Single-chain variable fragment (scFv)-modified lipid nanoparticle compositions and uses thereof

Abstract

Provided herein are pharmaceutical compositions comprising lipid nanoparticles (LNPs) and therapeutic nucleic acids (TNAs), wherein the LNPs comprise single-chain variable fragments (scFvs) linked to the LNPs, and at least one pharmaceutically acceptable excipient. The scFvs can bind to antigens present on the surface of cells, advantageously providing LNP compositions that target only cells or tissues that express the receptor. The LNP compositions described herein advantageously provide efficient covalent conjugation with minimal impact on particle size and stability.

Description

[Technical field]

[0001] (Related Applications) This application claims priority to U.S. Provisional Application No. 63 / 221,290, filed July 13, 2021, the contents of which are incorporated by reference herein in their entirety. [Background technology]

[0002] Ionizable lipid nanoparticles (LNPs) have been widely used for systemic delivery of RNA therapeutics. Various types of ionizable lipid materials, such as C12-200, cKK-E12, and DLin-MC3-DMA, have been previously reported for LNP formulations, demonstrating efficient gene silencing in the liver at a dosage level of 0.002 mg siRNA / kg (Dong, et al., Proc. Natl. Acad. Sci. USA 111, 3955-3960 (2014)). Inclusion of targeting ligands has been shown to enhance the delivery and therapeutic efficiency of mRNA-LNPs, but it has been recognized that conjugating targeting moieties can add complexity, cost, and regulatory difficulties to the process of manufacturing LNP systems (Cheng et al., Science. 2012 Nov 16; 338 (6109): 903-10). In addition, it has been demonstrated that the targeting specificity of some targeting ligands can be lost when lipid nanoparticles are exposed to biological fluids, resulting in interaction with proteins in the medium and the formation of a protein corona (Salvati et al., Nat Nanotechnol. 2013 Feb;8(2):137-43). Thus, there is a trade-off between the potential clinical benefit and the complexity and cost of targeted RNA-LNP manufacturing.

[0003] Antibodies work by targeting specific antigens that are either expressed exclusively on the surface of diseased cells or highly overexpressed on these cells compared to healthy cells. Because these antigens are present alone or abundantly on the surface of targeted diseased cells, antibodies can conceptually be utilized to transport nanoparticles and their cargo (e.g., therapeutic agents) through the body, allowing for selective delivery / targeting. This approach was first explored in the 1980s, but had considerable limitations, such as inadequate methods for generating and evaluating antibody-decorated nanoparticles, which prevented significant progress in this area. Advances in both antibody expression techniques and nanoparticle design over the past few decades have allowed for a more thorough exploration of nanoparticle-antibody conjugates, leading to a rapid expansion of the field. Early development focused almost entirely on using full antibodies as targeting ligands, primarily due to the wealth of information available on both their generation and modification. However, several issues associated with the use of full antibody ligands have become apparent, such as immunogenicity, rapid clearance, poor stability, and less than expected efficacy.

[0004] The modularity of antibodies, both structurally and functionally, allows the generation of smaller antigen-binding fragments, such as fragment antigen-binding (Fab), single-chain variable fragments (scFv), single-domain antibodies, and fragment crystallizable (Fc) domains, by molecular cloning, antibody engineering, and even enzymatic methods. Antigen-binding fragments of antibodies have considerable potential to overcome the shortcomings of traditional mAbs, such as poor penetration into solid tumors and Fc-mediated bystander activation of the immune system. Antibody fragments can be used by themselves or linked to other molecules, creating numerous possibilities for bispecific, multispecific, multimeric, or multifunctional molecules to achieve various biological effects. Antibody fragments can offer several advantages over the use of traditional antibodies. For example, they can generally be easily produced using microbial expression systems, resulting in faster culture, higher yields, and lower production costs (Fernandes JC, Drug Discov Today. 2018 Dec; 23(12): 1996-2002). Their small size allows access to challenging cryptic epitopes and tumor penetration, they have reduced immunogenicity, and the lack of Fc limits bystander activation of the immune system (Kholodenko et al. Curr Med Chem. 2019;26(3):396-426). On the other hand, their smaller size results in faster renal excretion, which may require higher doses and / or more frequent dosing regimens in vivo.

[0005] Although LNPs have been shown to be advantageous for in vivo delivery, systemic delivery of RNA therapeutics outside of hepatocytes remains extremely challenging. The relatively large size of these LNPs reduces the therapeutic index for liver indications by several mechanisms: (1) larger LNPs cannot efficiently bypass the fenestrae of endothelial cells lining the hepatic sinusoids, preventing access to target cells (hepatocytes), (2) larger LNPs cannot be efficiently internalized by hepatocytes via clathrin-mediated endocytosis by several different receptors (e.g., asialoglycoprotein receptor (ASGPR), low-density lipoprotein (LDL) receptor), and (3) LNPs above a certain threshold size tend to be preferentially taken up by cells of the reticuloendothelial system, which may trigger a dose-limiting immune response. Despite these advances, LNP-mediated delivery of larger, rigid polynucleotide cargoes (e.g., double-stranded linear DNA, plasmid DNA, closed-end double-stranded DNA (ceDNA)) presents additional challenges for smaller and / or flexible cargoes (e.g., siRNA). One such challenge involves the size of the LNPs that can be obtained when large, rigid cargoes are encapsulated. To fully realize the potential of LNP-targeted nucleic acid therapeutics, an efficient in vivo delivery system is required. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Dong,et al.,Proc.Natl.Acad.Sci.USA111,3955-3960(2014) [Non-Patent Document 2] Cheng et al.,Science.2012 Nov 16;338(6109):903-10 [Non-Patent Document 3] Salvati et al.,Nat Nanotechnol.2013 Feb;8(2):137-43 [Non-Patent Document 4] Fernandes JC,Drug Discov Today.2018 Dec;23(12):1996-2002 [Non-Patent Document 5] Kholodenko et al.Curr Med Chem.2019;26(3):396-426 Summary of the Invention

[0007] The present disclosure provides a pharmaceutical composition comprising a lipid nanoparticle (LNP), a therapeutic nucleic acid (TNA), and at least one pharma- ceutically acceptable excipient, wherein the LNP comprises a single chain variable fragment (scFv) linked to the LNP, and the scFv targets an antigen present on the surface of a cell (e.g., a tumor cell). The LNP compositions described herein advantageously provide efficient covalent conjugation with minimal impact on particle size and stability. It is the discovery of the present disclosure that maleimide conjugation of scFv to LNP, together with other thiol-based crosslinking methods, results in robust conjugation to LNP, and importantly, maintains the size and integrity of the LNP.

