Nanoparticles for delivery of nucleic acid cargo

Abstract

Nanoparticles suitable for delivery of linear DNA molecules are provided. Nanoparticles suitable for delivery of mRNA or DNA are provided. Further provided are uses of nanoparticles, including the use of nanoparticles to treat disease and the use of nanoparticles in vaccines.

Description

[Technical Field]

[0001] Nanoparticles suitable for delivery of linear DNA molecules are provided. Nanoparticles suitable for delivery of mRNA or DNA are provided. Uses of nanoparticles are further provided, including the use of nanoparticles to treat disease and the use of nanoparticles in vaccines. [Background technology]

[0002] DNA and mRNA vaccines use nucleic acid material that encodes the spike protein of a specific pathogen or a cancer antigen to generate an immune response.

[0003] Nucleic acid vaccines are significantly easier and more cost-effective to produce than conventional vaccine platforms, including attenuated or inactivated proteins, or viral vectors.

[0004] While the success of mRNA COVID vaccines from BioNTech and Moderna has demonstrated the safety and efficacy of mRNA vaccines, they come with limitations such as the requirement for cold chain logistics. DNA vaccines, including the ZyCoV-D vaccine approved in India, are more temperature stable than mRNA vaccines, but DNA vaccines present their own challenges: they require electroporation as a delivery method, which is difficult to deploy in a pandemic situation.

[0005] Alternatively, viruses as delivery agents have the advantages of high efficiency and high cell selectivity, but the disadvantages of toxicity, risk of insertional mutagenesis, generation of inflammatory responses, the potential to elicit a host immune response, or limited packaging capacity.

[0006] Non-viral gene delivery systems are based on the compression of genetic material into nanometer-sized particles through electrostatic interactions between the negatively charged phosphate backbone of nucleic acids and cationic lipids, peptides, or other polymers (Erbacher, P. et al., Gene Therapy, 1999, 6, 138-145).Known complexes for delivery include lipoplexes, which refer to lipid-based nucleic acid complexes, polyplexes, which refer to peptide- or polymer-based complexes, and lipopolyplexes, which refer to hybrid systems (Felgner et al., Human Gene Therapy 8, 1997, 511-512).

[0007] Non-viral lipid vector formulations that complex nucleic acids with cationic lipids have problems such as poor tissue penetration, non-specific charge-mediated cell binding, and interactions with serum proteins that can lead to inflammatory responses. The use of ionizable lipids as an alternative is attractive because they offer the potential for relatively low cytotoxicity and relatively weak interactions with serum components.

[0008] Known non-viral vectors can deliver DNA or RNA molecules to cells. In the context of DNA vaccines, plasmid DNA molecules have many drawbacks, including bacterial backbones, antibiotic resistance genes, and bacterial contaminants that can be toxic to cells or elicit unwanted immune responses. Summary of the Invention [Problem to be solved by the invention]

[0009] There is a need for improved delivery systems for nucleic acid vaccines and other applications. [Means for solving the problem]

[0010] The present invention relates to nanoparticles suitable for delivering cargo, particularly for use in DNA or RNA vaccines, for example, to deliver cargo intramuscularly. The present invention provides nanoparticles comprising: (a) cargo; (b) lipid components, the lipid components being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers. Preferably, the nanoparticles do not contain a targeting moiety. Preferably, the nanoparticles do not contain a targeting peptide. The nanoparticles may further comprise a PEG-lipid.

[0011] The cargo comprises a nucleic acid cargo such as DNA or RNA. Preferably, the cargo comprises a linear DNA product or mRNA that has enhanced resistance to nuclease digestion (e.g., exonuclease digestion).

[0012] The nanoparticles can be non-viral transfection complexes.

[0013] The present inventors have developed nanoparticles (e.g., non-viral transfection complexes) suitable for use in therapy (such as part of a vaccine). The nanoparticles of the present invention have several advantages over viral delivery systems. First, the nanoparticles of the present invention are less likely to provoke an immune response, which is particularly important since repeated doses may be required. In addition, the nanoparticles of the present invention can be used to deliver much larger cargoes (i.e., nucleic acids) with reduced lipid content due to the presence of cationic polymers.

[0014] The nanoparticles of the present invention offer the additional benefit of being cost-effective and simple to manufacture on a large scale.

[0015] The nanoparticles of the present invention comprise nucleic acid cargo (e.g., linear DNA molecules or closed linear DNA molecules) that have enhanced resistance to nuclease (e.g., exonuclease) digestion, thereby resulting in extended in vivo life of the cargo molecules, along with cationic polymers that aid in the stable loading of relatively large cargoes into the nanoparticles without interfering with the size, homogeneity, and efficacy of the nanoparticles (e.g., non-viral transfection complexes).

[0016] Importantly, the nanoparticles of the present invention allow efficient transfection of linear DNA molecules into cells and therefore have applications in therapy as vaccines.

[0017] Nanoparticles and non-viral transfection complexes The present invention provides nanoparticles comprising: (a) a cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid; (d) a steroid lipid; and (e) a cationic polymer. The nanoparticles may not comprise a targeting peptide or targeting moiety. Preferably, the cargo comprises a nucleic acid cargo such as linear DNA or RNA.

[0018] The lipid component may comprise an ionizable lipid and / or a cationic lipid. Preferably, the lipid component comprises DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LPO1, lipid 5 (ionizable lipid), DOTMA, DTDTMA, or DHDTMA (cationic lipid). The lipid component may be ALC-0315 and DOTMA. The lipid component may be DLin-MC3-DMA and DOTMA. The lipid component may be ALC-0315 and DLin-MC3-DMA. The nanoparticles of the present invention may comprise a combination of one or more ionizable lipids. The nanoparticles of the present invention may comprise one or more cationic lipids. The nanoparticles may comprise one or more ionizable lipids and one or more cationic lipids.

[0019] The phospholipid can include DOPE, DOPC, DSPC, DPPC, DMPC or POPC. Preferably, the phospholipid is DOPE. The phospholipid can be one or more of DOPE, DOPC, DSPC, DPPC, DMPC or POPC.

[0020] The nanoparticles comprise steroid lipids. The steroid lipids can comprise cholesterol or its derivatives (cholesterol derivatives), such as β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids. The steroid lipids can be one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids.

[0021] The nanoparticles of the present invention comprise a cationic polymer. The cationic polymer can comprise oligolysine (linear or branched), such as K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or a combination of oligolysine, oligohistidine, and oligoarginine, or PEI. Preferably, the cationic polymer is an oligolysine (K16, K17, or K30) containing 16, 17, or 30 lysine residues. The cationic polymer can consist of K16, K17, or K30.

[0022] The present invention provides the following embodiments:

[0023] 1. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0024] 2. The nanoparticles of the present invention are (a) a cargo comprising a linear deoxyribonucleic acid (DNA) molecule containing one or more nuclease-resistant nucleotides; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0025] 3. The nanoparticles of the present invention are (a) a cargo comprising a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0026] 4. The nanoparticles of the present invention are (a) cargo, the cargo comprising mRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0027] 5. The nanoparticles of the present invention are (a) a cargo, the cargo comprising an siRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0028] 6. The nanoparticles of the present invention are (a) a cargo comprising samRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0029] 7. The nanoparticles of the present invention are (a) a cargo comprising samRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0030] 8. The nanoparticles of the present invention are (a) a cargo comprising samRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0031] 9. The nanoparticles of the present invention are (a) cargo, the cargo comprising an miRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0032] 10. The nanoparticles of the present invention are (a) cargo, the cargo comprising snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0033] 11. The nanoparticles of the present invention are (a) a cargo, the cargo comprising a tRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0034] 12. The nanoparticles of the present invention are (a) a cargo comprising ribosomal RNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0035] 13. The nanoparticles of the present invention are (a) a cargo, the cargo comprising an antisense RNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0036] 14. The nanoparticles of the present invention are (a) cargo, the cargo comprising snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0037] 15. The nanoparticles of the present invention are (a) a cargo, the cargo comprising an shRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0038] 16. The nanoparticles of the present invention are (a) Cargo, the cargo comprising a gRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0039] In any of the above embodiments 1-16, the nanoparticles may be non-viral transfection complexes.

[0040] In any of embodiments 1-16, the phospholipid can include DOPE, DOPC, DSPC, DPPC, DMPC, or POPC. In any of embodiments 1-16, the steroid lipid can include cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (an alkyl steroid), a secosteroid (vitamin D2, D3), or a pentacyclic steroid. In any of embodiments 1-16, the cationic polymer includes oligolysine (linear or branched), such as K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI.

[0041] In any of embodiments 1-16, the nanoparticles can be free of a targeting moiety. In any of embodiments 1-16, the nanoparticles can be free of a targeting peptide.

[0042] In any of the embodiments 1 to 16, the phospholipid is preferably DOPE. Preferably, in the embodiments 1 to 16, the steroid lipid comprises cholesterol. Preferably, the above nanoparticles 1 to 16 comprise an oligolysine, for example, K16 or K30.

[0043] The nanoparticles can further comprise a PEG lipid. The PEG lipid can be a PEGylated phospholipid. The PEG lipid can be DMG-PEG.

[0044] The present invention further provides the following embodiments:

[0045] 17. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) steroid lipids; and (e) Cationic polymer may include:

[0046] 18. The nanoparticles of the present invention are (a) a cargo comprising a linear deoxyribonucleic acid (DNA) molecule containing one or more nuclease-resistant nucleotides; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) steroid lipids; and (e) Cationic polymer may include:

[0047] 19. Nanoparticles are (a) a cargo comprising a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) steroid lipids; and (e) Cationic polymer may include:

[0048] 20. The nanoparticles of the present invention are (a) cargo, the cargo comprising mRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) steroid lipids; and (e) Cationic polymer may include:

[0049] 21. The nanoparticles of the present invention are (a) a cargo, the cargo comprising an siRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) steroid lipids; and (e) Cationic polymer may include:

[0050] 24. The nanoparticles of the present invention are (a) a cargo comprising samRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) steroid lipids; and (e) Cationic polymer may include:

[0051] 25. The nanoparticles of the present invention are (a) cargo, the cargo comprising an miRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) steroid lipids; and (e) Cationic polymer may include:

[0052] 26. The nanoparticles of the present invention are (a) cargo, the cargo comprising snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) steroid lipids; and (e) Cationic polymer may include:

[0053] 27. The nanoparticles of the present invention are (a) a cargo, the cargo comprising a tRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) steroid lipids; and (e) Cationic polymer may include:

[0054] 28. The nanoparticles of the present invention are (a) a cargo comprising ribosomal RNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) steroid lipids; and (e) Cationic polymer may include:

[0055] 29. The nanoparticles of the present invention are (a) a cargo, the cargo comprising an antisense RNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) steroid lipids; and (e) Cationic polymer may include:

[0056] 30. The nanoparticles of the present invention are (a) cargo, the cargo comprising snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) steroid lipids; and (e) Cationic polymer may include:

[0057] 31. The nanoparticles of the present invention are (a) a cargo, the cargo comprising an shRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid comprising DOPE, DOPC, DSPC, DPPC, DMPC, POPC, or SM; (d) steroid lipids; and (e) Cationic polymer may include:

[0058] 32. The nanoparticles of the present invention are (a) Cargo, the cargo comprising a gRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) steroid lipids; and (e) Cationic polymer may include:

[0059] In any of embodiments 17-32, the lipid component can include DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA. In any of embodiments 2-32, the steroid lipid can include cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (an alkyl steroid), secosteroid (vitamin D2, D3), or pentacyclic steroid. In any of embodiments 1-16, the cationic polymer comprises oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI.

[0060] In any of embodiments 7-32, the nanoparticles can be free of a targeting moiety. In any of embodiments 2-32, the nanoparticles can be free of a targeting peptide.

[0061] In any of embodiments 7-32, the lipid component preferably comprises at least one ionizable lipid.

[0062] Preferably, in embodiments 7 to 32, the nanoparticles contain cholesterol. Preferably, in embodiments 7 to 32, the nanoparticles contain oligolysine, for example, K16 or K30.

[0063] The nanoparticles can further comprise a PEG lipid. The PEG lipid can be a PEGylated phospholipid. The PEG lipid can be DMG-PEG.

[0064] The present invention further provides the following embodiments:

[0065] 33. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0066] 34. The nanoparticles of the present invention are (a) a cargo comprising a linear deoxyribonucleic acid (DNA) molecule containing one or more nuclease-resistant nucleotides; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0067] 35. The nanoparticles of the present invention are (a) a cargo comprising a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids, including one or more of β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3), or pentacyclic steroids; and (e) Cationic polymer may include:

[0068] 36. The nanoparticles of the present invention are (a) cargo, the cargo comprising mRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0069] 37. The nanoparticles of the present invention are (a) a cargo, the cargo comprising an siRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0070] 38. The nanoparticles of the present invention are (a) a cargo comprising samRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0071] 39. The nanoparticles of the present invention are (a) cargo, the cargo comprising an miRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0072] 40. The nanoparticles of the present invention are (a) cargo, the cargo comprising snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0073] 41. The nanoparticles of the present invention are (a) a cargo, the cargo comprising a tRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids, including one or more of β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3), or pentacyclic steroids; and (e) Cationic polymer may include:

[0074] 42. The nanoparticles of the present invention are (a) a cargo comprising ribosomal RNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0075] 43. The nanoparticles of the present invention are (a) a cargo, the cargo comprising an antisense RNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0076] 44. The nanoparticles of the present invention are (a) cargo, the cargo comprising snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0077] 45. The nanoparticles of the present invention are (a) a cargo, the cargo comprising an shRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0078] 46. ​​The nanoparticles of the present invention are (a) Cargo, the cargo comprising a gRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0079] In any of the above embodiments 33-46, the nanoparticles may be non-viral transfection complexes.

[0080] In any of embodiments 33-46, the phospholipid can include DOPE, DOPC, DSPC, DPPC, DMPC, or POPC. In any of embodiments 33-46, the lipid component can include DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA. In any of embodiments 33-46, the cationic polymer can include oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI.

[0081] In any of embodiments 33-46, the nanoparticles can be free of a targeting moiety.In any of embodiments 33-46, the nanoparticles can be free of a targeting peptide.

[0082] In any of embodiments 33-46, the lipid preferably comprises at least one ionizable lipid. In embodiments 33-46, the phospholipid is preferably DOPE. In embodiments 33-46, the cationic polymer preferably comprises an oligolysine, such as K16 or K30.

[0083] The nanoparticles can further comprise a PEG lipid. The PEG lipid can be a PEGylated phospholipid. The PEG lipid can be DMG-PEG.

[0084] The present invention further provides the following embodiments:

[0085] 47. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0086] 48. Nanoparticles are (a) a cargo comprising a linear deoxyribonucleic acid (DNA) molecule containing one or more nuclease-resistant nucleotides; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0087] 49. The nanoparticles of the present invention are (a) a cargo comprising a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0088] 50. The nanoparticles of the present invention are (a) cargo, the cargo comprising mRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0089] 51. The nanoparticles of the present invention are (a) a cargo, the cargo comprising an siRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0090] 52. The nanoparticles of the present invention are (a) a cargo comprising samRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0091] 53. The nanoparticles of the present invention are (a) cargo, the cargo comprising an miRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0092] 54. The nanoparticles of the present invention are (a) cargo, the cargo comprising snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0093] 55. The nanoparticles of the present invention are (a) a cargo, the cargo comprising a tRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0094] 56. The nanoparticles of the present invention are (a) a cargo comprising ribosomal RNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0095] 57. The nanoparticles of the present invention are (a) a cargo, the cargo comprising an antisense RNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0096] 58. The nanoparticles of the present invention are (a) cargo, the cargo comprising snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0097] 59. The nanoparticles of the present invention are (a) a cargo, the cargo comprising an shRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0098] 60. The nanoparticles of the present invention are (a) Cargo, the cargo comprising a gRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0099] In any of the above embodiments 47-60, the nanoparticles may be non-viral transfection complexes.

[0100] In any of embodiments 47-60, the phospholipid can include DOPE, DOPC, DSPC, DPPC, DMPC, or POPC. In any of embodiments 47-60, the lipid component can include DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LPO1, lipid 5, DOTMA, DTDTMA, or DHDTMA. In any of embodiments 47-60, the steroid lipid can include cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (an alkyl steroid), secosteroid (vitamin D2, D3), or pentacyclic steroid.

[0101] In any of embodiments 47-60, the nanoparticles can be free of a targeting moiety. In any of embodiments 47-60, the nanoparticles can be free of a targeting peptide.

[0102] In any of embodiments 47-60, the lipid preferably comprises at least one ionizable lipid. In embodiments 47-60, the phospholipid preferably is DOPE. In embodiments 47-60, the cationic polymer preferably comprises an oligolysine, such as K16 or K30.

[0103] Preferably, the cationic polymer is a nucleic acid-binding cationic polymer (e.g., a DNA-binding cationic polymer, a closed linear DNA-binding cationic polymer, a partially closed linear DNA-binding cationic polymer or a linear DNA-binding cationic polymer, or an RNA, e.g., mRNA, cationic binding polymer).

[0104] The cationic polymer can comprise at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 32, at least 34, at least 36, at least 38, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 cationic monomers. Preferably, the nucleic acid-binding cationic polymer comprises at least 16, at least 17 or at least 30 cationic monomers.

[0105] The cationic polymer can include lysine, histidine, or arginine. The cationic polymer can include lysine, histidine, or arginine. The cationic polymer can include oligolysine (linear or branched), oligohistidine (linear or branched), or oligoarginine (linear or branched). For example, the cationic polymer can comprise at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 32, at least 34, at least 36, at least 38, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 lysine residues. Preferably, the cationic polymer contains at least 16, at least 17, or at least 30 lysine residues. Also more preferably, the nucleic acid-binding polycationic component contains at least 16 lysine residues. The polycationic polymer can consist of 16 lysine residues or 30 lysine residues.

[0106] An alternative cationic polymer is linear or branched polyethyleneimine (PEI) of MW 500-10,000.

[0107] The nanoparticles can further comprise a PEG lipid. The PEG lipid can be a PEGylated phospholipid. The PEG lipid can be DMG-PEG.

[0108] The present invention further provides the following embodiments:

[0109] 61. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising DLin-MC3-DMA; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0110] 62. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising DLin-KC2-DMA; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0111] 63. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising DLin-DMA; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0112] 64. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising TCL053; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0113] 65. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, and comprising SM-102; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0114] 66. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising ALC-0315; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0115] 67. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising C12-200; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0116] 68. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising DODMA; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0117] 69. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising DODAP; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0118] 70. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising lipid A9; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0119] 71. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising 9A1P9; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0120] 72. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising lipid C24; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0121] 73. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising lipid LPO1; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0122] 74. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising lipid 5; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0123] 75. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising DOTMA; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0124] 76. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising DTDTMA; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0125] 77. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising DHDTMA; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer may include:

[0126] In any of the above embodiments 61-77, the nanoparticles may be non-viral transfection complexes.

[0127] In any of embodiments 61-77, the phospholipid can include DOPE, DOPC, DSPC, DPPC, DMPC, or POPC. In any of embodiments 61-77, the steroid lipid can include cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (an alkyl steroid), a secosteroid (vitamin D2, D3), or a pentacyclic steroid. In any of embodiments 61-77, the cationic polymer can include oligolysine (linear or branched), such as K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI.

[0128] In any of embodiments 61-77, the nanoparticles can be free of a targeting moiety. In any of embodiments 61-77, the nanoparticles can be free of a targeting peptide.

[0129] In embodiments 61-77, the cargo preferably comprises linear DNA (e.g., closed linear DNA, linear DNA comprising one or more nuclease-resistant nucleotides at a first end and closed at a second end, or linear DNA comprising one or more nuclease-resistant nucleotides at a first end and a second end) or mRNA. In embodiments 61-77, the phospholipid is preferably DOPE. In embodiments 61-77, the steroid lipid preferably comprises cholesterol. In embodiments 61-77, the cationic polymer is preferably an oligolysine, e.g., K16 or K30.

