Lipid nanoparticles containing DNA-binding proteins

Abstract

The present disclosure relates to lipid nanoparticles and uses thereof for delivering DNA, wherein the lipid nanoparticles contain therein a DNA-binding protein or peptide bound to DNA.

Description

[Technical Field]

[0001] Related application data This application claims priority to U.S. Patent Application No. 63 / 371,914, entitled "Lipid nanoparticle comprising a DNA-binding protein," filed August 19, 2022, the entire contents of which are incorporated herein by reference. The present disclosure relates to lipid nanoparticles and uses thereof for delivering DNA, wherein the lipid nanoparticles contain therein a DNA-binding protein or peptide bound to DNA. [Background technology]

[0002] Nucleic acid therapeutics, including DNA vaccines and gene therapy, are promising approaches for treating and preventing various diseases by targeting genetic "blueprints" in vivo. Clinical application of DNA-based therapeutics relies on the use of delivery vehicles that improve stability, promote internalization of DNA into the nucleus, and increase target affinity. Efficient DNA delivery requires translocation through both the cytoplasm and nuclear membranes to the nucleus. This is a major limitation of current DNA and gene therapy approaches.

[0003] Existing nonviral delivery systems, such as lipid nanoparticles, which effectively deliver RNA, do not work well for DNA delivery due to the large size of the delivered molecules and the need to pass through the nuclear envelope. DNA delivery has traditionally required the use of viral vectors or physical DNA delivery via electroporation or needleless injection systems. However, these methods only allow delivery of short lengths of linear DNA (approximately 200–300 base pairs) into the nucleus.

[0004] In addition to the physical barriers to DNA delivery, current non-viral lipid delivery vehicles are composed of cationic lipids and other ionizable lipid components, such as neutral lipids, cholesterol, and PEGylated lipids. Cationic lipids are amphiphilic molecules with a lipophilic region containing one or more hydrocarbon groups and a hydrophilic region containing at least one positively charged polar head group. Cationic lipids and nucleic acids form a positively charged complex, which facilitates the nucleic acid's passage through the cell membrane.

[0005] However, several adverse cytotoxic effects of cationic lipids are known, including the generation of reactive oxygen species and their accumulation in plasma due to insufficient degradation in humans. Therefore, much effort has been devoted to identifying novel lipids or specific lipid compositions that can protect nucleic acids from degradation and removal and provide effective intracellular delivery and / or DNA expression / transcription. Furthermore, these lipid-nucleic acid particles must be well tolerated and provide an appropriate therapeutic index so that treatment with an effective dose of nucleic acid does not involve unacceptable toxicity and / or risks to patients.

[0006] It will therefore be apparent to those skilled in the art that there is a continuing need for improved delivery systems for delivering nucleic acids, and in particular, improved delivery vehicles for delivering DNA therapeutics and gene therapy. Summary of the Invention

[0007] The present inventors have determined that incorporating a DNA-binding protein or peptide, or a lipidated DNA-binding protein or peptide, into lipid nanoparticles can increase the stability of the associated DNA, promote the nucleation of the lipid nanoparticles, and / or reduce the toxicity and / or adverse side effects of the lipid nanoparticles. In some embodiments of the present disclosure, the present inventors have determined that incorporating a DNA-binding protein with a nuclear localization sequence enables the uptake of DNA into the nucleus. The present inventors have also determined that incorporating a DNA-binding protein or peptide into lipid nanoparticles can prevent toll-like receptor (TLR) stimulation / induction. Based on these findings, the present inventors have created reagents and methods for DNA delivery, e.g., non-viral delivery of DNA into cells, e.g., within a subject.

[0008] Thus, the present disclosure provides lipid nanoparticles for delivering DNA, the lipid nanoparticles comprising a DNA-binding protein or peptide therein that is bound to DNA.

[0009] In one example, the DNA binding protein or peptide is a lipidated DNA binding protein or peptide.

[0010] Thus, the present disclosure provides lipid nanoparticles for delivering DNA, the lipid nanoparticles comprising a lipidated DNA-binding protein or peptide therein that is bound to DNA.

[0011] In one example, the DNA-binding protein or peptide is lipidated before binding to the DNA, hi another example, the DNA-binding protein or peptide is lipidated after binding to the DNA.

[0012] In one example, the DNA binding protein or peptide is lipidated with a lipid moiety selected from the group consisting of fatty acids, isoprenoids, and combinations thereof.

[0013] In one example, DNA binding protein or peptide is lipidated with fatty acid.For example, fatty acid is triglyceride, phospholipid or cholesteryl ester.In one example, fatty acid is triglyceride.In another example, fatty acid is phospholipid.In another example, fatty acid is cholesteryl ester.

[0014] In one example, the DNA-binding protein or peptide is lipidated with an isoprenoid, for example, the isoprenoid is isoprene.

[0015] In one example, the DNA-binding protein or peptide is lipidated on a nucleophilic side chain at the N-terminus and / or C-terminus.

[0016] In one example, the DNA-binding protein or peptide is lipidated on a nucleophilic side chain. For example, it is lipidated on a cysteine, serine, threonine, tyrosine, and / or lysine amino acid residue. In one example, the nucleophilic side chain is a cysteine residue. In another example, the nucleophilic side chain is a serine residue. In a further example, the nucleophilic side chain is a threonine residue. In one example, the nucleophilic side chain is a tyrosine residue. In another example, the nucleophilic side chain is a lysine residue.

[0017] In one example, the DNA-binding protein or peptide is lipidated at the N-terminus of the protein or peptide.

[0018] It will be apparent to one skilled in the art that the N-terminus of the DNA binding protein or peptide may comprise a nuclear localization signal(s) (or sequence) and / or a nuclear export signal.

[0019] In one example, the DNA-binding protein or peptide is lipidated at the C-terminus of the protein or peptide.

[0020] In one example, the DNA-binding protein or peptide is lipidated by palmitoylation, myristoylation, fatty acid acylation, esterification, prenylation, or a combination thereof.

[0021] In one example, the DNA-binding protein or peptide is lipidated by palmitoylation, e.g., by N-terminal cysteine palmitoylation.

[0022] In one example, the DNA-binding protein or peptide is lipidated by myristoylation, e.g., by N-terminal glycine myristoylation.

[0023] In one example, the DNA-binding protein or peptide is lipidated by fatty acid acylation, e.g., by lysine N-acylation, or in another example, by serine O-acylation.

[0024] In one example, the DNA-binding protein or peptide is lipidated by esterification, e.g., by C-terminal cholesterol esterification.

[0025] In one example, the DNA-binding protein or peptide is lipidated by prenylation. For example, the prenylation is farnesylation or geranylgeranylation. In one example, the prenylation is cysteine prenylation.

[0026] In one example, the DNA-binding protein or peptide is lipidated by N-terminal cysteine palmitoylation, N-terminal glycine myristoylation, lysine N-acylation, C-terminal cholesterol esterification, cysteine prenylation, serine O-acylation, or a combination thereof.

[0027] In one example, the lipid moiety is linked to the DNA-binding protein or peptide by a thioether bond, an ester bond, a thioester bond, and / or an amide bond.

[0028] In one example, the lipid moiety is linked to the DNA-binding protein or peptide by a thioether bond.

[0029] In one example, the lipid moiety is linked to the DNA-binding protein or peptide by an ester bond.

[0030] In one example, the lipid moiety is linked to the DNA-binding protein or peptide by a thioester bond.

[0031] In one example, the lipid moiety is linked to the DNA-binding protein or peptide by an amide bond.

[0032] In one example, the DNA-binding protein or peptide is lipidated using chemical lipidation or enzymatic lipidation. For example, the DNA-binding protein or peptide is lipidated using chemical lipidation. In one example, the chemical lipidation is selected from the group consisting of chemical ligation, click chemistry, expressed protein ligation, and combinations thereof. In another example, the DNA-binding protein or peptide is lipidated using enzymatic lipidation. For example, the enzymatic lipidation is selected from the group consisting of sortase A-mediated lipidation, transglutaminase-mediated lipidation, and combinations thereof. In one example, the enzymatic lipidation is performed in vivo or in vitro. For example, the enzymatic lipidation is performed in vivo. In another example, the enzymatic lipidation is performed in vitro.

[0033] In one example, DNA binding protein or peptide directly binds to DNA.In another example, DNA binding protein or peptide binds to DNA before DNA is formulated into lipid nanoparticles.In another example, DNA binding protein or peptide binds to DNA in lipid nanoparticles after DNA is formulated into lipid nanoparticles, where DNA binding protein or peptide is in lipid nanoparticles.For example, DNA binding protein or peptide binds to the DNA that is encapsulated in lipid nanoparticles.

[0034] In one example, the DNA-binding protein or peptide further binds to the DNA on the surface of the lipid nanoparticle.In this situation, the DNA-binding protein or peptide is present both within the lipid nanoparticle and on the surface of the lipid nanoparticle.The DNA-binding protein or peptide on the surface of the lipid nanoparticle does not need to be the same as the DNA-binding protein or peptide within the lipid nanoparticle.

[0035] For example, lipid nanoparticles can be formed with a DNA-binding protein or peptide that binds to DNA therein, and the formed lipid nanoparticles can then be coated with a DNA-binding protein or peptide to bind to any unencapsulated and / or partially encapsulated DNA.

[0036] In one example, the DNA-binding protein or peptide encapsulates DNA. In another example, the DNA-binding protein or peptide binds to nucleophilic side chains at the N-terminus and / or C-terminus of DNA. In one example, the DNA-binding protein or peptide binds to nucleophilic side chains at the DNA. In another example, the DNA-binding protein or peptide binds at the 5'-terminus and / or 3'-terminus of DNA. For example, it binds at the 5'-terminus of DNA. In another example, it binds at the 3'-terminus of DNA. For example, the DNA-binding protein or peptide does not encapsulate DNA. In one example, the DNA binding protein or peptide is a) reduce the toxicity of lipid nanoparticles; b) stabilizes DNA; c) promotes uptake through the nuclear membrane; d) protects DNA from degradation; e) promoting the nucleation of lipid nanoparticles; f) increasing the immunogenicity of the DNA, and / or g) inhibiting the induction of signal transduction by one or more Toll-like receptors.

[0037] In one example, the DNA-binding protein or peptide reduces the toxicity of the lipid nanoparticle.

[0038] In one example, the DNA binding protein or peptide stabilizes the DNA.

[0039] In one example, the DNA binding protein or peptide promotes integration through the nuclear membrane.

[0040] In one example, the DNA binding protein or peptide protects DNA from degradation.

[0041] In one example, DNA-binding proteins or peptides promote the nucleation of lipid nanoparticles.

[0042] In one example, the DNA binding protein or peptide increases the immunogenicity of the DNA.

[0043] In one example, the DNA binding protein or peptide inhibits the induction of signaling by one or more Toll-like receptors. In one example, the DNA binding protein or peptide does not inhibit the induction of signaling by one or more Toll-like receptors.