[0008] According to a first aspect, the present disclosure provides a pharmaceutical composition comprising a lipid nanoparticle (LNP), a therapeutic nucleic acid (TNA), and at least one pharma- ceutically acceptable excipient, wherein the LNP comprises a single chain variable fragment (scFv) linked to the LNP, and the scFv is directed to an antigen present on the surface of a cell. In some embodiments, the scFv is covalently linked to the LNP. In some embodiments, the scFv is chemically conjugated to the LNP. In some embodiments, the scFv is chemically conjugated to the LNP via a non-cleavable linker. In some embodiments, the non-cleavable linker is a maleimide-containing linker. In some embodiments, the scFv is chemically conjugated to the LNP via a cleavable linker. In some embodiments, the cleavable linker is a pyridyl disulfide (PDS)-containing linker. In some embodiments, the scFv is linked to the LNP via transglutaminase-mediated conjugation. In some embodiments of any of the above aspects and embodiments, the antigen is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). In further embodiments, the antigen is human epidermal growth factor receptor 2 (HER2). In some embodiments of any of the above aspects and embodiments, the scFv is bivalent. In some embodiments of any of the above aspects and embodiments, the LNP is capable of being internalized into a cell. In some embodiments of any of the above aspects and embodiments, the scFV comprises an amino acid sequence of SEQ ID NO:2 or has at least 99% sequence similarity to the amino acid sequence set forth in SEQ ID NO:2. In some embodiments of any of the above aspects and embodiments, the scFV comprises an amino acid sequence of SEQ ID NO:3 or has at least 99% sequence similarity to the amino acid sequence set forth in SEQ ID NO:3. In some embodiments of any of the above aspects and embodiments, the LNP comprises a lipid selected from the group consisting of a cationic lipid, a sterol or derivative thereof, a non-cationic lipid, and a PEGylated lipid. In some embodiments of any of the above aspects and embodiments, the TNA is encapsulated in the LNP.In some embodiments of any of the above aspects and embodiments, the TNA is selected from the group consisting of a minigene, a plasmid, a minicircle, a small interfering RNA (siRNA), a microRNA (miRNA), an antisense oligonucleotide (ASO), a ribozyme, a closed end (ceDNA), a ministring, a doggybone™, a protelomeric closed end DNA, or a dumbbell linear DNA, a dicer substrate dsRNA, a small hairpin RNA (shRNA), an asymmetric interfering RNA (aiRNA), a microRNA (miRNA), an mRNA, a tRNA, a rRNA, a DNA viral vector, a viral RNA vector, a non-viral vector, and any combination thereof. In some embodiments, the TNA is a ceDNA. In some embodiments, the ceDNA is a linear double stranded DNA. In some embodiments, the TNA is an mRNA. In some embodiments, the TNA is an siRNA. In some embodiments, the TNA is a plasmid. In some embodiments of any of the above aspects and embodiments, the pharmaceutical composition is administered to a subject. In some embodiments, the subject is a human patient in need of treatment with a TNA encapsulated LNP. In some embodiments of any of the above aspects and embodiments, the composition targets cells expressing the cell surface antigen to which the scFv is directed. In some embodiments of any of the above aspects and embodiments, the composition targets tumor cells. In some embodiments of any of the above aspects and embodiments, the composition targets liver cells. In some embodiments of any of the above aspects and embodiments, the composition targets liver cells in the liver.

[0009] In some embodiments of any of the above aspects and embodiments, the cationic lipid is represented by formula (I):

[0010] [ka] or a pharma- ceutically acceptable salt thereof, wherein: R 1 and R 1’each independently represents an optionally substituted straight or branched chain C 1~3 is alkylene, R 2 and R 2’ each independently represents an optionally substituted straight or branched chain C 1~6 is alkylene, R 3 and R 3’ each independently represents an optionally substituted straight or branched chain C 1~6 Is it an alkyl group? or alternatively, R 2 optionally substituted branched chain C 1~6 When R is alkylene, 2 and R 3 together with the intervening N atom form a 4- to 8-membered heterocyclyl, or or alternatively, R 2’ optionally substituted branched chain C 1~6 When R is alkylene, 2’ and R 3’ together with the intervening N atom form a 4- to 8-membered heterocyclyl; R 4 and R 4’ However, each independently, -CR a , -C(R a )2CR a , or -[C(R a )2]2CR a and R a for each occurrence, independently, H or C 1~3 Is it an alkyl group? or alternatively, R 4 But -C(R a )2CR a , or -[C(R a )2]2CR a If R a C 1~3 If it is alkyl, R 3 and R 4 together with the intervening N atom form a 4- to 8-membered heterocyclyl, or or alternatively, R4’ But -C(R a )2CR a , or -[C(R a )2]2CR a If R a C 1~3 If it is alkyl, R 3’ and R 4’ together with the intervening N atom form a 4- to 8-membered heterocyclyl; R 5 and R 5’ are independently hydrogen, C 1~20 Alkylene or C 2~20 alkenylene, R 6 and R 6’ But for each occurrence, independently, C 1~20 Alkylene, C 3~20 Cycloalkylene or C 2~20 alkenylene, m and n are each independently an integer selected from 1, 2, 3, 4, and 5.

[0011] In some embodiments of any of the above aspects and embodiments, the cationic lipid is represented by formula (II):

[0012] [ka] or a pharma- ceutically acceptable salt thereof, wherein: a is an integer ranging from 1 to 20; b is an integer ranging from 2 to 10; R 1 does not exist or (C2~C 20 ) alkenyl, -C(O)O(C2-C 20 ) alkyl, and (C2-C 20 ) cyclopropyl substituted with alkyl; R 2 However, (C2~C 20 ) alkyl.

[0013] In some embodiments of any of the above aspects and embodiments, the lipid is represented by formula (V):

[0014] [ka] or a pharma- ceutically acceptable salt thereof, wherein: R 1 and R 1’ However, each independently, R a is a (C1-C6) alkylene optionally substituted with one or more groups selected from R 2 and R 2’ are each independently (C1-C2) alkylene; R 3 and R 3’ However, each independently, R b is a (C1-C6) alkyl optionally substituted with one or more groups selected from or alternatively, R 2 and R 3 and / or R 2’ and R 3’ together with the intervening N atom form a 4- to 7-membered heterocyclyl; R 4 and R 4 ' is a (C2-C6) alkylene interrupted by -C(O)O-, R 5 and R 5 ' are each independently, (C2~C 30 ) alkyl or (C2-C 30 ) alkenyl, each of which is optionally interrupted by -C(O)O- or (C3-C6)cycloalkyl; R a and R b are each halo or cyano.