[0130] The nanoparticles can further comprise a PEG lipid. The PEG lipid can be a PEGylated phospholipid. The PEG lipid can be DMG-PEG.

[0131] 78. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising DLin-MC3-DMA; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) steroid lipids; and (e) Cationic polymer may include:

[0132] 79. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising DLin-KC2-DMA; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) steroid lipids; and (e) Cationic polymer may include:

[0133] 80. The nanoparticles of the present invention are a) Cargo; b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising DLin-DMA; c) a phospholipid comprising DOPE, DOPC, DSPC, DPPC, DMPC or POPC; d) steroid lipids; and e) Cationic polymer may include:

[0134] 81. The nanoparticles of the present invention are a) Cargo; b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, and comprising TCL053; c) a phospholipid comprising DOPE, DOPC, DSPC, DPPC, DMPC or POPC; d) steroid lipids; and e) Cationic polymer may include:

[0135] 82. The nanoparticles of the present invention are a) Cargo; b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, and comprising SM-102; c) a phospholipid comprising DOPE, DOPC, DSPC, DPPC, DMPC or POPC; d) steroid lipids; and e) Cationic polymer may include:

[0136] 83. The nanoparticles of the present invention are a) Cargo; b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, and comprising ALC-0315; c) a phospholipid comprising DOPE, DOPC, DSPC, DPPC, DMPC or POPC; d) steroid lipids; and e) Cationic polymer may include:

[0137] 84. The nanoparticles of the present invention are a) Cargo; b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising C12-200; c) a phospholipid comprising DOPE, DOPC, DSPC, DPPC, DMPC or POPC; d) steroid lipids; and e) Cationic polymer may include:

[0138] 85. The nanoparticles of the present invention are a) Cargo; b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DODMA; c) a phospholipid comprising DOPE, DOPC, DSPC, DPPC, DMPC or POPC; d) steroid lipids; and e) Cationic polymer may include:

[0139] 86. The nanoparticles of the present invention are a) Cargo; b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DODAP; c) a phospholipid comprising DOPE, DOPC, DSPC, DPPC, DMPC or POPC; d) steroid lipids; and e) Cationic polymer may include:

[0140] 87. The nanoparticles of the present invention are a) Cargo; b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising lipid A9; c) a phospholipid comprising DOPE, DOPC, DSPC, DPPC, DMPC or POPC; d) steroid lipids; and e) Cationic polymer may include:

[0141] 88. The nanoparticles of the present invention are a) Cargo; b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising 9A1P9; c) a phospholipid comprising DOPE, DOPC, DSPC, DPPC, DMPC or POPC; d) steroid lipids; and e) Cationic polymer may include:

[0142] 89. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising lipid C24; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) steroid lipids; and (e) Cationic polymer may include:

[0143] 90. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising lipid LPO1; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) steroid lipids; and (e) Cationic polymer may include:

[0144] 91. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising lipid 5; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) steroid lipids; and (e) Cationic polymer may include:

[0145] 92. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising DOTMA; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) steroid lipids; and (e) Cationic polymer may include:

[0146] 93. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising DTDTMA; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) steroid lipids; and (e) Cationic polymer may include:

[0147] 94. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising DHDTMA; (c) a phospholipid comprising one or more of DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) steroid lipids; and (e) Cationic polymer may include:

[0148] In any of embodiments 78 to 94, the nanoparticles may be non-viral transfection complexes.

[0149] In any of embodiments 78-94, the cargo can comprise a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, mRNA, siRNA, samRNA, miRNA, gRNA, shRNA, antisense RNA, ribosomal RNA, tRNA, or snRNA. In any of embodiments 1-16, the steroid lipid can comprise cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (an alkyl steroid), a secosteroid (vitamin D2, D3), or a pentacyclic steroid. In any of embodiments 78-94, the cationic polymer can comprise oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI.

[0150] In any of embodiments 78-94, the nanoparticles can be free of a targeting moiety.In any of embodiments 78-94, the nanoparticles can be free of a targeting peptide.

[0151] In any of embodiments 78-94, the cargo preferably comprises linear DNA or mRNA. In any of embodiments 78-94, the steroid lipid preferably comprises cholesterol. In any of embodiments 78-94, the cationic polymer preferably is oligolysine.

[0152] The nanoparticles can further comprise a PEG lipid. The PEG lipid can be a PEGylated phospholipid. The PEG lipid can be DMG-PEG.

[0153] The present invention further provides the following embodiments:

[0154] 95. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising DLin-MC3-DMA; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0155] 96. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising DLin-KC2-DMA; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0156] 97. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising DLin-DMA; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0157] 98. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising TCL053; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0158] 99. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, and comprising SM-102; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0159] 100. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising ALC-0315; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0160] 101. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising C12-200; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0161] 102. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising DODMA; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0162] 103. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising DODAP; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0163] 104. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising lipid A9; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0164] 105. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising 9A1P9; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0165] 106. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising lipid C24; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0166] 107. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising lipid LPO1; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0167] 108. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising lipid 5; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0168] 109. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising DOTMA; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0169] 110. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising DTDTMA; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0170] 111. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising DHDTMA; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0171] In any of the above embodiments 95-111, the nanoparticles may be non-viral transfection complexes.

[0172] In any of embodiments 95-111, the phospholipid can comprise DOPE, DOPC, DSPC, DPPC, DMPC, or POPC. In any of embodiments 95-111, the cargo can comprise a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, mRNA, siRNA, samRNA, miRNA, gRNA, shRNA, antisense RNA, ribosomal RNA, tRNA, or snRNA. In any of embodiments 95-111, the cationic polymer comprises oligolysine (linear or branched), such as K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI.

[0173] In any of embodiments 95-111, the nanoparticles may not comprise a targeting moiety.In any of embodiments 95-111, the nanoparticles may not comprise a targeting peptide.

[0174] In any of embodiments 95-111, the cargo preferably comprises linear DNA or mRNA. In any of embodiments 95-111, the phospholipid is preferably DOPE. In any of embodiments 95-111, the cationic polymer is an oligolysine, such as K16 or K30.

[0175] The nanoparticles can further comprise a PEG lipid. The PEG lipid can be a PEGylated phospholipid. The PEG lipid can be DMG-PEG.

[0176] The present invention further provides the following embodiments:

[0177] 112. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising DLin-MC3-DMA; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0178] 113. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising DLin-KC2-DMA; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0179] 114. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising DLin-DMA; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0180] 115. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising TCL053; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0181] 116. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, and comprising SM-102; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0182] 117. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising ALC-0315; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0183] 118. Nanoparticles are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising C12-200; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0184] 119. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising DODMA; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0185] 120. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising DODAP; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0186] 121. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising lipid A9; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0187] 122. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising 9A1P9; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0188] 123. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising lipid C24; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0189] 124. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising lipid LPO1; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0190] 125. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising lipid 5; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0191] 126. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising DOTMA; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0192] 127. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising DTDTMA; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0193] 128. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, the lipid component comprising DHDTMA; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0194] In any of the above embodiments 112-128, the nanoparticles may be non-viral transfection complexes.

[0195] In any of embodiments 112-128, the phospholipid can include DOPE, DOPC, DSPC, DPPC, DMPC, or POPC. In any of embodiments 112-128, the steroid lipid can include cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (an alkyl steroid), a secosteroid (vitamin D2, D3), or a pentacyclic steroid. In any of embodiments 112-128, the cargo can comprise a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, wherein the partially closed linear DNA molecule comprises one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, mRNA, siRNA, samRNA, miRNA, gRNA, shRNA, antisense RNA, ribosomal RNA, tRNA, or snRNA.

[0196] In any of embodiments 112-128, the nanoparticles can be free of a targeting moiety.In any of embodiments 112-128, the nanoparticles can be free of a targeting peptide.

[0197] In embodiments 112-128, the phospholipid is preferably DOPE. In embodiments 112-128, the steroid is preferably cholesterol. In embodiments 112-128, the cargo preferably comprises linear DNA or mRNA.

[0198] The nanoparticles can further comprise a PEG lipid. The PEG lipid can be a PEGylated phospholipid. The PEG lipid can be DMG-PEG.

[0199] The present invention further provides the following embodiments:

[0200] 129. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid that is a phosphatidylethanolamine; (d) steroid lipids; and (e) Cationic polymer may include:

[0201] 130. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, the phospholipid being 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); (d) steroid lipids; and (e) Cationic polymer may include:

[0202] 131. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid that is a phosphatidylcholine; (d) steroid lipids; and (e) Cationic polymer may include:

[0203] 132. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid that is 1,2-dioleoyl-sn-glycero-3-phosphoethanoltrimethylamine (DOPC); (d) steroid lipids; and (e) Cationic polymer may include:

[0204] 133. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, the phospholipid being DSPC; (d) steroid lipids; and (e) Cationic polymer may include:

[0205] 134. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, the phospholipid being DPPC; (d) steroid lipids; and (e) Cationic polymer may include:

[0206] 135. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, the phospholipid being DMPC; (d) steroid lipids; and (e) Cationic polymer may include:

[0207] 136. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid that is a POPC; (d) steroid lipids; and (e) Cationic polymer may include:

[0208] 137. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid that is an SM; (d) steroid lipids; and (e) Cationic polymer may include:

[0209] In any of the above embodiments 129-137, the nanoparticles may be non-viral transfection complexes.

[0210] In any of embodiments 129-137, the lipid component can include DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA. In any of embodiments 129-137, the steroid lipid can include cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (an alkyl steroid), a secosteroid (vitamin D2, D3), or a pentacyclic steroid. In any of embodiments 129-137, the cationic polymer comprises oligolysine (linear or branched), such as K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI.

[0211] In any of embodiments 129-137, the nanoparticles can be free of a targeting moiety. In any of embodiments 129-137, the nanoparticles can be free of a targeting peptide.

[0212] In embodiments 129-137, the lipid preferably comprises at least one ionizable lipid. In embodiments 129-137, the steroid lipid preferably comprises cholesterol. In embodiments 129-137, the cationic polymer is preferably an oligolysine, such as K16 or K30.

[0213] The nanoparticles can further comprise a PEG lipid. The PEG lipid can be a PEGylated phospholipid. The PEG lipid can be DMG-PEG.

[0214] 138. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) a phospholipid that is a phosphatidylethanolamine; (d) steroid lipids; and (e) Cationic polymer may include:

[0215] 139. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) a phospholipid, the phospholipid being 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); (d) steroid lipids; and (e) Cationic polymer may include:

[0216] 140. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) a phospholipid that is a phosphatidylcholine; (d) steroid lipids; and (e) Cationic polymer may include:

[0217] 141. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) a phospholipid that is 1,2-dioleoyl-sn-glycero-3-phosphoethanoltrimethylamine (DOPC); (d) steroid lipids; and (e) Cationic polymer may include:

[0218] 142. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) a phospholipid, the phospholipid being DSPC; (d) steroid lipids; and (e) Cationic polymer may include:

[0219] 143. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) a phospholipid, the phospholipid being DPPC; (d) steroid lipids; and (e) Cationic polymer may include:

[0220] 144. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) a phospholipid, the phospholipid being DMPC; (d) steroid lipids; and (e) Cationic polymer may include:

[0221] 145. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) a phospholipid that is a POPC; (d) steroid lipids; and (e) Cationic polymer may include:

[0222] 146. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) a phospholipid that is an SM; (d) steroid lipids; and (e) Cationic polymer may include:

[0223] In any of the above embodiments 138-146, the nanoparticles may be non-viral transfection complexes.

[0224] In any of embodiments 138-146, the cargo can comprise a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, mRNA, siRNA, samRNA, miRNA, gRNA, shRNA, antisense RNA, ribosomal RNA, tRNA, or snRNA. In any of embodiments 138-146, the steroid lipid can comprise cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (an alkyl steroid), a secosteroid (vitamin D2, D3), or a pentacyclic steroid. In any of embodiments 138-146, the cationic polymer comprises oligolysine (linear or branched), such as K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI.

[0225] In any of embodiments 138-146, the nanoparticles can be free of a targeting moiety. In any of embodiments 138-146, the nanoparticles can be free of a targeting peptide.

[0226] In embodiments 138-146, the cargo preferably comprises linear DNA or mRNA. In embodiments 138-146, the steroid lipid preferably comprises cholesterol. In embodiments 138-146, the cationic polymer is preferably an oligolysine, such as K16 or K30.

[0227] The nanoparticles can further comprise a PEG lipid. The PEG lipid can be a PEGylated phospholipid. The PEG lipid can be DMG-PEG.

[0228] 147. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid that is a phosphatidylethanolamine; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0229] 148. The nanoparticles of the present invention are (a) Cargo; (b) lipid components that are one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, the phospholipid being 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0230] 149. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid that is a phosphatidylcholine; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0231] 150. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid that is 1,2-dioleoyl-sn-glycero-3-phosphoethanoltrimethylamine (DOPC); (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0232] 151. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, the phospholipid being DSPC; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0233] 152. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, the phospholipid being DPPC; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0234] 153. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, the phospholipid being DMPC; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0235] 154. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid that is a POPC; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0236] 155. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid that is an SM; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) Cationic polymer may include:

[0237] In any of embodiments 147 to 155, the nanoparticles may be non-viral transfection complexes.

[0238] In any of embodiments 147-155, the cargo can comprise a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, wherein the partially closed linear DNA molecule comprises one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, mRNA, siRNA, samRNA, miRNA, gRNA, shRNA, antisense RNA, ribosomal RNA, tRNA, or snRNA. In any of embodiments 147-155, the lipid component can be DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA. In any of embodiments 1-16, the cationic polymer comprises oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI.

[0239] In any of embodiments 147-155, the nanoparticles can be free of a targeting moiety.In any of embodiments 147-155, the nanoparticles can be free of a targeting peptide.

[0240] In embodiments 147-155, the cargo preferably comprises linear DNA or mRNA. In embodiments 147-155, the lipid preferably comprises at least one ionizable lipid. In embodiments 147-155, the cationic polymer is preferably an oligolysine, such as K16 or K30.

[0241] The nanoparticles can further comprise a PEG lipid. The PEG lipid can be a PEGylated phospholipid. The PEG lipid can be DMG-PEG.

[0242] 156. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid that is a phosphatidylethanolamine; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0243] 157. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, the phospholipid being 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0244] 158. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid that is a phosphatidylcholine; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0245] 159. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid that is 1,2-dioleoyl-sn-glycero-3-phosphoethanoltrimethylamine (DOPC); (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0246] 160. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, the phospholipid being DSPC; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0247] 161. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, the phospholipid being DPPC; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0248] 162. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, the phospholipid being DMPC; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0249] 163. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid that is a POPC; (d) steroid lipids; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0250] In any of embodiments 156 to 163, the nanoparticles may be non-viral transfection complexes.

[0251] In any of embodiments 156-163, the cargo is a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, mRNA, siRNA, samRNA, miRNA, gRNA, shRNA, antisense RNA, ribosomal RNA, tRNA, or snRNA. In any of embodiments 156-163, the steroid lipid can comprise cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (an alkyl steroid), a secosteroid (vitamin D2, D3), or a pentacyclic steroid. In any of embodiments 156-163, the lipid component can include DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA.

[0252] In any of embodiments 156-163, the nanoparticles can be free of a targeting moiety. In any of embodiments 156-163, the nanoparticles can be free of a targeting peptide.

[0253] In embodiments 156-163, the cargo preferably comprises linear DNA or mRNA. In embodiments 156-163, the lipid preferably comprises at least one ionizable lipid. In embodiments 156-163, the steroid lipid preferably comprises cholesterol.

[0254] The nanoparticles can further comprise a PEG lipid. The PEG lipid can be a PEGylated phospholipid. The PEG lipid can be DMG-PEG.

[0255] 164. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) a steroid lipid comprising one or more β-sitosterols; and (e) Cationic polymer may include:

[0256] 165. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) a steroid lipid, the steroid lipid comprising one or more of fucosterol; and (e) Cationic polymer may include:

[0257] 166. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) a steroid lipid, the steroid lipid comprising one or more of campesterol; and (e) Cationic polymer may include:

[0258] 167. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) a steroid lipid, the steroid lipid comprising one or more of stigmastanol; and (e) Cationic polymer may include:

[0259] 168. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, and comprising SM-102; (c) phospholipids; (d) a steroid lipid, the steroid lipid comprising one or more of: cholesterol; and (e) Cationic polymer may include:

[0260] In any of embodiments 164 to 168, the nanoparticles may be non-viral transfection complexes.

[0261] In any of embodiments 164-168, the phospholipid can include DOPE, DOPC, DSPC, DPPC, DMPC, or POPC. In any of embodiments 164-168, the steroid lipid can include cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (an alkyl steroid), a secosteroid (vitamin D2, D3), or a pentacyclic steroid. In any of embodiments 164-168, the cationic polymer can include oligolysine (linear or branched), such as K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI.

[0262] In any of embodiments 164-168, the nanoparticles can be free of a targeting moiety.In any of embodiments 164-168, the nanoparticles can be free of a targeting peptide.

[0263] In embodiments 164-168, the cargo preferably comprises linear DNA or mRNA. In embodiments 164-168, the lipid preferably comprises at least one ionizable lipid. In embodiments 164-168, the steroid lipid preferably comprises cholesterol.

[0264] The nanoparticles can further comprise a PEG lipid. The PEG lipid can be a PEGylated phospholipid. The PEG lipid can be DMG-PEG.

[0265] 169. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) phospholipids; (d) a steroid lipid comprising one or more β-sitosterols; and (e) Cationic polymer may include:

[0266] 170. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) phospholipids; (d) a steroid lipid, the steroid lipid comprising one or more of fucosterol; and (e) Cationic polymer may include:

[0267] 171. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) phospholipids; (d) a steroid lipid, the steroid lipid comprising one or more of campesterol; and (e) Cationic polymer may include:

[0268] 172. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) phospholipids; (d) a steroid lipid, the steroid lipid comprising one or more of stigmastanol; and (e) Cationic polymer may include:

[0269] 173. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) phospholipids; (d) a steroid lipid, the steroid lipid comprising one or more of: cholesterol; and (e) Cationic polymer may include:

[0270] In any of embodiments 169 to 173, the nanoparticles may be non-viral transfection complexes.

[0271] In any of embodiments 169-173, the phospholipid can comprise DOPE, DOPC, DSPC, DPPC, DMPC or POPC. In any of embodiments 169-173, the cargo can comprise a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, mRNA, siRNA, samRNA, miRNA, gRNA, shRNA, antisense RNA, ribosomal RNA, tRNA or snRNA. In any of embodiments 169-173, the cationic polymer can comprise oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI.

[0272] In any of embodiments 169-173, the nanoparticles can be free of a targeting moiety. In any of embodiments 169-173, the nanoparticles can be free of a targeting peptide.

[0273] In embodiments 169 to 173, the cargo preferably comprises linear DNA or mRNA. In embodiments 169 to 173, the phospholipid is preferably DOPE. In embodiments 169 to 173, the cationic polymer is preferably an oligolysine, such as K16 or K30.

[0274] The nanoparticles can further comprise a PEG lipid. The PEG lipid can be a PEGylated phospholipid. The PEG lipid can be DMG-PEG.

[0275] 174. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) a steroid lipid comprising one or more β-sitosterols; and (e) Cationic polymer may include:

[0276] 175. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) a steroid lipid, the steroid lipid comprising one or more of fucosterol; and (e) Cationic polymer may include:

[0277] 176. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) a steroid lipid, the steroid lipid comprising one or more of campesterol; and (e) Cationic polymer may include:

[0278] 177. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) a steroid lipid, the steroid lipid comprising one or more of stigmastanol; and (e) Cationic polymer may include:

[0279] 178. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) a steroid lipid, the steroid lipid comprising one or more of: cholesterol; and (e) Cationic polymer may include:

[0280] In any of the above embodiments 174-178, the nanoparticles may be non-viral transfection complexes.