[0044] Those skilled in the art will appreciate that there are a series of Toll-like receptors, namely endosomal Toll-like receptors, including TLR-3, TLR-7, TLR-8, and TLR-9, that recognize and bind nucleic acids such as DNA. Activation of these receptors results in the production of proinflammatory cytokines and type I interferons (interferon type I).

[0045] In one example, the DNA-binding protein or peptide inhibits the induction of signaling by one or more endosomal Toll-like receptors. For example, the DNA-binding protein or peptide inhibits the induction of signaling by one or more Toll-like receptors selected from the group consisting of TLR-3, TLR-7, TLR-8, and TLR-9. In one example, the DNA-binding protein or peptide inhibits the induction of signaling by TLR-3. In another example, the DNA-binding protein or peptide inhibits the induction of signaling by TLR-7. In another example, the DNA-binding protein or peptide inhibits the induction of signaling by TLR-9. In a further example, the DNA-binding protein or peptide inhibits the induction of signaling by TLR-8.

[0046] In one example, the DNA-binding protein or peptide is two DNA-binding proteins or peptides (i.e., a first and a second DNA-binding protein or peptide) linked by a linker. For example, the first and second DNA-binding proteins or peptides are covalently linked by an amide bond. The present disclosure encompasses other forms of covalent and non-covalent bonds. For example, the DNA-binding protein or peptide can be linked by a chemical linker.

[0047] In one example, the linker is a flexible linker, such as a flexible peptide linker, e.g., a first DNA-binding protein or peptide is linked to a second DNA-binding protein via a flexible linker.

[0048] In one example, the linker is a peptide linker. For example, a first DNA-binding protein or peptide is linked to a second DNA-binding protein or peptide via a linker, where the linker is a peptide linker of 2 to 31 amino acids in length. In one example, the linker has the sequence (Gly4Ser) nwhere n is 1 to 6. For example, the linker comprises the sequence SGGGGS (GS6) or the sequence SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGGS(GS31). In another example, the linker comprises the sequence (Ala) n where n is 2 to 31.

[0049] In one example, the linker is a rigid linker. For example, the rigid linker has the sequence (EAAAK) n where n is 1 to 3. In one example, the rigid linker comprises (EAAAK) n where n is 1 to 10 or about 1 to 100. For example, n is at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10. In one example, n is less than 100. For example, n is less than 90, or less than about 80, or less than about 60, or less than about 50, or less than about 40, or less than about 30, or less than about 20, or less than about 10.

[0050] In one example, the DNA binding protein or peptide is a viral or non-viral DNA binding protein or peptide.

[0051] In one example, the DNA-binding protein or peptide is a viral DNA-binding protein. For example, the viral DNA-binding protein or peptide is derived from a class I, class II, and / or class VII virus. In one example, the viral DNA-binding protein or peptide is derived from a class I virus. In another example, the viral DNA-binding protein or peptide is derived from a class II virus. In a further example, the viral DNA-binding protein or peptide is derived from a class VII virus.

[0052] In one example, the viral DNA binding protein or peptide is derived from a respiratory virus selected from the group consisting of influenza virus, respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, coronavirus, adenovirus, and bocavirus.

[0053] In one example, the viral DNA-binding protein or peptide is derived from an influenza virus, for example, the influenza virus is influenza A. In another example, the influenza virus is influenza B.

[0054] In one example, the viral DNA binding protein or peptide is selected from adenovirus, herpesvirus, poxvirus, adeno-associated virus, geminivirus, bacteriophage, parvovirus, heparnavirus, hepadnavirus, circoviridae virus, and papovaviridae virus.

[0055] In one example, the viral DNA binding protein or peptide is derived from an adenovirus virus. For example, the adenovirus is a human adenovirus. In another example, the adenovirus is an avian adenovirus. In another example, the adenovirus is adenovirus type 5 (Ad5). In this example, the viral DNA binding protein is protein VII derived from Ad5.

[0056] In one example, the viral DNA-binding protein or peptide is derived from a herpesvirus. For example, the herpesvirus is herpes simplex virus-1 (HSV-1). In another example, the herpesvirus is HSV-2. In a further example, the herpesvirus is varicella-zoster virus (VZV). In one example, the herpesvirus is Epstein-Barr virus (EBV). In another example, the herpesvirus is cytomegalovirus (CMV). In yet another example, the herpesvirus is roseolovirus. In a further example, the herpesvirus is Kaposi's sarcoma-associated herpesvirus (KSHV). In a still further example, the herpesvirus is cercopithecine herpesvirus 1 (simian B virus). In one example, the herpesvirus is murine herpesvirus 68 (MHV-68).

[0057] In one example, the viral DNA-binding protein or peptide is derived from a poxvirus. For example, the poxvirus is a smallpox virus. In another example, the poxvirus is a vaccinia virus. In a further example, the poxvirus is a cowpox virus. In one example, the poxvirus is a monkeypox virus. In another example, the poxvirus is a rabbitpox virus. In yet another example, the poxvirus is an orf virus. In a further example, the poxvirus is a pseudopox virus. In a still further example, the poxvirus is a bovine papular stomatitis virus. In one example, the poxvirus is a tanapox virus. In a further example, the poxvirus is a yabasa tumor virus. In one example, the poxvirus is a molluscum contagiosum virus (MCV).

[0058] In one example, the viral DNA binding protein or peptide is derived from an adeno-associated virus.

[0059] In one example, the viral DNA-binding protein or peptide is derived from a geminivirus. For example, the geminivirus is a begomovirus. In another example, the geminivirus is a vecurtovirus. In a further example, the geminivirus is a capravirus. In one example, the geminivirus is a kurtovirus. In another example, the geminivirus is an elagrovirus. In yet another example, the geminivirus is a grablovirus. In a further example, the geminivirus is a mastrevirus. In a still further example, the geminivirus is a topovirus. In one example, the geminivirus is a turncurtovirus.

[0060] In one example, the viral DNA binding protein or peptide is derived from a bacteriophage.

[0061] In one example, the viral DNA-binding protein or peptide is derived from a parvovirus, for example, the parvovirus is a bocavirus.

[0062] In one example, the viral DNA binding protein or peptide is derived from a hepadnavirus. For example, the hepadnavirus is an avihepadnavirus. In another example, the hepadnavirus is an orthohepadnavirus. In one example, the viral DNA binding protein or peptide is derived from a hepadnavirus. For example, the hepadnavirus is a hepatitis B virus (HBV).

[0063] In one example, the viral DNA-binding protein or peptide is from a Circoviridae virus, for example, the Circoviridae virus is Beak and Feather Disease Virus (BFDV).

[0064] In one example, the viral DNA-binding protein or peptide is derived from a Papovaviridae virus. For example, the Papovaviridae virus is a human papillomavirus (HPV). In another example, the human papillomavirus is HPV16.

[0065] In one example, the viral DNA-binding protein or peptide is a nucleoprotein, a nonstructural protein, a matrix protein, and / or a nucleocapsid protein. For example, the viral DNA-binding protein or peptide is a nucleoprotein. In another example, the viral DNA-binding protein or peptide is a matrix protein. In a further example, the viral DNA-binding protein or peptide is a nucleocapsid protein. In another example, the viral DNA-binding protein or peptide is a nonstructural protein.

[0066] In one example, the viral DNA binding protein or peptide is a non-structural (NS) protein derived from herpesvirus.For example, the viral DNA binding protein or peptide is HSV-1.In one example, the HSV-1 DNA binding domain is a full-length binding domain.In another example, the HSV-1 DNA binding domain is a truncated binding domain.In a further example, the HSV-1 DNA binding domain is a modified binding domain.For example, the 3' end of the first HSV-1 DNA binding domain is linked to the 5' end of the second HSV-1 DNA binding domain.

[0067] In one example, the viral DNA-binding protein or peptide is a capsid protein from beak and feather disease virus. In one example, the viral DNA-binding protein or peptide is an L2 protein from HPV16. In one example, the viral DNA-binding protein or peptide is protein VII from Ad5.

[0068] In one example, the viral DNA-binding protein or peptide is a nucleoprotein, where the DNA-binding protein or peptide encapsulates the DNA, stabilizes the DNA, and inhibits the induction of signaling by one or more endosomal Toll-like receptors (e.g., TLR-3, TLR-7, TLR-8, and / or TLR-9).

[0069] In one example, the viral DNA-binding protein or peptide is a nucleocapsid, where the DNA-binding protein or peptide encapsulates the DNA, stabilizes the DNA, and inhibits the induction of signaling by one or more endosomal Toll-like receptors (e.g., TLR-3, TLR-7, TLR-8, and / or TLR-9).

[0070] In one example, the viral DNA-binding protein or peptide is a substrate protein, where the DNA-binding protein or peptide binds to and stabilizes DNA but does not inhibit the induction of signaling by one or more endosomal Toll-like receptors (e.g., TLR-3, TLR-7, TLR-8 and / or TLR-9).

[0071] In one example, the DNA-binding protein or peptide is a non-viral DNA-binding protein or peptide. For example, the DNA-binding protein or peptide is a non-viral protein or peptide derived from a cellular protein. In one example, the DNA-binding protein or peptide is derived from a cellular protein associated with cell proliferation, cell signaling, and / or antiviral pathways.

[0072] In one example, the cellular protein is selected from the group consisting of TAR DNA binding protein (TRBP), Y-box binding protein, Z-DNA binding protein, and combinations thereof.

[0073] In one example, the cellular protein is TAR DNA-binding protein (TRBP). In one example, the TRBP is TRBP43.

[0074] In one example, the cellular protein is a Y-box binding protein. In one example, the Y-box binding protein is Y-box binding protein 1.

[0075] In one example, the cellular protein is a Z-DNA binding protein. In one example, the cellular protein is Z-DNA binding protein 1.

[0076] In one example, the lipid nanoparticles further comprise PEG lipids, structured lipids, and / or neutral lipids. For example, the lipid nanoparticles further comprise PEG lipids, structured lipids, and neutral lipids. In another example, the lipid nanoparticles further comprise PEG lipids, structured lipids, or neutral lipids. In one example, the lipid nanoparticles further comprise PEG lipids, structured lipids, ionizable lipids, and / or neutral lipids. For example, the lipid nanoparticles further comprise PEG lipids, structured lipids, ionizable lipids, and neutral lipids. In another example, the lipid nanoparticles further comprise PEG lipids, structured lipids, ionizable lipids, or neutral lipids.

[0077] In one example, the lipid nanoparticle further comprises a PEG lipid, for example, the PEG lipid is selected from the group consisting of PEG-c-DMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE lipids, and combinations thereof.

[0078] In one example, the lipid nanoparticle further comprises a structured lipid, for example, the structured lipid is selected from the group consisting of cholesterol, campesterol, and combinations thereof.

[0079] In one example, the lipid nanoparticle further comprises a neutral lipid, for example, the neutral lipid is selected from the group consisting of DSPC, DOPE, DLPC, DMPC, DOPC, DPPC, and combinations thereof.