[0015] In some embodiments of any of the above aspects and embodiments, the cationic lipid is represented by formula (XV):

[0016] [ka] or a pharma- ceutically acceptable salt thereof, wherein: R' is absent, hydrogen, or C1-C6 alkyl, provided that when R' is hydrogen or C1-C6 alkyl, R', R 1 , and R 2 The nitrogen atoms to which all are attached are protonated, R 1 and R 2 are each independently hydrogen, C1-C6 alkyl, or C2-C6 alkenyl; R 3 But, C1~C 12 Alkylene or C2-C 12 alkenylene, R 4 But, C1~C 16 Unbranched alkyl, C2-C 16 unbranched alkenyl, or

[0017] [ka] where: R 4a and R 4b However, each is independent, C1~C 16 Unbranched alkyl or C2-C 16 is an unbranched alkenyl; R 5 is absent, C1-C8 alkylene, or C2-C8 alkenylene; R 6a and R 6b However, each is independent, C7~C 16 Alkyl or C7-C 16 alkenyl, where R 6a and R 6b the total number of carbon atoms in the X 1 and X 2 are each independently -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -SS-, -C(R a )=N-, -N=C(R a )-, -C(R a )=NO-, -ON=C(R a )-, -C(=O)NR a -, -NR a C(=O)-, -NR a C(=O)NR a -, -OC(=O)O-, -OSi(R a )2O-, -C(=O)(CR a 2) C(=O)O- or OC(=O)(CR a 2) C(=O)-, wherein R a is, for each occurrence, independently, hydrogen or C1-C6 alkyl; n is an integer selected from 1, 2, 3, 4, 5, and 6.

[0018] In some embodiments of any of the above aspects and embodiments, the cationic lipid is represented by formula (XX):

[0019] [ka] or a pharma- ceutically acceptable salt thereof, wherein: R' is absent, hydrogen, or C1-C3 alkyl, provided that when R' is hydrogen or C1-C3 alkyl, R', R 1 , and R 2 The nitrogen atoms to which all are attached are protonated, R 1 and R 2 are each independently hydrogen or C1-C3 alkyl; R 3 But C3~C 10 Alkylene or C3-C 10 alkenylene, R 4 But, C1~C 16 Unbranched alkyl, C2-C 16 unbranched alkenyl, or

[0020] [ka] where: R 4a and R 4b However, each is independent, C1~C 16 Unbranched alkyl or C2-C 16 is an unbranched alkenyl; R 5 is absent, C1-C6 alkylene, or C2-C6 alkenylene; R 6a and R 6b However, each is independent, C7~C 14 Alkyl or C7-C 14 alkenyl, X is -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -SS-, -C(R a )=N-, -N=C(R a )-, -C(R a )=NO-, -ON=C(R a )-, -C(=O)NR a -, -NR a C(=O)-, -NR a C(=O)NR a -, -OC(=O)O-, -OSi(R a )2O-, -C(=O)(CR a 2) C(=O)O- or OC(=O)(CR a 2) C(=O)-, wherein R a is, for each occurrence, independently, hydrogen or C1-C6 alkyl; n is an integer selected from 1, 2, 3, 4, 5, and 6.

[0021] In some embodiments of any of the above aspects and embodiments, the cationic lipid is selected from any of the lipids in Table 2, Table 5, Table 6, Table 7, or Table 8. In some embodiments, the cationic lipid is a lipid having the following structure:

[0022] [ka] or a pharma- ceutically acceptable salt thereof. In some embodiments, the cationic lipid is MC3(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA or MC3) having the following structure:

[0023] [ka] or a pharma- ceutically acceptable salt thereof.

[0024] In some embodiments of any of the above aspects and embodiments, the sterol or derivative thereof is cholesterol or beta-sitosterol. In some embodiments, the non-cationic lipid is distearoyl-sn-glycero-phosphoethanolamine (DSPE), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidyl 1-Oleoyl-2-oleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine (e.g., 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (e.g., 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine) (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), dierucoyl phosphatidylcholine (DEPC), palmitoyl oleyl phosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE); 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE);The non-cationic lipid is selected from the group consisting of lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. In some embodiments, the non-cationic lipid is selected from the group consisting of dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), and dioleoyl-phosphatidylethanolamine (DOPE). In some embodiments, the PEGylated lipid is PEG-dilauryloxypropyl; PEG-dimyristyloxypropyl; PEG-dipalmityloxypropyl, PEG-distearyloxypropyl; 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (DMG-PEG); PEG-dilaurylglycerol; PEG-dipalmitoylglycerol; PEG-disterylglycerol; PEG-dilaurylglycamide; PEG-dimyristylglycamide; PEG-dipalmitoylglycamide; PEG-disterylglycamide; (1-[8'-(cholest-5-ene-3[beta]-oxy)carboxamido-3',6'-dioxaotanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol) (PEG-cholesterol);The PEGylated lipid is selected from the group consisting of 3,4-ditetradecaoxylbenzyl-[omega]-methyl-poly(ethylene glycol)ether (PEG-DMB), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol) (DSPE-PEG), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-hydroxyl (DSPE-PEG-OH). In some embodiments, the PEGylated lipid is DMG-PEG, DSPE-PEG, DSPE-PEG-OH, or a combination thereof. In some embodiments of any of the above aspects and embodiments, at least one PEGylated lipid is DMG-PEG2000, DSPE-PEG2000, DSPE-PEG2000-OH, DMG-PEG5000, DSPE-PEG5000, DSPE-PEG5000-OH, or a combination thereof. In some embodiments of any of the above aspects and embodiments, the scFv is chemically conjugated or covalently linked to a PEGylated lipid of the LNP to form a PEGylated lipid conjugate. In some embodiments of any of the above aspects and embodiments, the PEGylated lipid to which the scFv is chemically conjugated or covalently linked is DSPE-PEG. In some embodiments, the PEGylated lipid to which the scFv is chemically conjugated or covalently linked is DSPE-PEG2000. In some embodiments, the PEGylated lipid to which the scFv is chemically conjugated or covalently linked is DSPE-PEG5000. In some embodiments of any of the above aspects and embodiments, the cationic lipid is present at a molar percentage of about 30% to about 80%. In some embodiments, the sterol is present at a molar percentage of about 20% to about 50%. In some embodiments of any of the above aspects and embodiments, the non-cationic lipid is present at a molar percentage of about 2% to about 20%. In some embodiments of any of the above aspects and embodiments, the at least one PEGylated lipid is present at a molar percentage of about 2.1% to about 10%;

[0025] In some embodiments of any of the above aspects and embodiments, the scFv is present in a total amount of about 0.02 μg / μg TNA to about 0.1 μg / μg TNA.

[0026] In some embodiments of any of the above aspects and embodiments, the pharmaceutical composition further comprises dexamethasone palmitate.