[0281] In any of embodiments 174-178, the lipid component can include DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA. In any of embodiments 174-178, the cargo can comprise a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, wherein the partially closed linear DNA molecule comprises one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, mRNA, siRNA, samRNA, miRNA, gRNA, shRNA, antisense RNA, ribosomal RNA, tRNA, or snRNA. In any of embodiments 174-178, the cationic polymer comprises oligolysine (linear or branched), such as K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI.

[0282] In any of embodiments 174-178, the nanoparticles can be free of a targeting moiety.In any of embodiments 174-178, the nanoparticles can be free of a targeting peptide.

[0283] In embodiments 174-178, the cargo preferably comprises linear DNA or mRNA. In embodiments 174-178, the lipid preferably comprises at least one ionizable lipid. In embodiments 174-178, the cationic polymer preferably is an oligolysine, such as K16 or K30.

[0284] The nanoparticles can further comprise a PEG lipid. The PEG lipid can be a PEGylated phospholipid. The PEG lipid can be DMG-PEG.

[0285] 179. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) a steroid lipid comprising one or more β-sitosterols; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0286] 180. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) a steroid lipid, the steroid lipid comprising one or more of fucosterol; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0287] 181. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) a steroid lipid, the steroid lipid comprising one or more of campesterol; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0288] 182. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) a steroid lipid, the steroid lipid comprising one or more of stigmastanol; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0289] 183. Nanoparticles are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) a steroid lipid, the steroid lipid comprising one or more of: cholesterol; and (e) cationic polymers comprising oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI may include:

[0290] In any of the above embodiments 179-183, the nanoparticles may be non-viral transfection complexes.

[0291] In any of embodiments 179-183, the phospholipid can comprise DOPE, DOPC, DSPC, DPPC, DMPC or POPC. In any of embodiments 179-183, the cargo can comprise a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, mRNA, siRNA, samRNA, miRNA, gRNA, shRNA, antisense RNA, ribosomal RNA, tRNA or snRNA. In any of embodiments 179-183, the lipid component can include DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA.

[0292] In any of embodiments 179-183, the nanoparticles may not comprise a targeting moiety. In any of embodiments 179-183, the nanoparticles may not comprise a targeting peptide.

[0293] In embodiments 179 to 183, the cargo preferably comprises linear DNA or mRNA. In embodiments 179 to 183, the lipid preferably comprises at least one ionizable lipid. In embodiments 179 to 183, the phospholipid is preferably DOPE.

[0294] The nanoparticles can further comprise a PEG lipid. The PEG lipid can be a PEGylated phospholipid. The PEG lipid can be DMG-PEG.

[0295] 184. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers, including oligolysines (linear or branched), such as K16, K17, or K30; may include:

[0296] 185. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids; and (e) a cationic polymer containing oligohistidine (linear or branched chain); may include:

[0297] 186. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids; and (e) a cationic polymer containing oligoarginine (linear or branched chain) may include:

[0298] 187. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids; and (e) a cationic polymer containing oligolysine and oligohistidine may include:

[0299] 188. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids; and (e) a cationic polymer comprising oligohistidine and oligoarginine; may include:

[0300] 189. Nanoparticles are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids; and (e) a cationic polymer comprising oligoarginine and oligolysine; may include:

[0301] 190. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers, including oligolysines, oligohistidines, and oligoarginines; may include:

[0302] 191. The nanoparticles of the present invention are (a) a cargo comprising a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, an mRNA, an siRNA, an samRNA, an miRNA, a gRNA, an shRNA, an antisense RNA, a ribosomal RNA, an tRNA, or an snRNA; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids; and (e) a cationic polymer, the cationic polymer including PEI may include:

[0303] In any of the above embodiments 184-191, the nanoparticles may be non-viral transfection complexes.

[0304] In any of embodiments 184-191, the phospholipid can include DOPE, DOPC, DSPC, DPPC, DMPC, or POPC. In any of embodiments 184-191, the steroid lipid can include cholesterol, β-sitosterol, fucosterol, sterl3, or stigmastanol. In any of embodiments 184-191, the lipid component can include DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LPO1, lipid 5, DOTMA, DTDTMA, or DHDTMA.

[0305] In any of embodiments 184-191, the nanoparticles may not comprise a targeting moiety.In any of embodiments 184-191, the nanoparticles may not comprise a targeting peptide.

[0306] In embodiments 184-191, the lipid component preferably comprises at least one ionizable lipid. In embodiments 184-191, the phospholipid is preferably DOPE. In embodiments 184-191, the steroid lipid comprises cholesterol.

[0307] The nanoparticles can further comprise a PEG lipid. The PEG lipid can be a PEGylated phospholipid. The PEG lipid can be DMG-PEG.

[0308] 192. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers, including oligolysines (linear or branched), such as K16, K17, or K30; may include:

[0309] 193. Nanoparticles are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) phospholipids; (d) steroid lipids; and (e) a cationic polymer containing oligohistidine (linear or branched chain); may include:

[0310] 194. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) phospholipids; (d) steroid lipids; and (e) a cationic polymer containing oligoarginine (linear or branched chain) may include:

[0311] 195. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) phospholipids; (d) steroid lipids; and (e) a cationic polymer containing oligolysine and oligohistidine may include:

[0312] 196. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) phospholipids; (d) steroid lipids; and (e) a cationic polymer comprising oligohistidine and oligoarginine; may include:

[0313] 197. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) phospholipids; (d) steroid lipids; and (e) a cationic polymer comprising oligoarginine and oligolysine; may include:

[0314] 198. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) phospholipids; (d) steroid lipids; and (e) cationic polymers, including oligolysines, oligohistidines, and oligoarginines; may include:

[0315] 199. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids, comprising DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA; (c) phospholipids; (d) steroid lipids; and (e) a cationic polymer, the cationic polymer including PEI may include:

[0316] In any of embodiments 192 to 199, the nanoparticles may be non-viral transfection complexes.

[0317] In any of embodiments 192-199, the phospholipid can comprise DOPE, DOPC, DSPC, DPPC, DMPC, or POPC. In any of embodiments 192-199, the steroid lipid can comprise cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (an alkyl steroid), a secosteroid (vitamin D2, D3), or a pentacyclic steroid. In any of embodiments 192-199, the cargo can comprise a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, wherein the partially closed linear DNA molecule comprises one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, mRNA, siRNA, samRNA, miRNA, gRNA, shRNA, antisense RNA, ribosomal RNA, tRNA, or snRNA.

[0318] In any of embodiments 192-199, the nanoparticles can be free of a targeting moiety.In any of embodiments 192-199, the nanoparticles can be free of a targeting peptide.

[0319] In embodiments 192 to 199, the cargo preferably comprises linear DNA or mRNA. In embodiments 192 to 199, the phospholipid is preferably DOPE. In embodiments 192 to 199, the steroid lipid comprises cholesterol.

[0320] The nanoparticles can further comprise a PEG lipid. The PEG lipid can be a PEGylated phospholipid. The PEG lipid can be DMG-PEG.

[0321] 200. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) steroid lipids; and (e) cationic polymers, including oligolysines (linear or branched), such as K16, K17, or K30; may include:

[0322] 201. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) steroid lipids; and (e) a cationic polymer containing oligohistidine (linear or branched chain); may include:

[0323] 202. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) steroid lipids; and (e) a cationic polymer containing oligoarginine (linear or branched chain) may include:

[0324] 203. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) steroid lipids; and (e) a cationic polymer containing oligolysine and oligohistidine may include:

[0325] 204. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) steroid lipids; and (e) a cationic polymer comprising oligohistidine and oligoarginine; may include:

[0326] 205. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) steroid lipids; and (e) a cationic polymer comprising oligoarginine and oligolysine; may include:

[0327] 206. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) steroid lipids; and (e) cationic polymers, including oligolysines, oligohistidines, and oligoarginines; may include:

[0328] 207. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid, comprising DOPE, DOPC, DSPC, DPPC, DMPC, or POPC; (d) steroid lipids; and (e) a cationic polymer, the cationic polymer including PEI may include:

[0329] In any of embodiments 200-207, the nanoparticles can be non-viral transfection complexes.

[0330] In any of embodiments 200-207, the cargo can comprise a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, mRNA, siRNA, samRNA, miRNA, gRNA, shRNA, antisense RNA, ribosomal RNA, tRNA, or snRNA. In any of embodiments 200-207, the steroid lipid can comprise cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (an alkyl steroid), a secosteroid (vitamin D2, D3), or a pentacyclic steroid. In any of embodiments 200-207, the lipid component can include DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA.

[0331] In any of embodiments 200-207, the nanoparticles can be free of a targeting moiety.In any of embodiments 200-207, the nanoparticles can be free of a targeting peptide.

[0332] In embodiments 200-207, the cargo preferably comprises linear DNA or mRNA. In embodiments 200-207, the lipid component preferably comprises at least one ionizable lipid. In embodiments 200-207, the steroid lipid comprises cholesterol.

[0333] The nanoparticles can further comprise a PEG lipid. The PEG lipid can be a PEGylated phospholipid. The PEG lipid can be DMG-PEG.

[0334] 208. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) cationic polymers, including oligolysines (linear or branched), such as K16, K17, or K30; may include:

[0335] 209. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) a cationic polymer containing oligohistidine (linear or branched chain); may include:

[0336] 210. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) a cationic polymer containing oligoarginine (linear or branched chain) may include:

[0337] 211. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) a cationic polymer containing oligolysine and oligohistidine may include:

[0338] 212. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) a cationic polymer comprising oligohistidine and oligoarginine; may include:

[0339] 213. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) a cationic polymer comprising oligoarginine and oligolysine; may include:

[0340] 214. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) cationic polymers, including oligolysines, oligohistidines, and oligoarginines; may include:

[0341] 215. The nanoparticles of the present invention are (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids comprising one or more of cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids; and (e) a cationic polymer, the cationic polymer including PEI may include:

[0342] In any of embodiments 208-215, the nanoparticles may be non-viral transfection complexes.

[0343] In any of embodiments 208-215, the phospholipid can include DOPE, DOPC, DSPC, DPPC, DMPC, or POPC. In any of embodiments 208-215, the lipid component can include DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA. In any of embodiments 208-215, the cargo can comprise a closed linear deoxyribonucleic acid (DNA) molecule, a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides, a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, wherein the partially closed linear DNA molecule comprises one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end, mRNA, siRNA, samRNA, miRNA, gRNA, shRNA, antisense RNA, ribosomal RNA, tRNA, or snRNA.

[0344] In any of embodiments 208-215, the nanoparticles may not include a targeting moiety.In any of embodiments 208-215, the nanoparticles may not include a targeting peptide.

[0345] In embodiments 208-215, the cargo preferably comprises linear DNA or mRNA. In embodiments 208-215, the lipid preferably comprises at least one ionizable lipid. In embodiments 208-215, the phospholipid is DOPE.

[0346] The nanoparticles can further comprise a PEG lipid. The PEG lipid can be a PEGylated phospholipid. The PEG lipid can be DMG-PEG.

[0347] Specific nanoparticles of the present invention include:

[0348] Nanoparticles comprising closed linear DNA; ALC-0315; DOPE; cholesterol; DMG-PEG; and K16 (an oligolysine consisting of 16 lysine residues), the nanoparticles not containing a targeting peptide.

[0349] Nanoparticles comprising closed linear DNA; DLin-MC3-DMA; DOPE; cholesterol; DMG-PEG; and K16 (an oligolysine consisting of 16 lysine residues), the nanoparticles not containing a targeting peptide.

[0350] Nanoparticles comprising closed linear DNA; DOTMA; DOPE; cholesterol; DMG-PEG; and K16 (oligolysine consisting of 16 lysine residues), the nanoparticles not containing a targeting peptide.

[0351] Nanoparticles comprising closed linear DNA; ALC-0315 and DOTMA; DOPE; cholesterol; DMG-PEG; and K16 (an oligolysine consisting of 16 lysine residues), the nanoparticles not containing a targeting peptide.

[0352] Nanoparticles comprising mRNA; ALC-0315; DOPE; cholesterol; DMG-PEG; and K16 (an oligolysine consisting of 16 lysine residues), the nanoparticles not containing a targeting peptide.

[0353] Nanoparticles comprising mRNA; DLin-MC3-DMA; DOPE; cholesterol; DMG-PEG; and K16 (an oligolysine consisting of 16 lysine residues), the nanoparticles not containing a targeting peptide.

[0354] Nanoparticles comprising mRNA; DOTMA; DOPE; cholesterol; DMG-PEG; and K16 (an oligolysine consisting of 16 lysine residues), the nanoparticles not containing a targeting peptide.

[0355] Nanoparticles comprising mRNA; ALC-0315 and DOTMA; DOPE; cholesterol; DMG-PEG; and K16 (an oligolysine consisting of 16 lysine residues), the nanoparticles not containing a targeting peptide.

[0356] Nanoparticles comprising closed linear DNA; ALC-0315; DOPE; cholesterol; DMG-PEG; and K30 (oligolysine consisting of 30 lysine residues), the nanoparticles not containing a targeting peptide.

[0357] Nanoparticles comprising closed linear DNA; DLin-MC3-DMA; DOPE; cholesterol; DMG-PEG; and K30 (oligolysine consisting of 30 lysine residues), the nanoparticles not containing a targeting peptide.

[0358] Nanoparticles comprising closed linear DNA; DOTMA; DOPE; cholesterol; DMG-PEG; and K16 (oligolysine consisting of 16 lysine residues), the nanoparticles not containing a targeting peptide.

[0359] Nanoparticles comprising closed linear DNA; ALC-0315 and DOTMA; DOPE; cholesterol; DMG-PEG; and K30 (oligolysine consisting of 30 lysine residues), the nanoparticles not containing a targeting peptide.

[0360] Nanoparticles comprising mRNA; ALC-0315; DOPE; cholesterol; DMG-PEG; and K30 (oligolysine consisting of 30 lysine residues), the nanoparticles not containing a targeting peptide.

[0361] Nanoparticles comprising mRNA; DLin-MC3-DMA; DOPE; cholesterol; DMG-PEG; and K30 (oligolysine consisting of 30 lysine residues), the nanoparticles not containing a targeting peptide.

[0362] Nanoparticles comprising mRNA; DOTMA; DOPE; cholesterol; DMG-PEG; and K30 (oligolysine consisting of 30 lysine residues), the nanoparticles not containing a targeting peptide.

[0363] Nanoparticles comprising mRNA; ALC-0315 and DOTMA; DOPE; cholesterol; DMG-PEG; and K30 (oligolysine consisting of 30 lysine residues), the nanoparticles not containing a targeting peptide.

[0364] The term "targeting peptide" or "targeting moiety" refers to a peptide sequence or other moiety that has the ability to target, bind to, and / or interact with a specific cell type. For example, a brain cell targeting sequence can target, bind to, and / or interact with brain cells or biologically active molecules present on the surface of brain cells. The term "specific" refers to the ability to preferentially target, bind to, and / or interact with a given target, such as a receptor on the brain, compared to other cell types. The nanoparticles of the present invention may not contain such targeting peptides or targeting moieties.

[0365] In the nanoparticles of the invention, including embodiments 1 to 215, the cargo may be a nucleic acid. The cargo may be an RNA molecule or a DNA molecule.

[0366] The nucleic acid may be a DNA molecule. The DNA molecule may be a linear DNA molecule or a DNA molecule containing a linear portion. The nucleic acid may be single-stranded, double-stranded, or partially single-stranded and partially double-stranded.

[0367] A nucleic acid (e.g., a closed linear DNA molecule) can comprise at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 45, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, or at least 50,000 nucleotides. Preferably, the nucleic acid (eg, a closed linear DNA molecule) comprises at least 500 nucleotides.

[0368] A nucleic acid (e.g., a closed linear DNA molecule) can comprise at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 45, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, or at least 50,000 base pairs. Preferably, the nucleic acid (eg, a closed linear DNA molecule) comprises at least 500 base pairs.

[0369] The linear DNA molecule may be a chromosome. The linear DNA molecule may be a linear double-stranded DNA molecule. The linear double-stranded DNA molecule may contain one or more protected nucleotides (i.e., nucleotides resistant to nuclease (e.g., exonuclease) digestion) (e.g., phosphorothioated nucleotides). The linear double-stranded DNA molecule may contain a first adapter molecule at a first end and a second adapter molecule at a second end. The first adapter molecule and the second adapter may contain one or more protected nucleotides (i.e., nucleotides resistant to nuclease (e.g., exonuclease) digestion). The presence of the protected nucleotides may confer resistance to nuclease (e.g., exonuclease) digestion. A linear double-stranded DNA molecule can be a DNA molecule having a double-stranded portion comprising a double-stranded linear adaptor comprising protected nucleotides (i.e., nucleotides that are resistant to nuclease (e.g., exonuclease) digestion) at a first end and a double-stranded linear adaptor comprising protected nucleotides (i.e., nucleotides that are resistant to nuclease (e.g., exonuclease) digestion) at a second end.

[0370] The linear DNA molecule can be a closed linear DNA molecule. The closed linear DNA molecule can include a double-stranded DNA portion closed at a first end by a first single-stranded portion (i.e., can include a first hairpin at the first end) and closed at a second end by a second single-stranded portion (i.e., can include a second hairpin at the second end). The closed DNA molecule can be a covalently closed linear DNA molecule. The covalently closed linear DNA molecule can include a first adapter molecule at a first end and a second adapter molecule at a second end. The first adapter molecule and the second adapter molecule can each include a hairpin. The hairpin can confer resistance to nuclease (e.g., exonuclease) digestion. The closed linear DNA molecule can include one or more protected nucleotides [i.e., nucleotides resistant to nuclease (e.g., exonuclease) digestion]. The closed linear DNA molecule can be (i) a DNA molecule processed by TelN protelomerase; or (ii) a DNA molecule having a double-stranded portion that is closed at a first end by ligation of a first adaptor to the first end and at a second end by ligation of a second adaptor to the second end.

[0371] The linear DNA molecule may be a partially closed linear DNA molecule. A partially closed linear DNA molecule may comprise a double-stranded DNA portion that is closed at a first end and open at a second end. A partially closed linear DNA molecule may comprise a double-stranded DNA portion that is closed at a first end by a single-stranded portion (i.e., may comprise a first hairpin at the first end) and open at a second end. A partially closed linear DNA molecule may comprise one or more nuclease-resistant nucleotides in an open-ended region adjacent to the second end. The open-ended region adjacent to the second end may be at the 3' or 5' end of the molecule. The open-ended region adjacent to the second end can comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 nucleotides located at the second end of the partially closed linear DNA molecule. That is, the open-ended region adjacent to the second end can comprise any nucleotide between and including the terminal nucleotide of the second end and the nucleotide at position 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 from the terminal nucleotide of the second end.

[0372] A partially closed linear DNA molecule can include a hairpin loop at the 5' or 3' end. A partially closed linear DNA molecule can include a first adapter molecule at a first end and a second adapter molecule at a second end. The first adapter molecule can include a hairpin, and the second adapter can include one or more protected nucleotides (i.e., nucleotides resistant to nuclease (e.g., exonuclease) digestion). The hairpin can confer resistance to nuclease (e.g., exonuclease) digestion. The presence of protected nucleotides can confer resistance to nuclease (e.g., exonuclease) digestion. A partially closed linear DNA molecule can be a DNA molecule having a double-stranded portion that is closed at a first end by ligation of a first adapter (e.g., a hairpin adapter) to the first end and includes a double-stranded linear adapter at a second end that includes protected nucleotides (i.e., nucleotides resistant to nuclease (e.g., exonuclease) digestion).

[0373] The RNA molecule can be messenger RNA (mRNA), transfer RNA, ribosomal RNA, small interfering RNA (siRNA), antisense RNA (antisense oligonucleotide), small nuclear RNA (snRNA), double-stranded RNA, microRNA (miRNA), short hairpin RNA (shRNA), guide RNA (gRNA), self-amplifying RNA (samRNA), or circular RNA.

