure time was set to ensure that the obtained signal was within the effective detection range (open filter, bin 8, aperture 1). The bioluminescent signal was quantified by measuring the photon flux (photons / s) in the defined region of interest (ROI) using Living Image Software (Perkin Elmer). After whole-body imaging in vivo, mice were euthanized, heart blood was drawn, and tissues were extracted for ex vivo imaging.

[0442] Each tissue was placed into individual wells of a 24-well imaging plate (black side, 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. Fig.12 Given in.

[0443] Example 7 - Nanoparticle size, PDI and surface charge characterization

[0444] Dynamic Light Scattering

[0445] The hydrodynamic size is measured using the Dynamic Light Scattering (DLS) technique. DLS is a very sensitive, non-invasive method for measuring the size and size distribution of nanoparticles in liquids. The Brownian motion of the nanoparticles in suspension causes the laser light to be scattered with varying intensities. Analyzing these intensity fluctuations allows us to calculate the speed of the Brownian motion. The size of the nanoparticles can be determined by utilizing the Stokes-Einstein relationship. With the latest technology, nanoparticles smaller than 1nm can be measured.

[0446] Particle size can also be measured by the following methods:

[0447] Nanoparticle tracking analysis

[0448] Atomic force microscopy

[0449] Electron microscopy

[0450] Disc centrifuge

[0451] Adjustable resistive pulse sensor

[0452] Particle scattering diffusion measurement method

[0453] Polydispersity Index (PDI)

[0454] PDI is used to estimate the average uniformity of a particle solution, and the cumulant method is a standard technique for analyzing DLS data about sample polydispersity. PDI is a value calculated by a 2-parameter fit (cumulant analysis) to the correlation data. Cumulant analysis is used to evaluate the autocorrelation function produced by a DLS experiment. The calculation is defined in ISO 13321 and ISO 22412. A PDI value greater than 0.7 indicates that the size distribution of the sample is very wide and is not suitable for the DLS technique. The calculation of these parameters is defined in ISO standard documents 13321:1996E and ISO 22412:2008.

[0455] Zeta potential

[0456] Zeta potential is a measure of the magnitude of the electrostatic or charge repulsion / attraction between particles. This can be determined by analyzing particle mobility and charge (zeta potential) using the electrophoretic light scattering (ELS) technique.

[0457] The nanoparticle size, PDI and surface charge characteristics of nanoparticles of the invention comprising the second generation dendrimer alone, and the characteristics of nanoparticles of the invention comprising the second generation dendrimer and a further PGA dendrimer derivatized with mannose are shown in Table 2:

[0458]

[0459] Table 2. Size, polydispersity index (PDI) and zeta potential of nanocarriers with G1,2-RHL (N:P 0.6:1) and DOTMA:DOPE (w / w 10:1 for mRNA) with or without G1-EEEE coated with mannose ((Ac-EEEE)2KGSGGSGGSC[(SS)-α-D-thiomannose]). 3 equivalents of G1-EEEE with mannose were added to the mRNA to coat the nanocarriers.

[0460] Example 8 - In vivo delivery of mRNA using nanoparticles with or without mannose coating

[0461] The present inventors investigated the in vivo distribution of nucleic acids via nanoparticles of the present invention coated or not coated with mannose-G1-EEEE dendrimers.

[0462] method

[0463] Six-week-old female C57Bl / 6 mice were provided by Envigo UK. Mice were acclimatized in the animal facility for at least 7 days before use. MC38 cells were cultured at 1×10 per mouse. 7 The cells were implanted on the left side. Tumors were measured with a caliper three times a week once they were palpable. When tumors reached 0.15 cm3 (caliper measurement), mice were randomly divided into 4 groups, n=3 per group, and IV dosed with 2.25 mg / kg or 3 mg / kg.

[0464] Four hours after the formulation was administered, the animals were killed and the tissues were collected. The terminal blood was collected in EDTA tubes and treated with erythrocyte lysis buffer HYBRI-MAX (Merck) before antibody staining. The spleen was dissociated by pushing through a 70uM cell filter using a syringe plunger. The cell suspension was then treated with erythrocyte lysis buffer HYBRI-MAX (Merck) before antibody staining. The lung and tumor samples were cut into small pieces and the lung and tumor dissociation kit (mouse) and gentleMACS with heater (Miltenyi) were used according to the manufacturer's instructions.TM Octo dissociator for enzymatic digestion.

[0465] Before antibody staining, the final cell suspension was blocked with mouse Fc blocking solution. The cells were incubated with the antibody master mix at 4C for 20 minutes and then fixed with 4% PFA for 30 minutes at room temperature. They were analyzed using an Attune NxT flow cytometer (Thermo Fisher) within 24 hours of fixation and data were analyzed using FlowJo_v10.8.1 software. Compensation was adjusted using Attune compensation beads.

[0466] result

[0467] Cell Type % of live cells containing Alexafluor488 CD45+CD11b+ (myeloid cells) 36.8±11.9 CD45+CD11b+F4 / 80+(macrophages) 50.4±11.8

[0468] Table 3. In vivo delivery of mRNA to myeloid cells in tumors has GSC Percentage of myeloid cells targeted by Alexafluor488-labeled mRNA of G1,2-RL,3-LR (N:P=0.16:1) and DOTMA:DOPE (w / w 10:1, lipid to mRNA ratio) (dose 3 mg / kg, mRNA to body weight ratio). Cells were isolated 4 hours after injection of the formulation for flow cytometry analysis. N=3, SEM is shown.

[0469] in conclusion

[0470] like Fig.13 As shown in , 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 coating of dendritic PGA leads to interactions with serum components, which may allow the nanocarriers to be more easily directed to lung tissue.

[0471] All CD206+ cells took up nanocarriers coated with mannose-G1-EEEE more efficiently than uncoated nanocarriers and LPX-mRNA alone. This may indicate that mannose targeting is specific to cells expressing CD206+, including M2 macrophages.

[0472] Table 3 shows that mRNA with G1,2-RL,3-LR (N:P 0.16:1) and DOTMA:DOPE (w / w 10:1 for mRNA) mediates efficient uptake by myeloid cells such as macrophages, neutrophils and dendritic cells within tumors.

[0473] like Fig.39As shown in , CD206+M2 macrophages took up nanocarriers coated with mannose-G1-EEEE more efficiently than uncoated nanocarriers and LPX-mRNA alone. This suggests that mannose targeting is specific to cells expressing CD206+, including M2 macrophages.

[0474] The above examples emphasize that peptide dendrimer / lipid nanoparticles are very effective in delivering nucleic acids to myeloid cells in tissues, and in some cases non-myeloid cells (e.g., non-immune cells in the lung). Cell specificity can be further improved by adding targeting motifs to the nanocarriers (e.g., mannose for CD206 targeting).

[0475] Example 9 - Transfection efficiency of third generation dendrimers for mRNA and DNA compared to commercially available transfection reagents Comparison

[0476] In vitro transfection efficiency for mRNA and DNA delivery was assessed using certain dendrimers from Table 1 (as shown above in Example 1) and discussed in the following Examples:

[0477] Cationic lipid-based nucleic acid delivery systems are one of the most studied and effective non-viral vector platforms described to date, and the rational design and development of peptide vectors with natural amino acids is particularly attractive for therapeutic applications due to the non-toxic nature of amino acids. The present inventors have developed a structural framework for nucleic acid delivery using peptide dendrimers. The structural framework involves layers of peptide (or dipeptide) motifs bound to lysine residues. The present inventors have found that the distribution of cationic amino acid residues (Lys or Arg) in each generation (layer) makes peptide dendrimers more efficient at transfection than dendrimers whose charge is only localized on the surface (Kwok et al., 2013). Using a solid-phase peptide dendrimer synthesis procedure, the present inventors can precisely manipulate the position of each amino acid residue incorporated into the dendritic scaffold. This allows for better control of the structure and function of the dendrimer, which is generally not possible with previously studied systems such as polymers or other dendrimers where modifications are primarily performed on the surface of the molecule. The peptide dendrimer / lipid vectors show high transfection efficiency, good reproducibility of results, and low toxicity.

[0478] The inventors compared the in vitro transfection efficiency of these novel peptide dendrimer systems with other known transfection reagents. GSC A combination of G1,2-RL,3-LR (NP=0.16:1 for mRNA) and DOTMA / DOPE (w / w 10:1 for mRNA) or commercially available Lipofectamine TM 2000, HeLa cells were transfected with mRNA encoding luciferase. 14As shown in the upper left figure, the commercially available transfection reagent Lipofectamine TM Compared with 2000, including GSC The combination of G1,2-RL,3-LR (NP=0.16:1 for mRNA) and DOTMA / DOPE increased the in vitro transfection efficiency by about one order of magnitude. Similarly, compared with mRNA transfected using the DLin-MC3-DMA:cholesterol:DSPC:DMG-PE G lipid nanoparticle delivery system, the use of GSC G1,2-RL,3-LR (NP = 0.16:1 for mRNA) and DOTMA / DOPE increased eGFP expression by approximately 4-fold in HeLa cells transfected with mRNA encoding eGFP ( Fig.14 , upper right).

[0479] To further validate the peptide dendrimer system, C2C12 cells were transfected for 24 h with: 1) mRNA alone; 2) GSC G1,2-RL,3-LR (NP=8:1 for mRNA) and DOTMA / DOPE (w / w=10:1 for mRNA) with mRNA; 3) DOTMA / DOPE (w / w=10:1 for mRNA) with mRNA; 4) polyethyleneimine with mRNA; and 5) Lipofectamine 2000 with mRNA. Fig.14 As shown in the figure below, the peptide dendrimer formulation significantly improved the delivery of mRNA to C2C12 cells compared to commercially available lipid-based transfection reagents.

[0480] Using third generation peptide dendrimers GSC The inventors also compared the DNA transfection efficiency of G1,2,3-RL with that of commercially available transfection reagents. GSC G1,2,3-RL and DOTMA / DOPE; 2) DOTM A / DOPE alone; 3) polyethyleneimine; or 4) Lipofectamine 2000, H eLA cells were transfected with DNA ( Fig.15 , upper panel) or Nuero2A cells ( Fig.15 , lower figure). In serum-free conditions ( Fig.15 , left) and serum conditions ( Fig.15 , right) below, dendrimer GSCG1,2,3-RL outperformed all commercial transfection reagents tested. The third-generation dendrimers performed 2-600 times better in transfection of HeLa and Neuro2A cells than some widely used commercial reagents such as polyethyleneimine (PEI), Lipofectin (also known as DOTMA / DOPE), and Lipofectamine 2000 ( Fig.15 ).