[0080] In one example, the lipid nanoparticle further comprises an ionizable lipid. For example, the ionizable lipid may be 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminopropane (DLin-DMA), or 2,2-dilinoleyl-4-dimethylaminopropane (DLin-DMA). N,N-dimethyl-3-aminopropane (DSDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA). ), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2S)), 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), 2,5-bis((9z,12z)-octadeca-9,12,Dien-1-yloxyl)benzyl-4-(dimethylamino)butanoate (LKY750), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (also known as heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate) (SM-102), 2-hexyldecanoic acid, 1,1'-[[(4-hydroxybutyl)imino]di-6,1-hexanediyl] ester (((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis( 2-hexyldecanoate) (ALC-0315), 4-(dimethylamino)-butanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl ester (DLin-MC3-DMA or MC3), ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester, and combinations thereof.

[0081] In one example, the lipid nanoparticles do not include ionizable lipids.

[0082] In one example, the lipid nanoparticles do not include cationic lipids.

[0083] In one example, the lipid nanoparticles comprise about 25 mol% to about 60 mol% of a compound ionizable lipid, about 2 mol% to about 25 mol% of a neutral lipid, about 18.5 mol% to about 60 mol% of a structural lipid, and about 0.2 mol% to about 10 mol% of a PEG-lipid, wherein the total mol% does not exceed 100%.

[0084] In one example, the lipid nanoparticles comprise about 30 mol% to about 50 mol% of a compound ionizable lipid, about 5 mol% to about 20 mol% of a neutral lipid, about 30 mol% to about 55 mol% of a structural lipid, and about 1 mol% to about 5 mol% of a PEG-lipid, wherein the total mol% does not exceed 100%.

[0085] In one example, the lipid nanoparticles comprise a compound of about 30 mol% to about 50 mol% ionizable lipid, about 5 mol% to about 20 mol% neutral lipid, about 30 mol% to about 55 mol% structural lipid, and about 1 mol% to about 5 mol% PEG-lipid, wherein the total mol% does not exceed 100%.

[0086] In one example, the lipid nanoparticles comprise about 40 mol% ionizable lipids, about 10 mol% neutral lipids, about 48 mol% structural lipids, and about 2.0 mol% PEG-lipids.

[0087] In one example, the lipid nanoparticles have an average particle size of about 80 nm to 200 nm. For example, the lipid nanoparticles have an average particle size of about 100 nm to 200 nm. In one example, the lipid nanoparticles have an average particle size of about 100 nm to 190 nm, or about 100 nm to 180 nm, or about 110 nm to 180 nm, or about 110 nm to 150 nm, or about 110 nm to 140 nm, or about 110 nm to 130 nm. For example, the lipid nanoparticles have an average particle size of about 125 nm. In one example, the lipid nanoparticles have an average particle size of about 150 to 200 nm. In one example, the lipid nanoparticles have an average particle size of about 160 to 200 nm. For example, the lipid nanoparticles have an average particle size of about 160 nm, or about 165 nm, or about 170 nm, or about 175 nm, or about 180 nm, or about 185 nm, or about 190 nm, or about 200 nm. In one example, the average particle size is determined by measuring the Z-average diameter of the lipid nanoparticles.

[0088] In one example, the lipid nanoparticles have a nitrogen-to-phosphate ratio of about 2 to about 10. For example, the lipid nanoparticles have a nitrogen-to-phosphate ratio of about 2, or about 2.5, or about 3, or about 3.5, or about 4, or about 4.5, or about 5, or about 5.5, or about 6, or about 6.5, or about 7, or about 7.5, or about 8, or about 8.5, or about 9, or about 9.5, or about 10. In one example, the lipid nanoparticles have a nitrogen-to-phosphate ratio of about 3. In another example, the lipid nanoparticles have a nitrogen-to-phosphate ratio of about 4.5. In a further example, the lipid nanoparticles have a nitrogen-to-phosphate ratio of about 6.

[0089] In one example, at least 50% of the RNA is encapsulated in the lipid nanoparticle. For example, at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% of the RNA is encapsulated in the lipid nanoparticle. In one example, at least 80% of the RNA is encapsulated. In another example, at least 85% of the RNA is encapsulated. It will be clear to those skilled in the art that the encapsulation efficiency (or encapsulation percentage) can be determined by measuring the escape or activity of the pharmaceutical composition or mRNA of the present disclosure using fluorescence (e.g., using RiboGreen) and / or electron micrographs.

[0090] In one example, the DNA is linear DNA. In one example, the DNA is non-linear DNA. For example, the non-linear DNA is circular DNA, such as a plasmid, or covalently closed DNA. For example, as described in WO2010 / 086626 or WO2012 / 017210. In one example, the circular DNA is a plasmid DNA. In another example, the DNA is covalently closed DNA.

[0091] The present disclosure also provides a composition comprising lipid nanoparticles of the present disclosure.For example, when the composition of the present disclosure is administered, it can deliver DNA to the nucleus of cell, for example, the cell in subject.For example, this composition can deliver DNA to the nucleus of cell, for example, the cell in subject, non-viral delivery.

[0092] The present disclosure also provides an immunogenic composition comprising the lipid nanoparticles of the present disclosure. For example, when the composition of the present disclosure is administered, it can induce an immune response in a subject. For example, administration of the composition induces a humoral immune response and / or a cellular immune response. In one example, the composition induces a humoral immune response in a subject. For example, the humoral immune response is an antibody-mediated immune response. In another example, the composition induces a cellular immune response. For example, the cellular immune response includes the activation of antigen-specific cytotoxic T cells.

[0093] The present disclosure also provides pharmaceutical compositions comprising an immunogenic composition of the present disclosure and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers suitable for use in the present disclosure will be apparent to those of skill in the art and / or are described herein.

[0094] The present disclosure also provides compositions, immunogenic compositions or pharmaceutical compositions of the present disclosure for use in therapy.In one example, the therapy is gene therapy.For example, the immunogenic compositions or pharmaceutical compositions of the present disclosure are suitable for use as vaccines.

[0095] In one example, the composition, immunogenic composition, or pharmaceutical composition of the present disclosure is supplied in a vial, hi another example, the immunogenic composition or pharmaceutical composition of the present disclosure is supplied in a syringe.

[0096] The present disclosure also provides a method for delivering DNA to the nucleus of a cell, the method comprises contacting the cell with lipid nanoparticles described herein.For example, the present disclosure provides a method for delivering DNA to a cell in a subject, the method comprises administering to the subject lipid nanoparticles, compositions or pharmaceutical compositions of the present disclosure.

[0097] As those skilled in the art will understand, DNA delivery to subject can be used for gene therapy.Therefore, the present disclosure provides a method for treating subject, comprising administering lipid nanoparticles, compositions or pharmaceutical compositions of the present disclosure to subject, wherein the DNA in lipid nanoparticles, compositions or pharmaceutical compositions of the present disclosure encodes therapeutic protein or antigen. [Brief explanation of the drawings]

[0098] [Figure 1] 1 is a graphical representation showing SDS-PAGE of four nuclear proteins from influenza A, beak and feather disease virus (BFDV), human papillomavirus (HPV), and hepatitis B (HBV). [Figure 2] 1 is a graphical representation showing HPLC-SEC of four nucleoproteins from influenza A, BFDV, HPV, and HBV. [Figure 3] 1 is a graphical representation showing EMSA of four nucleoproteins and ssDNA from influenza A, BFDV, HPV, and HBV. DETAILED DESCRIPTION OF THE INVENTION

[0099] General Throughout this specification, unless specifically stated otherwise or the context requires otherwise, references to a single step, a single composition, a group of steps, or a group of compositions should be interpreted as encompassing one and more (i.e., one or more) of that step, composition, step, or composition.

[0100] As will be apparent to those skilled in the art, the present disclosure is readily susceptible to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The present disclosure also includes all steps, features, compositions, and compounds referred to or indicated herein, individually or collectively, and any and all combinations of such steps or features, or any two or more thereof.

[0101] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for illustrative purposes only, and functionally equivalent objects, compositions, and methods are expressly intended to be within the scope of the present disclosure.

[0102] Any example of the present disclosure should be construed as applicable mutatis mutandis to any other example of the present disclosure, unless specifically stated otherwise.

[0103] Unless specifically defined otherwise, all technical and scientific terms used herein shall be understood to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0104] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in this disclosure are standard procedures, well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M.G. Lover and B.D.H. Memes (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M.A. Usubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988 (including all updates to date)), Ed. Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J.E. Coligan et al. al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates to date), and other sources.

[0105] The descriptions and definitions of variable regions and portions thereof, immunoglobulins, antibodies and fragments thereof herein may be further clarified by a discussion in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991; Bork et al., J. Mol. Biol. 242, 309-320, 1994; Chothia and Lesk J. Mol. Biol. 196:901-917, 1987; Chothia et al. Nature 342, 877-883, 1989; and / or Al-Lazikani et al., J. Mol. Biol. 273, 927-948, 1997.

[0106] Any reference herein to a protein or antibody should be understood to include any variations of the protein or antibody produced during production and / or storage. For example, during production and / or storage, an antibody may be deamidated (e.g., at asparagine or glutamine residues), and / or have altered glycosylation, and / or have glutamine residues converted to pyroglutamic acid, and / or have N- or C-terminal residues removed or "truncated," and / or have some or all of an incompletely processed signal sequence remaining at the end of the antibody. It is understood that a composition comprising a particular amino acid sequence may be a heterogeneous mixture of the described or encoded sequence and / or variants of the described or encoded sequence.

[0107] The term "and / or," e.g., "X and / or Y," shall be understood to mean either "X and Y" or "X or Y," and shall be interpreted as providing explicit support for both meanings or either meaning.

[0108] Throughout this specification the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0109] As used herein, the term "derived from" shall be construed to indicate that the specified item may be obtained from a particular source, but not necessarily directly from that source.

[0110] Selected Definitions As used herein, the term "lipid nanoparticle" or "LNP" refers to a lipid-based particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) and comprising a compound of any formula described herein. In some instances, LNPs are formulated into compositions for delivering polynucleotides to desired targets, such as cells, tissues, organs, or tumors. For example, lipid nanoparticles or LNPs can be selected from, but are not limited to, liposomes or vesicles in which the aqueous volume is encapsulated by an amphiphilic lipid bilayer (e.g., single, unilamellar, or multiple, multilamellar), micelle-like lipid nanoparticles with a non-aqueous core, and solid lipid nanoparticles (solid lipid nanoparticles lack a lipid bilayer).

[0111] As used herein, the term "lipidated" or "lipidation" refers to the process of covalently modifying a protein (i.e., a DNA-binding protein or peptide) with one or more lipids.

[0112] As used herein, the term "DNA-binding protein or peptide" or "DBP" shall be understood to refer to proteins and peptides that bind to double-stranded or single-stranded DNA and participate in the formation of deoxyribonucleoprotein complexes.