[0374] The RNA molecule can contain at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100, at least 150, at least 200 nucleotides, at least 500 nucleotides, at least 1,000 nucleotides, at least 2,000 nucleotides, at least 5,000 nucleotides, at least 10,000 nucleotides, at least 15,000 nucleotides, or at least 16,000 nucleotides. The RNA molecule can contain 5 to 20,000, 6 to 19,000, 7 to 18,000, 8 to 17,000, 9 to 16,000, 10 to 15,000, 10 to 13,000, 15 to 10,000, 20 to 5,000, 20 to 1,000, 20 to 500, or 25 to 300 nucleotides. An RNA molecule can include one or more protected nucleotides [ie, nucleotides that are resistant to nuclease (eg, exonuclease) digestion] (eg, phosphorothioated nucleotides).

[0375] The RNA molecule can be an mRNA molecule containing at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100, at least 150, or at least 200 nucleotides. The mRNA molecule can contain 5 to 20,000, 6 to 19,000, 7 to 18,000, 8 to 17,000, 9 to 16,000, 10 to 15,000, 10 to 13,000, 15 to 10,000, 20 to 5,000, 20 to 1,000, 20 to 500, or 25 to 300 nucleotides. The mRNA molecule can contain one or more protected nucleotides (i.e., nucleotides that are resistant to nuclease (e.g., exonuclease) digestion) (e.g., phosphorothioated nucleotides).

[0376] The RNA molecule can be a self-amplifying mRNA (samRNA) molecule containing at least 3,000, at least 4,000, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, at least 15,000, at least 16,000, at least 17,000, at least 18,000, at least 19,000, or at least 20,000 nucleotides. The samRNA molecule can contain 3,000 to 22,000, 5,000 to 21,000, 7,000 to 20,000, or 8,000 to 17,000 nucleotides. A samRNA molecule can include one or more protected nucleotides [ie, nucleotides that are resistant to nuclease (eg, exonuclease) digestion] (eg, phosphorothioated nucleotides).

[0377] The siRNA can comprise a double-stranded portion of at least 17 base pairs, at least 18 base pairs, or preferably at least 19 base pairs. The siRNA can comprise a double-stranded portion of 17-30 base pairs, 18-27 base pairs, 19-24 base pairs, or preferably 19-21 base pairs. The siRNA can comprise one or more protected nucleotides [i.e., nucleotides that are resistant to nuclease (e.g., exonuclease) digestion] (e.g., phosphorothioated nucleotides).

[0378] An miRNA can comprise at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, or at least 100 nucleotides. An miRNA molecule can comprise between 10 and 200, 12 and 150, 15 and 125, 17 and 100, 18 and 75, 20 and 75, 20 and 50, or 20 and 30 nucleotides.

[0379] The miRNA can comprise a double-stranded portion of at least 15 base pairs, at least 17 base pairs, or preferably at least 20 base pairs. The miRNA can comprise a double-stranded portion of 15-30 base pairs, 17-27 base pairs, 20-25 base pairs, or preferably 21-23 base pairs. The miRNA can comprise one or more protected nucleotides [i.e., nucleotides that are resistant to nuclease (e.g., exonuclease) digestion] (e.g., phosphorothioated nucleotides).

[0380] The antisense RNA can comprise at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 nucleotides. The antisense RNA can comprise 18 to 24 nucleotides, or preferably 19 to 23 nucleotides. The antisense RNA can comprise one or more protected nucleotides [i.e., nucleotides that are resistant to nuclease (e.g., exonuclease) digestion] (e.g., phosphorothioated nucleotides).

[0381] In the nanoparticles of the present invention, including embodiments 1 to 215, the term "phospholipid" refers to a lipid containing a fatty acid chain and a phosphate group. Phospholipids are typically neutral molecules in that they have no overall charge, unlike cationic lipids, which are positively charged. Phospholipids are typically zwitterionic molecules that contain both positively and negatively charged components but no overall charge. For example, the phospholipid can be DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphoethanoltrimethylamine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (dipalmitoylphosphatidylcholine), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), or POPC (1-palmitoyl-2-oleoylphosphatidylcholine).

[0382] In the nanoparticles of the present invention, including embodiments 1-215, the steroid lipid can be cholesterol or a derivative thereof (ie, a cholesterol derivative).

[0383] The lipid components (one or more ionizable lipids and / or one or more cationic lipids) together with phospholipids and steroid lipids (and optionally PEG lipids) can form liposomes.

[0384] The liposomes can comprise at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55% cholesterol or a derivative thereof (as defined by the molar amount of cholesterol or a derivative thereof). That is, the liposomes contain at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, or at least 25% cholesterol or derivatives thereof and at least 99%, at least 98%, at least 97%, at least 96%, at least 98%. %, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 89%, at least 88%, at least 87%, at least 86%, at least 85%, at least 84%, at least 83%, at least 82%, at least 81%, at least 80%, at least 79%, at least 78%, at least 77%, at least 76%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, or at least 45% of other lipids in the liposome (as defined by molar ratio). The liposome can comprise 10% cholesterol or a derivative thereof. The liposome can comprise 20% cholesterol or a derivative thereof.The liposomes may contain 30% cholesterol or a derivative thereof. The liposomes may contain 40% cholesterol or a derivative thereof.

[0385] In the nanoparticles of the present invention, including embodiments 1 to 215, the cholesterol derivative can be cholesterol, β-sitosterol, fucosterol, campesterol, stigmastanol (an alkyl steroid), a secosteroid (vitamin D2, D3), or a pentacyclic steroid. The structures of fucosterol, campesterol, and stigmastanol are shown below (from Nano Lett. 2020, 20, 6, 4543-4549).

[0386] [ka]

[0387] In the nanoparticles of the present invention, including those of embodiments 1 to 215, the cationic polymer can be a nucleic acid-binding cationic polymer. The cationic component of the cationic polymer can be used to achieve a desired charge (i.e., and negative / positive ratio) of the nanoparticle. A specific charge (i.e., nitrogen / phosphate ratio) may be required to promote or enhance cell transfection.

[0388] The nucleic acid-binding cationic polymer can contain at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 32, at least 34, at least 36, at least 38, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 cationic monomers. Preferably, the nucleic acid-binding cationic polymer contains at least 16, at least 17, or at least 30 cationic monomers. The nucleic acid-binding cationic polymer can contain fewer than 10 or fewer than 9 cationic monomers. The nucleic acid-binding cationic polymer can include eight cationic monomers.

[0389] The cationic polymer can include lysine, histidine, or arginine. The nucleic acid-binding cationic polymer can include oligolysine (linear or branched), oligohistidine (linear or branched), or oligoarginine (linear or branched). For example, the nucleic acid-binding cationic polymer can contain at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 32, at least 34, at least 36, at least 38, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 lysine residues. Preferably, the nucleic acid-binding cationic polymer can contain at least 16, at least 17, or at least 30 lysine residues. Even more preferably, the nucleic acid-binding cationic polymer can contain at least 17 lysine residues. The nucleic acid-binding cationic polymer can contain fewer than 10 lysine residues, or fewer than 9. The nucleic acid-binding cationic polymer can contain 8 lysine residues.

[0390] The cationic polymer can be linear or branched. The nucleic acid-binding cationic polymer can be linear or branched. For example, the nucleic acid-binding cationic polymer can contain at least 16, at least 17, or at least 30 lysine residues in the linear chain. Alternatively, the nucleic acid-binding cationic polymer can contain at least 16, at least 17, or at least 30 lysine residues in the branched chain. The nucleic acid can be the nucleic acid-binding cationic polymer. The nucleic acid-binding cationic polymer can be linear or branched. For example, the nucleic acid-binding cationic polymer (e.g., DNA-binding cationic polymer) can contain at least 16, at least 17, or at least 30 lysine residues in the linear chain. Alternatively, the nucleic acid-binding cationic polymer (e.g., DNA-binding cationic polymer) can contain at least 16, at least 17, or at least 30 lysine residues in the branched chain.

[0391] The lipid components (one or more ionizable lipids and / or one or more cationic lipids) together with phospholipids and steroid lipids (and optionally PEG lipids) can form liposomes.

[0392] The lipid component comprises a cationic lipid or an ionizable lipid (or a combination thereof).

[0393] In the nanoparticles of the present invention, including those of embodiments 1 to 215, the liposomes comprise at least one lipid component. The liposomes may comprise at least one cationic lipid. The liposomes may comprise at least one ionizable lipid. The liposomes may comprise at least one ionizable lipid, at least one steroid lipid, and at least one phospholipid. The liposomes may comprise at least one PEG-lipid. The liposomes may comprise at least one cationic lipid, at least one steroid lipid, and at least one phospholipid. The liposomes may comprise at least one ionizable lipid, at least one steroid lipid, at least one phospholipid, and at least one PEG-lipid. The liposomes may comprise at least one cationic lipid, at least one steroid lipid, at least one phospholipid, and at least one PEG-lipid. The liposomes may comprise at least one cationic lipid, at least one steroid lipid, at least one phospholipid, and at least one PEG-lipid. The liposomes may comprise one or more of the ionizable lipids listed in Table 1.

[0394] The ionizable lipid can be any one or more of those listed in the table below.

[0395] TIFF2026506360000002.tif163155

[0396] Liposomes can contain one or more of the following cationic lipids: DTDTMA (ditetradecyltrimethylammonium), DOTMA (2,3-dioleyloxypropyl-1-trimethylammonium), or DHDTMA (dihexadecyltrimethylammonium). In addition to the cation, the cationic lipid can contain a counter anion, for example, an inorganic counter ion, particularly a pharmaceutically acceptable anion such as chloride or bromide. Preferably, the cationic lipid is DOTMA.

[0397] The nanoparticles of the present invention comprise phospholipids. The term "phospholipid" refers to a lipid containing a fatty acid chain and a phosphate group. Phospholipids are typically neutral molecules, in that they do not have an overall charge, unlike cationic lipids, which are positively charged. Phospholipids are typically zwitterionic molecules, containing both positively and negatively charged components but not an overall charge. For example, the phospholipid may be DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphoethanoltrimethylamine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (dipalmitoylphosphatidylcholine), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), or POPC (1-palmitoyl-2-oleoylphosphatidylcholine). Preferably, the phospholipid is DOPE.

[0398] The nanoparticles of the present invention can include PEGylated lipids.

[0399] In the nanoparticles of the present invention, including embodiments 1-215, the PEG lipid can be provided by a phospholipid comprising a PEG (polyethylene glycol) moiety. The PEG moiety can have a molecular weight of about 100 to about 10,000, the PEG moiety can have a molecular weight of about 250 to about 7,500, the PEG moiety can have a molecular weight of about 500 to about 5,000, the PEG moiety can have a molecular weight of about 750 to about 4,000, the PEG moiety can have a molecular weight of about 1,000 to about 3,000, or the PEG moiety can have a molecular weight of approximately 2,000.

[0400] The lipid can be a PEGylated lipid, for example, DMG-PEG.

[0401] The lipid components (one or more ionizable lipids and / or one or more cationic lipids) together with phospholipids and steroid lipids (and optionally PEG lipids) can form liposomes.

[0402] In the nanoparticles of the present invention, including embodiments 1-215, the steroid lipid can be cholesterol or a derivative thereof (i.e., a cholesterol derivative). The nanoparticles of the present invention can comprise at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55% cholesterol or a derivative thereof (as defined by the molar amount of cholesterol or a derivative thereof). That is, the nanoparticles contain at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, or at least 25% cholesterol and at least 99%, at least 98%, at least 97%, at least 96%, at least At least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 89%, at least 88%, at least 87%, at least 86%, at least 85%, at least 84%, at least 83%, at least 82%, at least 81%, at least 80%, at least 79%, at least 78%, at least 77%, at least 76%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, or at least 45% of the other lipids in the liposomes (when defined by molar ratio).The cholesterol derivative may be β-sitosterol, fucosterol, campesterol or stigmastanol.

[0403] The molar ratio of lipid components, including ionizable lipids and / or cationic lipids, to phospholipids in the nanoparticles (or non-viral transfection complexes) can be 1:1, 1:2, 1:3, 1:4, 1:5, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. Preferably, the molar ratio of at least one cationic lipid to at least one phospholipid in the nanoparticles (or non-viral transfection complexes) is 1:1 or 2:1. For example, the molar ratio of DOTMA to DOPE in the nanoparticles (or non-viral transfection complexes) can be 1:1. That is, the molar amounts of DOTMA and DOPE in the nanoparticles are the same. The molar ratio of DOTMA to DOPE in the nanoparticles (or non-viral transfection complexes) can be 2:1. That is, the molar amount of DOTMA is twice the molar amount of DOPE. The molar ratio of ALC-0315 to DOPE can be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. Accordingly, the molar ratio of ALC-0315 to DOTMA to DOPE can be 1:1:1, 2:1:1, 3:1:1, 4:1:1, 5:1:1, 6:1:1, 7:1:1, 8:1:1, 9:1:1, or 10:1:1. The molar ratio of ALC-0315 to DOTMA to DOPE can be 1:2:1, 2:2:1, 3:2:1, 4:2:1, 5:2:1, 6:2:1, 7:2:1, 8:2:1, 9:2:1, or 10:2:1. The molar ratio of ALC-0315 to DOTMA to DOPE can be 1 to 10:1 to 2:1. Preferably, the molar ratio of ALC-0315 to DOTMA is 1:1 to 3:1. The ratio of ionizable lipid + cationic lipid (e.g., ALC-0315 + DOTMA) to DOPE can be 3:1 to 10:1, 4:1 to 8:1, or 5:1 to 6:1. For example, the ratio of ALC-0315:DOTMA:DOPE can be 3:3:1.

[0404] The nanoparticles can comprise an ionizable lipid and a cationic lipid. The nanoparticles of the present invention can comprise an ionizable lipid, a phospholipid, and a cationic lipid. The nanoparticles can further comprise cholesterol. The nanoparticles of the present invention can comprise an ionizable lipid, a phospholipid, and cholesterol. The nanoparticles of the present invention can comprise a cationic lipid, a phospholipid, and cholesterol. The nanoparticles of the present invention can comprise an ionizable lipid, a phospholipid, cholesterol, and a PEG-lipid. The nanoparticles of the present invention can comprise a cationic lipid, a phospholipid, cholesterol, and a PEG-lipid. The nanoparticles of the present invention can comprise an ionizable lipid, a phospholipid, cholesterol, and a cationic polymer. For example, the nanoparticles can comprise ALC-0315, DMG-PEG, cholesterol, DOPE, and peptide K16. The nanoparticles of the present invention can comprise DLin-MC3-DMA, DMG-PEG, cholesterol, DOPE, and peptide K30. The nanoparticles of the present invention can comprise ALC-0315, DMG-PEG, cholesterol, DOPE, DOTMA, and PEI. The nanoparticles of the present invention can comprise DLin-MC3-DMA, DOTMA, DMG-PEG, cholesterol, DOPE, and peptide K16.

[0405] The mass ratio of cationic polymer to DNA molecules described herein in the nanoparticles can be 0.1:1 to 9:1 (cationic polymer:nucleic acid cargo). The mass ratio can be 1:1 to 6:1. The mass ratio can be 2:1 to 4:1. For example, the mass ratio of cationic polymer to DNA or RNA molecules described herein can be about 0.1:1, about 0.5:1, about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, about 5.5:1, about 6:1, about 6.5:1, about 7:1, about 7.5:1, about 8:1, about 8.5:1, or about 9:1 [cationic polymer:nucleic acid cargo].

[0406] The molar ratio of cationic polymer to DNA molecules described herein in the nanoparticles can be at least 50:1, at least 100:1, at least 150:1, at least 200:1, at least 250:1, at least 300:1, at least 350:1, at least 400:1, at least 450:1, at least 500:1, at least 550:1, at least 600:1, at least 650:1, at least 700:1, at least 750:1, at least 800:1, at least 850:1, at least 900:1, at least 950:1, at least 1000:1, at least 1050:1, at least 1100:1, at least 1150:1, at least 1200:1, at least 1250:1, or at least 1300:1. The molar ratio can be between 100:1 and 1500:1, between 200:1 and 1200:1, between 300:1 and 1100:1, between 400:1 and 1000:1, between 500:1 and 900:1, between 600:1 and 800:1 or about 700:1.

[0407] The cationic polymer can be an oligopeptide. The oligopeptide can contain at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, or at least 40 amino acids. The amount of positively charged amino acids in the cationic polymer can be at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, or at least 31.

[0408] The molar ratio of cationic polymer to DNA or RNA molecules in the nanoparticles can be at least 350:1 or at least 650:1, and the N / P ratio [charge ratio (i.e., nitrogen / phosphate molar ratio)] of the nanoparticles can be about 4. The molar ratio of cationic polymer to nucleic acid molecules can be 100:1 to 650:1 or 200:1 to 400:1, and the N / P ratio of the nanoparticles can be about 4.

[0409] The molar ratio of cationic polymer to DNA molecules in the nanoparticles can be at least 500:1 or at least 900:1, and the N / P ratio of the nanoparticles can be about 6. The molar ratio of cationic polymer to nucleic acid molecules can be 200:1 to 1100:1 or 350:1 to 700:1, and the N / P ratio of the nanoparticles can be about 6.

[0410] The molar ratio of cationic polymer to DNA molecules in the nanoparticles can be at least 750:1 or at least 1400:1, and the N / P ratio of the nanoparticles can be about 8.

[0411] The molar ratio of cationic polymer to DNA molecules in the nanoparticles can be 550:1 to 1400:1, or 650:1 to 1100:1, and the N / P ratio of the nanoparticles is 9.

[0412] The cationic polymer can contain at least 30 (e.g., 31) positively charged amino acids. In such cases, the peptide:DNA or RNA cargo molar ratio can be 30:1 to 1000:1, 75:1 to 750:1, or 100:1 to 500:1.

[0413] The ratios of each of the nanoparticle components can be as in any of the nanoparticle formulations described in Tables 4 and 5. Although these tables show ALC-0315 and DOTMA, the ratios can be equally applied to alternative ionizable and / or cationic lipids, respectively.

[0414] The cationic polymer can include at least 15 (e.g., 16) positively charged amino acids. In such cases, the peptide:DNA or RNA cargo (e.g., nucleic acid) molar ratio can be 50:1 to 2000:1, 75:1 to 1000:1, or 200:1 to 1400:1.

[0415] A specific ratio of cationic polymer to DNA or RNA cargo allows for the effective and stable formulation of nanoparticles.

[0416] The molar ratio of DNA molecules:lipids:cationic polymer in the nanoparticles can be 1 (DNA molecules):1000-6000 (lipids):30-2000 (peptides).

[0417] The lipid components (one or more ionizable lipids and / or one or more cationic lipids) together with phospholipids and steroid lipids (and optionally PEG lipids) can form liposomes.

[0418] The mass ratio of nucleic acid (e.g., DNA or mRNA) to liposomes can be 1:5 to 1:15, or about 1:11. The mass ratio of nucleic acid to liposomes can be 1:16 to 1:25, for example, 1:22.

[0419] The nanoparticles (e.g., non-viral transfection complexes) can have a particle size of less than 300 nm, for example, less than 200 nm, or less than 100 nm, or about 80 nm. Although there will be some variation in particle size within a population of particles, the above criteria will be considered met if at least 70%, at least 80%, or at least 90% of the particles are less than 300 nm, for example, less than 200 nm or less than 100 nm. Preferably, within a population of particles, at least 80% of the particles are less than 300 nm, for example, less than 200 nm or less than 100 nm. Preferably, the nanoparticles are self-assembled nanoparticles. Within a population of self-assembled nanoparticles (e.g., non-viral transfection complexes), the size of the particles may be lower than the size of particles produced by methods other than self-assembly (e.g., methods in which lipid components are conjugated to cationic polymers prior to encapsulation of the cargo). For example, the size of the self-assembled nanoparticles may be at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% smaller than the size of particles produced by methods other than the self-assembly method. Preferably, the self-assembly of the nanoparticles is carried out using a microfluidic device.

[0420] In a population or library of nanoparticles described herein, each nanoparticle can have substantially the same size as at least 9, at least 99, at least 999, at least 9,999, at least 99,999 other nanoparticles in the library.