[0481] In general, the tested G3 dendrimers efficiently transfected both mRNA and DNA in HeLa cells, and efficiently transfected at least mRNA in C2C12 cells, and DNA in Neuro2A cells.

[0482] Example 10 - Comparison of transfection efficiency of 1st, 2nd and 3rd generation dendrimers

[0483] The number of generations from G1 to G2 to G3 based on KL repeating units in delivery efficiency was explored. The KL units were also replaced with RL repeating units to compare the impact of protonated basic groups with different pKa. Interestingly, a relationship was observed between the generations and transfection in cells ( Fig.16 ). Generation dependence on transfection reveals first-generation dendrimers GSC G1-KL or GSC G1-RL did not transfect HeLa or Neuro2A cells with lysine or arginine as charged residues. This may be due to the fact that the G1 peptide is not physically large enough to fully encapsulate the plasmid DNA to form stable nanoparticles, as shown by complex stability assays (data not shown).

[0484] However, for the second generation, the arginine-containing GSC G1,2-RL and GSC G1,2-KL showed higher transfection efficiency (N / P ratio of 10:1 or 20:1 relative to mRNA in HeLa cells and N / P ratio of 20:1 relative to mRNA in Neuro2A cells) ( Fig.16 ). GSC G1,2-RL targets GSC This transfection advantage of G1,2-KL is GSC G1,2-RL ratio GSC This was consistent with the ability of G1,2-KL to form more stable transfection complexes with DNA (data not shown).

[0485] Example 11 - Comparison of a single dendrimer system with a hybrid dendrimer system

[0486] Hybrid dendrimer systems were investigated to determine whether transfection efficiency could be further improved compared to single dendrimer systems.

[0487] Materials and methods

[0488] 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 of 5% CO2 and 37°C. eGFP mRNA was purchased from Trilink ( EGFP mRNA (5 moU)-(L-7201)). DOTMA:DOPE, 1:1 (w / w) was obtained from Invitrogen (Lipofectin TM Transfection reagent) (Fisher-18292037) or Encapsula NanoSciences LLC.

[0489] Nanoparticle Formulation Procedure: To formulate hybrid dendrimer nanoparticles, peptide dendrimer 1 was mixed with peptide dendrimer 2 in a molar ratio relative to the contributed N, and the final N / P ratio was typically 8 overall. For example, a 10 mg / ml solution of peptide dendrimer 2 (1.886 μl, 18.9 μg, 7.50×10-6 mmol dendrimer, 5.25×10-5 mmol N, N / P 5.33) in sterile water (2.90 μl) and 200 mM HEPES buffer (0.814 μl) was added to tube A containing peptide dendrimer 1 (10 mg / ml solution of peptide dendrimer 1 (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 for 10-15 seconds using a microcentrifuge to ensure that all the liquid was at the bottom of the tube. 16.25 μl of mRNA (200.0 μg / ml, 3.25 μg, in 25 mM 9.85×10-6 mmol of mRNA phosphate in HEPES buffer) was added to the tube to allow the formation of N / P=8 complexes by rapid up and down pipetting 10-15 times. It was allowed to stand for 2-5 minutes. 796.2 μg / ml DOTMA / DOPE liposome solution (42.2 μl, 32.5 μg) was mixed into the tube by rapid up and down pipetting 10-15 times.

[0490] Transfection procedure: 24 hours before 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 at 25°C and then with DOTMA:DOPE in 25 mM HEPES buffer. All transfection mixtures were formulated with a final dendrimer:mRNA NP ratio of 8:1. DOTMA:DOPE was added to the mRNA complex at w / w=10:1. The transfection complexes were then overlaid onto the cells in complete growth medium. Cells were harvested 24 hours after transfection for reporter gene assays.

[0491] Transgene expression assay: Cells were washed twice with PBS and incubated with 50ul of 1x M-PER lysis buffer (Thermo 11874111). Plates were protected from light and gently agitated for 15 minutes at room temperature to aid cell lysis. 40ul of lysate from each well was transferred to all black 96-well plates to quantify eGFP relative fluorescence units (RFU), absorbance at 535nm using a molecular device SpectraMax iD5.

[0492] Protein content determination: Lysate (25 μL) was mixed with Pierce TM The protein content of each cell lysate was determined by mixing with a BCA protein assay kit (200ul, Thermo Scientific). After incubation at 37°C in the dark for 30 minutes, the absorbance at 562nm was measured using a molecular device SpectraMax iD5 and converted to protein concentration using a BSA standard curve. RFU per mg protein represents eGFP expression. Transfection The values ​​shown in the figure are expressed after normalization to control transfection experiments with DOTMA / DOPE and are shown as percentages.

[0493] Size, Zeta Potential and Polydispersity Index (PDI) Measurements: Hydrodynamic size was measured using the dynamic light scattering (DLS) technique using a Zetasizer Advance Series–Pro (Malvern Panalytical Ltd, Malvern, UK) according to the manufacturer's instructions. DLS is a very sensitive, non-invasive method for measuring nanoparticle size and size distribution in liquids. The Brownian motion of the nanoparticles in suspension causes the laser light to be scattered with varying intensities. Analysis of these intensity fluctuations allows us to calculate the speed of the Brownian motion. The size of the nanoparticles can be determined by exploiting the Stokes-Einstein relationship. With the latest technology, nanoparticles smaller than 1 nm can be measured.

[0494] The data obtained by DLS measurement can also be used to calculate the PDI of particles in solution. PDI is used to estimate the average uniformity of particle solutions. The cumulant method is a standard technique for analyzing DLS data on sample polydispersity. PDI is a numerical value calculated by a 2-parameter fit (cumulant analysis) to the correlation data. Cumulant analysis is used to evaluate the autocorrelation function generated by the DLS experiment. The calculation is defined in ISO 13321 and ISO 22412. A PDI value greater than 0.7 indicates that the size distribution of the sample is very wide and is not suitable for DLS technology. The calculation of these parameters is defined in ISO standard documents 13321:1996E and ISO 22412:2008.

[0495] For size and PDI measurements, 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 measured in nanoparticles containing a dendrimer:nucleic acid NP ratio of 8:1.

[0496] The zeta potential is a measure of the magnitude of the electrostatic or charge repulsion / attraction between particles. This can be determined by analyzing particle mobility and charge (zeta potential) using the electrophoretic light scattering (ELS) technique. To measure the zeta potential, the sample was diluted 150 times in 25 mM HEPES buffer (10 μl sample + 690 μl buffer) and added to a clean DTS1070 cell. 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.

[0497] result

[0498] The PDI of nanoparticles containing mRNA, lipids and a single peptide dendrimer was studied. The polydispersity index (PDI) can be used to measure the ability of a particular composition containing a particular peptide dendrimer to form a monodisperse population of nanoparticles. A higher PDI is associated with a reduced formation of a monodisperse population of nanoparticles. As can be seen in Table 5, nanoparticles containing, for example, 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 NP ratio of 8:1. This inability to form a monodisperse population indicates that the dendrimer-mRNA complexes with a PDI>0.35 in the nanoparticles are relatively unstable compared to the dendrimer-mRNA complexes with a PDI less than or equal to 0.35 in the nanoparticles.

[0499]

[0500]

[0501] Table 5. Polydispersity index (PDI) of peptide dendrimer, lipid and mRNA complexes. The N:P ratio of the dendrimer was 8:1 and the w / w ratio of lipid DOTMA / DOPE to mRNA was 10:1. The NH2 group present at the C-terminus of the core sequence is a result of the peptide synthesis method. Acp or X = 6-aminohexanoic acid, B = β-alanine, Dab = 2,4-diaminobutyric acid. Lowercase letters refer to D-form amino acids, while uppercase letters refer to L-form amino acids. l is D-form leucine. Glycine does not have D or L forms.

[0502] In view of the above, we investigated whether including a dendrimer with a PDI>0.35 in a composition comprising a stable dendrimer-mRNA complex (ie, PDI less than or equal to 0.35) could increase transfection efficiency by increasing the rate of mRNA dissociation after entry into cells.

[0503] When using a separate GSC G1,2-RL,3-LR or 1:2 ratio GSC G1,2-RL,3-LR and GSC The first comparison was made between HeLa cells transfected with the G1-LRLR mixture and with GSC Compared with the single dendrimer composition of G1,2-RL,3-LR, the dendrimer composition of G2 GSC The composition of G1,2-RL,3-LR contains G1 dendrimer GSC G1-LRLR significantly increased the mRNA transfection efficiency ( Fig.17 A) The transfection efficiency of the hybrid dendrimer composition was increased by -100% compared to the single dendrimer composition.

[0504] A second G1 dendrimer was investigated for use in hybrid dendrimer systems. GSC G1, 2-RL, 3-LR or 2:1 or 1:2 ratio GSC G1,2-RL,3-LR and RHC The transfection mixtures of G1-R were compared. GSC G1,2-RL,3-LR: RHC G1-R resulted in an increase in transfection efficiency of ∼100% ( Fig.17B). A further increase in transfection efficiency was obtained by changing the G2:G1 ratio to 1:2, with a transfection efficiency of -300% compared to the G2 dendrimer mixture alone.

[0505] Next, use RHC G1,2-R, RHC G1-RLR or GSC G1-RLR studied RHC Dendrimer transfection mixture of G1-RL, 2-LR (respectively Fig.17 CE). It can be seen that using a 1:1 G2:G1 ratio resulted in a modest increase in transfection efficiency between -20-50% compared to the G2 dendrimer alone ( Fig.17 D and E, compare first and second columns). Based on the results discussed previously, using a 1:2 ratio further increased transfection efficiency by ∼60%-100% ( Fig.17 D and E, compare first and third columns).

[0506] Considering Fig.17 As can be seen from the results presented in AB and 17D-E, the addition of G1 dendrimers to form hybrid dendrimer systems with G2 or G3 dendrimers improved mRNA transfection compared to mRNA complexes formed with either dendrimer alone. Fig.17 A shows that the G1 dendrimer GSC G1-RLRL and G3 dendrimers GSC The combination of G1,2-RL,3-LR increased transfection by 200%. Fig.17 B, DE also show that the addition of G1 dendrimer to G2 dendrimer can enhance transfection by up to 300%.