[0113] The term "protein" is intended to include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains linked to each other covalently or non-covalently (i.e., a polypeptide complex). For example, a series of polypeptide chains can be covalently linked by suitable chemical bonds or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0114] As used herein, the term "peptide" is intended to include compounds composed of amino acid residues linked by amide bonds. Peptides can be natural or non-natural, ribosomal encoded, or synthetically derived. Typically, peptides consist of 2 to 200 amino acids. For example, peptides can have lengths ranging from 10 to 20 amino acids, or 10 to 30 amino acids, or 10 to 40 amino acids, or 10 to 50 amino acids, or 10 to 60 amino acids, or 10 to 70 amino acids, or 10 to 80 amino acids, or 10 to 90 amino acids, or 10 to 100 amino acids, including any length within the above range(s).

[0115] As used herein, the term "recombinant" is understood to mean the product of artificial genetic recombination.

[0116] As used herein, the term "subject" shall be taken to mean any animal, including a human, e.g., a mammal. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, the subject is a human.

[0117] lipid nanoparticles The present disclosure provides lipid nanoparticles for delivering DNA, the lipid nanoparticles comprising a lipidated DNA-binding protein or peptide bound to DNA.

[0118] DNA-binding proteins or peptides The present disclosure provides lipid nanoparticles comprising DNA-binding proteins or peptides. For example, the present disclosure provides lipid nanoparticles comprising lipidated DNA-binding proteins or peptides.

[0119] DNA-binding proteins regulate many aspects of co-transcriptional and post-transcriptional gene expression, including, for example, DNA replication, DNA recombination, DNA modification, DNA repair, DNA organization / compaction, DNA storage, and DNA stabilization. DNA-binding proteins or peptides bind to double-stranded or single-stranded DNA and participate in the formation of deoxyribonucleoprotein complexes. Those skilled in the art will understand that DNA-binding proteins or peptides can be viral or non-viral proteins or peptides.

[0120] Non-viral DNA-binding proteins or peptides In one example, the DNA-binding protein is a non-viral protein or peptide derived from a cellular protein, for example, the DNA-binding protein or peptide is derived from a cellular protein associated with cell proliferation, cell signaling and / or antiviral pathways.

[0121] Non-viral DNA-binding proteins or peptides contain numerous structural motifs or DNA-binding domains that promote DNA binding, including, for example, the helix-turn-helix (HTH) motif, basic helix-loop-helix (bHLH) domain, zinc finger (ZnF) domain, leucine zipper (bZIP) domain, winged helix (WH), winged helix-turn-helix (wHTH) domain, β-sheet motif, β-hairpin / ribbon motif, high mobility group (HMG) domain, Wor3 domain, OB-fold domain, immunoglobulin domain, B3 domain, TAL effector, homeodomain motif, histone fold, AT-hook domain, TATA-binding protein (TBP) domain, histone-like protein (HU) motif, POU domain, Zn-containing motif, and receptor DNA-binding domain (DBD).

[0122] In one example, the DNA-binding protein or peptide comprises an HTH motif. For example, the DNA-binding protein or peptide comprising an HTH motif is selected from the group consisting of 1LMB, 1LLI, 1PER, 1RPE, 2OR1, 3CRO, 6CRO, 6CRO, 3ORC, 1WT, 1BDH, 1BDI, 1PNR, 2PUA, 2PUB, 2PUC, 2PUD, 2PUE, 2PUF, 2PUG, QVPW, 1QPZ, 1ZAY, 1FOK, 1GDT, 1HCR, 1IGN, 1PDN, 1TC3, 1TRR, 1DDN, 1D3U, 1VOL, and 1C9B.

[0123] In one example, the DNA binding protein or peptide comprises a bHLH motif. For example, the DNA binding protein or peptide comprising a bHLH motif is selected from the group consisting of 1AM9, 1HLO, 1AN4, 1AN2, 1MDY, and 1A0A.

[0124] In one example, the DNA-binding protein or peptide comprises a ZnF motif. For example, the DNA-binding protein or peptide comprising a ZnF motif is selected from the group consisting of 1AAY, 1ZAA, 2DRP, 1UBD, 1MEY, 1A1G, 1A1H, 1A1I, 1A1J, 1A1K, 1A1L, 2GLI, 2NLL, 1HCQ, 1GLU, 1LAT, 1BY4, 1CIT, 1A6Y, 1TSR, 1TUP, 1ZME, and 1D66.

[0125] In one example, the DNA binding protein or peptide comprises a bZIP motif. For example, the DNA binding protein or peptide comprising a bZIP motif is selected from the group consisting of 2DGC, 1DGC, 1YSA, and 1A02.

[0126] In one example, the DNA-binding protein or peptide comprises a WH motif. For example, the DNA-binding protein or peptide comprising a WH motif is selected from the group consisting of 2IRF, 1IF1, 2CGP, 1BER, 1CGP, 1RUN, 1RUO, 3HTS, 1CF7, 1BC8, 1BC7, 1PUE, and 1AWC.

[0127] In one example, the DNA-binding protein or peptide comprises a homeodomain motif, for example, the DNA-binding protein or peptide comprising a homeodomain motif is selected from the group consisting of 1FJ1, 1HDD, 1APL, 1YRN, 1AU7, 1OCT, 2HDD, 3HDD, 9ANT, 6PAX, 1AKH, 1B72, 1B8I, and 1MNM.

[0128] In one example, the DNA-binding protein or peptide comprises a beta-sheet motif. For example, the DNA-binding protein or peptide comprising a beta-sheet motif is selected from the group consisting of 1TYB, 1YTF, 1AIS, 1CDW, 1TGH, 1VOL, 1D3Y, and 1C9B.

[0129] In one example, the DNA binding protein or peptide comprises a β-hairpin / ribbon motif. For example, the DNA binding protein or peptide comprising a β-hairpin / ribbon motif is selected from the group consisting of 1CMA, 1ECR, 1IHF, 1XBR, 1AZP, 1BNZ, 1BF4, 1BDT, 1BDV, and 1PAR.

[0130] Viral DNA-binding proteins In one example, the DNA binding protein or peptide is a viral DNA binding protein or peptide, e.g., the DNA binding protein is a nucleoprotein, matrix protein, nucleocapsid protein, and / or nonstructural protein from a DNA virus.

[0131] Those skilled in the art will appreciate that viruses are classified according to the Baltimore classification system, as shown in Table 1, which is primarily based on the transcription of the viral genome. [Table 1]

[0132] In one example, the DNA binding protein or peptide is derived from a DNA virus, e.g., the DNA binding protein or peptide is derived from a class I, class II, and / or class VII virus.

[0133] In one example, the DNA virus is a Class I virus (i.e., a double-stranded DNA virus). Class I viruses include, for example, all viruses in the Duplodnaviridae, Adnaviria, and Validnaviria domains, all viruses in the Papovaviridae (Monodnaviridae domain) and Naldaviricetes families, all viruses in the Ampullaviridae, Bycaudaviridae, Clavaviridae, Fuseroviridae, Globuloviridae, Guttaviridae, Halspiviridae, Ovaliviridae, Plasmaviridae, Polydnaviridae, Portogloboviridae, and Thaspiviridae families, and all viruses in the Dinodnavirus and Rigidiovirus genera. Exemplary Class I viruses include, but are not limited to, adenoviruses (e.g., Ad5), herpesviruses, human papillomaviruses (e.g., HPV16), and poxviruses.

[0134] In one example, the DNA virus is a Class II virus (i.e., a single-stranded DNA virus). Class II viruses include, for example, viruses from the Monodnaviridae domain (excluding the Papovaviridae class) and the Anelloviridae, Circoviridae, Spiraviridae, and Finnlakeviridae families. Exemplary Class II viruses include, but are not limited to, beak and feather disease viruses, adeno-associated viruses, geminiviruses, bacteriophages, and parvoviruses.

[0135] In one example, the DNA virus is a Class VII virus (i.e., a single-stranded DNA virus with an RNA intermediate during its life cycle). Class VI viruses include, for example, viruses of the Caulimoviridae family (phylum Artverviricota). Exemplary Class VII viruses include, but are not limited to, heparnaviruses (e.g., hepatitis A virus (HAV)) and hepadnaviruses (e.g., hepatitis B virus, hepatitis C virus).

[0136] Linker In one example, the DNA binding protein or peptide comprises a first DNA binding protein or peptide and a second DNA binding protein or peptide linked via a linker, e.g., the linker is a linker peptide.

[0137] In one example, the linker is a flexible linker.

[0138] A "flexible" linker is an amino acid sequence that does not have a fixed structure (secondary or tertiary structure) in solution. Thus, such a flexible linker can freely adopt a variety of conformations. Flexible linkers suitable for use in the present disclosure are known in the art. One example of a flexible linker for use in the present disclosure is the linker sequence SGGGGS / GGGGS / GGGGS or (Gly4Ser)3. Another example of a flexible linker is an alanine linker (e.g., Ala n )

[0139] The linker may comprise any amino acid sequence that does not substantially interfere with the interaction of the DNA-binding protein or peptide with the DNA. Preferred amino acid residues for flexible linker sequences include, but are not limited to, glycine, alanine, serine, threonine, proline, lysine, arginine, glutamine, and glutamic acid.

[0140] The linker sequence between the DNA-binding proteins or peptides preferably comprises 5 or more amino acid residues. Flexible linker sequences according to the present disclosure consist of 5 or more residues, preferably 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more residues. In one example, the flexible linker sequence consists of 5, 7, 10, or 16 residues.

[0141] In one example, the linker is a rigid linker. A "rigid linker" (including a "semi-rigid linker") refers to a linker that has limited flexibility. For example, a relatively rigid linker may have the sequence (EAAAK) n where n is 1 to 3. The value of n can be 1 to about 10, or about 1 to 100. For example, n is at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10. In one example, n is less than 100. For example, n is less than 90, or less than about 80, or less than about 70, or less than about 60, or less than about 50, or less than about 40, or less than about 30, or less than about 20, or less than about 10. A rigid linker need not be completely devoid of flexibility.

[0142] Lipidation of DNA-binding proteins The present disclosure provides lipid nanoparticles comprising lipidated DNA-binding proteins or peptides.

[0143] It will be apparent to those skilled in the art that lipidation of a protein or peptide is the covalent attachment of a lipid moiety to a protein or peptide (ie, a DNA-binding protein or peptide).

[0144] lipids Lipid moieties suitable for use in the present disclosure will be apparent to those skilled in the art and include, for example, fatty acids, isoprenoids, and combinations thereof. In one example, the lipid moiety is selected from the group consisting of isoprenoids, triglycerides, phospholipids, cholesteryl esters, and combinations thereof.

[0145] Isoprenoids Isoprenoids, also known as terpenoids or prenol lipids, are branched lipids, a type of organic compound composed of two or more hydrocarbon units, each consisting of five carbon atoms arranged in a specific pattern. These five carbon units are called isoprenes, which are synthesized from a common intermediate known as mevalonic acid, which is itself synthesized from acetyl-CoA. Isoprenoids may have one or more functional chemical groups, such as hydroxyl or carbonyl, attached to their carbon skeleton, which constitute the diversity of isoprenoids. Isoprenoids are classified into monoterpenes (C 10 H 16 ), sesquiterpenes (C 15 H 24 ), diterpenes (C 20 H 32 ), triterpenes (C 30 H 48 ), tetraterpenes (C 40 H 64 ), or other polyterpenes (C5H8) n It can be classified as:

[0146] Suitable isoprenoids for use in the present disclosure will be apparent to those of skill in the art and / or are described herein.