[0027] In some embodiments of any of the above aspects and embodiments, the LNPs have a total lipid to TNA ratio of about 10:1 to about 40:1.

[0028] In some embodiments of any of the above aspects and embodiments, LNPs have diameters ranging from about 40 nm to about 120 nm.

[0029] In some embodiments of any of the above aspects and embodiments, the nanoparticles have a diameter of less than about 100 nm.

[0030] In some embodiments of any of the above aspects and embodiments, The nanoparticles have a diameter of about 60 nm to about 80 nm.

[0031] In some embodiments of any of the above aspects and embodiments, the ceDNA comprises an expression cassette, the expression cassette comprising a promoter sequence and a transgene. In some embodiments, the expression cassette comprises a polyadenylation sequence. In some embodiments of any of the above aspects and embodiments, the ceDNA comprises at least one inverted terminal repeat (ITR) flanking either the 5' or 3' end of the expression cassette. In some embodiments, the expression cassette is flanked by two ITRs, the two ITRs comprising one 5'ITR and one 3'ITR. In some embodiments, the expression cassette is linked to the ITR at the 3' end (3'ITR). In some embodiments of any of the above aspects and embodiments, the expression cassette is linked to the ITR at the 5' end (5'ITR).

[0032] In some embodiments of any of the above aspects and embodiments, at least one ITR is an ITR from an AAV serotype, an ITR from a goose virus ITR, an ITR from a B19 virus ITR, or a wild-type ITR from a parvovirus, in some embodiments, the AAV serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

[0033] In some embodiments of any of the above aspects and embodiments, at least one of the 5'ITR and the 3'ITR is a wild-type AAV ITR.

[0034] In some embodiments of any of the above aspects and embodiments, at least one of the 5'ITR and the 3'ITR is a modified or mutated ITR.

[0035] In some embodiments of any of the above aspects and embodiments, the 5'ITR and the 3'ITR are symmetrical.

[0036] In some embodiments of any of the above aspects and embodiments, the 5'ITR and the 3'ITR are asymmetric.

[0037] In some embodiments of any of the above aspects and embodiments, the ceDNA further comprises a spacer sequence between the 5'ITR and the expression cassette.

[0038] In some embodiments of any of the above aspects and embodiments, the ceDNA further comprises a spacer sequence between the 3'ITR and the expression cassette. In some embodiments of any of the above aspects and embodiments, the spacer sequence is at least 5 base pairs in length.

[0039] In some embodiments of any of the above aspects and embodiments, the ceDNA has a nick or a gap.

[0040] In some embodiments of any of the above aspects and embodiments, the ceDNA is a CELiD, a DNA-based minicircle, a MIDGE, a ministring DNA, a dumbbell-shaped linear double-stranded closed-end DNA containing two hairpin structures of ITRs at the 5' and 3' ends of the expression cassette, or a doggybone™ DNA.

[0041] In some aspects, the disclosure provides a method of treating cancer in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition of any one of the aspects or embodiments herein. In some embodiments, the subject is a human.

[0042] In some aspects, the present disclosure provides a method of delivering a therapeutic nucleic acid (TNA) to a tumor in a subject or increasing the concentration of a TNA in a tumor in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition of any one of the aspects and embodiments herein.

[0043] In some aspects, the present disclosure provides a method of delivering a therapeutic nucleic acid (TNA) to the liver of a subject or increasing the concentration of a TNA in the liver of a subject, comprising administering to the subject an effective amount of a pharmaceutical composition of any one of the aspects or embodiments herein.

[0044] According to some embodiments, the LNP is internalized in the cell. According to some embodiments of the above aspects and embodiments, the LNP comprises a cationic lipid, a sterol or derivative thereof, a non-cationic lipid, or a PEGylated lipid. According to some embodiments of the above aspects and embodiments, the TNA is encapsulated in a lipid. According to some embodiments of the above aspects and embodiments, the TNA is selected from the group consisting of a minigene, a plasmid, a minicircle, a small interfering RNA (siRNA), a microRNA (miRNA), an antisense oligonucleotide (ASO), a ribozyme, a closed end (ceDNA), a ministring, a doggybone™, a protelomeric closed end DNA, or a dumbbell linear DNA, a dicer substrate dsRNA, a small hairpin RNA (shRNA), an asymmetric interfering RNA (aiRNA), a microRNA (miRNA), an mRNA, a tRNA, a rRNA, a DNA virus vector, a viral RNA vector, a non-viral vector, and any combination thereof. According to some embodiments, the TNA is a ceDNA. According to some embodiments, the ceDNA is a linear double-stranded DNA. According to some embodiments, the TNA is an mRNA. According to some embodiments, the TNA is an siRNA. According to some embodiments, the TNA is a plasmid.

[0045] According to some embodiments, the LNP comprises a PEGylated lipid, and the PEGylated lipid is linked to an amino acid sequence encoding an scFv (scFv polypeptide). According to some embodiments of the above aspects and embodiments, the pharmaceutical composition is administered to a subject. According to some embodiments of the above aspects and embodiments, the subject is a human patient in need of treatment with a TNA-encapsulated LNP. According to some embodiments of the above aspects and embodiments, the composition targets cells or tissues expressing a target antigen via binding of the scFv in the LNP to the antigen target. According to some embodiments of the above aspects and embodiments, the composition targets tumor cells. According to some embodiments, the tumor is a solid tumor. According to some embodiments, the tumor is a hematological tumor. According to some embodiments of the above aspects and embodiments, the composition targets liver cells.

[0046] According to some embodiments of the above aspects and embodiments, the cationic lipid is represented by formula (I):

[0047] [ka] or a pharma- ceutically acceptable salt thereof, wherein: R 1 and R 1’ each independently represents an optionally substituted straight or branched chain C 1~3 is alkylene, R 2 and R 2’ each independently represents an optionally substituted straight or branched chain C 1~6 is alkylene, R 3 and R 3’ each independently represents an optionally substituted straight or branched chain C 1~6 Is it an alkyl group? or alternatively, R 2 optionally substituted branched chain C 1~6 When R is alkylene, 2 and R 3together with the intervening N atom form a 4- to 8-membered heterocyclyl, or or alternatively, R 2’ optionally substituted branched chain C 1~6 When R is alkylene, 2’ and R 3’ together with the intervening N atom form a 4- to 8-membered heterocyclyl; R 4 and R 4’ However, each independently, -CR a , -C(R a )2CR a , or -[C(R a )2]2CR a and R a for each occurrence, independently, H or C 1~3 Is it an alkyl group? or alternatively, R 4 But -C(R a )2CR a , or -[C(R a )2]2CR a If R a C 1~3 If it is alkyl, R 3 and R 4 together with the intervening N atom form a 4- to 8-membered heterocyclyl, or or alternatively, R 4’ But -C(R a )2CR a , or -[C(R a )2]2CR a If R a C 1~3 If it is alkyl, R 3’ and R 4’ together with the intervening N atom form a 4- to 8-membered heterocyclyl; R 5 and R 5’ are independently hydrogen, C 1~20 Alkylene or C 2~20 alkenylene, R 6 and R 6’But for each occurrence, independently, C 1~20 Alkylene, C 3~20 Cycloalkylene or C 2~20 alkenylene, m and n are each independently an integer selected from 1, 2, 3, 4, and 5.