[0421] Thus, in a population or library of nanoparticles (e.g., non-viral transfection complexes) of the present invention, the nanoparticles can be monodisperse or substantially monodisperse. The nanoparticles (e.g., non-viral transfection complexes) can have a polydispersity index (PDI) of less than 0.4, less than 0.3, less than 0.2, or less than 0.15. The nanoparticles (e.g., non-viral transfection complexes) can have a polydispersity index similar to or equal to that of empty liposomes (controls).

[0422] In a population or library of self-assembled nanoparticles (e.g., non-viral transfection complexes), the polydispersity index can be lower than the polydispersity index of a population of nanoparticles produced by methods other than self-assembly (e.g., methods in which lipid components are conjugated to a cationic polymer prior to encapsulation of cargo). The polydispersity index of a population or library of self-assembled nanoparticles can be at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% lower than the polydispersity index of a population or library of nanoparticles produced by methods other than self-assembly.

[0423] Polydispersity index is a measure of the heterogeneity of a sample based on size.Polydispersity can be caused by the size distribution in the sample or the agglomeration or aggregation of the sample during isolation or analysis.Those skilled in the art know various methods for determining polydispersity index.For example, polydispersity index can be obtained from an instrument using dynamic light scattering (DLS) or determined from electron micrographs.In general, polydispersity index values ​​less than 0.3 are more typical for monodisperse or substantially monodisperse samples, and values ​​greater than 0.7 are typical for a wide size (e.g., polydisperse) distribution of particles.

[0424] The inventors of the present application have discovered that the production of monodisperse or substantially monodisperse nanoparticles of the present invention is facilitated by a particular ratio of charges between the cationic polymer, the lipid component and the cargo.

[0425] Thus, the present invention provides libraries containing two or more nanoparticles described herein. The libraries can include at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 1000, at least 10,000, or at least 100,000 nanoparticles described herein. The polydispersity index (PDI) of the nanoparticles in the library can be less than about 0.3, or less than about 0.2, e.g., 0.3-0.1, 0.25-0.15, or 0.22-0.13. The polydispersity index (PDI) of the nanoparticles in the library can be 0.22, 0.19, 0.17, or 0.13.

[0426] The nanoparticles (e.g., non-viral transfection complexes) can have a charge ratio (i.e., nitrogen / phosphate (N / P) molar ratio) of 2.0 to 13.0, 3.0 to 12.0, 4.0 to 11.0, or 4.0 to 12.0. The nanoparticles (e.g., non-viral transfection complexes) can have a charge ratio (i.e., N / P ratio) of about 4.0, about 4.2, about 4.5, about 4.7, about 5.0, about 5.2, about 5.5, about 5.7, about 6.0, about 6.5, about 7.5, about 8.5, about 9.0, about 9.5, or about 10.5. Preferably, the nanoparticles have a charge ratio (i.e., N / P ratio) of 7.0 to 11.0, 4.0 to 9.0, or 3.0 to 8.0.

[0427] Preferably, the nanoparticles have a charge ratio of about 4.0, or about 5.0, or about 6.0, or about 9.0.

[0428] For example, when the nucleic acid-binding component of the nanoparticle is a nucleic acid-binding cationic polymer containing 16 lysine residues, the nanoparticle can have a charge ratio (i.e., N / P ratio) of 3.0 to 12.0 or 4.0 to 12.0, preferably 7.0 to 11.0. For example, when the nucleic acid-binding component of the nanoparticle is a nucleic acid-binding cationic polymer containing 16 lysine residues, the nanoparticle can have a charge ratio (i.e., N / P ratio) of about 4.0, about 5.0, about 6.0, or about 9.0.

[0429] For example, when the cationic polymer contains 30 lysine residues, the nanoparticles can have a charge ratio (i.e., N / P ratio) of 3.0 to 8.0 or 4.0 to 8.0. For example, when the cationic polymer contains 30 lysine residues, the nanoparticles can have a charge ratio (i.e., N / P ratio) of about 4.2, about 4.5, about 4.7, about 5.2, about 5.5, about 5.7, about 6.5, or about 7.5. Preferably, when the cationic polymer contains 30 lysine residues, the N / P ratio can be about 4.0, about 5.0, about 6.0, or about 9.0.

[0430] The nanoparticles (e.g., non-viral transfection complexes) can have a charge ratio [i.e., nitrogen / phosphate (N / P) molar ratio] of 3.5-11.0, 4.0-11.0, 5.0-11.0, 9.0-11.0, 7.0-13.0, 7.2-13.0, 7.5-13.0, or 8.0-13.0.

[0431] The charge ratio (N / P ratio) is calculated from the molar amount of each free amine group (N) in the nanoparticle component relative to the phosphate group (P) in the nanoparticle component. For example, the free amine group can be derived from the cationic polymer and lipid component, and the phosphate group can be derived from the phosphate group (P) in a nucleic acid molecule (e.g., a DNA molecule). The charge ratio is typically determined by the mass of the cationic polymer. An N / P ratio of 1, for example, consists of one amine group per one phosphate group, and is commonly expressed as N / P=1. Similarly, the ratio N / P=5 means the ratio between one phosphate group and five amine groups.

[0432] For example, for nanoparticles consisting of lipid, peptide and mRNA components, the N / P ratio is calculated as follows:

[0433]

number

[0434] For three-component nanoparticles, the mass of each component to be formulated can be calculated from the desired mass of the nucleic acid of interest to be encapsulated and the desired charge ratio of peptide / mRNA to lipid / mRNA.

[0435] First, P must be calculated as follows:

[0436]

number

[0437] The required number of moles of each lipid and peptide in the final formulation can then be calculated as follows:

[0438]

number

[0439] In the formula, N pep is equal to the number of positively charged amino acids (lysine, histidine, and arginine) in the peptide sequence, and N lip is equal to the number of free amine groups in the cationic lipid component.

[0440] Therefore, the mass of peptide or lipid to formulate in the final nanoparticle formulation can be calculated as follows: Mass = moles x molecular weight

[0441] Finally, the N / P ratio of the final nanoparticle equation can be calculated using the formula above.

[0442] The term "about," as used herein with respect to a numerical parameter, means a value within 10% (i.e., plus or minus 10%) of the underlying parameter. For example, a charge ratio of "about 4.5" can include charge (N / P) ratios between 4.1 and 5.0, including charge ratios of 4.1 and 5.0.

[0443] Nanoparticles (e.g., non-viral delivery complexes) can deliver cargo, such as nucleic acid cargo, to cells with a transfection efficiency of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 52%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. Preferably, the transfection efficiency is at least 15%. Preferably, the transfection efficiency of nanoparticles containing closed linear DNA as cargo is better than that of nanoparticles containing plasmid DNA.

[0444] The nanoparticles of the present invention facilitate the delivery of DNA or RNA cargo into cells. Improved delivery can be determined, for example, by determining transfection efficiency (i.e., the percentage of transfected cells compared to untransfected cells). Nanoparticles containing cationic polymers (e.g., non-viral delivery complexes) can deliver DNA or RNA cargo into cells with a transfection efficiency of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. Nanoparticles containing cationic polymers (e.g., non-viral delivery complexes) can deliver DNA or RNA cargo into cells with a transfection efficiency higher than that of nanoparticles without cationic polymers (i.e., nanoparticles containing lipid components, phospholipids, steroid lipids, and cargo). Preferably, the transfection efficiency of nanoparticles containing cationic polymers, such as cationic oligopeptides, is higher than that of nanoparticles without cationic peptides.

[0445] Those skilled in the art will recognize various methods for determining transfection efficiency. For example, transfection efficiency can be determined by measuring or detecting the expression level of the gene encoded by the nucleic acid. For example, the nucleic acid can encode green fluorescent protein (GFP), and when expressed, GFP can be detected to determine transfection efficiency.

[0446] The nanoparticles are preferably self-assembled nanoparticles. They may be nanoparticles produced by a process in which pre-existing components (e.g., lipid components, cargo, and cationic polymers) form organized structures as a result of specific local interactions between the components themselves without external guidance. They may be nanoparticles prepared without pre-prepared liposomes using microfluidic methods such as the dilution cartridge method in a one-step process.

[0447] The DNA or RNA cargo, lipid components, steroid lipids, phospholipids, and cationic polymers can reversibly interact to form self-assembled nanoparticles. The DNA or RNA cargo, lipid components, steroid lipids, phospholipids, and cationic polymers can reversibly interact in the self-assembled nanoparticles through intermolecular forces. The DNA cargo, lipid components, steroid lipids, phospholipids, and cationic polymers can reversibly interact in the self-assembled nanoparticles through non-covalent interactions. The DNA or RNA cargo, lipid components, steroid lipids, phospholipids, and cationic polymers can reversibly interact in the self-assembled nanoparticles through hydrogen bonding, van der Waals interactions, hydrophobic interactions, and / or electrostatic interactions. The DNA or RNA cargo, lipid components, and cationic polymers can not be conjugated or linked in the self-assembled nanoparticles by forces other than intermolecular forces.

[0448] The cargo suitable for use in the nanoparticles (e.g., non-viral transfection complexes) described herein is a nucleic acid. Preferably, the cargo comprises a closed linear DNA molecule or a linear DNA molecule. The closed linear DNA molecule and / or the linear DNA molecule can have enhanced resistance to nuclease (e.g., exonuclease) digestion.

[0449] The nucleic acid can be a DNA molecule. The nucleic acid can be a closed linear DNA molecule (e.g., a covalently closed linear DNA molecule) or a linear DNA molecule (e.g., a linear double-stranded DNA molecule). The DNA molecule can be a linear DNA molecule or a partially linear DNA molecule (i.e., the DNA molecule can include a linear portion). The nucleic acid can be single-stranded, double-stranded, or partially single-stranded and partially double-stranded.

[0450] A nucleic acid (e.g., a closed linear DNA molecule) can comprise at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 45, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, or at least 50,000 nucleotides. Preferably, the nucleic acid (eg, a closed linear DNA molecule) comprises at least 500 nucleotides.

[0451] A nucleic acid (e.g., a closed linear DNA molecule) can comprise at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 45, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, or at least 50,000 base pairs. Preferably, the nucleic acid (eg, a closed linear DNA molecule) comprises at least 500 base pairs.

[0452] The linear DNA molecule can be a closed linear DNA molecule. The closed linear DNA molecule can include a double-stranded DNA portion closed at a first end by a first single-stranded portion (i.e., can include a first hairpin at the first end) and closed at a second end by a second single-stranded portion (i.e., can include a second hairpin at the second end). The closed DNA molecule can be a covalently closed linear DNA molecule. The covalently closed linear DNA molecule can include a first adapter molecule at a first end and a second adapter molecule at a second end. The first adapter molecule and the second adapter molecule can each include a hairpin. The hairpin can confer resistance to nuclease (e.g., exonuclease) digestion. The closed linear DNA molecule can include one or more protected nucleotides [i.e., nucleotides resistant to nuclease (e.g., exonuclease) digestion]. The closed linear DNA molecule can be (i) a DNA molecule processed by TelN protelomerase; or (ii) a DNA molecule having a double-stranded portion that is closed at a first end by ligation of a first adaptor to the first end and at a second end by ligation of a second adaptor to the second end.

[0453] The linear DNA molecule may be a portion of a chromosome or a gene. The linear DNA molecule may be a linear double-stranded DNA molecule. The linear double-stranded DNA molecule may contain one or more protected nucleotides (i.e., nucleotides resistant to nuclease (e.g., exonuclease) digestion) (e.g., phosphorothioated nucleotides). The linear double-stranded DNA molecule may contain a first adapter molecule at a first end and a second adapter molecule at a second end. The first adapter molecule and the second adapter may contain one or more protected nucleotides (i.e., nucleotides resistant to nuclease (e.g., exonuclease) digestion). The presence of the protected nucleotides may confer resistance to nuclease (e.g., exonuclease) digestion. A linear double-stranded DNA molecule can be a DNA molecule having a double-stranded portion comprising a double-stranded linear adaptor comprising protected nucleotides (i.e., nucleotides that are resistant to nuclease (e.g., exonuclease) digestion) at a first end and a double-stranded linear adaptor comprising protected nucleotides (i.e., nucleotides that are resistant to nuclease (e.g., exonuclease) digestion) at a second end.

[0454] The linear DNA molecule can be a closed linear DNA molecule. The closed linear DNA molecule can include a double-stranded DNA portion closed at a first end by a first single-stranded portion (i.e., can include a first hairpin at the first end) and closed at a second end by a second single-stranded portion (i.e., can include a second hairpin at the second end). The closed DNA molecule can be a covalently closed linear DNA molecule. The covalently closed linear DNA molecule can include a first adapter molecule at a first end and a second adapter molecule at a second end. The first adapter molecule and the second adapter molecule can each include a hairpin. The hairpin can confer resistance to nuclease (e.g., exonuclease) digestion. The closed linear DNA molecule can include one or more protected nucleotides [i.e., nucleotides resistant to nuclease (e.g., exonuclease) digestion]. The closed linear DNA molecule can be (i) a DNA molecule processed by TelN protelomerase; or (ii) a DNA molecule having a double-stranded portion that is closed at a first end by ligation of a first adaptor to the first end and at a second end by ligation of a second adaptor to the second end.

[0455] The linear DNA molecule may be a partially closed linear DNA molecule. A partially closed linear DNA molecule may comprise a double-stranded DNA portion that is closed at a first end and open at a second end. A partially closed linear DNA molecule may comprise a double-stranded DNA portion that is closed at a first end by a single-stranded portion (i.e., may comprise a first hairpin at the first end) and open at a second end. A partially closed linear DNA molecule may comprise one or more nuclease-resistant nucleotides in an open-ended region adjacent to the second end. The open-ended region adjacent to the second end may be at the 3' or 5' end of the molecule. The open-ended region adjacent to the second end can comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 nucleotides located at the second end of the partially closed linear DNA molecule. That is, the open-ended region adjacent to the second end can comprise any nucleotide between and including the terminal nucleotide of the second end and the nucleotide at position 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 from the terminal nucleotide of the second end.

[0456] The open-ended region adjacent to the second end can comprise the sense strand and the antisense strand. The open-ended region adjacent to the second end can comprise one or more nuclease-resistant nucleotides in the sense strand or the antisense strand. The open-ended region adjacent to the second end can comprise one or more nuclease-resistant nucleotides in both the sense strand and the antisense strand. The open-ended region adjacent to the second end can comprise at least 2, at least 3, at least 4, or at least 5 nuclease-resistant nucleotides in the sense strand and / or the antisense strand. Preferably, the open-ended region adjacent to the second end comprises five nuclease-resistant nucleotides in the sense strand and / or the antisense strand.

[0457] A partially closed linear DNA molecule can include a hairpin loop at the 5' or 3' end. A partially closed linear DNA molecule can include a first adapter molecule at a first end and a second adapter molecule at a second end. The first adapter molecule can include a hairpin, and the second adapter can include one or more protected nucleotides (i.e., nucleotides resistant to nuclease (e.g., exonuclease) digestion). The hairpin can confer resistance to nuclease (e.g., exonuclease) digestion. The presence of protected nucleotides can confer resistance to nuclease (e.g., exonuclease) digestion. A partially closed linear DNA molecule can be a DNA molecule having a double-stranded portion that is closed at a first end by ligation of a first adapter (e.g., a hairpin adapter) to the first end and includes a double-stranded linear adapter at a second end that includes protected nucleotides (i.e., nucleotides resistant to nuclease (e.g., exonuclease) digestion).

[0458] A partially closed linear DNA molecule can comprise (i) a cassette comprising a sense strand and an antisense strand; and (ii) one or more nuclease-resistant nucleotides in the open-ended region of the partially closed linear DNA molecule, wherein the open-ended region comprises a nuclease-resistant nucleotide that is 5' to the sense strand of the cassette. A partially closed linear DNA molecule can comprise (i) a cassette comprising a sense strand and an antisense strand; and (ii) one or more nuclease-resistant nucleotides in the open-ended region of the partially closed linear DNA molecule, wherein the open-ended region comprises a nuclease-resistant nucleotide that is 5' to the antisense strand of the cassette. A partially closed linear DNA molecule can comprise (i) a cassette comprising a sense strand and an antisense strand; and (ii) one or more nuclease-resistant nucleotides in the open-ended region of the partially closed linear DNA molecule, wherein the open-ended region comprises a nuclease-resistant nucleotide that is 3' to the sense strand of the cassette. The partially closed linear DNA molecule can comprise: (i) a cassette, the cassette comprising a sense strand and an antisense strand; and (ii) one or more nuclease-resistant nucleotides in an open-ended region of the partially closed linear DNA molecule, the open-ended region comprising a nuclease-resistant nucleotide that is 3' of the antisense strand of the cassette.

[0459] The partially closed linear DNA molecule can comprise: (i) a cassette, the cassette comprising a sense strand and an antisense strand; (ii) one or more nuclease-resistant nucleotides in the open-ended region of the partially closed linear DNA molecule, wherein the open-ended region is 5' of the sense strand of the cassette; and (iii) one or more nuclease-resistant nucleotides in the open-ended region of the partially closed linear DNA molecule, wherein the open-ended region is 3' of the antisense strand of the cassette.

[0460] The partially closed linear DNA molecule can comprise: (i) a cassette, the cassette comprising a sense strand and an antisense strand; (ii) one or more nuclease-resistant nucleotides in the open-ended region of the partially closed linear DNA molecule, wherein the open-ended region is 3' of the sense strand of the cassette; and (iii) one or more nuclease-resistant nucleotides in the open-ended region of the partially closed linear DNA molecule, wherein the open-ended region is 5' of the antisense strand of the cassette.

[0461] Closed linear DNA molecules have particular utility as therapeutic agents (i.e., DNA therapeutic agents) that can be used to express gene products in vivo. This is because their closed structure (e.g., covalently closed structure) prevents attack by enzymes such as exonucleases, thereby resulting in enhanced stability and longevity of gene expression compared to "open" DNA molecules with exposed DNA ends. Linear double-stranded open-ended cassettes have been demonstrated to be inefficient for gene expression when introduced into host tissues. This has been attributed to the instability of the cassettes due to the action of exonucleases in the extracellular space.

[0462] As shown in Figures 8 and 10 and Example 5, the nanoparticles described herein are highly stable over extended periods of time. The nanoparticles may exhibit no or very little variation in the PDI value and / or size of the nanoparticles when stored below 4°C for at least 20 weeks, at least 30 weeks, or at least 40 weeks. The nanoparticles may exhibit no or very little variation in transfection efficiency when stored below 4°C for at least 20 weeks, at least 30 weeks, or at least 40 weeks.

[0463] Isolating DNA ends within a closed structure also has other advantages. The DNA ends are prevented from integrating with genomic DNA, and therefore, closed linear DNA molecules offer improved safety. In addition, closed linear structures reduce concatamerization of DNA molecules within host cells, and therefore, gene product expression levels can be regulated in a more delicate manner.

[0464] A linear DNA molecule (e.g., a linear double-stranded DNA molecule, a closed linear DNA, or a partially closed linear DNA molecule) can contain a cassette. Thus, a linear DNA molecule (e.g., a linear double-stranded DNA molecule, a closed linear DNA, or a partially closed linear DNA molecule) can contain a sense strand and an antisense strand, and the linear DNA molecule contains a single cassette and one or more protected (e.g., phosphorothioated) nucleotides at internal positions in each strand (the linear DNA molecule may also contain one or more protected (e.g., phosphorothioated) nucleotides at internal positions in each strand outside the cassette).

[0465] The term "single cassette," as used herein in the context of a linear DNA molecule, is intended to encompass a linear DNA molecule that does not contain or consist of multiple cassettes. That is, a linear DNA molecule contains only a single cassette that can contain a single coding sequence of a gene of interest. A single cassette can contain or consist of multiple tandem repeat sequences and / or concatemeric DNA. The term "single cassette," as used herein, is intended to encompass a single copy of a DNA sequence of interest, for example, a single copy of a coding sequence. Thus, a "single cassette" may not encompass a cassette that contains or consists of multiple copies of the same DNA sequence linked in series. A single cassette can contain a collection of genes of interest. For example, a single cassette can contain sequences for at least two, three, four, or five genes of interest. The genes of interest may not be the same in a single cassette.