[0147] In one example, the isoprenoid is a monoterpene. Exemplary monoterpenes include citronellol, citronellal, citral, geraniol, metol, pseudoionone, and β-ionone.

[0148] In one example, the isoprenoid is a sesquiterpene. Exemplary sesquiterpenes include cadalene, eudalene, cadinene, and β-selinene.

[0149] In one example, the isoprenoid is a diterpene. Exemplary diterpenes include phytol and abietic acid.

[0150] In one example, the isoprenoid is a triterpene. Exemplary triterpenes include squalene and β-amyrin.

[0151] In one example, the isoprenoid is a tetraterpene. Exemplary tetraterpenes include carotenoids (e.g., β-carotene) and lycopene.

[0152] fatty acid Fatty acids are lipids containing long hydrocarbon chains terminated with a carboxylic acid functional group. Fatty acids can be saturated or unsaturated. In one example, a fatty acid contains a carbon chain having 6 to 22 carbons. Exemplary fatty acids include palmitic acid, myristic acid, oleic acid, α-linolenic acid, and stearic acid.

[0153] Fatty acids rarely occur in free form in nature, but generally exist as three major types of esters: triglycerides, phospholipids, and cholesteryl esters.

[0154] In one example, the fatty acid is a triglyceride. A triglyceride is a triester consisting of glycerol linked to three fatty acid molecules via ester bonds. The three fatty acids may be the same or different. An exemplary triglyceride is tristearin.

[0155] In one example, the fatty acid is a phospholipid. A phospholipid is a complex lipid comprising a hydrophilic polar head group containing one or more phosphate groups and a hydrophobic tail containing two fatty acyl chains. The polar head group is linked to the hydrophobic portion by a phosphodiester bond via a glycerol (i.e., phosphoglyceride) or sphingosine molecule (i.e., phosphosphingolipid). Phospholipids can be saturated or unsaturated. Exemplary phosphoglycerides include phosphatidic acid (phosphatidate), phosphatidylethanolamine (cephalin), phosphatidylcholine (lecithin), phosphatidylserine, phosphoinositides (e.g., phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP), phosphatidylinositol diphosphate (PIP2), and phosphatidylinositol triphosphate (PIP3)), phosphatidylglycerol, and cardiolipin. Exemplary phosphosphingolipids include ceramide phosphorylcholine (sphingomyelin), ceramide phosphorylethanolamine (sphingomyelin), ceramide phosphoryl lipid, galactocerebroside, glucocerebroside, and lactosylceramide.

[0156] In one example, the fatty acid is a cholesteryl ester. Cholesteryl esters are cholesterol esterified with a long-chain fatty acid. Exemplary cholesteryl esters include cholesteryl oleate, cholesteryl benzoate, and cholesteryl linoleate.

[0157] Lipidization Exemplary lipidations include palmitoylation, myristoylation, fatty acid acylation, esterification, prenylation, or a combination thereof.

[0158] Palmitoylation In one example, the lipid moiety is attached to the DNA-binding protein or peptide by palmitoylation.

[0159] In one example, palmitoylation is cysteine palmitoylation (also known as S-palmitoylation). Those skilled in the art will understand that cysteine palmitoylation is the addition of a 16-carbon palmitoyl group to a cysteine residue of a protein. In one example, the palmitoyl group is added via a thioester bond. In another example, the palmitoyl group is added via an amide bond.

[0160] Myristoylation In one example, the lipid moiety is attached to the DNA-binding protein or peptide by myristoylation.

[0161] In one example, the myristoylation is N-glycine myristoylation, which those skilled in the art will recognize refers to the co- or post-translational attachment of myristoyl, a saturated 14-carbon fatty acyl group, to the N-terminal glycine of a protein via an amide bond.

[0162] In one example, the myristoylation is lysine myristoylation.

[0163] Fatty acid acylation In one example, the lipid moiety is attached to the DNA-binding protein or peptide by fatty acid acylation.

[0164] Those skilled in the art will recognize that fatty acid acylation involves the covalent attachment of an acyl group to a protein.

[0165] In one example, fatty acid acylation is lysine N-acylation, which one of skill in the art will understand to refer to the transfer of an acetyl moiety from acetyl-CoA to the epsilon (ε)-amino group of a lysine residue on a protein.

[0166] Esterification In one example, the lipid moiety is attached to the DNA-binding protein or peptide by esterification.

[0167] In one example, the esterification is C-terminal sterol esterification, such as C-terminal cholesterol esterification. Those skilled in the art will understand that C-terminal cholesterol esterification is the replacement of at least one hydroxyl (-OH) group with an alkoxy (-O-alkyl) group.

[0168] Prenylation In one example, the lipid moiety is attached to the DNA-binding protein or peptide by prenylation.

[0169] In one example, the prenylation is cysteine prenylation, which those skilled in the art will appreciate is the addition of multiple isoprene units to a cysteine residue near the C-terminus of a protein.

[0170] In one example, the prenylation is farnesylation (ie, the addition of three isoprene units), or the prenylation is geranylgeranylation (ie, the addition of four isoprene units).

[0171] In one example, the bond between the farnesyl or geranylgeranyl group and the cysteine residue is a thioether bond. In another example, the bond is an ester bond. In a further example, the bond is a thioester bond.

[0172] Lipidation Method Lipid modifications typically occur at the nucleophilic side chains of proteins or peptides (eg, cysteine, serine, and lysine) at the N-terminus and / or C-terminus of the protein or peptide.

[0173] Various methods of lipidation will be apparent to those skilled in the art and / or are described herein. Suitable methods may include chemical lipidation or enzymatic lipidation.

[0174] chemical lipidation In one example, a lipid moiety is attached to a DNA-binding protein or peptide using chemical ligation. The lipid moiety can contain an amine, carboxylic acid, hydrazide, or maleimide group, and can be chemically attached to the DNA-binding protein or peptide via a primary amine group of lysine or a thiol group of cysteine. In one example, the lipid moiety contains a maleimide group, and the lipid moiety is attached to the DNA-binding protein or peptide via the formation of a thioether bond with a sulfhydryl group in the DNA-binding protein or peptide. In one example, the lipid moiety contains a carboxylic acid, and the carboxylic acid is activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS). The activated acylaminoester or sulfo-NHS ester then reacts with the primary amine of a lysine residue in the DNA-binding protein or peptide to form an amide bond.

[0175] In one example, the lipid moiety includes a maleimide group. For example, the lipid moiety is a phospholipid capped with a maleimide group. In one example, the lipid moiety is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-maleimide (DSPE-maleimide, DSPE-Mal).

[0176] In one example, lipid moieties are attached to DNA-binding proteins or peptides using various "click chemistry" strategies such as those disclosed in Kolb et al. (2001), WO2003 / 101972, and Malkoch et al. (2005).

[0177] In one example, lipid moiety is used to be linked to DNA-binding protein or peptide by expressed protein ligation.Expressed protein ligation involves the chemoselective ligation between the protein or peptide with C-terminal thioester and the protein or peptide with N-terminal cysteine in aqueous solution at physiological pH.In one example, C-terminal thioester is inserted into DNA-binding protein or peptide by genetic engineering, and lipid moiety is fused to the peptide with N-terminal cysteine residue.

[0178] Other chemical lipidation methods known to those skilled in the art may also be used, such as the method disclosed in Takahara & Kamiya (2020).

[0179] enzymatic lipidation In one example, the lipid moiety is attached to the DNA-binding protein or peptide using enzymatic lipidation. Enzymatic lipidation can be performed in vivo or in vitro. In some examples, the DNA-binding protein or peptide is genetically engineered using techniques known to those skilled in the art to include a consensus sequence recognized by the lipidation enzyme.

[0180] In one example, lipid moieties are attached to DNA-binding proteins or peptides using sortase A-mediated lipidation. Sortase A (e.g., SrtA from Staphylococcus aureus) binds to Ca 2+ In the presence of a sortase, secreted proteins are covalently linked to bacterial cell wall peptidoglycan via a transpeptidation reaction. In this example, the DNA-binding protein or peptide is engineered to contain an LPXTG motif (e.g., LPETG) at its C-terminus, and the lipid moiety contains a nucleophile and an oligo-glycine motif (e.g., triglycine, tetraglycine, or pentaglycine). Upon addition of a sortase, the DNA-binding protein or peptide is covalently linked to the lipid via a peptide bond.

[0181] In one example, lipid moieties are attached to DNA-binding proteins or peptides using transglutaminase-mediated lipidation. Transglutaminase (e.g., microbial transglutaminase: MTG) binds Ca 2+ In the absence of ATP, the reaction between glutamine and lysine residues in a peptide or protein is catalyzed to form an irreversible crosslink. In one example, the DNA-binding protein or peptide is engineered to contain an MTG lysine recognition sequence (e.g., MRHKGS), for example, at the N- or C-terminus, and the lipid portion contains an MTG glutamine recognition sequence (e.g., LLQG). In one example, the DNA-binding protein or peptide is engineered to contain an MTG glutamine recognition sequence (e.g., LLQG or LQ), for example, at the N- or C-terminus, and the lipid portion contains an MTG lysine recognition sequence (e.g., MRHKGS).

[0182] Other enzymatic lipidation methods known to those skilled in the art may also be used, such as the method disclosed in Takahara & Kamiya (2020).

[0183] Additional fats In one example, the lipid nanoparticles further comprise a PEG lipid, a sterol-structured lipid, and / or a neutral lipid. In one example, the lipid nanoparticles further comprise a PEG lipid, a sterol-structured lipid, an ionizable lipid, and / or a neutral lipid. In one example, the lipid nanoparticles do not comprise a cationic lipid.

[0184] PEG lipids In one example, the present disclosure provides lipid nanoparticles comprising PEGylated lipids.

[0185] It will be clear to those skilled in the art that the reference to PEGylated lipid refers to a lipid modified with polyethylene glycol.Exemplary PEGylated lipids include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.For example, PEG lipids include PEG-c-DMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE lipids, and combinations thereof.

[0186] neutral lipid In one example, the present disclosure provides lipid nanoparticles comprising a neutral lipid.

[0187] Neutral or zwitterionic lipids suitable for use in the present disclosure will be apparent to those of skill in the art and include, for example, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DL ...palmitoyl-sn-glycero-3-phosphocho 1-Oleoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin. The lipids may be saturated or unsaturated.

[0188] structural lipids In one example, the present disclosure provides lipid nanoparticles comprising structured lipids.

[0189] Exemplary structural lipids include, but are not limited to, cholesterol, fecosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, and α-tocopherol.

[0190] In one example, the structured lipid is a sterol, for example, cholesterol, or in another example, campesterol.

[0191] Ionizable lipids In one example, the present disclosure provides lipid nanoparticles comprising an ionizable lipid.