[0048] According to some embodiments of the above aspects and embodiments, the cationic lipid is represented by formula (II):

[0049] [ka] or a pharma- ceutically acceptable salt thereof, wherein: a is an integer ranging from 1 to 20; b is an integer ranging from 2 to 10; R 1 does not exist or (C2~C 20 ) alkenyl, -C(O)O(C2-C 20 ) alkyl, and (C2-C 20 ) cyclopropyl substituted with alkyl; R 2 However, (C2~C 20 ) alkyl.

[0050] According to some embodiments of the above aspects and embodiments, the lipid is represented by formula (V):

[0051] [ka] or a pharma- ceutically acceptable salt thereof, wherein: R 1 and R 1’ However, each independently, R a is a (C1-C6) alkylene optionally substituted with one or more groups selected from R 2 and R 2’ are each independently (C1-C2) alkylene; R 3 and R3’ However, each independently, R b is a (C1-C6) alkyl optionally substituted with one or more groups selected from or alternatively, R 2 and R 3 and / or R 2’ and R 3’ together with the intervening N atom form a 4- to 7-membered heterocyclyl; R 4 and R 4 ' is a (C2-C6) alkylene interrupted by -C(O)O-, R 5 and R 5 ' are each independently, (C2~C 30 ) alkyl or (C2-C 30 ) alkenyl, each of which is optionally interrupted by -C(O)O- or (C3-C6)cycloalkyl; R a and R b are each halo or cyano.

[0052] According to some embodiments of the above aspects and embodiments, the cationic lipid is represented by formula (XV):

[0053] [ka] or a pharma- ceutically acceptable salt thereof, wherein: R' is absent, hydrogen, or C1-C6 alkyl, provided that when R' is hydrogen or C1-C6 alkyl, R', R 1 , and R 2 The nitrogen atoms to which all are attached are protonated, R 1 and R 2 are each independently hydrogen, C1-C6 alkyl, or C2-C6 alkenyl; R 3 But, C1~C 12 Alkylene or C2-C 12 alkenylene, R 4 But, C1~C 16 Unbranched alkyl, C2-C 16 unbranched alkenyl, or

[0054] [ka] where: R 4a and R 4b However, each is independent, C1~C 16 Unbranched alkyl or C2-C 16 is an unbranched alkenyl; R 5 is absent, C1-C8 alkylene, or C2-C8 alkenylene; R 6a and R 6b However, each is independent, C7~C 16 Alkyl or C7-C 16 alkenyl, where R 6a and R 6b the total number of carbon atoms in the X 1 and X 2 are each independently -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -SS-, -C(R a )=N-, -N=C(R a )-, -C(R a )=NO-, -ON=C(R a )-, -C(=O)NR a -, -NR a C(=O)-, -NR a C(=O)NR a -, -OC(=O)O-, -OSi(R a )2O-, -C(=O)(CR a 2) C(=O)O- or OC(=O)(CR a 2) C(=O)-, wherein R a is, for each occurrence, independently, hydrogen or C1-C6 alkyl; n is an integer selected from 1, 2, 3, 4, 5, and 6.

[0055] According to some embodiments of the above aspects and embodiments, the cationic lipid is represented by formula (XX):

[0056] [ka] or a pharma- ceutically acceptable salt thereof, wherein: R' is absent, hydrogen, or C1-C3 alkyl, provided that when R' is hydrogen or C1-C3 alkyl, R', R 1 , and R 2 The nitrogen atoms to which all are attached are protonated, R 1 and R 2 are each independently hydrogen or C1-C3 alkyl; R 3 But C3~C 10 Alkylene or C3-C 10 alkenylene, R 4 But, C1~C 16 Unbranched alkyl, C2-C 16 unbranched alkenyl, or

[0057] [ka] where: R 4a and R 4b However, each is independent, C1~C 16 Unbranched alkyl or C2-C 16 is an unbranched alkenyl; R 5 is absent, C1-C6 alkylene, or C2-C6 alkenylene; R 6a and R 6b However, each is independent, C7~C 14 Alkyl or C7-C 14 alkenyl, X is -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -SS-, -C(R a )=N-, -N=C(R a )-, -C(R a )=NO-, -ON=C(R a )-, -C(=O)NR a -, -NR a C(=O)-, -NR a C(=O)NR a -, -OC(=O)O-, -OSi(R a )2O-, -C(=O)(CR a 2) C(=O)O- or OC(=O)(CR a 2) C(=O)-, wherein R a is, for each occurrence, independently, hydrogen or C1-C6 alkyl; n is an integer selected from 1, 2, 3, 4, 5, and 6.

[0058] According to some embodiments of the above aspects and embodiments, the cationic lipid is selected from any of the lipids in Table 2, Table 5, Table 6, Table 7, or Table 8.

[0059] According to some embodiments of the above aspects and embodiments, the cationic lipid is a lipid having the structure:

[0060] [ka] or a pharma- ceutically acceptable salt thereof.

[0061] According to some embodiments of the above aspects and embodiments, the cationic lipid is MC3(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA or MC3), having the following structure:

[0062] [ka] or a pharma- ceutically acceptable salt thereof.

[0063] According to some embodiments of the above aspects and embodiments, the sterol or derivative thereof is cholesterol or beta-sitosterol. According to some embodiments, the non-cationic lipid is selected from the group consisting of distearoyl-sn-glycero-phosphoethanolamine (DSPE), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPC), dioleo ... dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (e.g. 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (e.g. 16-O-dimethyl PE), 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleylphosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine ( ... sphoethanolamine, DLPE); 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE); lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. According to some embodiments, the non-cationic lipid is selected from the group consisting of dioleoylphosphatidylcholine (DOPC),Distearoylphosphatidylcholine (DSPC) and dioleoyl-phosphatidylethanolamine (DOPE). According to some embodiments, the PEGylated lipid is selected from the group consisting of PEG-dilauryloxypropyl; PEG-dimyristyloxypropyl; PEG-dipalmityloxypropyl, PEG-distearyloxypropyl; 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (DMG-PEG); PEG-dilaurylglycerol; PEG-dipalmitoylglycerol; PEG-disterylglycerol; PEG-dilaurylglycamide; PEG-dimyristylglycamide; PEG-dipalmitoylglycamide; PEG-disterylglycamide; (1-[8'-(cholest-5-ene-3[beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol) (PEG-cholesterol); 3,4 -ditetradecaoxylbenzyl-[omega]-methyl-poly(ethylene glycol)ether (PEG-DMB), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol) (DSPE-PEG), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-hydroxyl (DSPE-PEG-OH). According to some embodiments, the PEGylated lipid is DMG-PEG, DSPE-PEG, DSPE-PEG-OH, or a combination thereof. According to some embodiments, at least one PEGylated lipid is DMG-PEG2000, DSPE-PEG2000, DSPE-PEG2000-OH, or a combination thereof.