[0466] The linear DNA molecule (e.g., a linear double-stranded DNA molecule, a closed linear DNA, or a partially closed linear DNA molecule) can comprise a cassette. The cassette can comprise a coding sequence. The coding sequence can encode a gene of interest, for example, a gene encoding a protein. The cassette can comprise at least a portion of a promoter and a coding sequence. The cassette can comprise a promoter and a coding sequence. The cassette can comprise a promoter, a coding sequence, a ribosome binding site, and a translation termination sequence. The cassette can additionally comprise a sequence that supports protein expression, such as a cap-independent translation element. The cassette can comprise (or encode) a repair template (or editing template). The repair template (or editing template) can be for use in CRISPR-Cas-mediated homology-directed repair (HDR). The cassette can encode a CRISPR guide RNA. The cassette can be a mammalian expression cassette. The promoter can be a CMV promoter. The cassette can further comprise an enhancer. The cassette can further include a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette can further include a homopolymer sequence, such as a polyA, polyC, polyT, or polyG sequence. The homopolymer sequence can be 3 to 200 nucleotides in length. The homopolymer sequence can be used to facilitate purification of the cassette, in which case the homopolymer sequence can be 4 to 12 nucleotides in length, or 5 to 10 nucleotides in length. The homopolymer sequence can be used to improve mRNA expression, in which case the homopolymer sequence can be 10 to 200 nucleotides in length, preferably 80 to 150 nucleotides in length. The homopolymer sequence can be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides in length. Preferably, the homopolymer sequence can be at least 100 nucleotides in length. Also more preferably, the homopolymer sequence may be at least 120 nucleotides in length.For example, the homopolymer sequence can include a polyA sequence of at least 120 nucleotides.

[0467] The double-stranded linear DNA molecule can include a spacer region, which can include at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, or at least 200 base pairs.

[0468] The closed linear DNA molecule can include a spacer region, which can include at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, or at least 200 base pairs.

[0469] The partially closed linear DNA molecule can comprise a spacer region, which can comprise at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, or at least 200 base pairs.

[0470] The double-stranded linear DNA molecule may contain inverted terminal repeats.

[0471] The closed linear DNA molecule may comprise an inverted terminal repeat sequence.

[0472] The partially closed linear DNA molecule may comprise an inverted terminal repeat sequence.

[0473] The double-stranded linear DNA molecule can contain at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, or at least 50,000 base pairs. Preferably, the double-stranded linear DNA molecule contains at least 500 base pairs.

[0474] The closed linear DNA molecule can contain at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, or at least 50,000 base pairs. Preferably, the closed linear DNA molecule contains at least 500 base pairs.

[0475] A partially closed linear DNA molecule can contain at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, or at least 50,000 base pairs. Preferably, the partially closed linear DNA molecule contains at least 500 base pairs.

[0476] A closed linear DNA molecule can contain one or more protected nucleotides (i.e., nucleotides that are resistant to nuclease (e.g., exonuclease) digestion). A closed linear DNA molecule can contain one or more protected nucleotides (i.e., nucleotides that are resistant to nuclease (e.g., exonuclease) digestion) in each strand. For example, a closed linear DNA molecule can contain at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 protected nucleotides (e.g., phosphorothioated nucleotides) in each strand.

[0477] The linear DNA molecule may be a linear double-stranded DNA molecule. The linear double-stranded DNA molecule may contain one or more protected nucleotides [i.e., nucleotides resistant to nuclease (e.g., exonuclease) digestion] (e.g., phosphorothioated nucleotides). The protected nucleotides may be located at the 3' and / or 5' ends and / or 3' and / or 5' end regions of the linear double-stranded DNA molecule. Preferably, the protected nucleotides are located at the 3' and 5' ends and 3' and 5' regions of the linear double-stranded DNA molecule. The 3' region comprises at least 20 3'-terminal nucleotides of the linear DNA molecule. The 3' region comprises fewer than 30 3'-terminal nucleotides of the linear DNA molecule. The 5' region comprises 30 or fewer 5'-terminal nucleotides of the linear DNA molecule. That is, the last and / or first nucleotide in the linear DNA molecule may be a protected nucleotide. In other words, a linear DNA molecule can include a "cap" of protective nucleotides that protects the ends of the linear DNA molecule from nuclease (eg, exonuclease) digestion.

[0478] A partially closed linear DNA molecule can contain one or more protected nucleotides (i.e., nucleotides that are resistant to nuclease (e.g., exonuclease) digestion). A partially closed linear DNA molecule can contain one or more protected nucleotides (i.e., nucleotides that are resistant to nuclease (e.g., exonuclease) digestion) in each strand. For example, a partially closed linear DNA molecule can contain at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 protected nucleotides (e.g., phosphorothioated nucleotides) in each strand.

[0479] The protected nucleotide (i.e., a nucleotide resistant to nuclease (e.g., exonuclease) digestion) may be a phosphorothioated nucleotide. For example, the phosphorothioated nucleotide may be α-S-dATP (i.e., 2'-deoxyadenosine-5'-(α-thio)-triphosphate), α-S-dCTP (i.e., 2'-deoxycytidine-5'-(α-thio)-triphosphate), α-S-dGTP (i.e., 2'-deoxyguanosine-5'-(α-thio)-triphosphate), α-S-dTTP (i.e., 2'-deoxythymidine-5'-(α-thio)-triphosphate), α-S-dUTP (i.e., 2'-deoxyuridine-5'-(α-thio)-triphosphate), and / or uridine 2',3'-cyclophosphorothioate.

[0480] The phosphorothioated nucleotides can be Sp isomers, Rp isomers, or a mixture of both Sp and Rp isomers.

[0481] The protected nucleotides (i.e., nucleotides that are resistant to nuclease (e.g., exonuclease) digestion) can be 2'-O-methyl nucleotides or 2'-O-methoxyethyl (MOE) nucleotides. For example, the MOE nucleotides can be 2'-O-methoxyethyl guanosine, 2'-O-methoxyethyl cytidine, 2'-O-methoxyethyl adenosine, and / or 2'-O-methoxyethyl thymidine.

[0482] A linear double-stranded DNA molecule can include one or more protected nucleotides (eg, phosphorothioated nucleotides) at internal positions in each strand (ie, the sense and antisense strands).

[0483] The internal position can be any position in a linear double-stranded DNA molecule other than the last nucleotide on the 3'-end and 5'-end of the sense strand and antisense strand. The internal position can be located at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50, 75, or 100 nucleotides away from the 3'-end and / or 5'-end of each strand of the linear double-stranded DNA molecule. The internal position can be located at least 7 nucleotides away from the 3'-end and / or 5'-end of each strand of the linear double-stranded DNA molecule. Preferably, the internal position is located at least 10 nucleotides away from the 3'-end and / or 5'-end of each strand of the linear double-stranded DNA molecule. In a linear double-stranded DNA molecule, the internal position can be located at least 7 nucleotides away from the 3'-end and / or 5'-end of the sense strand. Preferably, in a linear double-stranded DNA molecule, the internal position is located at least 10 nucleotides away from the 3'-end and 5'-end of the sense strand. In a linear double-stranded DNA molecule, the internal position can be located at least 7 nucleotides away from the 3'-end and 5'-end of the antisense strand. Preferably, in a linear double-stranded DNA molecule, the internal position can be located at least 10 nucleotides away from the 3'-end and 5'-end of the antisense strand. In a linear double-stranded DNA molecule, the internal position can be located at least 7 nucleotides away from the 3'-end of each strand. Preferably, in a linear double-stranded DNA molecule, the internal position can be located at least 10 nucleotides away from the 3'-end of each strand. In a linear double-stranded DNA molecule, the internal position can be located at least 7 nucleotides away from the 5'-end of each strand. Preferably, in a linear double-stranded DNA molecule, the internal position can be located at least 10 nucleotides away from the 5'-end of each strand.

[0484] The linear double-stranded DNA molecule can contain a cassette. The positions of the protected (e.g., phosphorothioated) nucleotides in the linear double-stranded DNA molecule can be such that the cassette is protected from nuclease (e.g., exonuclease III) digestion. Thus, one or more phosphorothioated nucleotides at internal positions in each strand can be: (a) 5′-terminal nucleotide of the cassette; (b) the 3'-terminal nucleotide of the cassette; and (c) one or more nucleotides outside the cassette You can choose from:

[0485] The term "5'-terminal nucleotide of a cassette," as used herein, is intended to encompass the 5'-terminal nucleotide of each strand of the cassette. Thus, in a linear double-stranded DNA molecule, a cassette typically includes the 5'-terminal nucleotide of the sense strand and the 5'-terminal nucleotide of the antisense strand.

[0486] The term "3'-terminal nucleotide of a cassette," as used herein, is intended to encompass the 3'-terminal nucleotide of each strand of the cassette. Thus, in a linear double-stranded DNA molecule, a cassette typically includes the 3'-terminal nucleotide of the sense strand and the 3'-terminal nucleotide of the antisense strand.

[0487] The term "outside the cassette," as used herein, is intended to encompass any nucleotide that is not part of the cassette. This includes any nucleotide that is contained in a linear double-stranded DNA molecule and does not form part of the cassette. The term "N nucleotides outside the cassette" or "N nucleotides away from the cassette" is intended to describe a nucleotide that is located N nucleotides from the end of the cassette, toward the end of the linear double-stranded DNA molecule. For example, the term "2 nucleotides outside the cassette," in the context of an internal nucleotide position, is meant to describe a nucleotide that is outside the cassette and 2 nucleotides away from the last nucleotide of the cassette. For example, in the sequence 5'-AAAAAACATAAAA (SEQ ID NO: 3), where the cassette begins with nucleotide "T" (in the 5' to 3' direction), the term "2 nucleotides outside the cassette" refers to a "C" nucleotide. Thus, the term "at least 2 nucleotides outside the cassette" or "at least 2 nucleotides away from the cassette" is meant to describe a nucleotide that is outside the cassette and at least 2 nucleotides away from the last nucleotide of the cassette. In the above example, the nucleotide "at least two nucleotides away from the cassette" would be any nucleotide selected from 5'-AAAAAAC. Similarly, the term "at least two nucleotides away from the 5' end of the cassette" is meant to describe a nucleotide that is outside the cassette and at least two nucleotides away from the last nucleotide at the 5' end of the cassette. In the above example, the last nucleotide at the 5' end of the cassette is a "T," and the nucleotide "at least two nucleotides away from the 5' end of the cassette" would be any nucleotide selected from 5'-AAAAAAC.

[0488] The internal position may not be located between the second and penultimate nucleotides of the cassette. The internal position may be any position in the linear double-stranded DNA molecule other than the last nucleotide on the 3' and 5' ends of the sense and antisense strands and other than the nucleotide located between the second and penultimate nucleotides of the cassette. The internal position may be located outside the cassette and at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50, 75, or 100 nucleotides away from the 3' and / or 5' ends of each strand of the linear double-stranded DNA molecule, and / or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50, 75, or 100 nucleotides away from the 3' and / or 5' ends of each strand of the cassette (i.e., outside the cassette). Preferably, the internal positions are located outside the cassette and at least 6, at least 8, or at least 10 nucleotides away from the 3' and / or 5' ends of each strand of the linear double-stranded DNA molecule, and / or at least 6, at least 8, or at least 10 nucleotides away from the 3' and / or 5' ends of each strand of the cassette (i.e., at least 6, at least 8, or at least 10 nucleotides outside the cassette). Preferably, the cassette does not contain a phosphorothioated nucleotide at any one of positions 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 12, 1 to 14, 1 to 16, 1 to 18, or 1 to 20 of the sense and / or antisense strands away from the 3' and / or 5' ends of the cassette (i.e., outside the cassette). Preferably, the internal position in each strand is located at least 6 nucleotides from the end of the linear double-stranded DNA molecule and is not located between the second and penultimate nucleotides of the cassette. Preferably, the internal position in each strand is located at least 10 nucleotides from the end of the linear double-stranded DNA molecule and is not located between the second and penultimate nucleotides of the cassette.Preferably, the internal position in each strand is located at least 6 nucleotides away from the end of the linear double-stranded DNA molecule and at least 6 nucleotides away from the end of the cassette (i.e., at least 6 nucleotides outside the cassette). Preferably, the internal position in each strand is located at least 10 nucleotides away from the end of the linear double-stranded DNA molecule and at least 10 nucleotides away from the end of the cassette (i.e., at least 10 nucleotides outside the cassette). The linear double-stranded DNA molecule can contain a first phosphorothioated nucleotide at an internal position other than the last nucleotide on the 3'-end and 5'-end of the sense strand and antisense strand, as long as this position is located at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, or 50 nucleotides outside the cassette. For example, the linear double-stranded DNA molecule can comprise a first phosphorothioated nucleotide that is at least the 6th, 8th, or 10th nucleotide counting from the 3'-end and / or 5'-end of the sense strand and / or antisense strand, as long as these positions are not located between the second and penultimate nucleotide of the cassette. Preferably, the linear double-stranded DNA molecule can comprise a first phosphorothioated nucleotide that is at least the 6th, 8th, or 10th nucleotide counting from the 3'-end and / or 5'-end of the sense strand and / or antisense strand, as long as these positions are located outside the cassette. The linear double-stranded DNA molecule can include a first phosphorothioated nucleotide that is at least the 6th, 8th, or 10th nucleotide counting from the 3' end and / or 5' end of the sense and / or antisense strand, so long as these positions are located at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, or 50 nucleotides outside the cassette.Preferably, the linear double-stranded DNA molecule can include a first phosphorothioated nucleotide that is at least the 6th, 8th, or 10th nucleotide counting from the 3'-end and / or 5'-end of the sense and / or antisense strand, so long as these positions are located at least 6, 8, or 10 nucleotides outside the cassette.

[0489] A linear double-stranded DNA molecule can contain at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 protected (e.g., phosphorothioated) nucleotides at internal positions in each strand. Preferably, a linear double-stranded DNA molecule contains at least two protected (e.g., phosphorothioated) nucleotides at internal positions in each strand.

[0490] The position of the phosphorothioated nucleotides in the sense strand of the linear double-stranded DNA molecule can be such that one or more phosphorothioated nucleotides are located upstream of the cassette (i.e., toward the 5' end of the sense strand of the DNA molecule).

[0491] In the sense strand of a linear double-stranded DNA molecule, (a) one of the at least two phosphorothioated nucleotides can be the 5' terminal nucleotide of the cassette; and / or (b) One of the at least two phosphorothioated nucleotides can be in the first region of the sense strand, where the first region of the sense strand is 5' of the cassette.

[0492] The position of the phosphorothioated nucleotides in the sense strand of the linear double-stranded DNA molecule can be such that one or more phosphorothioated nucleotides are located downstream of the cassette (i.e., toward the 3' end of the sense strand of the DNA molecule).

[0493] In the sense strand of a linear double-stranded DNA molecule, (a) one of the at least two phosphorothioated nucleotides can be the 3' terminal nucleotide of the cassette; and / or (b) One of the at least two phosphorothioated nucleotides can be in the second region of the sense strand, where the second region of the sense strand is 3' of the cassette.

[0494] The position of the phosphorothioated nucleotides in the antisense strand of the linear double-stranded DNA molecule can be such that one or more phosphorothioated nucleotides are located upstream of the cassette (i.e., toward the 5' end of the antisense strand of the DNA molecule).

[0495] In the antisense strand of a linear double-stranded DNA molecule, (a) one of the at least two phosphorothioated nucleotides can be the 5' terminal nucleotide of the cassette; and / or (b) One of the at least two phosphorothioated nucleotides can be in the first region of the antisense strand, where the first region of the antisense strand is 5' to the cassette.

[0496] The position of the phosphorothioated nucleotides in the antisense strand of the linear double-stranded DNA molecule can be such that one or more phosphorothioated nucleotides are located downstream of the cassette (i.e., toward the 3' end of the antisense strand of the DNA molecule).

[0497] In the antisense strand of a linear double-stranded DNA molecule, (a) one of the at least two phosphorothioated nucleotides can be the 3' terminal nucleotide of the cassette; and / or (b) One of the at least two phosphorothioated nucleotides can be in the second region of the antisense strand, where the second region of the antisense strand is 3' to the cassette.

[0498] The positions of the phosphorothioated nucleotides in both the sense and antisense strands of the linear double-stranded DNA molecule can be such that at least one phosphorothioated nucleotide is located downstream of the cassette and at least one phosphorothioated nucleotide is located upstream of the cassette in each strand.

[0499] In a linear double-stranded DNA molecule, (a) in the sense strand, one of the at least two phosphorothioated nucleotides can be the 5' terminal nucleotide of the cassette and / or one of the at least two phosphorothioated nucleotides can be in a first region of the sense strand, wherein the first region of the sense strand is 5' of the cassette; (b) in the sense strand, one of the at least two phosphorothioated nucleotides can be the 3' terminal nucleotide of the cassette and / or one of the at least two phosphorothioated nucleotides can be in a second region of the sense strand, wherein the second region of the sense strand is 3' of the cassette; (c) in the antisense strand, one of the at least two phosphorothioated nucleotides can be the 5' terminal nucleotide of the cassette and / or one of the at least two phosphorothioated nucleotides can be in the first region of the antisense strand, wherein the first region of the antisense strand is 5' of the cassette; and (d) In the antisense strand, one of the at least two phosphorothioated nucleotides can be the 3' terminal nucleotide of the cassette, and / or one of the at least two phosphorothioated nucleotides can be in the second region of the antisense strand, where the second region of the antisense strand is 3' of the cassette.

[0500] The term "first region of the sense strand," as used herein, is intended to encompass a portion of the sense strand of a linear double-stranded DNA molecule that is between the 5' end of the linear double-stranded DNA molecule and the first 5' nucleotide of the cassette in the sense strand. For example, in the sense strand sequence 5'-AAAAAACATAAAA-3' (SEQ ID NO: 3), where the cassette begins with nucleotide "T" in the 5'-3' direction, the term "first region of the sense strand" refers to the 5'-AAAAAACA-3' region.

[0501] The term "first region of the antisense strand" is intended to encompass a portion of the antisense strand of a linear double-stranded DNA molecule that is between the 5' end of the linear double-stranded DNA molecule and the first 5' nucleotide of the cassette in the antisense strand. For example, in the antisense strand sequence 5'-AAAAAACATAAAA-3' (SEQ ID NO: 3), where the cassette begins with nucleotide "T" in the 5'-3' direction, the term "first region of the antisense strand" refers to the 5'-AAAAAACA-3' region.

[0502] The term "second region of the sense strand" is intended to encompass a portion of the sense strand of a linear double-stranded DNA molecule that is between the 3' end of the linear double-stranded DNA molecule and the first 3' nucleotide of the cassette in the sense strand. For example, in the sense strand sequence 5'-AAAAAACATAAAA-3' (SEQ ID NO: 3), where the cassette begins with nucleotide "T" in the 3'-5' direction, the term "second region of the sense strand" refers to the 5'-AAAA-3' region.

[0503] The term "second region of the antisense strand" is intended to encompass a portion of the antisense strand of a linear double-stranded DNA molecule that is between the 3' end of the linear double-stranded DNA molecule and the first 3' nucleotide of the cassette in the antisense strand. For example, in the antisense strand sequence 5'-AAAAAACATAAAA-3' (SEQ ID NO: 3), where the cassette begins with nucleotide "T" in the 3'-5' direction, the term "second region of the antisense strand" refers to the 5'-AAAA-3' region.

[0504] A linear double-stranded DNA molecule can comprise multiple phosphorothioated nucleotides upstream of the cassette (i.e., toward the 5' end of the sense strand of the DNA molecule).For example, a linear double-stranded DNA molecule can comprise at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphorothioated nucleotides upstream of the cassette.Preferably, at least two phosphorothioated nucleotides upstream of the cassette.Therefore, the position of phosphorothioated nucleotides in the sense strand of a linear double-stranded DNA molecule can be such that at least two phosphorothioated nucleotides are located upstream of the cassette.