[0507] Unexpectedly, a G2 dendrimer containing two RHC G1-RL, 2-LR and RHC The hybrid dendrimer mixture of G1,2-R can also increase the mRNA transfection efficiency ( Fig.17 C). Although it is unclear what causes this increase in transfection efficiency, one hypothesis is that the inclusion of the relatively unstable RHCG1,2-R in the nanoparticles increases the overall instability of the nanoparticles, promoting mRNA release once the nanoparticles have entered the cells.

[0508] As can be seen from Table 5, the PDI of RHCG1,2-R nanoparticles is relatively higher (0.252 vs. 0.109) compared to RHCG1-RL,2-LR. RHCThe nanoparticles of G1,2-R formed an acceptable monodisperse population, but compared with the RHC The increase in relative instability compared to the very stable nanoparticles formed by G1-RL,2-LR may contribute to the dissociation of mRNA from nanoparticles containing both dendrimers. This is supported by the results of PDI determination of mixed dendrimer compositions (Table 6, discussed further below), which demonstrate that the PDI of nanoparticles containing RHCG1-RL,2-LR and RHCG1,2-R in a 1:1 ratio is higher than that of nanoparticles containing RHCG1-RL,2-LR alone (0.155 vs. 0.109, respectively).

[0509] Example 12 - PDI determination of mixed dendrimer systems

[0510] While improving transfection efficiency is critical to developing improved nucleic acid therapeutics, it is also very important that any new formulation has pharmaceutically acceptable properties. For example, it is important that the new formulation will have a sufficiently monodisperse population of nanoparticles so that any in vivo results will be predictable. Therefore, the PDI of the above-mentioned "hybrid" nanoparticles was analyzed. Table 6 shows that most of the nanoparticles tested had a sufficiently monodisperse population of nanoparticles that would be suitable for pharmaceutical compositions, i.e., all combinations except one tested had a PDI of less than 0.35.

[0511] The PDI of nanoparticles containing mixed dendrimer populations can be tuned by varying the ratio of the dendrimers in the nanoparticles. For example, a 1:2 ratio versus a 2:1 ratio of dendrimers RHC G1-RL, 2-LR and RHC The PDIs of G1-R were 0.521 and 0.108, respectively. Similarly, the dendrimer combinations of 1:1 and 1:2 ratios RHC G1-RL, 2-LR and RHC The PDIs of G1-RLR were 0.0819 and 0.248, respectively. RHC G1-RL, 2-LR and GSC The PDIs of G1-LRLR were 0.141 and 0.289, respectively. Taken together, these results suggest that the PDI of hybrid dendrimer nanoparticles was increased by increasing the proportion of the dendrimer with the higher PDI when used alone.

[0512]

[0513] Table 6. Polydispersity index (PDI) and hydrodynamic size of mRNA complexes. The N / P ratio of the complexes was 8. The complexes were formulated according to the molar ratio relative to the N contributed by each dendrimer (i.e. the ratios in brackets represent the molar ratio relative to the N of each dendrimer). DOTMA:DOPE was added to the mRNA complexes at w / w=10:1.

[0514] It can be seen that increasing the proportion of dendrimers with higher PDI in the nanoparticles generally increases the transfection efficiency of the nanoparticles. However, as the proportion of dendrimers with higher PDI increases, the PDI also increases. Based on this observation, it is possible that the ratio of peptide dendrimers in the nanoparticles can be optimized to obtain the highest transfection efficiency while keeping the PDI within acceptable limits (e.g., less than or equal to 0.35).

[0515] Example 13 - Exemplary Dendrimers for Hybrid Peptide Dendrimer / Lipid Nanoparticles

[0516] name structure PDI RHCG1-R (R)2KRHC-NH2 0.566 RHCG1-LR (LR)2KRHC-NH2 0.577 RHCG1-RLR (RLR)2KRHC-NH2 0.766 GSCG1-LRLR (LRLR)2KGSC-NH2 0.581

[0517] Table 7. Exemplary first generation dendrimers for hybrid dendrimer nanoparticles.

[0518]

[0519]

[0520] Table 8. Exemplary second and third generation dendrimers for use in hybrid dendrimer nanoparticles.

[0521] Example 14 - Determination of transfection efficiency

[0522] Transfection of C2c12 cells:

[0523] 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-labeled mRNA expressing eGFP was purchased from RiboPro. 24 hours before transfection, C2c12 cells were seeded in 96-well plates to reach 70% confluence. The mRNA transfection complex was formed by mixing the mRNA with the dendrimer in 25mM HEPES buffer at 25°C and then mixing with DOTM A:DOPE (1:1 w / w) in 25mM HEPES buffer. The transfection complex ("NTX3") was then overlaid onto the cells in complete growth medium. Cells were harvested 4 hours after transfection for FACS analysis. Cell culture medium from each treatment and PBS used to wash each well were collected before adding 5uM EDTA in PBS. After incubation at 37°C for 10 minutes, cells were gently detached from the plate and precipitated at 400g for 8 minutes at 4°C. According to the manufacturer's instructions, cells were stained with LIVE / DEAD fixable aqueous fluorescent active dye (Invitrogen). Ar C beads were stained with 3uL pure Live / Dead Aqua for compensation. After staining, cells were precipitated at 400g for 8 minutes at 4°C and resuspended in 500uL / sample PBS washing solution before repeated centrifugation. Negative ArC beads were added to the compensation control. GFP compensation beads were distributed in separate tubes as GFP compensation controls. All samples (including beads) were fixed with 4% paraformaldehyde in PBS for 15 minutes at room temperature. After this, the sample was once again spun at 4°C for 8 minutes at 400g and resuspended in FACS buffer (PBS+2mM ED TA+0.5% w / v BSA). The samples were stored at 4°C and flow analyzed 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. Fig.18 Approximately 100% of C2c12 myocytes were successfully transfected.

[0524] Transfection of J774 cells:

[0525] The day before transfection, the J774 cells cultured in a 10 cm diameter culture dish were washed with PBS, scraped with 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 F BS and 2 mM GlutaMAX supplements) for counting. The cells were seeded into a 24-well plate format. The cells were transferred to an incubator overnight. For transfection, the culture medium covering the J774 cells was replaced with a fresh J774 complete medium of 480 ul / well. The cells were transfected in an incubator for 4 hours (i.e., the total volume during transfection was 600 u L) with 120 ul / well of 25 mM HEPES or a formulation (encapsulated AlexaFluor 488-eGFP mRNA of various concentrations). After 4 hours, J774 cells were washed with PBS, and harvested with 5mM EDTA and a cell scraper for 15 minutes incubated with cells at 37 degrees Celsius. J774 cells were then rotated. Supernatant was removed, cells were resuspended in 100ul / sample of LIVE / DEAD fixable red diluted (1:250) in PBS, and placed in a refrigerator for 30 minutes. The cells were then rotated for 5 minutes at 400g, and washed with PBS. The cells were rotated once more, resuspended in 4% PFA (diluted in PBS) of 100ul / sample, and fixed at room temperature for 10 minutes. After fixation, cells were rotated for 5 minutes at 500g, and washed with PBS. The cells were rotated once more, and resuspended in 400ul / sample of FACS buffer (PBS containing 0.5%w / v BSA and 2mM EDTA), and passed through a 70um 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.

[0526] Fig.19 It was shown that when 1.5ug of nucleic acid was applied using the composition of the present invention, approximately 100% of J774 macrophages were successfully transfected. When 0.015ug of nucleic acid was applied using the composition of the present invention, approximately 50% of J774 macrophages were successfully transfected.

[0527] Transfection of Jurkat cells:

[0528] On the day of transfection, Jurkat cells were harvested and spun at 300g for 5 minutes and resuspended in 10ml of Jurkat complete medium (DMEM containing 10% heat-inactivated FBS and 2mM GlutaMAX supplement) for cell counting. Cells were diluted to 4.17E+5 cells / ml and 480ul / well cells (i.e. 2E+5 cells / well) were inoculated into a 24-well format. Cells were left for 30 minutes before transfection. Cells were transfected in an incubator for 4 hours (i.e., the total volume during transfection was 600uL) with 120ul / well of 25mM HEPES or formulations (encapsulated AlexaFluor 488-eGFP mRNA of various concentrations). After transfection for 4 hours, cells were collected in 1.5ml Eppendorf tubes and spun at 300g for 5 minutes. The cells were then washed once with 750ul / sample of PBS and then spun. Dispose of the supernatant, resuspend the cells in 100ul / sample of LIVE / DEAD fixable red diluted in PBS (1:250), and place in a refrigerator for 30 minutes. Then the cells were spun at 400g for 5 minutes and washed with PBS. The cells were spun again, resuspended in 100ul / sample of 4% PFA (diluted in PBS), and fixed at room temperature for 10 minutes. After fixation, the cells were spun at 500g for 5 minutes and washed with PBS. The cells were spun again, resuspended in 400ul / sample of FACS buffer (PBS containing 0.5%w / v BSA and 2mM EDTA), and passed through a 70um filter before flow analysis. Data were collected using a BDLSFortessa I analyzer running FACS DIVA software (Beckton Dickinson). The collected data were analyzed using FlowJo 10.0 software.

[0529] Fig. 20 It was shown that when 1.5ug of nucleic acid was applied using the composition of the present invention, approximately 100% of Jurkat T cells were successfully transfected. When 0.15ug of nucleic acid was applied using the composition of the present invention, approximately 50% of Jurkat T cells were successfully transfected.

[0530] Transfection of HeLa cells:

[0531] 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 at 25°C and then with DOTMA:DOPE in 25 mM HEPES buffer. mRNA expressed eGFP and was labeled with Alexa Fluor 488. Transfection complexes were overlaid on cells in complete growth medium. Cells were harvested 2 hours after 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 Aqueous Fluorescent Active Dye (Invitrogen) according to the manufacturer's instructions. ArC beads were stained with 3uL of pure Live / Dead Aqua for compensation. After staining, cells were pelleted at 4°C and resuspended in 500uL / sample of PBS wash before repeating centrifugation. Negative ArC beads were added to compensation controls. GFP compensation beads are distributed in separate tubes as GFP compensation controls. All samples (including beads) are fixed with 4% paraformaldehyde in PBS for 15 minutes at room temperature. After this, the samples are rotated at 4°C once more and then resuspended in FACS buffer (PBS+2mM EDTA+0.5%w / v BSA). The samples are stored at 4°C and flow analyzed within 24 hours of fixation. Data are collected using the BD LSRFortessa I analyzer running FACSDIVA software (BecktonDickinson). FlowJo 10.0 software is used to analyze the collected data.