[0064] According to some embodiments of the above aspects and embodiments, the scFv is chemically conjugated or covalently linked to the PEGylated lipid of the LNP to form a PEGylated lipid conjugate. According to some embodiments, the PEGylated lipid to which the scFv is chemically conjugated or covalently linked is DSPE-PEG. According to some embodiments of the above aspects and embodiments, the scFv is covalently linked to the LNP via a non-cleavable linker. According to some embodiments, the non-cleavable linker is a maleimide-containing linker.

[0065] According to some embodiments of the above aspects and embodiments, the scFv is covalently linked to the LNP via a cleavable linker.

[0066] According to some embodiments of the above aspects and embodiments, the scFv is covalently linked to the LNP via a pyridyl disulfide (PDS)-containing linker.

[0067] According to some embodiments of the above aspects and embodiments, the cationic lipid is present at a molar percentage of about 30% to about 80%. According to some embodiments, the sterol is present at a molar percentage of about 20% to about 50%. According to some embodiments of the above aspects and embodiments, the non-cationic lipid is present at a molar percentage of about 2% to about 20%. According to some embodiments of the above aspects and embodiments, the at least one PEGylated lipid is present at a molar percentage of about 2.1% to about 10%. According to some embodiments, the scFv polypeptide is present in a total amount of about 0.02 μg / μg TNA to about 0.1 μg / μg TNA.

[0068] According to some embodiments of the above aspects and embodiments, the pharmaceutical composition further comprises dexamethasone palmitate. According to some embodiments of the above aspects and embodiments, the LNPs have a total lipid to TNA ratio of about 10:1 to about 40:1. According to some embodiments of the above aspects and embodiments, the LNPs have a diameter ranging from about 40 nm to about 120 nm. According to some embodiments of the above aspects and embodiments, the nanoparticles have a diameter of less than about 100 nm. According to some embodiments of the above aspects and embodiments, the nanoparticles have a diameter of about 60 nm to about 80 nm. According to some embodiments of the above aspects and embodiments, the ceDNA comprises an expression cassette, the expression cassette comprising a promoter sequence and a transgene. According to some embodiments, the expression cassette comprises a polyadenylation sequence.

[0069] According to some embodiments of the above aspects and embodiments, the ceDNA comprises at least one inverted terminal repeat (ITR) flanking either the 5' or 3' end of the expression cassette. According to some embodiments, the expression cassette is flanked by two ITRs, the two ITRs comprising one 5'ITR and one 3'ITR. According to some embodiments, the expression cassette is linked to an ITR at the 3' end (3'ITR). According to some embodiments of the above aspects and embodiments, the expression cassette is linked to an ITR at the 5' end (5'ITR). According to some embodiments of the above aspects and embodiments, the at least one ITR is from an AAV serotype, from a goose virus ITR, from a B19 virus ITR, or a wild-type ITR from a parvovirus. According to some embodiments of the above aspects and embodiments, the AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12. According to some embodiments of the above aspects and embodiments, at least one of the 5'ITR and the 3'ITR is a wild-type AAV ITR. According to some embodiments of the above aspects and embodiments, at least one of the 5'ITR and the 3'ITR is a modified or mutated ITR. According to some embodiments of the above aspects and embodiments, the 5'ITR and the 3'ITR are symmetric. According to some embodiments of the above aspects and embodiments, the 5'ITR and the 3'ITR are asymmetric. According to some embodiments of the above aspects and embodiments, the ceDNA further comprises a spacer sequence between the 5'ITR and the expression cassette. According to some embodiments of the above aspects and embodiments, the ceDNA further comprises a spacer sequence between the 3'ITR and the expression cassette. According to some embodiments of the above aspects and embodiments, the spacer sequence is at least 5 base pairs in length. According to some embodiments of the above aspects and embodiments, the ceDNA has a nick or a gap.According to some embodiments of the above aspects and embodiments, the ceDNA is a CELiD, a DNA-based minicircle, a MIDGE, a ministring DNA, a dumbbell-shaped linear double-stranded closed-end DNA containing two hairpin structures of ITRs at the 5' and 3' ends of the expression cassette, or a doggybone™ DNA.

[0070] According to another aspect, the disclosure features a method of treating, e.g.,

[0071] According to another aspect, the present disclosure provides a method of delivering a therapeutic nucleic acid (TNA) to a tumor in a subject or increasing the concentration of a TNA in a tumor in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition of any one of the aspects or embodiments herein. [Brief description of the drawings]

[0072]