[0505] In the sense strand of a linear double-stranded DNA molecule, (a) one of the at least two phosphorothioated nucleotides can be the 5' terminal nucleotide of the cassette and one of the at least two phosphorothioated nucleotides can be in a first region of the sense strand, wherein the first region of the sense strand is 5' of the cassette; or (b) At least two phosphorothioated nucleotides can be in the first region of the sense strand, where the first region of the sense strand is 5' of the cassette.

[0506] The linear double-stranded DNA molecule can comprise multiple phosphorothioated nucleotides downstream of the cassette (i.e., toward the 3' end of the DNA molecule).For example, the linear double-stranded DNA molecule can comprise at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphorothioated nucleotides downstream of the cassette, preferably at least two phosphorothioated nucleotides downstream of the cassette.Therefore, the position of the phosphorothioated nucleotides in the sense strand of the linear double-stranded DNA molecule can be such that at least two phosphorothioated nucleotides are located downstream of the cassette.

[0507] In the sense strand of a linear double-stranded DNA molecule, (a) one of the at least two phosphorothioated nucleotides can be the 3' terminal nucleotide of the cassette and one of the at least two phosphorothioated nucleotides can be in a second region of the sense strand, wherein the second region of the sense strand is 3' of the cassette; or (b) At least two phosphorothioated nucleotides can be in the first region of the sense strand, where the first region of the sense strand is 3' of the cassette.

[0508] The position of the phosphorothioated nucleotides in the antisense strand of the linear double-stranded DNA molecule can be such that at least two of the phosphorothioated nucleotides are located upstream of the cassette (i.e., toward the 5' end of the antisense strand of the DNA molecule).

[0509] In the antisense strand of a linear double-stranded DNA molecule, (a) one of the at least two phosphorothioated nucleotides is the 5' terminal nucleotide of the cassette and one of the at least two phosphorothioated nucleotides is in a first region of the antisense strand, wherein the first region of the antisense strand is 5' of the cassette; or (b) at least two phosphorothioated nucleotides are in the first region of the antisense strand, where the first region of the antisense strand is 5' to the cassette;

[0510] The position of the phosphorothioated nucleotides in the antisense strand of the linear double-stranded DNA molecule can be such that at least two of the phosphorothioated nucleotides are located downstream of the cassette (i.e., toward the 3' end of the antisense strand of the DNA molecule).

[0511] In the antisense strand of a linear double-stranded DNA molecule, (a) one of the at least two phosphorothioated nucleotides can be the 3' terminal nucleotide of the cassette and one of the at least two phosphorothioated nucleotides can be in a second region of the antisense strand, wherein the second region of the antisense strand is 3' of the cassette; or (b) At least two phosphorothioated nucleotides can be in the second region of the antisense strand, where the second region of the antisense strand is 3' to the cassette.

[0512] Each strand of linear double-stranded DNA molecule can contain multiple phosphorothioated nucleotides.For example, linear double-stranded DNA molecule can contain at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphorothioated nucleotides upstream and downstream of cassette.The position of phosphorothioated nucleotides in both sense strand and antisense strand of linear double-stranded DNA molecule can be such that in each strand, at least two phosphorothioated nucleotides are located downstream of cassette, and at least two phosphorothioated nucleotides are located upstream of cassette.

[0513] In a linear double-stranded DNA molecule, (a) In the sense strand, i. one of the at least two phosphorothioated nucleotides can be the 5' terminal nucleotide of the cassette and one of the at least two phosphorothioated nucleotides can be in a first region of the sense strand, wherein the first region of the sense strand is 5' of the cassette; or ii. at least two phosphorothioated nucleotides may be in the first region of the sense strand, wherein the first region of the sense strand is 5' of the cassette; (b) In the sense strand, i. one of the at least two phosphorothioated nucleotides can be the 3' terminal nucleotide of the cassette and one of the at least two phosphorothioated nucleotides can be in a second region of the sense strand, wherein the second region of the sense strand is 3' of the cassette; or ii. at least two phosphorothioated nucleotides may be in the second region of the sense strand, wherein the second region of the sense strand is 3' of the cassette; (c) In the antisense strand, i. one of the at least two phosphorothioated nucleotides can be the 5' terminal nucleotide of the cassette and one of the at least two phosphorothioated nucleotides can be in a first region of the antisense strand, wherein the first region of the antisense strand is 5' of the cassette; or ii. at least two phosphorothioated nucleotides may be in the first region of the antisense strand, wherein the first region of the antisense strand is 5' of the cassette; and (d) in the antisense strand, i. one of the at least two phosphorothioated nucleotides can be the 3' terminal nucleotide of the cassette and one of the at least two phosphorothioated nucleotides can be in a second region of the antisense strand, wherein the second region of the antisense strand is 3' of the cassette; or ii. At least two phosphorothioated nucleotides can be in the second region of the antisense strand, where the second region of the antisense strand is 3' of the cassette.

[0514] The amount of phosphorothioated nucleotides at the internal position in each strand can depend on the length of linear double-stranded DNA product.Therefore, linear double-stranded DNA product can comprise phosphorothioated nucleotides with the ratio of phosphorothioated nucleotides to total nucleotides being at least 0.0001, at least 0.0025, at least 0.01, at least 0.025, at least 0.05, at least 0.075, at least 0.10, at least 0.12, at least 0.15, at least 0.25, at least 0.35, at least 0.5 or at least 0.75.Linear double-stranded DNA product can comprise phosphorothioated nucleotides with the ratio of phosphorothioated nucleotides to total nucleotides being less than 1, less than 0.9, less than 0.8, less than 0.65, less than 0.5, less than 0.4, less than 0.3, less than 0.2, less than 0.1, less than 0.075 or less than 0.05. The linear double-stranded DNA product can contain phosphorothioated nucleotides at a ratio of phosphorothioated nucleotides to total nucleotides of 0.0001 to 1, 0.0025 to 0.75, 0.025 to 0.65, 0.025 to 0.15, or 0.25 to 0.50. Preferably, the ratio of phosphorothioated nucleotides to total nucleotides is 0.025 to 0.15. The linear double-stranded DNA product can contain phosphorothioated nucleotides at a ratio of phosphorothioated nucleotides to total nucleotides of 0.005 to 0.3, 0.0075 to 0.2, 0.01 to 0.15, 0.01 to 0.10, 0.02 to 0.08, 0.03 to 0.07, 0.04 to 0.06, or 0.05 to 0.075. Preferably, the ratio of phosphorothioated nucleotides to total nucleotides is 0.01 to 0.10. Preferably, the ratio of phosphorothioated nucleotides to total nucleotides is about 0.02, about 0.025, about 0.05, about 0.075, about 0.08, about 0.10, about 0.12, about 0.15, or about 0.25. Even more preferably, the ratio of phosphorothioated nucleotides to total nucleotides is about 0.025 or about 0.05.As shown in the examples, a ratio of phosphorothioated nucleotides to total nucleotides of about 0.025 is sufficient to provide enhanced resistance to exonuclease digestion. As shown in the examples, a ratio of phosphorothioated nucleotides to total nucleotides of about 0.05 or about 0.075 is particularly effective in providing enhanced resistance to exonuclease digestion (as shown by the level of expression; see Examples 7 and 8). Furthermore, as shown in the examples, a ratio of about 0.05 is particularly suitable for in vivo expression of a gene of interest (see Example 10).

[0515] The amount of phosphorothioated nucleotides at internal positions in each strand can be different.For example, if linear double-stranded DNA product comprises a total of 1000 nucleotides, and the ratio of phosphorothioated nucleotides to total nucleotides is 0.1, linear double-stranded DNA product will comprise 100 phosphorothioated nucleotides, of which 75 will be located in the sense strand, and 25 will be located in the antisense strand of DNA product.For example, 100% of the phosphorothioated nucleotides in linear double-stranded DNA product can be located in the sense strand.Alternatively, 100% of the phosphorothioated nucleotides in linear double-stranded DNA product can be located in the antisense strand.Sense strand can comprise at least 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% of the total number of phosphorothioated nucleotides in linear double-stranded DNA product. The antisense strand can comprise at least 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% of the total number of phosphorothioated nucleotides in linear double-stranded DNA product.The sense strand and the antisense strand can comprise the same number of phosphorothioated nucleotides.Preferably, each of the sense strand and the antisense strand comprises 50% of the total number of phosphorothioated nucleotides in linear double-stranded DNA product.

[0516] Linear double-stranded DNA molecules or closed linear DNA molecules (e.g., covalently closed linear DNA molecules) may be resistant to nuclease digestion or may have improved or enhanced resistance to nuclease digestion. Linear double-stranded DNA molecules or closed linear DNA molecules may be resistant to exonuclease digestion or may have improved or enhanced resistance to exonuclease digestion. Linear double-stranded DNA molecules or closed linear DNA molecules may be resistant to digestion by exonucleases that cleave the 3'-terminal nucleotide (e.g., exonuclease III) and / or exonucleases that cleave the 5'-terminal nucleotide (e.g., exonuclease VIII), or have improved or enhanced resistance to it. The terms "improved" or "enhanced," in the context of resistance to enzymatic digestion, refer to a higher resistance to enzymatic digestion compared to DNA molecules not produced by the methods described herein. For example, in the context of a linear double-stranded DNA molecule, as compared to a molecule that does not contain protected nucleotides such as phosphorothioated nucleotides.

[0517] A linear DNA molecule (eg, a linear double-stranded DNA molecule) or a closed linear DNA molecule (eg, a covalently closed linear DNA molecule) can contain a functional moiety.

[0518] The functional moiety can be at the 3'-end and / or 5'-end (or terminal region) of the polynucleotide chain. The functional moiety can be linked or attached to the 5'-overhang, 3'-overhang, and / or blunt end of a linear DNA molecule (e.g., a linear double-stranded DNA molecule) or a closed linear DNA molecule. The functional moiety can be linked or attached to the 5'-overhang, 3'-overhang, and / or blunt end of a linear DNA molecule (e.g., a linear double-stranded DNA molecule) or a closed linear DNA molecule by covalent or non-covalent bonds or by nucleic acid hybridization. The functional moiety can be linked or attached to the linear DNA molecule (e.g., a linear double-stranded DNA molecule) or a closed linear DNA molecule directly or indirectly (e.g., via a linker molecule). The functional moieties can be linked or attached by being bound to a linear DNA molecule (e.g., a linear double-stranded DNA molecule) or a closed linear DNA molecule, and / or by being bound or annealed to a linker molecule that is bound to the linear DNA molecule (e.g., a linear double-stranded DNA molecule) or a closed linear DNA molecule. The linker molecule can be a biopolymer (e.g., a nucleic acid molecule) or a synthetic polymer. The linker molecule can contain one or more units of ethylene glycol and / or poly(ethylene)glycol (e.g., hexaethylene glycol or pentaethylene glycol).

[0519] The DNA molecule can comprise two functional portions: a first functional portion at the 3' end of the polynucleotide chain and a second functional portion at the 5' end of the polypeptide chain. The first functional portion can be linked or attached to the 5' overhang, 3' overhang, and / or blunt end of the 3' end of the DNA molecule. The second functional portion can be linked or attached to the 5' overhang, 3' overhang, and / or blunt end of the 5' end of the DNA molecule. The two functional portions can be the same or different. For example, the first functional portion can be a barcode for facilitating detection and / or sequencing of the DNA molecule, and the second functional portion can be a nuclear targeting sequence.

[0520] The functional moiety can be a probe. As used herein, the term "probe" refers to a fragment of DNA, RNA, or DNA / RNA chimera of various lengths (e.g., 3 to 1,000 bases) used to detect the presence of a target nucleotide sequence complementary to the sequence in the probe. Typically, a probe hybridizes to a single-stranded nucleic acid whose base sequence allows probe-target base pairing due to complementarity between the probe and the target. Thus, the functional moiety can be a DNA sequence, an RNA sequence, or a DNA / RNA chimera sequence. As used herein, the term "complementary" refers to the pairing of nucleotide sequences according to the Watson-Crick pairing rules. For example, the sequence 5'-GCGGTCCCA-3' has a complementary sequence of 5'-TGGGACCGC-3'. The complementary sequence can also be an RNA sequence complementary to a DNA sequence.

[0521] The functional part can be a binding molecule. The term "binding molecule" refers to any molecule that can bind to the DNA product described herein and / or can bind to an additional molecule or target. The binding molecule can be a protein, polypeptide, or peptide. The binding molecule can be an antibody, for example, a monoclonal antibody or a polyclonal antibody. The binding molecule can be an antibody fragment.

[0522] The functional moiety can facilitate detection of the DNA molecule by binding to a capture molecule (e.g., a capture antibody bound by a protein-protein interaction). The functional moiety can bind to a cellular target, e.g., a cellular receptor.

[0523] The functional moiety can be a label. Thus, the sense strand of the DNA molecule can contain a label at the 5'-end or 3'-end for detection. Alternatively or additionally, the antisense strand of the DNA molecule can contain a label at the 5'-end or 3'-end for detection. A "label" can be any chemical entity that allows the detection of a double-stranded nucleic acid molecule through physical, chemical, and / or biological means. The label can be a chromophore, a fluorophore, and / or a radioactive molecule.

[0524] To facilitate detection and / or quantification of the DNA molecule, the functional moiety can include a fluorophore, a radioactive compound, or a barcode.

[0525] A signal corresponding to the presence, absence, and / or level of a DNA molecule can be measured using a barcode. The barcode can include at least one binding moiety linked to a barcoding portion, where the barcoding portion includes at least one nucleotide (i.e., the barcoding portion includes a nucleotide sequence at least one nucleotide long), and the binding moiety can bind to the 3' overhang, 5' overhang, or blunt end of the DNA molecule. The binding moiety can bind to the 3' and / or 5' end of the DNA molecule. The signal can be measured by determining the presence, absence, and / or level of the barcoding portion of the barcode (e.g., by sequencing or PCR). The barcoding portion can include at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. The barcode can include at least two binding moieties (e.g., a first binding moiety and a second binding moiety). For example, a first binding moiety linked to a first barcoded moiety can bind to the 3' end of a DNA molecule, and a second binding moiety linked to a second barcoded moiety can bind to the 5' end of a DNA molecule. The 3' or 5' end can include a 3' overhang, a 5' overhang, or a blunt end.

[0526] A signal corresponding to the presence, absence, and / or level of a DNA molecule can be measured using a fluorophore (i.e., a fluorescently labeled molecule) linked or attached to the 3' overhang, 5' overhang, or blunt end of the linear molecule. The signal can be measured by flow cytometry and / or fluorescence-activated cell sorting.

[0527] The functional moiety can also facilitate DNA sequencing. For example, the functional moiety can be a sequencing adaptor. The term "sequencing adaptor" is intended to encompass one or more nucleic acid domains comprising at least a portion of a nucleic acid sequence (or its complement) utilized by a sequencing platform of interest, such as a sequencing platform provided by Illumina® (e.g., HiSeq™, MiSeq™, and / or Genome Analyzer™ sequencing systems), Oxford Nanopore™ Technologies (e.g., MinION sequencing system), Ion Torrent™ (e.g., Ion PGM™ and / or Ion Proton™ sequencing system), Pacific Biosciences (e.g., PACBIO RS II sequencing system); Life Technologies™ (e.g., SOLiD sequencing system), Roche (e.g., 454 GS FLX+ and / or GS Junior sequencing system), or any other sequencing platform of interest.

[0528] 1. Pharmaceutical Composition The present invention provides pharmaceutical compositions comprising the nanoparticles (e.g., non-viral transfection complexes) described herein.The present invention provides pharmaceutical compositions comprising the nanoparticles (e.g., non-viral transfection complexes) described herein and a pharmaceutically suitable carrier or excipient.

[0529] The present invention provides (a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer The present invention provides a pharmaceutical composition comprising a nanoparticle (e.g., a non-viral transfection complex), the nanoparticle comprising:

[0530] The present invention provides i.(a) Cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer a nanoparticle (e.g., a non-viral transfection complex) comprising: ii. A pharmaceutically suitable carrier The present invention provides a pharmaceutical composition comprising:

[0531] The pharmaceutical composition comprises i. The nanoparticle (e.g., non-viral transfection complex) of any one of embodiments 1-215; and ii. A pharmaceutically suitable carrier may include:

[0532] The pharmaceutical composition comprises i.(a) Closed linear DNA molecule; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer a nanoparticle (e.g., a non-viral transfection complex) comprising: ii. A pharmaceutically suitable carrier may include:

[0533] The pharmaceutical composition comprises (a) a linear DNA molecule containing one or more nuclease-resistant nucleotides (e.g., phosphorothioated nucleotides); (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer a nanoparticle (e.g., a non-viral transfection complex) comprising: Pharmaceutically Suitable Carriers may include:

[0534] The pharmaceutical composition comprises (a) a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides (e.g., phosphorothioated nucleotides) in the open-ended region adjacent to the second end; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer a nanoparticle (e.g., a non-viral transfection complex) comprising: Pharmaceutically Suitable Carriers may include:

[0535] The pharmaceutical composition comprises (a)mRNA molecule; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) steroid lipids; and (e) Cationic polymer a nanoparticle (e.g., a non-viral transfection complex) comprising: Pharmaceutically Suitable Carriers may include:

[0536] In all embodiments described herein, the presence of a pharmaceutically suitable carrier is optional. The nanoparticles of the present invention can function as a pharmaceutically suitable carrier.

[0537] In all embodiments described herein, the lipid component may be one or more of DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5 (ionizable lipid), DOTMA, DTDTMA, or DHDTMA (cationic lipid).

[0538] The phospholipid may comprise DOPE, DOPC, DSPC, DPPC, DMPC or POPC. Preferably, the phospholipid is DOPE.

[0539] The steroid lipid may be one or more of cholesterol or its derivatives (cholesterol derivatives) such as β-sitosterol, fucosterol, campesterol, stigmastanol (alkyl steroids), secosteroids (vitamins D2, D3) or pentacyclic steroids.

[0540] The cationic polymer can comprise oligolysine (linear or branched), e.g., K16, K17, or K30, oligohistidine (linear or branched), or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine, and oligoarginine, or PEI. Preferably, the cationic polymer is an oligolysine (K16, K17, or K30) containing 16, 17, or 30 lysine residues.

[0541] The pharmaceutical composition can be formulated as a pill, tablet or capsule combined with one or more pharmaceutically acceptable solid carriers, or as a solution in one or more pharmaceutically acceptable solvents, or as an emulsion, suspension or dispersion in one or more pharmaceutically acceptable solvents or carriers.The formulation can also contain other pharmaceutically acceptable excipients, such as stabilizers, antioxidants, binders, coloring agents, or emulsifiers or taste modifiers, and sustained-release formulations.

[0542] The pharmaceutical composition can be administered orally, topically, parenterally, transdermally, intraarticularly, by IV or by inhalation.The pharmaceutical composition can be administered by injection or intravenous infusion using a suitable sterile solution.The topical dosage form can be a cream, ointment, patch, or similar vehicle suitable for transdermal and topical dosage forms.

[0543] Pharmaceutical compositions can be dissolved or suspended in a liquid medium or formulated as granules (small particles or grains), pellets (small sterile solid masses of highly pure composition, with or without excipients, produced by forming granules or by compressing and molding), or sustained-release coated pellets (solid dosage forms in which the composition itself is in the form of granules, coated with a variable amount of coating, and which release the composition in a manner that allows for reduced dosing frequency when compared to compositions presented in conventional dosage forms).

[0544] Other forms of pharmaceutical compositions include pills (small, round, solid dosage forms containing the composition intended for oral administration), powders (an intimate mixture of dry, finely divided composition with one or more pharmaceutically acceptable excipients that may be intended for internal or external use), elixirs (clear, flavored, sweetened, hydroalcoholic liquids containing the dissolved composition; intended for oral use), chewing gums (sweetened, flavored, insoluble, plastic substances of various shapes that release the composition in the mouth when chewed), syrups (oral solutions containing the composition and a high concentration of sucrose or other sugars; this term also refers to sweetened, viscous liquids, including oral suspensions). These include tablets (which have also been used to include any other liquid dosage form prepared in a vehicle), tablets (solid dosage forms containing the composition with or without a suitable diluent), chewable tablets (solid dosage forms containing the composition with or without a suitable diluent that are intended to produce a pleasant-tasting residue in the mouth upon chewing, which residue is easily swallowed and does not leave a bitter or unpleasant aftertaste), coated or delayed-release tablets, dispersible tablets, effervescent tablets, extended-release tablets, film-coated tablets, or film-coated extended-release tablets, where the tablet is formulated in such a way that the contained composition is available for an extended period of time after ingestion.