[0532] Fig.21 It was shown that when 1.5ug or 1.125ug of nucleic acid was applied using the composition of the present invention, about 100% of HeLa cells were successfully transfected. When 0.1875ug of nucleic acid was applied using the composition of the present invention, about 75% of HeLa cells were successfully transfected.

[0533] Example 15 - Lipid mixture (i)

[0534] To formulate nanocarriers with dendrimers and mRNA with different lipids, the individual lipids dissolved in solvents were mixed in the desired molar ratio and combined with the dendrimer and mRNA.

[0535] To form the exemplary nanoparticle compositions, prepare GSC G1,2-RHL ((RHL)4(KRHL)2KGSC-NH2) and EGFP mRNA were mixed in aqueous buffer. The liposome mixture disclosed below was then added.

[0536] 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 an ethanol solvent at a molar ratio of 1:1:1 and mixed with the solution of peptide and mRNA to obtain a turbid solution. DLS measurement (27-fold dilution in 25 mM HEPES buffer): 115.9 nm; PDI 0.11.

[0537] DODAP:DOTMA:DOPE 1:1:1. DODAP, DOTMA (1N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride) and DOPE were combined in an ethanol solvent at a molar ratio of 1:1:1 and mixed with the solution of peptide and mRNA to obtain a turbid solution. DLS measurement (27-fold dilution in 25 mM HEPES buffer): 116.9 nm; PDI 0.10.

[0538] DODAP:DOTAP:DOPE 1:1:2. DODAP, DOTAP and DOPE were combined in an ethanol solvent at a molar ratio of 1:1:2 and mixed with the solution of peptide and mRNA to obtain a turbid solution. DLS measurement (27-fold dilution in 25 mM HEPES buffer): 127.3 nm; PDI 0.09.

[0539] DODAP:DOTMA:DOPE 1:1:2. DODAP, DOTMA and DOPE were combined in an ethanol solvent at a molar ratio of 1:1:2 and mixed with the solution of peptide and mRNA to obtain a turbid solution. DLS measurement (27-fold dilution in 25 mM HEPES buffer): 136.9 nm; PDI 0.08.

[0540] DODAP:DOTMA:DOPE 2:1:1. DODAP, DOTMA and DOPE were combined in an ethanol solvent at a molar ratio of 2:1:1 and mixed with the solution of peptide and mRNA to obtain a turbid solution. DLS measurement (27-fold dilution in 25 mM HEPES buffer): 139.5 nm; PDI 0.07.

[0541] DODAP:DOTAP:DOPE 2:1:1. DODAP, DOTAP and DOPE were combined in an ethanol solvent at a molar ratio of 2:1:1 and mixed with the solution of peptide and mRNA to obtain a turbid solution. DLS measurement (27-fold dilution in 25 mM HEPES buffer): 160.3 nm; PDI 0.06.

[0542] DODAP:DOTAP 1:1. DODAP and DOTAP were combined in a 1:1 molar ratio in ethanol solvent and mixed with the solution of peptide and mRNA to obtain a turbid solution. DLS measurement (27-fold dilution in 25 mM HEPES buffer): 124.6 nm; PDI 0.12.

[0543] DODAP:DORI:DOPE 1:1:1. DODAP, DORI (N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(oleyloxy)propane-1-ammonium bromide) and DOPE were combined in an ethanol solvent at a molar ratio of 1:1:1 and mixed with the solution of peptide and mRNA to obtain a turbid solution. DLS measurement (27-fold dilution in 25 mM HEPES buffer): 148.9 nm; PDI 0.06.

[0544] DODAP:DORI:DOPE 1:1:2. DODAP, DORI and DOPE were combined in a 1:1:1 molar ratio in ethanol solvent and mixed with the solution of peptide and mRNA to obtain a turbid solution. DLS measurement (27-fold dilution in 25 mM HEPES buffer): 160.3 nm; PDI 0.06.

[0545] DOPE has a CAS number of 4004-05-1 and has the following formula: 41 H 78 NO8P); CAS number of DOTAP: 132172-61-3, and has the following formula: C 42 H 80 NO4Cl); CAS number of DODAP: 127512-29-2, and has the following formula: C 41 H 77 NO4. D ORI has the CAS number: 153312-59-5 and has the following formula: C 43 H 82 BrNO5.DO TMA has a CAS number of 104162-48-3 and has the following formula: 42 H 84 NO2Cl. DO TMA can also be named as 1,2-di-O-octadecenyl-3-trimethylammonium propane (chloride salt), (CAS No.: 104872-42-6).

[0546] The transfection efficacy of nanocarriers containing each lipid mixture was evaluated on human cancer cell lines (HeLa and A549).

[0547] The method described herein was used to transfect HeLa cells with nanocarriers prepared with different lipid components. The nanocarriers used comprised: (1) GSC G1,2-RL,3-LR (N:P=0.16:1 for mRNA) and DOTMA:DOPE (the ratio of the molar number of DOTMA to the molar number of DOPE is 1:1), the ratio of total lipid mass to mRNA is w / w=10:1; (2) GSC G1,2-RL,3-LR (N:P=8:1 for mRNA) and DOTMA:DOPE (the ratio of the molar number of DOTMA to the molar number of DOPE is 1:1), the ratio of total lipid mass to mRNA is w / w=10:1; (3) GSC G1,2-RHL (N:P = 0.6:1 for mRNA) and DOTMA:DOPE (the ratio of the molar number of DOTMA to the molar number of DOPE is 1:1), the ratio of total lipid mass to mRNA is w / w = 10:1; (4) GSC G1,2-RHL (N:P = 0.6:1 for mRNA) and DODAP:DOTAP (the ratio of the molar number of DODAP to the molar number of DOTAP is 1:1), the ratio of total lipid mass to mRNA is w / w = 10:1; (5) GSC G1,2-RHL (N:P = 0.6:1 for mRNA) and DODAP:DOTAP:DOPE (the ratio of the number of moles of DODAP to the number of moles of DOTAP to the number of moles of DOPE is 1:1:1), the ratio of total lipid mass to mRNA is w / w = 10:1, and (6) GSC G1,2-RHL (N:P = 0.6:1 for mRNA) and DODAP:DOTAP:DOPE (the ratio of the molar number of DODAP to the molar number of DOTAP to the molar number of DOPE is 1:1:2), and the ratio of total lipid mass to mRNA is w / w = 10:1. (7) GSC G1,2-RHL (N:P = 0.6:1 for mRNA) and DODAP:DOT MA:DOPE (the ratio of the molar number of DODAP to the molar number of DOTMA to the molar number of DOPE is 1:1:1), and the ratio of total lipid mass to mRNA is w / w = 10:1; and (8) GSCG 1,2-RHL (N:P=0.6:1 for mRNA) and DODAP:DOTMA:DOPE (the ratio of the moles of DO DAP to the moles of DOTMA to the moles of DOPE is 1:1:2), and the ratio of total lipid mass to mRNA is w / w=10:1. The mRNA used is the mRNA expressing eGFP, and the cells were harvested 24 hours after transfection to analyze the fluorescent protein expression by plate reader. Control: cells transfected with mRNA alone. Fig. 22 and 23 Shown are the increased transduction efficiencies achieved by the three lipid systems within nanoparticles comprising representative second generation peptide dendrimers when transfecting HeLa cells.

[0548] The method described herein was used to transfect A549 cells with nanocarriers formulated with different lipid components. The nanocarriers used comprised: (1) GSC G1,2-RL,3-LR (N:P=0.16:1 for mRNA) and DOTMA:DOPE (the ratio of the molar number of DOTMA to the molar number of DOPE is 1:1), the ratio of total lipid mass to mRNA is w / w=10:1; (2) GSC G1,2-RL,3-LR (N:P=8:1 for mRNA) and DOTMA:DOPE (the ratio of the molar number of DOTMA to the molar number of DOPE is 1:1), the ratio of total lipid mass to mRNA is w / w=10:1; (3) GSC G1,2-RHL (N:P = 0.6:1 for mRNA) and DOTMA:DOPE (the ratio of the molar number of DOTMA to the molar number of DOPE is 1:1), the ratio of total lipid mass to mRNA is w / w = 10:1; (4) GSC G1,2-RHL (N:P = 0.6:1 for mRNA) and DODAP:DOTAP:DOPE (the ratio of the number of moles of DODAP to the number of moles of DOTAP to the number of moles of DOPE is 2:1:1), and the ratio of total lipid mass to mRNA is w / w = 10:1; and (5) GSC G1,2-RHL (N:P = 0.6:1 for mRNA) and DODAP:DORI:DOPE (the ratio of the molar number of DODAP to the molar number of DORI to the molar number of DOPE is 1:1:1), and the ratio of total lipid mass to mRNA is w / w = 10:1; (6) GSC G1,2-R HL (N:P = 0.6:1 for mRNA) and DODAP:DORI:DOPE (the ratio of the molar number of DODAP to the molar number of DORI to the molar number of DOPE is 1:1:2), and the ratio of total lipid mass to mRNA is w / w = 10:1; (7) GSC G1,2-RHL (N:P=0.6:1 for mRNA) with DODAP:DOTMA:DOPE (the ratio of the moles of DODAP to the moles of DORI to the moles of DOPE is 2:1:1), the ratio of total lipid mass to mRNA is w / w=10:1; the mRNA used is the mRNA expressing eGFP, and the cells were harvested 24 hours after transfection to analyze the fluorescent protein expression by plate reader. Compared with formulations (1), (2) and (3), formulations (4), (5), (6) and (7) contain only 67% of the eGFP mRNA content. Control: cells transfected with mRNA alone. Fig.24 Shown are the increased transduction efficiencies achieved by the three lipid systems within nanoparticles comprising representative second generation peptide dendrimers when transfecting A549 cells.

[0549]

[0550]

[0551] Table 4. GSC Size and PDI of G1,2-RHL (N:P=0.6:1 for mRNA) nanocarriers. The ratios in brackets indicate the molar ratios between lipids. The total amount of lipids to mRNA ratio is w / w=10:1. (Following rows, DODAP:DOTM A:DOPE:DMG-PEG and DODAP:DOTAP:DOPE:DMG-PEG are exemplified in Example 26 below).