[0192] Ionizable lipids suitable for use in the present disclosure will be apparent to those of skill in the art and include, for example, 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA ... ), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), Heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 1,2-Dioleoyl-3-trimethylammoniumpropane (DOTAP), 1,2-Distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (D Lin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), ,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2R)), (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2S)), 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), 2,5-bis((9z,12z)-octadeca-9,12,Dien-1-yloxyl)benzyl-4-(dimethylamino)butanoate (LKY750), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (also known as heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate) (SM-102), 2-hexyldecanoic acid, 1,1'-[[(4-hydroxybutyl)imino]di-6,1-hexanediyl] ester (((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate)) (ALC-0315), 4-(dimethylamino)-butanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl ester (DLin-MC3-DMA or MC3), ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester, and combinations thereof.

[0193] Pharmaceutically acceptable carrier Suitably, in the composition or method for administering the lipid nanoparticles of the present disclosure to a subject, the lipid nanoparticles are combined with a pharmaceutically acceptable carrier, as understood in the art.Thus, one example of the present disclosure provides a composition (e.g., a pharmaceutical composition) comprising the lipid nanoparticles of the present disclosure combined with a pharmaceutically acceptable carrier.

[0194] Generally, a "carrier" refers to a solid or liquid filler, binder, diluent, encapsulating substance, emulsifier, wetting agent, solvent, suspending agent, coating, or lubricant that can be safely administered to any subject, for example, a human. Depending on the particular route of administration, a variety of acceptable carriers known in the art can be used, for example, as described in Remington's Pharmaceutical Sciences (Mack Publishing Co. NJUSA, 1991).

[0195] The lipid nanoparticles of the present disclosure are useful for parenteral administration, topical administration, oral or topical administration, intramuscular administration, aerosol administration, or transdermal administration for preventive or therapeutic treatment.In one example, lipid nanoparticles are administered parenterally, such as intramuscularly, subcutaneously, or intravenously.For example, lipid nanoparticles are administered intramuscularly.

[0196] The formulation of lipid nanoparticles to be administered varies depending on the route of administration and the selected formulation (e.g., solution, emulsion, capsule). Suitable pharmaceutical compositions containing lipid nanoparticles to be administered can be prepared in a physiologically acceptable carrier. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Various suitable aqueous carriers are known to those skilled in the art, including water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution, and glycine. Intravenous vehicles can include various additives, preservatives, fluid, nutrient, or electrolyte replenishers (see generally Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980). The compositions of the present invention can optionally contain pharmaceutically acceptable auxiliary substances (pH adjusters, buffers, and toxicity adjusters, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate) as needed to approximate physiological conditions. The lipid nanoparticles can be stored in the liquid phase or can be lyophilized for storage and reconstituted in a suitable carrier prior to use according to lyophilization and reconstitution techniques known in the art.

[0197] The optimal concentration of the active ingredient(s) (i.e., DNA) in the selected vehicle can be determined empirically according to procedures known to those skilled in the art and will depend on the final pharmaceutical formulation desired.

[0198] When formulated, the compositions of the present disclosure are administered in a manner compatible with the dosage formulation and in a therapeutically / prophylactically effective amount.The dosage range of the lipid nanoparticles of the present disclosure is an amount sufficient to produce the desired effect.For example, the composition comprises an effective amount of encapsulated DNA.In one example, the composition comprises a therapeutically effective amount of DNA.In another example, the composition comprises a prophylactically effective amount of DNA.

[0199] The dosage should not be so large as to cause adverse side effects. Generally, the dosage will vary depending on the age, condition, sex, and extent of the disease in the patient, but can be determined by those skilled in the art. The dosage can be adjusted by an individual physician in the event of any complications.

[0200] DNA The present disclosure provides lipid nanoparticles for DNA delivery, wherein DNA-binding protein or peptide is bound to DNA.For example, the present disclosure provides lipid nanoparticles for DNA delivery, wherein lipidated DNA-binding protein or peptide is bound to DNA.

[0201] The DNA of the present disclosure may be natural or non-naturally occurring DNA, or may contain one or more modified nucleobases, nucleosides, nucleotides, promoters, enhancers (e.g., cytomegalovirus), poly(A) sequences, or polyadenylation signals.

[0202] Preparation method Suitable methods for preparing lipid nanoparticles of the present disclosure will be clear to those skilled in the art and / or are described herein.For example, lipid nanoparticles of the present disclosure can be produced using approaches well known in the formulation field.For example, suitable LNPs can be formed using mixing processes such as microfluidics, including herringbone micromixing and T-junction mixing of two fluid streams (one containing DNA, typically in aqueous solution, and the other containing various necessary lipid components, typically in ethanol).

[0203] LNPs can then be prepared by mixing, for example, a phospholipid (such as DOPE or DSPC, available from commercial sources including Avanti Polar Lipids, Alabaster, AL), a PEGylated lipid (such as 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol, also known as PEG-DMG, available from commercial sources including Avanti Polar Lipids, Alabaster, AL), and a structural lipid / sterol (such as cholesterol, available from commercial sources including Sigma-Aldrich) at a concentration of, for example, about 50 mM in ethanol. The solution should be refrigerated, for example, at -20°C during storage. Various lipids can be combined to produce the desired molar ratio and diluted with water and ethanol to the desired final lipid concentration, for example, about 5.5 mM to about 25 mM.

[0204] LNP compositions containing DNA (including, but not limited to, ssDNA or dsDNA) can be prepared by mixing the lipid solution described above with a DNA-containing solution, for example, at a lipid component to DNA weight ratio of about 5:1 to about 50:1. Using a NanoAssemblr microfluidic system, the lipid solution can be rapidly injected into the DNA solution at a flow rate of about 3 ml / min to about 18 ml / min to produce a suspension with a water to ethanol ratio of about 1:1 to about 4:1.

[0205] For LNP compositions containing ssDNA or dsDNA, a DNA solution at a concentration of 1.0 mg / ml in deionized water can be diluted with 50 mM sodium citrate buffer at pH 3-6 to form a stock solution.

[0206] As known in the art, the LNP composition can be further processed by diluting 10-fold into 50 mM citrate buffer at pH 6 and then subjected to tangential flow filtration (TFF) using a 300,000 molecular weight cutoff membrane (mPES) until concentrated to the original volume. In one example, the citrate buffer can then be replaced with a buffer containing 20 mM Tris buffer at pH 7.5, 80 mM sodium chloride, and 3% sucrose using diafiltration with 10 volumes of fresh buffer. The LNP solution can be concentrated, for example, to a volume of 5-10 mL, filtered using a 0.2 micron PES syringe filter, aliquoted into vials, and frozen at 1°C / min using a Corning® CoolCell® LX Cell Freezing Container until the sample reaches -80°C. Samples can be stored at -80°C until needed.

[0207] The above method results in nanoprecipitation and particle formation. Alternative processes, including but not limited to T-junction and direct injection, can be used to achieve the same nanoprecipitation.

[0208] In some examples, the lipid component of the LNP formulation comprises about 2 mol% to about 25 mol% phospholipids (neutral lipids), about 18.5 mol% to about 60 mol% structural lipids (sterols), and about 0.2 mol% to about 10 mol% PEGylated lipids, provided that the total mol% does not exceed 100%. In some examples, the lipid component of the LNP formulation comprises about 5 mol% to about 20 mol% phospholipids, about 30 mol% to about 55 mol% structural lipids, and about 1 mol% to about 5 mol% PEGylated lipids. In particular examples, the lipid component comprises about 10 mol% phospholipids, about 48 mol% structural lipids, and about 2.0 mol% PEGylated lipids. In some examples, the phospholipid may be DOPE or DSPC. In other examples, the PEG lipid may be PEG-DMG and / or the structural lipid may be cholesterol.

[0209] The encapsulation efficiency of DNA within the LNP can be at least 50%, for example, about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some examples, the encapsulation efficiency can be at least 80%. In certain examples, the encapsulation efficiency can be at least 90%.

[0210] Assays for Lipid Nanoparticles of the Present Disclosure Lipid nanoparticles of the present disclosure are readily screened for physical and biological activity and / or stability using methods known in the art and / or described below.

[0211] Assessment of DNA degradation In one example, the level of DNA degradation by DNase is assessed, e.g., DNA is treated with DNase, either alone or in combination with a DNA-binding protein or peptide.

[0212] In one example, the level of DNA is assessed in DNAse-treated and untreated samples using real-time PCR. In one example, the cycle threshold (CT) value in DNA samples without DNA-binding proteins or peptides is increased compared to DNA samples with DNA-binding proteins or peptides, indicating DNA degradation.

[0213] Assessment of DNA translation In one example, DNA translation is assessed using a cell-based reporter system. Systems suitable for use in the present disclosure will be apparent to those of skill in the art, and include, for example, the use of a cell-based fluorescent reporter system or the histochemical expression of alkaline phosphatase.

[0214] In one example, a reported cell-based system involves the delivery of a DNA reporter for the expression of an enzyme that results in the conversion of a substrate that can be visualized by microscopy or quantified by colorimetric analysis.

[0215] In one example, DNA is assessed in the presence or absence of a DNA binding protein or peptide.

[0216] In one example, the DNA is nanoluciferase DNA, and the amount of DNA translation is measured by the amount of luciferase produced, assessed by measuring luminescence in relative light units (RLU). In one example, the assay is performed at 4°C, 24°C, and / or 37°C. In another example, the assay is performed after incubating the sample for 0 hours, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 hours, or 96 hours.

[0217] Assessment of TLR induction In one example, the level of TLR-3, TLR-7, TLR-8, and / or TLR9 induction is assessed. For example, the level of TLR-3, TLR-7, TLR-8, and / or TLR9 induction by DNA alone or DNA in combination with a DNA-binding protein or peptide is assessed. In one example, TLR-3, TLR-7, TLR-8, and / or TLR9 induction is assessed using a TLR-induced NfKB reporter assay. In this assay, NfKB is operatively linked to secreted alkaline phosphatase (SEAP). DNA is introduced into any cell type (conditionally transduced TLR-3, conditionally transduced TLR-7, conditionally transduced TLR-8, or conditionally transduced TLR-9). TLR receptor binding induces NfKB activation, which in turn induces SEAP. In one example, SEAP levels are determined by chemical reaction and calorific readout.

[0218] Uses of Lipid Nanoparticles of the Present Disclosure The lipid nanoparticles of the present disclosure can be used to deliver DNA to cells by any of several methods and strategies known in the art, such as transient transfection, stable transfection, and viral transduction.

[0219] In one example, the lipid nanoparticles of the present disclosure are used to deliver DNA to genetically modify cells.

[0220] In one example, the lipid nanoparticles of the present disclosure are used in combination with a second lipid nanoparticle comprising a nucleic acid encoding a programmable nuclease and a nucleic acid encoding a guide RNA (gRNA) to genetically modify cells. In another example, the lipid nanoparticles of the present disclosure are used in combination with a second lipid nanoparticle comprising a nucleic acid encoding a programmable nuclease and a third lipid nanoparticle comprising a nucleic acid encoding a gRNA to genetically modify cells.

[0221] In one example, the nucleic acid is RNA, for example, the RNA is mRNA.