[0545] In other forms of pharmaceutical compositions, tablets for liquid solutions, tablets for suspensions, multilayer tablets, sustained-release multilayer tablets, which are formulated in a manner that allows at least a reduction in the frequency of administration compared to compositions presented in conventional dosage forms, can be provided. Orally disintegrating tablets, orally disintegrating sustained-release tablets, soluble tablets, sugar-coated tablets, osmotic agents, etc. are also suitable.

[0546] Orally administered pharmaceutical compositions can contain, in addition to the composition, one or more inactive pharmaceutical ingredients, such as diluents, solubilizers, alcohols, binders, controlled-release polymers, enteric polymers, disintegrants, excipients, colorants, flavorings, sweeteners, antioxidants, preservatives, dyes, additives, fillers, suspending agents, surfactants (e.g., anionic, cationic, amphoteric, and nonionic), etc. A variety of FDA-approved topical inactive ingredients can be found in the FDA's "Inactive Ingredients Database," which includes inactive ingredients specifically intended as such by the manufacturer.

[0547] The pharmaceutical composition can be administered by injection (e.g., intravenous or intramuscular injection). The pharmaceutical composition can be formulated as an emulsion consisting of a sterile, pyrogen-free preparation or a lipid-complex preparation.

[0548] For example, pharmaceutical compositions can be administered by intratympanic injection (e.g., into the middle ear) and / or injection into the outer, middle, and / or inner ear. Such methods are routinely used in the art, for example, for the administration of steroids and antibiotics into the human ear. Injection can be, for example, via the round window of the ear or via the cochlear capsule.

[0549] The pharmaceutical composition can be administered by intra-articular injection (i.e., into the joint). Such a method is routinely used in the art, for example, for administering hydrocortisone into the joint.

[0550] Other forms of pharmaceutical compositions include powder for solution injection, which is a sterile preparation intended for reconstitution to form a solution for parenteral use; powder for suspension injection, which is a sterile preparation intended for reconstitution to form a suspension for parenteral use; lyophilized powder for liposomal suspension injection, which is a sterile freeze-dried preparation intended for reconstitution for parenteral use, formulated in a manner that allows for the formation of liposomes (lipid bilayer vesicles usually composed of phospholipids used to encapsulate compositions either within the lipid bilayer or in an aqueous space) upon reconstitution; or lyophilized powder for solution injection, where lyophilization ("freeze-drying") is a process involving the removal of water from a product in a frozen state at extremely low pressure.

[0551] In another mode of administration, the pharmaceutical composition can be administered in situ via a catheter or pump, which can, for example, direct the composition into the selected tissue where the pharmaceutical composition is to be administered.

[0552] Parenteral delivery systems (intravenous or intramuscular) include one or more pharmaceutically suitable excipients, such as solvents and cosolvents, solubilizing agents, wetting agents, suspending agents, thickening agents, emulsifying agents, chelating agents, buffers, pH adjusters, antioxidants, reducing agents, antimicrobial preservatives, bulking agents, protectants, osmolality adjusters, and special additives. Formulations suitable for parenteral administration conveniently comprise a sterile oily or aqueous preparation of the composition, preferably isotonic with the blood of the recipient, although this is not required.

[0553] As used herein, inhalation dosage forms include, but are not limited to, aerosols (products filled under pressure and containing a composition intended for topical application to the skin and for localized application within the nose (intranasal aerosols), mouth (lingual and sublingual aerosols), or lungs (inhalation aerosols) that are released upon actuation of an appropriate valve system). Foam aerosols are dosage forms containing a composition, surfactant, an aqueous or non-aqueous liquid, and a propellant that release a stable foam when the propellant is in the internal (discontinuous) phase (i.e., oil-in-water), or a spray or rapidly disintegrating foam when the propellant is in the external (continuous) phase (i.e., water-in-oil). Metered-dose aerosols are pressurized dosage forms consisting of a metered-dose valve that allows the delivery of a uniform amount of spray with each actuation. Powder aerosols are products containing a composition in powder form that are filled under pressure and released upon actuation of an appropriate valve system. Aerosol sprays are aerosol products that utilize compressed gas as a propellant to provide the necessary pressure to eject the product as a wet spray and are applicable to solutions of the composition in aqueous solvents.

[0554] Transdermal dosage forms include, but are not limited to, patches (drug delivery systems, often including an adhesive backing, that are typically applied to an external site on the body, through which ingredients (including compositions) are passively diffused through or actively transported from portions of the patch, and depending on the patch, ingredients (including compositions) are delivered to or within the body.) Various types of transdermal patches, such as matrix, reservoir, and others, are known in the art.

[0555] Topical dosage forms include various dosage forms known in the art, such as lotions (emulsion liquid dosage forms whereby the dosage form is generally for external application to the skin), enhanced lotions (lotion dosage forms whereby enhanced does not refer to the concentration of the composition in the dosage form, and the delivery of the composition is enhanced), gels (semi-solid dosage forms containing a gelling composition that provides rigidity to a solution or colloidal dispersion whereby the gel may contain suspended particles), and ointments (semi-solid dosage forms whereby the dosage form is generally for external application to the skin or mucous membranes, and usually containing less than 20% water and volatile materials and more than 50% hydrocarbons, waxes, or polyols as a vehicle). Further embodiments include enriched ointments (whereby enriched does not refer to the concentration of the composition in the formulation, but rather an ointment formulation that enhances composition delivery), creams (whereby the formulation is generally for external application to the skin or mucous membranes, an emulsion semisolid formulation that typically contains more than 20% water and volatile materials and / or less than 50% hydrocarbons, waxes, or polyols that may also be used as vehicles), and enriched creams (whereby enriched does not refer to the concentration of the composition in the formulation, but rather a cream formulation that enhances composition delivery). As used herein, "emulsion" means a formulation consisting of a two-phase system composed of at least two immiscible liquids, one of which is dispersed as droplets, an internal or dispersed phase, within the other liquid, an external or continuous phase, generally stabilized with one or more emulsifiers, whereby emulsion is used as the formulation term unless a more specific term is applicable (e.g., cream, lotion, ointment). Further embodiments include suspensions (liquid dosage forms containing solid particles dispersed in a liquid medium), sustained-release suspensions, pastes (semi-solid dosage forms containing a large proportion, 20-50%, of finely dispersed solids in a fatty medium whereby the dosage form is generally for external application to the skin or mucous membranes), solutions (clear, homogeneous liquid dosage forms containing one or more chemicals dissolved in a solvent or mutually miscible solvents), and powders.

[0556] Topical dosage forms of compositions contain one or more inactive pharmaceutical ingredients, such as compositions and excipients, colorants, pigments, additives, fillers, emollients, surfactants (e.g., anionic, cationic, amphoteric, and nonionic), penetration enhancers (e.g., alcohols, fatty alcohols, fatty acids, fatty acid esters, and polyols), etc. A variety of FDA-approved topical inactive ingredients can be found in the FDA's "Inactive Ingredients Database," which includes inactive ingredients specifically intended as such by the manufacturer.

[0557] 2. Use and Application General Therapeutic and Diagnostic Uses The present invention provides nanoparticles as described herein for use in therapy.

[0558] The present invention also provides pharmaceutical compositions described herein for use in therapy.

[0559] The nanoparticles can include nucleic acid cargoes as described herein that encode sequences for therapeutic proteins, portions of vaccines, or elements of genetic engineering mechanisms used to treat disease or infection in a subject.

[0560] The nanoparticles described herein or the pharmaceutical compositions described herein can be used in the treatment of a disease or disorder, which can be a disease or illness caused by a pathogen, or cancer, or a viral infection (e.g., coronavirus (e.g., COVID-19), herpes simplex virus type 2, HIV, influenza, and / or measles).

[0561] Preferably, the nanoparticles described herein or the pharmaceutical compositions described herein can be used to treat pathogens or cancers / tumors using cancer antigens or spike proteins, coats of pathogens.

[0562] The nanoparticles described herein or the pharmaceutical composition described herein can be used as a medicament. The present invention provides the nanoparticles described herein or the pharmaceutical composition described herein for use in the manufacture of a medicament for the prevention of a condition caused by a pathogen or cancer in a subject by stimulating an immune response.

[0563] The present invention provides a method for treating a disease or disorder caused in a subject by a pathogen or cancer, comprising administering to the subject the nanoparticles described herein or the pharmaceutical composition described herein. Preferably, the amount of nanoparticles or pharmaceutical composition administered to the subject is a therapeutically active amount. Thus, the present invention also provides the nanoparticles described herein or the pharmaceutical composition described herein, which can be used in cancer therapy.

[0564] The subject to be treated with the nanoparticles described herein or the pharmaceutical compositions described herein can be administered the nanoparticles described herein or the pharmaceutical compositions described herein together with other forms of treatment for the disorder involved, including treatment with drugs commonly used for the treatment of the disorder.The drug can be administered in one or several dosage units.Those skilled in the art (e.g., physicians) can fully determine the appropriate dosage regimen for the subject according to the specific condition of the subject.

[0565] As used herein, "administering" means introducing a nanoparticle described herein, or a pharmaceutical composition described herein, into the body of a subject, as described in more detail above (see "Pharmaceutical Compositions"). Examples include, but are not limited to, oral, topical, buccal, sublingual, pulmonary, transdermal, transmucosal, as well as subcutaneous, intraperitoneal, intravenous, and intramuscular injection, or in the form of a liquid or solid dose via the digestive tract.

[0566] As used herein, the phrase "therapeutically active amount" refers to an amount of nanoparticles described herein or pharmaceutical compositions described herein that, when administered to a subject for treating a disease, is sufficient to achieve such treatment of the disease. A "therapeutically active amount" will vary depending on factors such as the specific product used, the severity of the subject's disease, the age and relative health of the subject, and the route and form of administration. It is routine for a person skilled in the art (e.g., a physician) to determine the relevant therapeutically active amount for a particular subject based on such factors. Treatment of a disease as described herein should be understood to mean an improvement in one or more of the symptoms of the disease.

[0567] The present invention also provides the use of a nanoparticle as described herein, or a pharmaceutical composition as described herein, in a method of diagnosing a disease and / or disorder.

[0568] The present invention provides the use of the nanoparticles described herein, or the pharmaceutical compositions described herein, in the "in vitro" diagnosis of disease.

[0569] The present invention also provides a nanoparticle as described herein, or a pharmaceutical composition as described herein, for use in a method for the "in vivo" diagnosis of disease.

[0570] The nanoparticles described herein or the pharmaceutical compositions described herein can be used to diagnose a disease or disorder. The disease or disorder can be a viral infection (e.g., coronavirus (e.g., COVID-19), herpes simplex virus type 2, HIV, influenza, and / or measles), or a cancer, such as breast cancer, colon cancer, ALL, AML, bone cancer, bladder cancer, cervical cancer, CLL, CML, gastric cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, skin cancer, or testicular cancer.

[0571] The diagnostic and therapeutic methods described herein can be in vitro or in vivo methods.

[0572] Methods of diagnosis may rely on the detection and / or quantification of the nanoparticles described herein, or the pharmaceutical compositions described herein.

[0573] To facilitate detection and / or quantification of the nanoparticles described herein, the nanoparticles can be linked or bound to a functional moiety. For example, the functional moiety can be a probe. The functional moiety can include a fluorophore, a radioactive compound, or a barcode. The functional moiety can be a protein, such as an antibody.

[0574] Diagnosis can rely on detecting a signal corresponding to the presence, absence, and / or level of nanoparticles. For example, the signal can be measured by flow cytometry and / or fluorescence-activated cell sorting of nanoparticles linked to fluorescent probes.

[0575] The nanoparticles can be detected by binding to a capture moiety, for example, in a lateral flow assay. In this example, the functional moiety is a protein, e.g., an antibody specific for the capture moiety. Capture of the antibody linked to the nanoparticle can result in a visual signal (e.g., a band of a different color).

[0576] The present invention also provides a method for combining disease diagnosis with disease treatment.

[0577] cell therapy The nanoparticles described herein, or the pharmaceutical compositions described herein, can be used in cell therapy.

[0578] The present invention provides the use of the nanoparticles described herein or the pharmaceutical composition described herein in the manufacture of cell therapy. The present invention also provides a method of cell therapy, comprising contacting the nanoparticles described herein or the pharmaceutical composition described herein with cells. Preferably, the cell therapy is ex vivo cell therapy. The cells can be animal cells, preferably mammalian cells, such as human cells.

[0579] The present invention also provides cells obtainable by any of the methods described herein. For example, the cells may be suitable for use in cell therapy.

[0580] vaccine The products of the present invention are particularly suitable for use in vaccine production.The nanoparticles described herein, the cationic polymers described herein, or the pharmaceutical compositions described herein can be used to produce vaccines, preferably mRNA-based vaccines.For example, vaccines such as BioNTech and Moderna mRNA vaccines against COVID-19.

[0581] The nanoparticles described herein, or the pharmaceutical compositions described herein, can be used as a vaccine.

[0582] The nanoparticles described herein, the cationic polymers described herein, or the pharmaceutical compositions described herein can be used in therapeutic or prophylactic immunization.

[0583] The nanoparticles described herein, or the pharmaceutical compositions described herein, can be used in the therapeutic treatment of cancer, such as in neoantigen vaccines.

[0584] CRISPR delivery Nanoparticles and cationic polymers are particularly suitable for use in delivering CRISPR machinery to cells, for example, in in vivo therapy.

[0585] The nanoparticles can include a cargo that is a nucleic acid cargo (e.g., a nucleic acid cargo described herein). The nucleic acid cargo can include (or encode) a repair template (or editing template). The repair template (or editing template) can be for editing a genome, for example, using a CRISPR-Cas system. The repair template (or editing template) can include or consist of a homology region (e.g., a homology arm) that is homologous to a desired DNA region (i.e., a target molecule). The repair template (or editing template) can be for use in CRISPR-Cas-mediated homology-directed repair (HDR). The repair template (or editing template) can be used to repair a target molecule that has a strand break (e.g., a single-strand break or a double-strand break). The strand break can be created by a nuclease (e.g., Cas9, Cpf1, or MAD7) of the CRISPR system. The repair template (or editing template) is capable of introducing at least one mutation (eg, an insertion, deletion, and / or substitution) into a desired DNA region (ie, a target molecule). The repair template (or editing template) can comprise at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000, at least 5500, at least 6000, at least 6500, at least 7000, at least 7500, at least 8000, at least 8500, at least 9000, at least 9500, 10000, at least 11000, at least 12000, at least 13000, at least 14000, or at least 15000 base pairs.

[0586] Thus, the present invention provides nanoparticles for use in delivering a CRISPR system to a cell. The present invention provides a method of delivering a CRISPR system to a cell, comprising contacting a nanoparticle with the cell.

[0587] The cell may be an animal cell, preferably a mammalian cell, such as a human cell.

[0588] The present invention also provides cells obtainable by the methods described herein. The cells are particularly suitable for use in cell therapy and / or in vivo therapy.

[0589] The use of the products described herein can be in vivo or in vitro use.

[0590] 3. Methods for producing nanoparticles The present invention provides a method for producing (or forming) the nanoparticles described herein, the method comprising: (a) contacting a lipid component, a phospholipid, a steroid lipid, with a linear DNA molecule and a cationic polymer that is resistant to nuclease (e.g., exonuclease) digestion; and (b) forming nanoparticles; Includes:

[0591] The step of contacting the lipid component, phospholipid, steroid lipid with the linear DNA molecule and cationic polymer can be carried out in a single step or in multiple steps.

[0592] The present invention provides a method for producing (or forming) the nanoparticles described herein, the method comprising: (a) contacting a lipid component, a phospholipid, a steroid lipid, with a closed linear DNA molecule and a cationic polymer to form a single continuous aqueous volume; and (b) forming nanoparticles; Includes:

[0593] The present invention also provides a method for producing (or forming) the nanoparticles described herein, the method comprising: (a) contacting a lipid component, a phospholipid, a steroid lipid, with a linear DNA molecule containing one or more protected nucleotides and a cationic polymer to form a single continuous aqueous volume; and (b) forming nanoparticles; Includes:

[0594] The present invention also provides a method for producing (or forming) the nanoparticles described herein, the method comprising: (a) contacting a lipid component, a phospholipid, a steroid lipid with a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, the partially closed linear DNA molecule comprising one or more nuclease-resistant nucleotides in the open-ended region adjacent to the second end; and a cationic polymer to form a single contiguous aqueous volume; and (b) forming nanoparticles; Includes:

[0595] The present invention provides a method for producing (or forming) the nanoparticles described herein, the method comprising: (a) contacting a lipid component, a phospholipid, a steroid lipid, with a closed linear DNA molecule and a cationic polymer; and (b) forming nanoparticles; Includes:

[0596] The present invention also provides a method for producing (or forming) the nanoparticles described herein, the method compr...

Claims

1. (a) cargo; (b) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (c) phospholipids; (d) a steroid lipid; and (e) Cationic polymer Nanoparticles comprising:

2. The nanoparticle of claim 1 , which does not comprise a targeting moiety or a targeting peptide.

3. 3. The nanoparticle of claim 1 or claim 2, further comprising a PEG lipid.

4. The nanoparticle of any one of claims 2 to 3, which is a non-viral transfection complex.

5. The nanoparticle of claim 1 , wherein the cargo is RNA, which may be mRNA.

6. 5. The nanoparticle of claim 1, wherein the cargo is DNA, and the DNA may be (i) a closed linear DNA molecule; (ii) a linear deoxyribonucleic acid (DNA) molecule comprising one or more nuclease-resistant nucleotides and a cassette, wherein the one or more nuclease-resistant nucleotides in the linear DNA molecule are located outside the cassette; or (iii) a partially closed linear deoxyribonucleic acid (DNA) molecule comprising a double-stranded DNA portion that is closed at a first end and open at a second end, wherein the partially closed linear DNA molecule comprises one or more nuclease-resistant nucleotides in the open-end region adjacent to the second end.

7. The nanoparticle of claim 1 , wherein the cationic polymer is a polycationic peptide.

8. The nanoparticle of claim 7 , wherein the polycationic peptide comprises a nucleic acid-binding cationic moiety, which may be an oligolysine.

9. 9. The nanoparticle of any one of claims 1 to 8, wherein the lipid component comprises one or more of DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, lipid A9, 9A1P9, lipid C24, lipid LP01, lipid 5, DOTMA, DTDTMA, or DHDTMA.

10. 10. The nanoparticles of any one of claims 1 to 9, wherein the cationic polymer comprises oligolysine (linear or branched), such as K16, K17 or K30, oligohistidine (linear or branched) or oligoarginine (linear or branched), or a combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine and oligoarginine, or PEI.

11. A pharmaceutical composition comprising nanoparticles according to any one of claims 1 to 10 and an appropriate pharmaceutically suitable carrier, which may be a vaccine.

12. Nanoparticles according to any one of claims 1 to 10 or a pharmaceutical composition according to claim 11 for use in therapy.

13. (a) contacting a cell with the nanoparticles of any one of claims 1 to 10 or the pharmaceutical composition of claim 11; and (b) transfecting the nanoparticles or pharmaceutical composition into cells; 1. A method for transfecting a cell, comprising:

14. A library comprising two or more nanoparticles according to any one of claims 1 to 10, wherein the polydispersity index (PDI) of the nanoparticles in the library is less than 0.2, preferably less than 0.

15.

15. (a) contacting (i) a cargo; (ii) a lipid component, the lipid component being one or more ionizable lipids and / or one or more cationic lipids; (iii) a phospholipid; (iv) a steroid lipid; and (v) a cationic polymer; and (b) forming nanoparticles 11. A method for forming nanoparticles according to any one of claims 1 to 10, comprising:

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