[0552] Example 16 - Comparison of Linear and Dendritic PGA

[0553] Human T cells were transfected with naked mRNA expressing eGFP (enhanced GFP) or with nanocarriers containing mRNA expressing eGFP using the methods described herein. GSC G1,2-RHL (N:P=0.6:1 for mRNA) and DOTMA / DOPE (w / w=10:1 for mRNA) and mRNA.

[0554] In the first experiment, the nanocarriers were coated with equal numbers of linear PGA or dendritic PGA molecules ( Fig.25 A). Linear PGA is the linear PGA with 100 glutamic acids; dendritic PGA is GSEGSEGSEC(OH)G1-(Ac)ESGESGESG.

[0555]

[0556] Table 9. Exemplary calculations for coating nanoparticles with linear PGA molecules or the same number of dendritic PGA molecules (see Fig.25 A).

[0557] ratio Size(nm) PDI Zeta potential (mV) Linear PGA-coated particles 180 0.12 -59 Dendritic PGA-coated particles 184 0.16 -32

[0558] Table 10. Hydrodynamic size, polydispersity index (PDI) and zeta potential of nanoparticles coated with linear PGA molecules or the same number of dendritic PGA molecules (see Fig.25 A).

[0559] In the second experiment, the nanocarriers were coated with equimolar charges of linear PGA or dendritic PGA ( Fig.25 B). Linear PGA is the linear PGA with 100 glutamic acids; dendritic PGA is G1-EEEE.

[0560]

[0561] Table 11. Exemplary calculations for coating nanoparticles with linear PGA molecules or dendritic PGA molecules of equimolar charge (see Fig.25 B).

[0562] ratio Size(nm) PDI Zeta potential (nmV) Linear PGA-coated particles 175 0.14 -46 Dendritic PGA-coated particles 187 0.10 -22

[0563] Table 12. Hydrodynamic size, polydispersity index (PDI) and zeta potential of nanoparticles coated with linear PGA molecules or dendritic PGA molecules of equimolar charge (see Fig.25 B).

[0564] The expression of intracellular eGFP protein was quantified by flow cytometry. Fig.25 A and 25B show expression levels normalized to the levels achieved by the preparation coated with linear PGA ("Normalized relative to transfection"). Negative control cells were either not transfected or transfected with mRNA only.

[0565] Example 17 - Muscle-Targeted Nanocarriers

[0566] The nanoparticles of the present invention are coated with dendrimers containing muscle targeting peptides to improve targeting and transfection of muscle cells, even differentiated muscle cells.

[0567] First, prepare a GSCNanoparticles of G1,2-RHL (N:P=0.6:1 for mRNA) with DOTMA / DOPE (w / w=10:1 for mRNA) and mRNA. The nanoparticles were then coated with dendrimers with muscle targeting domains Myo-G1-EEEE (Ac-EEEE)2K GSCGAASSLNIA-(Acp)-NH2) at different mRNA:coating molar ratios between 0.5:1 and 5:1, preferably around 2.74:1. As a control, nanoparticles were coated with dendrimers G1-EEEE without muscle targeting domains at different mRNA:coating molar ratios between 0.5:1 and 5:1, preferably around 2.74:1.

[0568] In the first experiment, mouse muscle cells (C2c12 cells) were transfected with a preparation containing mRNA expressing eGFP. The preparation used contained GSC G1,2-RHL (N:P=0.6:1 for mRNA) was co-transfected with DOTMA / DOPE (w / w=10:1 for mRNA) and mRNA and coated with the same molar equivalent of dendrimer PGA with or without muscle targeting domain. Cells were harvested 24 hours after transfection. Control: cells were not transfected with mRNA. The dendrimer with muscle targeting domain was (Ac-EEEE)2KGSCGAASSLNIA-(Acp)-NH2 (designated as "muscle-G1-EEEE". ASSLNIA, SEQ ID NO: 1 is the muscle targeting motif). The dendrimer without muscle targeting domain was (Ac-EEEE)2KGSGGSGGSC-NH2 ("GSGGSGGSCG1-EEEE"). Fig.26 showed that eGFP expression was dramatically increased in myocytes transfected with nanocarriers containing the muscle targeting domain.

[0569] In a second experiment, differentiated mouse myocytes (C2c12 cells) were transfected with a formulation containing mRNA expressing eGFP. Myocyte differentiation was performed as follows: C2c12 cells were seeded and allowed to grow to confluence in growth medium. Once the cells were confluent, the medium on the cells was replaced with differentiation medium for 2 days to allow the cells to develop myotube characteristics. Thirty minutes prior to transfection, the differentiation medium was replaced with growth medium. Fig. 27 Cells were transfected with the nanocarriers shown in for 24 hours and harvested to determine eGFP reporter gene expression using a plate reader. The mRNA used expressed eGFP. The growth medium contained high glucose DMEM (Gibco), 10% FBS (Gibco), 2mM L-glutamine (Gibco). Differentiation medium contained high glucose DMEM (Gibco), 2% horse serum (Gibco), 2mM L-glutamine (Gibco), 1uM insulin.

[0570] As in the first experiment, the preparation used contained GSC G1,2-RHL (N:P=0.6:1 for mRNA) was co-transfected with DOTMA / DOPE (w / w=10:1 for mRNA) and mRNA and coated with the same molar equivalent of dendrimer PGA with or without muscle targeting domain. Cells were harvested 24 hours after transfection. Control: cells were not transfected with mRNA, or transfected with mRNA alone. The dendrimer with muscle targeting domain is muscle-G1-EEEE. The dendrimer without muscle targeting domain is GSGGSGGSCG1-EEEE. Fig. 27 showed that eGFP expression was dramatically increased in differentiated myocytes transfected with nanocarriers containing the muscle targeting domain.

[0571] Nanoparticles can also be coated with dendrimers at different molar ratios of Myo-G1-EEEE (e.g., 2.72:1 and 2.06:1, dendrimer to mRNA ratio) and show improved mRNA transfection on differentiated C2c12 cells compared to nanoparticles coated with dendrimers without targeting domains. Myo-G1-EEEE coated at 2.72:1 had a PDI of 0.14 and a zeta potential of -44.0 mV, with a size of 172 nm. Myo-G1-EEEE coated at 2.06:1 had a PDI of 0.15 and a zeta potential of -38.7 mV, with a size of 176 nm. GSGGSGGSCG1-EEEE coated at 2.72:1 had a PDI of 0.13 and a zeta potential of -35.4 mV, with 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.

[0572] Example 18 - Tumor Targeting Nanocarriers

[0573] A549 is a cancer cell that highly expresses integrins (Guo et al., 2009). A549 cells were transfected with dendritic PGA coated with or without the integrin targeting domain ACDCRGDCFCG (SEQ ID NO: 5) and containing mRNA expressing eGFP. The nanocarrier also contained GSCG1,2-RHL (N:P=0.6:1 for mRNA) and DOTMA / DOPE (w / w=10:1 for 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 transfected with mRNA only. The dendrimer with integrin targeting domain is (Ac-EEEE)2KGSGGSGGSACDCRGDCFCG-NH2 (disulfide bonds: C1-C4, C2-C3). The dendrimer without integrin targeting domain is (Ac-EEEE)2KGSGGSGGSC-NH2 ("GSGGSGGSCG1-EEEE").

[0574] Methods: First, an initial solution of GSCG1,2-RHL and DOTMA:DOPE nanoparticles in 25 mM HEPES buffer (150 ug / mL mRNA) was prepared as follows: a 10 mg / ml aqueous solution of peptide (RHL)4(KRHL)2KGSC-NH2 (4912 g / mol inc. TFA counterion, 10 N / peptide) was added to a sterile polypropylene tube (4.240 uL, 42.4 ug, 8.632 × 10 -6 mmol, 8.632×10 -5 mmol N), sterile water (37.34 uL) and 200 mM HEPES buffer (5.94 uL) and mixed. To the second tube was added 1 mM sodium citrate pH 6.4 (46.44 uL, 46.44 ug, 1.407 × 10 -4 1mg / mL in millimoles P) EGFP mRNA (5 moU), sterile water (48.37 uL) and 200 mM HEPES buffer (13.54 uL) were mixed. DOTMA:DOPE liposomes (1:1 molar ratio) in a 25 mM (17.68 mg / mL) aqueous solution (16.27 uL, 287.7 ug, 3.287 × 10 -4 mmol N), sterile water (109.2 uL) and 200 mM HEPES buffer (19.34 uL) and mixed. 43.20 uL of the peptide mixture (7.847 × 10 -6 mmol, 7.847×10 -5 mRNA P = 0.60 equivalents) were transferred to a fresh polypropylene tube. 100.8ul of the mRNA mixture (1.309×10 -4 mmol P) was added to the tube containing the peptide and mixed rapidly up and down with a pipette. The solution was incubated for 2 minutes. 144.0 uL of the liposome mixture (3.057 × 10-4 100 mM N) was added to the tube containing the peptide and mRNA and mixed quickly up and down with a pipette to give a turbid solution. DLS measurement (40-fold dilution in 25 mM HEPES buffer): 184 nm, PdI 0.13). Zeta potential measurement (3-fold dilution in 25 mM HEPES and then further 60-fold dilution in water): +33.5 mV.

[0575] Coating Material Stock Solution:

[0576] Int-GSGGSGGSCG1-EEEE(Ac-EEEE)2KGSGGSGGSACDCR GDCFCG (disulfide bridge between C1 and C4 and disulfide bridge between C2 and C3, 3081 g / mol, 8 negative charges / peptide). 2.00 mg was dissolved in 133.3 uL 50 mM ammonium carbonate solution, followed by 266.7 uL 25 mM HEPES buffer to give a final concentration of 5.0 mg / mL. The tube was centrifuged at 11,000 g for 3 minutes and aliquoted before storage at -80°C.

[0577] GSGGSGGSCG1-EEEE ((Ac-EEEE)2KGSGGSGGSC-NH2, 1912 g / mol, 8 negative charges / peptide). 10 mg was dissolved in 156.2 uL 50 mM ammonium carbonate solution, followed by 468.8 uL 25 mM HEPES buffer to give a final concentration of 16.0 mg / mL. The tube was centrifuged at 11,000 g for 3 minutes and aliquoted before storage at -80°C.