[0013] Embodiments of the present disclosure, briefly summarized above and discussed in more detail below, can be understood by reference to the exemplary embodiments of the present disclosure, which are depicted in the accompanying drawings. The accompanying drawings, however, depict only typical embodiments of the present disclosure and therefore should not be considered as limiting in scope, since the present disclosure may admit of other equally effective embodiments. [Figure 1] Figures 1A-1F show that trastuzumab-derived α-HER2 scFv showed clear HER2 bispecific membrane targeting and internalization in vitro. Using Alexa-fluor 488-(AF488)-labeled anti-HER2 scFv, we demonstrated HER2 receptor binding in SkBR3 (Figure 1A) and SkOV3 (Figure 1B) Her2-expressing (HER2+) cell lines, but not in MCF7 cells (Figure 1C), which do not express the HER2 receptor (HER2-). A second immunofluorescence labeling (pHrhodo) was used to demonstrate ligand internalization. As shown in Figures 1D-1F, SkBR3 and SkOV3 cells expressing the HER2 receptor showed ligand internalization (Figures 1D and 1E), but not the MCF7 HER2 cell line (Figure 1F). [Figure 2A] FIG. 1 shows a schematic diagram of an exemplary primary pathway for conjugation using thiol-based crosslinking. [Figure 2B] FIG. 1 shows a schematic diagram of an exemplary primary pathway for conjugation using thiol-based crosslinking. [Figure 3A] It shows that the scFv-LNP conjugation process demonstrated excellent conjugation yield and LNP particle stability. The results of the conjugation process including initial TCEP reduction, fresh MAL-LNP (maleimide conjugated LNP) preparation, 0.5% MAL-PEG2K, scFv: 0.5, 0.25, 0.1, and 0.05 equimolar equivalents are shown in Figure 3A. Next, the PEG chain length was increased to PEG5K, and a dialysis step was performed to remove unreacted scFv without destroying particle size and stability. The results are shown in Figure 3B. [Figure 3B] It shows that the scFv-LNP conjugation process demonstrated excellent conjugation yield and LNP particle stability. The results of the conjugation process including initial TCEP reduction, fresh MAL-LNP (maleimide conjugated LNP) preparation, 0.5% MAL-PEG2K, scFv: 0.5, 0.25, 0.1, and 0.05 equimolar equivalents are shown in Figure 3A. Next, the PEG chain length was increased to PEG5K, and a dialysis step was performed to remove unreacted scFv without destroying particle size and stability. The results are shown in Figure 3B. [Figure 4A] FIG. 13 is a graph showing that LNP size and encapsulation efficiency were maintained after scFv conjugation (±10 nm) through the conjugation process. [Figure 4B] FIG. 13 is a graph showing that LNP size and encapsulation efficiency were maintained after scFv conjugation (±10 nm) through the conjugation process. [Diagram 5] 1 shows that the maleimide conjugation process resulted in robust conjugation. [Figure 6A]6A-6B are graphs showing that only Tras-scFv conjugated LNPs (FIG. 6A), but not 0.5% DSPE control LNPs (FIG. 6B), demonstrated HER2 binding, thereby confirming ligand function on the LNPs. [Figure 6B] 6A-6B are graphs showing that only Tras-scFv conjugated LNPs (FIG. 6A), but not 0.5% DSPE control LNPs (FIG. 6B), demonstrated HER2 binding, thereby confirming ligand function on the LNPs. [Figure 7] We show that maleimide-conjugated LNPs (MAL-LNPs) demonstrated Her2-specific enhanced cellular uptake, specifically demonstrating that uptake of conjugated Tras-scFv Lipid A LNPs (mCherry) was mediated by HER2. [Figure 8A] This shows that ligand presentation on the LNP surface significantly affected bioactivity. The graph in Figure 8A compares LNP uptake (mCherry) in maleimide-conjugated LNPs where the PEG chain length was either 2000 Da (PEG2K) or 5000 Da (PEG5K), normalized to cell viability. As shown in Figure 8A, the maleimide-conjugated LNPs with PEG5K exhibited greater bioactivity as assessed by LNP cellular uptake. The graph in Figure 8B shows that a dose-dependent decrease in LNP uptake (mCherry) was observed as the maleimide concentration (conjugated to PEG5K) was increased from 0.5% to 1.25%. [Figure 8B]This shows that ligand presentation on the LNP surface significantly affected bioactivity. The graph in Figure 8A compares LNP uptake (mCherry) in maleimide-conjugated LNPs where the PEG chain length was either 2000 Da (PEG2K) or 5000 Da (PEG5K), normalized to cell viability. As shown in Figure 8A, the maleimide-conjugated LNPs with PEG5K exhibited greater bioactivity as assessed by LNP cellular uptake. The graph in Figure 8B shows that a dose-dependent decrease in LNP uptake (mCherry) was observed as the maleimide concentration (conjugated to PEG5K) was increased from 0.5% to 1.25%. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0073] The present disclosure provides lipid nanoparticle (LNP) compositions (e.g., pharmaceutical compositions) comprising therapeutic nucleic acids (TNAs), wherein the LNP comprises a single chain variable fragment (scFv) linked to the LNP, and the scFv targets an antigen present on the surface of a cell (e.g., a tumor cell). It is an advantageous feature of the present disclosure that any scFv can be linked to the LNP, and is useful for targeting any cell or tissue expressing the antigen of interest of the scFv. The LNP compositions described herein advantageously provide efficient covalent conjugation with minimal impact on particle size and stability.

[0074] According to one aspect, the present disclosure provides a pharmaceutical composition comprising a lipid nanoparticle (LNP), a therapeutic nucleic acid (TNA), and at least one pharma- ceutically acceptable excipient, wherein the LNP comprises a single chain variable fragment (scFv) linked to the LNP, and the scFv targets an antigen present on the surface of a cell. It is the discovery of the present disclosure that maleimide conjugation of the scFv to the LNP results in robust conjugation to the LNP, and importantly, maintains the size and integrity of the LNP. According to some embodiments, the scFv is covalently linked to the LNP. As used herein, the term "covalent bond" refers to a chemical bond involving the sharing of electron pairs between atoms. According to some embodiments, the scFV is chemically conjugated to the LNP. As used herein, the term "conjugation" when referring to a conjugation chemistry or system refers to a system of overlapping p-orbitals with delocalized electrons from multiple atoms. According to some embodiments, the scFV is chemically conjugated to the LNP via a non-cleavable linker. According to some embodiments, the non-cleavable linker is a maleimide-containing linker. According to some embodiments, the scFV is chemically conjugated to the LNP via a cleavable linker. According to some embodiments, the cleavable linker is a pyridyl disulfide (PDS)-containing linker. According to some embodiments, the scFV is linked to the LNP via transglutaminase-mediated conjugation. As used herein, "transglutaminase-mediated conjugation" refers to conjugation as defined herein that is mediated by microbial transglutaminase (MTGase).MTGase catalyzes the site-specific modification (i.e., transpeptidation) between a primary amine in the linker and the side chain of a specific glutamine residue of an antibody or single chain variable fragment (scFv), such as glutamine 295 in deglycosylated chimeric, humanized IgG1 and human IgG1 (see, e.g., Anami Y., Tsuchikama K. (2020) Transglutaminase-Mediated Conjugations. In: Tumey L. (eds) Antibody-Drug Conjugates. Methods in Molecular Biology, vol 2078. Humana, New York, NY., incorporated herein by reference in its entirety). This method can be performed by mutation of asparagine 297, insertion of a glutamine-containing peptide tag, and use of a branched linker. Such modifications facilitate the conjugation process and provide flexibility in adjusting the conjugation site and drug-to-antibody ratio (DAR) (Yasuaki Anami and Kyoji Tsuchikama, "Transglutaminase-Mediated Conjugations," in Methods in Molecular Biology, Antibody Drug Conjugates (2020), incorporated herein by reference in its entirety). In some embodiments, conjugation can be enhanced by the insertion of a glutamine-containing peptide tag and / or the use of a branched linker. In one embodiment, the glutamine-containing peptide tag is LLQGA (Leu-Leu-Gln-Glu-Ala or SEQ ID NO: 4). In some embodiments, the glutamine-containing peptide tag comprises SEQ ID NO: 4. In some embodiments, the glutamine-containing peptide tag consists of SEQ ID NO: 4.