[0222] In one example, genetically modifying a cell involves repairing DNA breaks by endogenous cellular processes such as homology-directed repair (HDR) and non-homologous end joining (NHEJ).

[0223] In one example, the lipid nanoparticles of the present disclosure are used to deliver DNA to genetically modify cells using HDR.

[0224] HDR is essentially an error-free mechanism for repairing double-stranded DNA breaks in the presence of homologous DNA sequences.The most common form of HDR is homologous recombination.It uses homologous sequences as templates for inserting or replacing specific DNA sequences at the breakpoint.The template for homologous DNA sequences can be endogenous sequences (e.g., sister chromatids), or exogenous or supplied sequences (e.g., plasmids or oligonucleotides).Therefore, HDR can be used to introduce precise modifications, such as replacement or insertion, in desired regions.

[0225] In one example, the DNA-binding protein or peptide bound to the DNA is a template for a homologous DNA sequence. In one example, the template includes a homology arm. In some examples, the homology arm is 100 nucleotides in length.

[0226] In contrast, NHEJ is an error-prone repair mechanism that directly joins the DNA ends resulting from a double-strand break, potentially resulting in the loss, addition, or mutation of several nucleotides at the break site. The resulting small deletions or insertions (called "indels") or mutations can disrupt or enhance gene expression. Furthermore, when there are two breaks in the same DNA, NHEJ can result in the deletion or inversion of the intervening segment. Therefore, NHEJ can be used to introduce insertions, deletions, or mutations at the break site.

[0227] In one example, the gRNA targets a globin locus. For example, the gRNA targets an α-globin locus. In another example, the gRNA targets a β-globin locus.

[0228] Programmable nucleases that can be used in accordance with the present disclosure include, but are not limited to, bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-cas, (CRISPR-related) systems, zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALENs), meganucleases, and RNA-guided engineered nucleases (RGENs) derived from Argonaute.

[0229] In one example, the cells are ex vivo, for example, as a cultured cell population. In another example, the cells are in vivo, for example, in a mouse or human. In one example, the genetically modified cells are mammalian cells. For example, the mammalian cells are human cells.

[0230] In one example, the cells are hematopoietic stem cells. In one example, the cells are hematopoietic progenitor cells. In one example, the cells are hematopoietic stem and progenitor cells. For example, the cells are CD34+ hematopoietic stem and / or progenitor cells.

[0231] The invention is further disclosed in the following numbered paragraphs: 1. A lipid nanoparticle for delivering DNA, said lipid nanoparticle comprising a DNA-binding protein or peptide therein that is bound to said DNA.

[0232] 2. The lipid nanoparticle described in paragraph 1, wherein the DNA-binding protein or peptide is a lipidated DNA-binding protein or peptide.

[0233] 3. Lipid nanoparticles for delivering DNA, comprising a lipidated DNA-binding protein or peptide therein that is bound to said DNA.

[0234] 4. The lipid nanoparticle of paragraph 2 or 3, wherein the DNA-binding protein or peptide is lipidated before binding to the DNA.

[0235] 5. The lipid nanoparticle of any one of paragraphs 2 to 4, wherein the DNA-binding protein or peptide is lipidated with a lipid moiety selected from the group consisting of fatty acids, isoprenoids, and combinations thereof.

[0236] 6. The lipid nanoparticles of paragraph 5, wherein the fatty acid is a triglyceride, a phospholipid, or a cholesteryl ester.

[0237] 7. The lipid nanoparticle of any one of paragraphs 2 to 6, wherein the DNA-binding protein or peptide is lipidated on a nucleophilic side chain at the N-terminus and / or C-terminus.

[0238] 8. The lipid nanoparticle described in paragraph 7, wherein the nucleophilic side chain is a cysteine, serine, threonine, tyrosine and / or lysine amino acid residue.

[0239] 9. The lipid nanoparticle of any one of paragraphs 2 to 8, wherein the DNA-binding protein or peptide is lipidated by palmitoylation, myristoylation, fatty acid acylation, esterification, prenylation, or a combination thereof.

[0240] 10. The lipid nanoparticle of paragraph 9, wherein the DNA-binding protein or peptide is lipidated by N-terminal cysteine palmitoylation, N-terminal glycine myristoylation, lysine N-acylation, C-terminal cholesterol esterification, cysteine prenylation, serine O-acylation, or a combination thereof.

[0241] 11. The lipid nanoparticle of paragraph 9 or 10, wherein the prenylation is farnesylation or geranylgeranylation.

[0242] 12. The lipid nanoparticle of any one of paragraphs 5 to 11, wherein the lipid moiety is linked to the DNA-binding protein or peptide by a thioether bond, an ester bond, a thioester bond and / or an amide bond.

[0243] 13. The lipid nanoparticle of any one of paragraphs 2 to 12, wherein the DNA-binding protein or peptide is lipidated using chemical or enzymatic lipidation.

[0244] 14. The lipid nanoparticle described in paragraph 13, wherein the DNA-binding protein or peptide is lipidated using a chemical lipidation method selected from the group consisting of chemical ligation, click chemistry, expressed protein ligation, and combinations thereof.

[0245] 15. The lipid nanoparticle of paragraph 13, wherein the DNA-binding protein or peptide is lipidated using enzymatic lipidation selected from the group consisting of sortase A-mediated lipidation, transglutaminase-mediated lipidation, and combinations thereof.

[0246] 16. The lipid nanoparticles described in paragraph 15, wherein the enzymatic lipidation is carried out in vivo or in vitro.

[0247] 17. The lipid nanoparticle of any one of paragraphs 1 to 16, wherein the DNA-binding protein or peptide encapsulates the DNA.

[0248] 18. The lipid nanoparticle of any one of paragraphs 1 to 17, wherein the DNA-binding protein or peptide directly binds to the DNA.

[0249] 19. The DNA-binding protein or peptide is a) reducing the toxicity of said lipid nanoparticles, and / or b) stabilizing the DNA, and / or c) promotes uptake through the nuclear membrane, and / or d) protecting the DNA from degradation, and / or e) promoting the nucleation of said lipid nanoparticles; f) increasing the immunogenicity of said DNA, and / or g) A lipid nanoparticle according to any one of paragraphs 1 to 18, which inhibits the induction of signal transduction by one or more Toll-like receptors.

[0250] 20. The lipid nanoparticle of any one of paragraphs 1 to 20, wherein the DNA-binding protein or peptide is a viral or non-viral DNA-binding protein or peptide.

[0251] 21. The lipid nanoparticle described in paragraph 20, wherein the viral DNA-binding protein is derived from a class I, class II, and / or class VII virus.

[0252] 22. The lipid nanoparticle of paragraph 21, wherein the viral DNA-binding protein or peptide is a virus selected from the group consisting of adenovirus, herpesvirus, poxvirus, adeno-associated virus, geminivirus, bacteriophage, parvovirus, heparnavirus, hepadnavirus, circoviridae virus, and papovaviridae virus.

[0253] 23. The lipid nanoparticle described in paragraph 22, wherein the viral DNA-binding protein or peptide is a nucleoprotein, a nonstructural protein, a matrix protein, and / or a nucleocapsid protein.

[0254] 24. Lipid nanoparticles described in paragraph 20, wherein the non-viral DNA-binding protein or peptide is derived from a cellular protein associated with cell proliferation, cell signaling and / or antiviral pathways.

[0255] 25. The lipid nanoparticle described in paragraph 24, wherein the cellular protein is selected from the group consisting of TAR DNA binding protein (TRBP), Y-box binding protein, Z-DNA binding protein, and combinations thereof.

[0256] 26. The lipid nanoparticle of any one of paragraphs 1 to 25, wherein the lipid nanoparticle further comprises a PEG lipid, a structured lipid, an ionizable lipid, and / or a neutral lipid.

[0257] 27. The lipid nanoparticles of any one of paragraphs 1 to 26, which do not contain cationic lipids.

[0258] 28. The lipid nanoparticles described in any one of paragraphs 1 to 27, wherein the DNA is non-linear DNA.

[0259] 29. The lipid nanoparticle described in paragraph 28, wherein the DNA is a plasmid.

[0260] 30. A composition comprising the lipid nanoparticles of any one of paragraphs 1 to 29.

[0261] 31. A pharmaceutical composition comprising the lipid nanoparticles of any one of paragraphs 1 to 29 or the composition of paragraph 30, and a pharmaceutically acceptable carrier.

[0262] 32. The lipid nanoparticles according to any one of paragraphs 1 to 29, the composition according to paragraph 30, or the pharmaceutical composition according to paragraph 31, for use in therapy.

[0263] 33. A method for delivering DNA to the nucleus of a cell, the method comprising contacting the cell with a lipid nanoparticle described in any one of paragraphs 1 to 29.

[0264] 34. A method for delivering DNA to cells in a subject, the method comprising administering to the subject a lipid nanoparticle described in any one of paragraphs 1 to 29, a composition described in paragraph 30, or a pharmaceutical composition described in paragraph 31. [Example]

[0265] Example 1: DNA-free NPs bind to DNA To assess whether DNA-free nucleoproteins (NPs) protect DNA, NPs were combined with nanoluciferase, and samples were heated to 40°C or incubated at room temperature and then analyzed by agarose gel electrophoresis.

[0266] Samples containing four nucleoproteins from influenza A, beak and feather disease virus (BFDV), human papillomavirus (HPV), and hepatitis B virus (HBV) were analyzed using SDS-PAGE, electrophoretic mobility shift assay (EMSA), and size-exclusion high-performance liquid chromatography (HPLC-SEC).

[0267] HPLC-SEC was performed on a Superdex 200 Increase column in PBS at 4 mL / min.

[0268] EMSA was performed in 10% glycerol, 0.1 mM DTT, 100 mM KCl, 0.1 mg / mL BSA (Mg + Native PAGE gels were run at 180V for 40 minutes using a binding buffer of 40 mM Tris, 1 mM EDTA, containing TEA (without EDTA). 33 ng of DNA was complexed with various amounts of DNA in a total volume of 20 μL.

[0269] Influenza A binds well to ssDNA as shown in Figures 1 and 3 and Table 2. Figure 2 shows that influenza A elutes well.

[0270] As shown in Figure 1, BFDV contains some impurities and appears to oligomerize, which can be recovered mostly under reducing conditions. Figure 2 shows that BFDV elutes poorly under non-reducing conditions. Figure 3 and Table 2 show that BFDV binds to ssDNA, but only above 500 nM protein to DNA (10 nM DNA) or an approximately 10:1 w / w ratio. 2+ indicates that it does not include

[0271] As shown in Figure 1, HPV16 has many impurities and either oligomerizes or degrades, making it unlikely to be recoverable under reducing conditions. The data in Figure 2 reflect this. However, as shown in Figure 3 and Table 2, HPV16 binds to ssDNA in a gel shift similar to BFDV.