[0578] Coated Nanoparticles:

[0579] The above basic nanoparticles were diluted twice in 25 mM HEPES to an mRNA concentration of 75 ug / mL. 134.0 uL (10.05 ug mRNA, 3.045×10 -5 To these nanoparticles (1000 mmol P) was added 67.0 uL of coating material dissolved in 25 mM HEPES and mixed quickly up and down with a pipette. The final mRNA concentration of the solution was 50 ug / mL. The following table provides a breakdown of the coating:

[0580]

[0581] Table 13. Examples of coating calculations and size, zeta potential and PDI of the resulting nanoparticles. The size (nm), zeta potential (mV) and PDI of the uncoated nanocarriers were 190 nm, +37.1 mV and 0.21, respectively.

[0582] Fig.28showed that eGFP expression was dramatically increased in A549 cells transfected with nanocarriers containing the tumor-targeting integrin-binding domain.

[0583] Example 19 - Targeted Nanocarriers via Antibody-Conjugated Dendrimers

[0584] A 10 mg / mL aqueous solution of GSEGSEGSEC(OH)G1-(Ac)ESGESGESG ([(Ac-ESGESGESG)2K]GSEGSEGSEC, 2793 g / mol, 9 negative charges / peptide) (180 ug, 6.446×10 -5 mmol, 5.801×10 -4 mmol COOH) was added to a 500uL polypropylene tube. A 2mg / mL aqueous solution of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC.HCl, 191.7g / mol) (6.80uL, 13.59ug, 7.090×10 -5 mmol), and the solution was allowed to incubate at room temperature for 2 minutes. A 2 mg / mL aqueous solution of N-hydroxysulfosuccinimide sodium salt (Sulfo-NHS, 217.1 g / mol) (8.47 u L, 16.94 ug, 7.799×10 -5 mmol), and the solution was allowed to incubate at room temperature for 15 minutes. -6 7.19 mg / mL of InVivoMAb anti-human CD3 (BioXCell BE0001-2, 150 kDa) was added to 400 mL of PBS (0.529 mg / mL GSEGSEGS EC(OH)G1-(Ac)ESGESGESG peptide, 2.844 mg / mL antibody). 182.2 uL of PBS was added, and 350 uL of the solution was dialyzed (MWCO 50 kDa) in 500 mL PBS at 4°C overnight. The theoretical concentration of the resulting conjugate in PBS was 3.373 mg / mL (0.529 mg / mL GSEGSEGS EC(OH)G1-(Ac)ESGESGESG peptide, 2.844 mg / mL antibody).

[0585] An initial solution of lipid nanoparticles containing mRNA is prepared, and then they are coated with the antibody-conjugated dendrimer, for example by mixing in a volume ratio of 2: 1. Adding coating material will reduce the zeta potential, indicating that the nanoparticles are coated.

[0586] Example 20 - T cell targeting nanocarriers comprising CD3 binding antibodies

[0587] Coating of nanocarriers with human CD3 targeting antibodies improves human T cell targeted mRNA delivery. Jurkat cells were untransfected or transfected with the following composition:

[0588] (1) mRNA expressing eGFP alone; (2) GSC G1,2-RHL (N:P=0.6 for mRNA) and DOTMA / DOPE (w / w=10 for mRNA) and mRNA (controls for (4) and (7)); (3) GSC G1,2-RHL (N:P=0.6 for mRNA) and DOTM A / DOPE (w / w=10 for mRNA) with mRNA, coated with 1 equivalent of isotype antibody control (ITC) conjugated dendrimer (control for (4)); (4) GSC G1,2-RHL (N:P=0.6 for mRNA) and DOTMA / DOPE (w / w=10 for mRNA) and mRNA, coated with dendrimer conjugated with 1 equivalent of anti-CD3 antibody; (5) GSC G1,2-R HL (N:P=0.6 for mRNA) and DOTMA / DOPE (w / w=10 for mRNA) with mRNA, coated with dendrimer alone (control for (7)); (6) GSC G1,2-RHL (N:P=0.6 for mRNA) and DOTMA / DOPE (w / w=10 for mRNA) and mRNA, coated with 3 equivalents of isotype antibody control (ITC) conjugated dendrimer (control for (7)); (7) GSC G1,2-RHL (N:P=0.6 for mRNA) and DOT MA / DOPE (w / w=10 for mRNA) with mRNA, coated with 3 equivalents of anti-CD3 antibody-conjugated dendrimer. D / D indicates DOTMA:DOPE (w / w=10:1 for mRNA).

[0589] T cells were transfected in 24-well plates for 2 hours using 4.5 ug mRNA. Formulations were analyzed for eGFP expression by cytometry 24 hours post transfection. Isotype Control (ITC): InVivoM Ab Mouse IgG2a Isotype Control (BioXCell BE0085, Clone C1.18.4).

[0590] Example 21 - In vitro delivery of multiple nucleic acids to cells in a single formulation

[0591] HeLa cells were transfected with a formulation comprising two nucleic acids; a first mRNA expressing eGFP, a second mRNA expressing mCherry, and a third mRNA expressing luciferase. The nanocarrier also comprises GSCG1,2-RHL (N:P=0.6:1 for mRNA) and DOTM A / DOPE (w / w=10:1 for mRNA). The mRNA molecules are mRNA expressing eGFP and mRNA expressing mCherry or mRNA expressing luciferase (Fluc). Cells were harvested 24 hours after transfection to analyze fluorescent protein expression by flow cytometry. Only eGFP+Fluc mRNA or with nanocarriers consisting of 1:1w / w eGFP mRNA and Fluc mRNA; only mCherry+Fluc mRNA or with nanocarriers consisting of 1:1w / w mCherry mRNA and Fluc mRNA; only eGFP+mCherry mRNA or with nanocarriers consisting of 1:1w / w eGFP mRNA and mCherry mRNA. Fig.30 showed that nanocarriers can encapsulate and deliver more than one mRNA for functional protein expression.

[0592]

[0593] Table 14. Proteins with 2 mRNAs GSC Size and PDI of G1,2-RHL (N:P=0.6:1) nanocarriers. All nanocarriers also contained DOTMA:DOPE.

[0594] Example 22 - Delivery of multiple nucleic acids to a subject in a single formulation

[0595] Prepared as previously described GSC In order to prepare nanocarriers of G1,2-RHL (N:P=0.6:1 for mRNA), DOT MA:DOPE (10:1 for mRNA) and mRNA. G SC G1,2-RHL (N:P=0.6:1 for mRNA) and CpG, DOTMA:DOPE (10:1 for mRNA and CpG) and mRNA nanocarriers were first mixed with mRNA and CpG at a ratio of mRNA to CpG of w / w=9:1. This mixture was then added to GSC G1,2-RHL (N:P=0.6:1), followed by the addition of DOTMA:DOPE (10:1 for mRNA and CpG). The CpG used was CpG ODN 2006 (CpG 7909) purchased from InvivoGen.

[0596] Luciferase expression in mouse tissues after intravenous administration of a composition comprising the following substances: GSCG1,2-RHL (N:P=0.6:1 for mRNA) with DOTMA / DOPE (w / w=10:1 for mRNA), mRNA expressing luciferase and CpG molecules with DOTMA / DOPE (w / w=10:1 for nucleic acids). CpG oligonucleotides (ODNs) are synthetic ODNs containing unmethylated CpG dinucleotides (CpG motifs). Individual mRNA treatments were used as controls. 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.

[0597] Fig.31 It is shown that high levels of luciferase were detected in the lung and spleen 6 hours after administration of nanocarriers containing luciferase mRNA and CpG (white bars).

[0598]

[0599] Table 15. GSC Size, zeta potential, and PD I of G1,2-RHL (N:P = 0.6:1) nanocarriers with DOTMA:DOPE, coated with GSGGSGGSCG1-EEEE, and with / without CpG nanocarriers.

[0600] Example 23 - Functional delivery to primary leukocytes using nanoparticles carrying two nucleic acids

[0601] Bone marrow cells were isolated from BALB / c mice, resuspended in complete DC medium (RPMI-1640+10% heat-inactivated FBS+1% P / S+40ng / ml mouse GM-CSF, from Peprotech) at 2-3E+5 cells / ml, and seeded into 10 cm diameter culture dishes (2-3E+6 cells / dish). 2-3 days after seeding, an equal volume of complete DC medium was added. Half of the medium was exchanged on days 5-6 and 8 after seeding.

[0602] Transfection and staining: 13-day-old suspension cells (moDC) were spun down and resuspended in complete DC medium at a concentration of 5.68E+5 cells / ml and seeded in a 24-well plate format at 2.5E+5 cells / well (i.e., 440ul / well). The cells were then transfected with the following substances for 2, 4, or 22 hours: (1) GSC G1,2-RHL (N:P=0.6:1 for mRNA) with DOT MA / DOPE (w / w=10:1 for mRNA) and mRNA encoding eGFP, or (2) GSCG1,2-RHL (N:P=0.6:1 for mRNA) with DOTMA / DOPE (w / w=10:1 for mRNA), mRNA encoding eGFP, and CpG nucleotides. Transfection efficiency (% eGF P+) of moDCs was measured 22-24 hours after the start of transfection by flow cytometry. moDCs were distinguished by surface expression of CD11c and lack of expression of F4 / 80 (macrophage marker). D / D refers to DOTMA / DOPE (w / w=10:1 for mRNA).

[0603] The cells were then spun and resuspended in 1 ml of fresh complete medium and re-seeded into 24-well plates. The cells were harvested the next day and stained with live / dead aqua (1:250 in PBS) for 30 minutes. After PBS washing, the cells were blocked for 5 minutes with 2x TruStain human FcX blocking solution of 50ul / sample diluted in FACS buffer (1:20; final concentration 1:40). The cells were then stained for 20 minutes with a 2x mixture of surface marker antibodies of 50ul / sample diluted in FACS buffer. The final concentrations of these antibodies used were: 1:100CD11c-PerCP-Cy5.5 (Biolegend; 117328), 1:200CD11b-APC (Biolegend; 101212) and 1:100F4 / 80-PE (Biolegend; 123110). After washing with FACS buffer, the 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. Fig.32 It was shown that eGFP expression was achieved at similar levels by all three transfection times.

[0604] Example 24 - Functional repolarization of primary macrophages

[0605] The nanocarriers of the present invention are used to deliver modified IRF5 to polarize M2 macrophages to M1 macrophages. The modified IRF5 is a protein with a mutation that acts as an activated form of wild-type IRF5. The nanocarrier formulation used comprises GSC G1,2-RHL (N:P=0.6:1 for mRNA) and DOTMA / DOPE (w / w=10:1 for mRNA) and mRNA expressing modified IRF5.