[0272] As shown in Figure 1, HBV appears to dimerize, which can be partially reversed under reducing conditions. Figure 2 shows aggregation under non-reducing conditions. Figure 3 and Table 2 show that HBV does not bind to ssDNA at low MW. [Table 2]

[0273] Example 2: DNA-free NPs protect DNA from degradation To assess whether DNA-free NPs protect DNA, NP:DNA and DNA alone were evaluated in a DNase assay. Briefly, NP:DNA or DNA was treated with DNase and incubated at 30°C for 5–10 min. Samples were further treated with or without 1 μl of heat-labile proteinase K (PK). Reactions were incubated at 37°C for 15–30 min, followed by incubation at 60°C for 10–20 min to inactivate PK. When necessary, 1–2 μl of DNase was added. Real-time PCR was used to assess the levels of DNA present in treated and untreated samples.

[0274] Example 3: DNA-free NPs protect DNA from degradation at 4°C, 24°C, and 37°C To assess whether the ability of NPs to protect nanoluciferase DNA (nLuc DNA) is temperature dependent, NP:nLuc DNA and nLuc DNA were incubated at 4°C, 24°C, and 37°C for up to 96 h, and luciferase production was assessed by measuring luminescence in RLU.

[0275] Example 4: DNA-free NPs protect TLR induction To assess whether the presence of NPs inhibits DNA induction of TLR-3, TLR-7, TLR-8, and / or TLR-9 induction with dsDNA or ssDNA, a TLR-induced NfKB reporter assay was used. In this assay, NfKB is operatively linked to secreted alkaline phosphatase (SEAP). DNA is introduced into any cell type (conditionally transduced TLR-3, conditionally transduced TLR-7, conditionally transduced TLR-8, or conditionally transduced TLR-9). TLR receptor binding induces NfKB activation, which in turn induces SEAP. SEAP levels can be determined by chemical reaction and calorific readout.

[0276] Example 5: Conjugation of DNA-free NPs with maleimide-DSPE lipids NPs were conjugated with maleimide-DSPE by incubating NPs (0.85 mg / ml) with DSPE (1,2-distearoyl-sn-glycero-3-phosphorylethanolamine, 8 mM) in the presence of 10% ethanol. Labeled and unlabeled NPs were run on a non-reducing gel to assess the molecular weight of the protein and confirm the binding of the protein to the NPs with a molecular weight shift. Furthermore, because DSPE forms a suspension (i.e., liposomes), the composition was centrifuged and the supernatant was evaluated.

[0277] Example 6: Preparation of DNA-binding proteins and peptides Cellular and viral protein sequences are examined to identify protein domains and peptide sequences that have the potential to bind to DNA. Sequences from cellular proteins correlate with proteins involved in cell proliferation, cell signaling, and / or antiviral pathways, while sequences from viral proteins are derived from nonstructural and nucleoproteins.

[0278] Proteins are designed from sequences derived from, for example, Hepatitis B Virus (HBV). Peptides are designed from sequences derived from cellular proteins, including, but not limited to, TAR DNA binding protein (TDBP), Y-box binding protein, and Z-DNA binding protein.

[0279] Example 7: NP binding to DNA at different ratios To further evaluate the four nuclear proteins derived from influenza A, BFDV, HPV, and HBV, HPLC sizing, SDS-PAGE, and EMSA were performed. The four nuclear proteins were mixed with ssDNA at five different molar ratios (protein:DNA molar ratios ranging from 0.5:1 to 100:1).

[0280] Sixteen nanograms of ssDNA was used per complexation step, and all were loaded onto a Native PAGE gel using TEA buffer (40 mM Tris, 1 mM EDTA) containing 10% glycerol, 0.1 mM DTT, 100 mM KCl, and 0.1 mg / mL BSA. A buffer containing 100 mM Tris-HCl, pH 8, 300 mM KCl, 25 mM MgCl, 20% glycerol, and 500 μg / mL BSA was also used for BFDV NP.

[0281] Electroporate 0.5 μg of ssDNA into 5E5 cells using 10 μl Neon tips per condition. For example, use a molar ratio of 3:1 or 9:1 protein to ssDNA. Controls include electroporated ssDNA, mock electroporation, and protein alone electroporated at the highest dose.

[0282] Example 8: Optimal protein-to-ssDNA ratio for binding and nuclear delivery To assess the optimal protein:ssDNA ratio for binding and nuclear delivery, four nucleoproteins derived from influenza A, BFDV, HPV, and HBV are evaluated. These NPs are mixed with 528-nt-long ssDNA containing no viral sequences to form deoxyribonucleoproteins (DNPs). The optimal protein:ssDNA ratio is determined within the range of 2-100 mol / mol based on electrophoretic mobility shift assays.

[0283] The identified optimal ratio is used to form nucleoprotein-ssDNA complexes before electroporation into K562 signaling reporter cells. Improved levels of homology-directed repair (HDR) insertions are determined based on insertions and NHEJ-induced indels.

[0284] Example 9: Encapsulation of ssDNA:nucleoprotein complexes into lipid nanoparticles To evaluate the encapsulation of ssDNA:NP complexes into lipid nanoparticles, four nucleoproteins from influenza A, BFDV, HPV, and HBV were evaluated. These NPs were mixed with 528-nt ssDNA containing no viral sequences using the optimal ratios identified in Example 8 to form DNPs. DNPs were encapsulated into lipid nanoparticles (LNPs) using a standard microfluidic mixer and formulated with a mixture of ionizable lipid / cationic lipid helper lipid (DSPC), cholesterol, and DSPE-PEG lipids in ratios ranging from 10-50:10-30:10-50:0.5-2.5, respectively. LNPs were purified by standard processes.

[0285] The degree of encapsulation is determined by BCA and OliGreen ssDNA assays. Size and polydispersity are measured by dynamic light scattering (DLS). Zeta potential is measured by electrophoretic light scattering.

[0286] Example 10: Improvement of DNP-LNP-mediated homology-directed repair (HDR) To evaluate HDR, the optimal DNP-LNP formulation from Example 9 is used for encapsulation. K562 signaling light reporter cells are transfected with increasing doses of DNP-encapsulated LNPs in parallel with LNPs containing mRNA encoding Cas9 and sgRNA. A control of ssDNA alone without nucleoprotein is also included along with the DNP-LNPs. Five days after transfection, the levels of HDR and non-homologous end joining (NHEJ) are determined by fluorescence-activated cell sorting (FACS). The optimal dose and formulation for improving HDR with nucleoprotein are identified.

[0287] Example 11: Improved HDR in CD34+ hematopoietic stem and progenitor cells To evaluate HDR in CD34+ cells, the optimal DNP-LNP formulation from Example 9 is used. DNP HDR template and ssDNA alone are evaluated for HDR efficiency in ex vivo CD34+ hematopoietic stem and progenitor cells from three donors.

[0288] A guide targeting the globin locus is used with a short ssDNA HDR template containing 100-nt homology arms. The addition of ApoE3 to the culture medium is used to ensure proper LNP transfection of cells. Readout is performed via Sanger sequencing 3 days after transfection.

Claims

1. Lipid nanoparticles for delivering DNA, wherein the lipid nanoparticles contain a DNA-binding protein or peptide bound to the DNA.

2. The lipid nanoparticle according to claim 1, wherein the DNA-binding protein or peptide is a lipidized DNA-binding protein or peptide.

3. Lipid nanoparticles according to claim 2, wherein the DNA-binding protein or peptide is a) Lipidized before binding to the DNA, b) Lipidized with a lipid moiety selected from the group consisting of fatty acids, isoprenoids and combinations thereof, however optionally, i) The fatty acid is a triglyceride, a phospholipid, or a cholesteryl ester, or ii) The lipid portion is linked to the DNA-binding protein or peptide by thioether bonds, ester bonds, thioester bonds and / or amide bonds, c) Lipidized on the N-terminal and / or C-terminal nucleophilic side chains, wherein the nucleophilic side chains are optionally cysteine, serine, threonine, tyrosine, and / or lysine amino acid residues. d) Lipidization by palmitoylation, myristoylation, fatty acid acylation, esterification, prenylation, or a combination thereof, However, optionally, the DNA-binding protein or peptide may be lipid-modified by N-terminal cysteine ​​palmitoylation, N-terminal glycine myristoylation, lysine N-acylation, C-terminal cholesterol esterification, cysteine ​​prenylation, serine O-acylation, or a combination thereof. However, optionally, the prenylation may be farnesylation or geranylgeranylation, and / or e) Lipidized using chemical or enzymatic lipidization, wherein the DNA-binding protein or peptide is optionally i) Lipidized using chemical ligation selected from the group consisting of chemical ligation, click chemistry, expression protein ligation, and combinations thereof, or ii) Lipidization is performed using enzymatic lipidization selected from the group consisting of saltase A-mediated lipidization, transglutaminase-mediated lipidization, and combinations thereof. However, optionally, the enzymatic lipidization may be performed in vivo or in vitro. Lipid nanoparticles.

4. Lipid nanoparticles according to claim 1, i) The DNA-binding protein or peptide encapsulates the DNA, ii) The DNA-binding protein or peptide directly binds to the DNA and / or iii) The DNA-binding protein or peptide is a) Reduce the toxicity of the lipid nanoparticles, and / or b) Stabilize the DNA and / or c) Promotes uptake across the nuclear membrane, and / or d) Protect the DNA from degradation, and / or e) Promote nucleation of the lipid nanoparticles, f) Increase the immunogenicity of the DNA, and / or g) Lipid nanoparticles that inhibit the induction of signal transduction by one or more Toll-like receptors.

5. The lipid nanoparticle according to claim 1, wherein the DNA-binding protein or peptide is a viral or non-viral DNA-binding protein or peptide.

6. The lipid nanoparticles according to claim 5, wherein the viral DNA-binding protein is derived from a class I, class II, and / or class VII virus.

7. The lipid nanoparticle according to claim 6, wherein the viral DNA-binding protein or peptide is a virus selected from the group consisting of adenovirus, herpesvirus, poxvirus, adeno-associated virus, geminivirus, bacteriophage, parvovirus, heparnavirus, hepadnavirus, circoviridae viruses, and papovaviridae viruses.

8. The lipid nanoparticle according to claim 7, wherein the viral DNA-binding protein or peptide is a nucleoprotein, a non-structural protein, a substrate protein, and / or a nucleocapsid protein.

9. The lipid nanoparticle according to claim 5, wherein the nonviral DNA-binding protein or peptide is derived from a cellular protein associated with cell proliferation, cell signaling and / or antiviral pathways, wherein the cellular protein is optionally selected from the group consisting of TAR DNA-binding protein (TRBP), Y-box-binding protein, Z-DNA-binding protein, and combinations thereof.

10. The aforementioned lipid nanoparticles a) further comprising PEG lipids, structural lipids, ionizable lipids and / or neutral lipids, and / or b) Lipid nanoparticles according to claim 1, which do not contain cationic lipids.

11. The lipid nanoparticle according to claim 1, wherein the DNA is non-linear DNA.

12. A pharmaceutical composition comprising lipid nanoparticles according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

13. A pharmaceutical composition comprising lipid nanoparticles according to any one of claims 1 to 11, for use in therapeutic purposes.

Images