[0606] Primary murine bone marrow-derived macrophages were polarized to an M2 phenotype and then transfected with the nanocarrier formulations for 24 h.

[0607] The M1 signature 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 above shows the change in gene expression on the x-axis versus the -Log10 P value on the y-axis. P values ​​were adjusted using the Benjamini-Hoc hberg method. Fold changes were adjusted using the visualization method described by Love et al., 2014.

[0608] Fig.33 A shows that many genes are significantly differentially expressed (DE) compared to the luciferase control. Genes upregulated in activated M1 macrophages (interleukins: Il27, Il12b, Il1b and nitric oxide synthesis gene Nos2) or M2 macrophages (Arg1, Cd163, Mrc1) are marked, showing that M1 genes are significantly upregulated and M2 genes are downregulated. Fig.33 B shows the results of gene set enrichment analysis (GSEA) performed on the RNA sequencing dataset to find the DE gene sets that were significantly enriched in cells transfected with modified IRF5 mRNA (FDR < 0.05). The cells were highly enriched for genes related to cell killing, cytokine activity, and interleukin production. GSEA was performed in WebGestalt with Wald statistical ranking as input.

[0609] IL12 secretion in primary mouse macrophages transfected with IRF5 using the nanocarriers of the present invention: Primary mouse bone marrow-derived macrophages were polarized to an M2 phenotype. The cells were then transfected with mRNA expressing a modified form of the IRF5 protein for 24 hours, which can polarize the M2 cells to M1 cells. The secretion of cytokines was measured 24 hours after transfection. Control: Cells were transfected with mRNA expressing luciferase (control mRNA). The modified IRF5 is a protein with a mutation that acts as an activated form of WT IRF5. The formulation used comprises GSC G1,2-RHL (N:P=0.6:1 for mRNA) and DOTMA / DOPE (w / w=10:1 for mRNA) and mRNA. IL12p70 and IL12p40 secretion of cells transfected with modified IRF5 mRNA was significantly increased (see Fig.34 A and 34B).

[0610] IL12 and TNF secretion in primary human macrophages transfected with IRF5 using the nanocarriers of the present invention: Primary human macrophages are polarized to an M2 phenotype. The cells are then transfected with mRNA expressing a modified form of the IRF5 protein for 24 hours, which can polarize M2 cells to M1 cells. The secretion of cytokines is measured 24 hours after transfection. Control: Cells are transfected with mRNA expressing luciferase (control mRNA). The modified IRF5 is a protein with a mutation that acts as an activated form of WT IRF5. The formulation used comprises GSC G 1,2-RHL (N:P = 0.6:1 for mRNA) and DOTMA / DOPE (w / w = 10:1 for mRNA) and mRNA. Cells transfected with modified IRF5 mRNA showed a significant increase in IL12p70 and TNFα secretion (see Fig.34 C and 34D).

[0611] Method for cytokine secretion experiment: A standard 6-day protocol was used to differentiate mouse (bone marrow-derived) and human (peripheral blood-derived) monocytes by cytokine exposure. Transfection was perfo...

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 branch residue and two first peptide motifs, wherein the nanoparticles are targeted to target cells or tissues, such as myeloid cells, lymphoid cells, muscle cells, lung cells, CD206+ cells and / or tumor cells, and / or myeloid tissue, lymphoid tissue, muscle tissue, lung tissue and / or tumor tissue, And wherein 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 of claim 1, wherein the nanoparticle comprises a myeloid cell, a lymphoid cell, a muscle cell, a lung cell, a CD206+ cell, or a tumor cell targeting motif; optionally selected from; a. a muscle targeting motif, optionally comprising an ASSLNIA (SEQ ID NO: 1), PYDQLRH (SEQ ID NO: 2) or 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 mannose or maltotriose; or e. an antibody or a target binding fragment thereof, optionally wherein the antibody specifically binds to CD3 f. Lipids linked to a bisphosphonate (BP) group.

3. The nanoparticle 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. The nanoparticle of any one of claims 2 or 3, wherein the myeloid cell, lymphoid cell, muscle cell, lung cell, CD206+ cell and / or tumor cell targeting motif is bound to a negatively charged or neutrally charged polymer or lipid.

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 cholesterol hemisuccinate / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, an electrically neutral zwitterionic polymer or lipid or a glutamic acid-containing peptide.

6. Nanoparticles according to claim 4 or claim 5, wherein the polymer is linear or dendritic PGA.

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

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

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

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

11. A nanoparticle according to claim 10, wherein the second peptide comprises a linear PGA comprising 2 to 10, 2 to 25, 2 to 15 or 2 to 100 glutamate residues, or wherein the second peptide comprises a branched PGA comprising 2 to 10, 2 to 25, 2 to 15 or 2 to 100 glutamate residues in each branch and / or in the core sequence, optionally wherein the second peptide comprises the structure (Ac-EEEE)2KGSGGSGGSC[(SS)-α-D-thiomannose].

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

13. The nanoparticle of any one of the preceding claims, wherein the lipid comprises two lipids, three lipids, four lipids, five lipids, or more than five lipids, optionally wherein the lipid comprises DOPE, DOTMA, DODAP, DORI, DMG-PEG, and / or DOTAP.

14. The nanoparticle of claim 13, wherein the lipids comprise DOTMA and DOPE.

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

16. The nanoparticle of claim 13 or claim 15, wherein the lipids comprise DODAP, DOTMA, and DOPE; or wherein the lipids comprise DODAP, DOTAP, and DOPE; or wherein the lipids comprise DODAP, DORI, and DOPE; or wherein the lipids comprise DODAP, DOTMA, DOPE, and DMG-PEG; or wherein the lipids comprise DODAP, DOTAP, DOPE, and DMG-PEG.

17. A nanoparticle according to any of the preceding claims, wherein the peptide dendrimer further comprises two second branch residues and four second peptide motifs, wherein one of the second branch residues is covalently bound to one of the first peptide motifs, and another second branch residue is covalently bound to another first peptide motif, and wherein each second branch residue is covalently bound to two second peptide motifs.

18. The nanoparticle of claim 17, wherein the peptide dendrimer further comprises at least four third branch residues and eight third peptide motifs, wherein each second peptide motif is covalently bound to one of the third branch residues, respectively, such that each third branch residue is covalently bound to one second peptide motif, and wherein each third branch residue is covalently bound to two third peptide motifs.

19. The nanoparticle of any of the preceding claims, wherein the peptide dendrimer is selected from Table 1 or Table 1B.

20. The nanoparticle of any of the preceding claims, wherein the peptide dendrimer has a structure selected from the group consisting of: G1,2-RL,3-LR; G1,2-R; G1-RL,2-LR; G1,2-RHL; G1-LRLR; G1,2-RF,3-HL; G1-R; GSC G1,2-RL,3-LR; RHC G1,2-R; RHC G1-RL,2-LR; GSC G1,2-RHL; GSC G1-LRLR; GSC G1,2-RF,3-HL; or GSC G1-R.

21. The nanoparticle of any one of the preceding claims, wherein the nucleic acid is: 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.

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

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. 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), CCAAT enhancer binding protein alpha (CEBPA).

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

26. A nanoparticle according to any one of claims 1 to 24 for use in a method of treating cancer, autoimmune diseases, immune cell related diseases, lung diseases and / or myopathies.

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

28. A nanoparticle 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, GI tract tumors, head and neck squamous cell carcinoma (HNSCC), renal cancer, myelofibrosis, CD206+ cancer, melanoma, prostate cancer or anal cancer.

29. A nanoparticle 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 wherein the immune-related disease is Gaucher disease, graft-versus-host disease, allogeneic transplant rejection, acute rejection after transplantation, chronic rejection after transplantation, primary graft dysfunction or chronic granulomatous disease (CGD).

30. Nanoparticles for use according to claim 26, 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 hypertension, alveolar proteinosis, fibrotic lung disease, chronic lung disease or respiratory tract infection; or wherein the muscle disease is muscular dystrophy or muscular dystrophy.

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

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

33. The method of claim 32(a), wherein the peptide dendrimer comprises PGA or a glutamic acid-containing peptide, wherein the glutamic acid-containing peptide comprises a glutamic acid-rich domain, wherein the glutamic acid-rich domain comprises 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 wherein at least 20% of the amino acid residues of the glutamic acid-rich domain are glutamic acid, or the method of claim 31(d), wherein the peptide comprises linear PGA, wherein the linear PGA comprises more than 2 and less than 100 glutamic acid residues.

34. The method of claim 32 or 33, wherein the peptide dendrimer comprises a myeloid cell, lymphoid cell, muscle cell, lung cell, CD206+ cell, tumor cell, bone marrow, pancreas, neuronal tissue, kidney tissue, heart tissue, liver tissue, eye, joint or prostate or a stem cell, pancreatic cell, neuronal cell, kidney cell, heart cell, liver cell, eye cell, synoviocyte or prostate cell targeting motif; optionally selected from; a. a muscle targeting motif, optionally comprising an ASSLNIA (SEQ ID NO: 1), PYDQLRH (SEQ ID NO: 2) or 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 mannose or maltotriose; e. an antibody or a target binding fragment thereof, optionally wherein 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 comprising a bisphosphonate (BP) group; g. a pancreatic targeting motif, optionally comprising a glucagon-like peptide-1 (GLP-1) homolog, such as exenatide-4 (SEQ ID NO: 9) or a variant or binding fragment thereof; h. a kidney targeting motif, optionally comprising (KKEEE)3K; i. a neuronal tissue or cell targeting motif, optionally comprising YTIWMPENPRPGTP CDIFTNSRGKRASNGGGG (SEQ ID NO: 11) or KSVRTWNEIIPSKG CLRVGGRCHPHVNGGG (SEQ ID NO: 12) or a neuronal targeting peptide comprising a Phe-Arg-Trp (FRW) motif; j. a cardiac targeting or cardiac cell targeting motif, optionally comprising APWHLSSQYSRT (SEQ ID NO: 13); k. a liver targeting or hepatocyte targeting motif, optionally comprising an asialoglycoprotein receptor binder, such as an N-acetylgalactosamine (Gal-NAc) sugar or a vitamin A moiety; 1. an eye or ocular cell targeting motif, optionally comprising CARSKNKDC (SEQ ID NO: 14); m. a joint or synovial cell targeting motif, optionally comprising an antibody that specifically binds to CD44 and / or a hyaluronic acid saccharide; or n. A prostate targeting motif, optionally comprising PKRGFQD (SEQ ID NO: 15) or SNTRVAP (SEQ ID NO: 16).

35. A coated nanoparticle capable of transfecting a target cell, wherein the coated nanoparticle comprises a peptide on its surface, wherein: a) the nanoparticle is a nanoparticle according to any one of claims 1 to 24; and / or b) the peptide is a linear PGA comprising more than 2 and less than 100 glutamic acid residues, or the peptide is a branched PGA; and / or c) the peptide is a glutamate-containing peptide comprising a glutamate-rich domain, the glutamate-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 wherein at least 20% of the amino acid residues of the glutamate-rich domain are glutamic acid; and / or d) the nanoparticles are coated with a dendritic polymer selected from Table 1 or Table 1B; or e) the nanoparticles are coated with a peptide selected from Table 1A, wherein the peptide is not E100.

36. The coated nanoparticle of claim 35(b), (c), (d) or (e), wherein the PGA and / or glutamic acid-containing peptide comprises a myeloid cell, lymphoid cell, muscle cell, lung cell, CD206+ cell, tumor cell, neuronal tissue, renal tissue, cardiac tissue, neuronal cell, renal cell or cardiac cell targeting motif; optionally selected from; a. a muscle targeting motif, optionally comprising an ASSLNIA (SEQ ID NO: 1), PYDQLRH (SEQ ID NO: 2) or 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 mannose or maltotriose; e. an antibody or a target binding fragment thereof, optionally wherein 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 comprising a bisphosphonate (BP) group; g. a pancreatic targeting motif, optionally comprising a glucagon-like peptide-1 (GLP-1) homolog, such as exenatide-4 (SEQ ID NO: 9) or a variant or binding fragment thereof; h. a kidney targeting motif, optionally comprising (KKEEE)3K; i. a neuronal tissue or cell targeting motif, optionally comprising YTIWMPENPRPGTP CDIFTNSRGKRASNGGGG (SEQ ID NO: 11) or KSVRTWNEIIPSKG CLRVGGRCHPHVNGGG (SEQ ID NO: 12) or a neuronal targeting peptide comprising a Phe-Arg-Trp (FRW) motif; j. a cardiac targeting or cardiac cell targeting motif, optionally comprising APWHLSSQYSRT (SEQ ID NO: 13); k. a liver targeting or hepatocyte targeting motif, optionally comprising an asialoglycoprotein receptor binder, such as an N-acetylgalactosamine (Gal-NAc) sugar or a vitamin A moiety; 1. an eye or ocular cell targeting motif, optionally comprising CARSKNKDC (SEQ ID NO: 14); m. a joint or synovial cell targeting motif, optionally comprising an antibody that specifically binds to CD44 and / or a hyaluronic acid saccharide; or n. A prostate targeting motif, optionally comprising PKRGFQD (SEQ ID NO: 15) or SNTRVAP (SEQ ID NO: 16).

37. A composition comprising a nanoparticle, the nanoparticle 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 branch residue, and two first peptide motifs.

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

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, wherein one of the second branch residues is covalently bound to one of the first peptide motifs and another second branch residue is covalently bound to another first peptide motif, and wherein each second branch residue is covalently bound to two second peptide motifs.

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, 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 wherein each third branch residue is covalently bound to two third peptide motifs.

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

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 single amino acid, dipeptide, tripeptide or tetrapeptide motifs.

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

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

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

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

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

1.

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

1.

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

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; GSC G1-LRLR; RHC G1-R; RHC G1,2-R; or RHC G1-RLR; and / or the second peptide dendrimer comprises the structure G1,2-RL,3-LR; G1-RL,2-LR; GSC G1,2-RL,3-LR; or RHC G1-RL,2-LR.

55. A composition according to any one of claims 37 to 54, wherein the nucleic acid is selected from: 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.

56. A composition according to 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 interferon.

57. The composition of any one of claims 37 to 56, wherein delivery of the 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 a lipid, a nucleic acid and only 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. A composition according to any one of claims 37 to 57 for use in a method of treating cancer, an autoimmune disease and / or a lung disease.

61. A composition for use according to 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, hematological tumors, esophageal cancer, breast cancer, prostate cancer, bladder cancer, GI tract tumors, head and neck squamous cell carcinoma (HNSCC), kidney cancer, CD206+ cancer, melanoma, prostate cancer or anal cancer.

62. A composition for use according to 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 wherein the immune-related disease is Gaucher disease, graft-versus-host disease, allogeneic transplant rejection, acute rejection after transplantation, chronic rejection after transplantation, primary graft dysfunction or chronic granulomatous disease (CGD), or 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 hypertension, alveolar proteinosis, fibrotic lung disease, chronic lung disease or respiratory tract infection; or wherein the muscle disease is muscular dystrophy or muscular dystrophy.

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 branch residue and two first peptide motifs, The nanoparticles are targeted to bone marrow, pancreas, neuronal tissue, kidney tissue, heart tissue, liver tissue, eyes, joints or prostate, or to stem cells, pancreatic cells, neuronal cells, kidney cells, heart cells, liver cells, eye cells, synovial cells or prostate cells.

64. The nanoparticle of claim 63, wherein the nanoparticle comprises a targeting motif selected from: a. a bone marrow or stem cell targeting motif, optionally comprising an antibody that specifically binds to CD34 and / or a lipid comprising a bisphosphonate (BP) group; b. a pancreatic targeting motif, optionally comprising a glucagon-like peptide-1 (GLP-1) homolog, such as exenatide-4 (SEQ ID NO: 9) or a variant or binding fragment thereof; c. a kidney targeting motif, optionally comprising (KKEEE)3K; d. a neuronal tissue or cell targeting motif, optionally comprising YTIWMPENPRPGTP CDIFTNSRGKRASNGGGG (SEQ ID NO: 11) or KSVRTWNEIIPSKG CLRVGGRCHPHVNGGG (SEQ ID NO: 12) or a neuronal targeting peptide comprising a Phe-Arg-Trp (FRW) motif; e. a cardiac targeting or cardiac cell targeting motif, optionally comprising APWHLSSQYSRT (SEQ ID NO: 13); f. a liver targeting or hepatocyte targeting motif, optionally comprising an asialoglycoprotein receptor binder, such as an N-acetylgalactosamine (Gal-NAc) sugar or a vitamin A moiety; g. an eye or ocular cell targeting motif, optionally comprising CARSKNKDC (SEQ ID NO: 14); h. a joint or synovial cell targeting motif, optionally comprising an antibody that specifically binds to CD44 and / or a hyaluronic acid saccharide; or i. A prostate targeting motif, optionally comprising PKRGFQD (SEQ ID NO: 15) or SNTRVAP (SEQ ID NO: 16).

65. The nanoparticle of claim 63 or claim 64, wherein the nanoparticle comprises a cell targeting motif that targets a stem cell, a pancreatic cell, a neuronal cell, a kidney cell, a cardiac cell, a liver cell, an ocular cell, a synoviocyte, or a prostate cell.

66. according to the nano-particle described in any one in claim 63 to 65, wherein said nano-particle can be with 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% efficiency in vitro transfection institute's targeted stem cell, pancreatic cell, neuronal cell, kidney cell, heart cell, hepatocyte, eye cell, synovial cell or prostate cell.

67. according to the nano-particle described in any one in claim 63 to 66, wherein said bone marrow, pancreas, neuronal tissue, kidney tissue, cardiac tissue, liver tissue, eyes, joints or prostate, stem cell, pancreatic cell, neuronal cell, kidney cell, cardiac cell, liver cell, eye cell, synoviocyte or prostate cell targeting motif is combined with negatively charged or electroneutral polymer or lipid.

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 cholesterol hemisuccinate / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, an electrically neutral zwitterionic polymer or lipid, or a glutamic acid-containing peptide.

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

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

71. The nanoparticle of any one of claims 63 to 70, wherein the bone marrow, pancreas, neuronal tissue, kidney tissue, cardiac tissue, liver tissue, eye, joint or prostate, stem cell, pancreatic cell, neuronal cell, kidney cell, cardiac cell, liver cell, eye cell, synoviocyte or prostate cell targeting motif is covalently bound to the peptide dendrimer, or wherein the nanoparticle further comprises a second peptide comprising a bone marrow, pancreas, neuronal tissue, kidney tissue, cardiac tissue, liver tissue, eye, joint or prostate, stem cell, pancreatic cell, neuronal cell, kidney cell, cardiac cell, liver cell, eye cell, synoviocyte or prostate cell targeting motif.

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

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

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

75. The nanoparticle of claim 74, wherein the lipids comprise DOTMA and DOPE.

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

77. The nanoparticle of claim 75 or claim 76, wherein the lipids comprise DODAP, DOTMA, and DOPE; or wherein the lipids comprise DODAP, DOTAP, and DOPE; or wherein the lipids comprise DODAP, DORI, and DOPE; or wherein the lipids comprise DODAP, DOTMA, DOPE, and DMG-PEG; or wherein the lipids comprise DODAP, DOTAP, DOPE, and DMG-PEG.

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

79. The nanoparticle of claim 78, wherein the peptide dendrimer further comprises at least four third branch residues and eight third peptide motifs, Each second peptide motif is covalently bound to one of the third branch residues, so 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. The nanoparticle of any of the preceding claims, wherein the peptide dendrimer is selected from Table 1 or Table 1B, or wherein the nanoparticle is coated with a peptide selected from Table 1A.

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

82. The nanoparticle of any one of claims 63 to 81, wherein the nucleic acid is: 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.

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

84. A nanoparticle according to 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. 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), CCAAT enhancer binding protein alpha (CEBPA).

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

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

88. A nanoparticle according to any one of claims 63 to 85 for use in a method of treating cancer, autoimmune diseases, immune cell related diseases, lung diseases and / or myopathies.

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

90. A nanoparticle 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, GI tract tumors, head and neck squamous cell carcinoma (HNSCC), renal cancer, myelofibrosis, CD206+ cancer, melanoma, prostate cancer or anal cancer.

91. A nanoparticle 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 wherein the immune-related disease is Gaucher disease, graft-versus-host disease, allogeneic transplant rejection, acute rejection after transplantation, chronic rejection after transplantation, primary graft dysfunction or chronic granulomatous disease (CGD).

92. Nanoparticles 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 hypertension, alveolar proteinosis, fibrotic lung disease, chronic lung disease or respiratory tract infection; or wherein the muscle disease is muscular dystrophy or muscular dystrophy.

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