[0075] As an additional advantage, the LNPs described herein provide more efficient delivery of therapeutic nucleic acids, better tolerability and improved safety profile. The therapeutic nucleic acid-lipid particles (e.g., lipid nanoparticles) described herein are free of packaging constraints imposed by the space within the viral capsid, so theoretically, the only size limitation of the therapeutic nucleic acid-lipid particles (e.g., lipid nanoparticles) resides in the DNA replication efficiency of the host cell. As described and exemplified herein, in some embodiments, the therapeutic nucleic acid is a therapeutic nucleic acid (TNA), such as double-stranded DNA (e.g., ceDNA). As described and exemplified herein, in some embodiments, the therapeutic nucleic acid is ceDNA. Also as described herein, in some embodiments, the therapeutic nucleic acid is an mRNA.

[0076] I. Definition Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those skilled in the art to which this disclosure belongs. It is to be understood that this disclosure is not limited to the specific methodology, protocols, and reagents, etc. described herein, and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the disclosure, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3), Robert S. Porter et al. (eds.), Fields Virology, 6th Edition, published by Lippincott Williams & Wilkins, Philadelphia, PA, USA (2013), Knipe, DM and Howley, PM (ed.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908), and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, VCH Publishers, Inc.Printed: Immunology by Werner Luttmann, Elsevier: Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver(Point),Taylor & Francis Limited,2014(ISBN 0815345305,9780815345305),Lewin's Genes XI,Jones & Bartlett Publishers Sambrook,Molecular Cloning:A Laboratory Manual,4th ed.,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY,USA(2012)(ISBN 1936113414); 044460149X)、Laboratory Methods in Enzymology:DNA,Jon Lorsch(Post)Elsevier,2013(ISBN 0124199542)、Current Protocols in Molecular Biology(CPMB),Frederick M.Ausubel(Post),John Wiley and Sons, 2014(ISBN047150338X,9780471503385)、Current Protocols in Protein Science(CPPS),John E.Coligan(Power),John Wiley and Sons,Inc.,2005;Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of all of which are incorporated herein by reference in their entireties.

[0077] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0078] The abbreviation "eg" comes from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with "for example."

[0079] The use of the alternative (eg, "or") should be understood to mean either one, both, or any combination thereof.

[0080] As used herein, the term "about" when referring to a measurable value, such as an amount, temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value as appropriate to perform the disclosed methods.

[0081] As used herein, any concentration range, percentage range, ratio range, or integer range, unless otherwise specified, should be understood to include any integer value within the recited range, and fractions thereof, where appropriate (such as tenths and hundredths of integers).

[0082] As used herein, "comprise," "comprising," and "comprises," and "comprised of" are meant to be synonymous with the terms "include," "including," "includes," or "contain," "containing," "contains," e.g., are inclusive or open-ended terms specifying the presence of subsequent components, and do not exclude or preclude the presence of additional, unrecited components, features, elements, members, steps that are known in the art or disclosed therein.

[0083] The term "consisting of" refers to compositions, methods, processes, and their respective components described herein, excluding any elements not recited in the description of the embodiments.

[0084] As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristics of that embodiment of the present disclosure.

[0085] As used herein, the terms "such as," "for example," and the like are intended to refer to example embodiments and are not intended to limit the scope of the disclosure.

[0086] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice for testing of this disclosure, suitable materials and methods are described herein.

[0087] As used herein, the terms "administration", "administering" and variations thereof refer to the introduction of a composition or agent (e.g., a nucleic acid, particularly ceDNA) into a subject, including simultaneous and sequential introduction of one or more compositions or agents. "Administration" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods. "Administration" also encompasses in vitro and ex vivo treatments. The introduction of a composition or agent into a subject is by any suitable route, including oral, pulmonary, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, intratumoral, or topical. Administration includes self-administration and administration by another person. Administration can be performed by any suitable route. A suitable route of administration allows the composition or agent to perform its intended function. For example, if the suitable route is intravenous, the composition is administered by introducing the composition or agent into the subject's vein.

[0088] The term "antibody", as used herein, encompasses any naturally occurring, recombinant, modified or engineered immunoglobulin or immunoglobulin-like structure or antigen-binding fragment or portion thereof, or derivatives thereof, as further described elsewhere herein. Thus, the term refers to an immunoglobulin molecule that specifically binds to a target antigen, including, for example, chimeric antibodies, humanized antibodies, fully human antibodies, and bispecific antibodies. An intact antibody generally comprises at least two full-length heavy chains and two full-length light chains, but in some cases may comprise fewer chains, such as naturally occurring antibodies in camelids, which may comprise only heavy chains. An antibody may be derived from only a single source, or may be "chimeric", i.e., different portions of the antibody may be derived from two different antibodies. An antibody, or antigen-binding portions thereof, may be produced in a hybridoma, by recombinant DNA techniques, or by enzymatic or chemical cleavage of an intact antibody. The term antibody, as used herein, includes each of monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, human antibodies, and antibody fusions (sometimes referred to herein as "antibody conjugates").

[0089] The term "antigen-binding portion" or "antigen-binding fragment" of an antibody, as used herein, is meant to refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., TGFβ1). Antigen-binding portions include, but are not limited to, any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. In some embodiments, an antigen-binding portion of an antibody can be derived from a complete antibody molecule, for example, using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable domains and, optionally, constant domains. Non-limiting examples of antigen-binding portions include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a single-chain Fv (scFv) molecule (see, e.g., Bird et al. (1988) SCIENCE 242:423-426, and Huston et al. (1988) PROC. NAT'L. ACAD. SCI. USA 85:5879-5883); (vi) a dAb fragment (see, e.g., Ward et al. (1989) NATURE 341:544-546), and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable regions of an antibody (e.g., isolated complementarity determining regions (CDRs)). Other forms of single chain antibodies, such as diabodies, are also encompassed.The term antigen-binding portion of an antibody includes a "single-chain Fab fragment", also known as "scFab", which comprises an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein the antibody domains and the linker have one of the following orders from N-terminal to C-terminal: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL, and wherein the linker is a polypeptide of at least 30 amino acids, preferably 32-50 amino acids.

[0090] As used herein, the term "single-chain variable fragment" or "scFv" is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulins covalently linked to form a VH::VL heterodimer. The heavy (VH) and light (VL) chains are either directly linked or linked by a peptide-encoded linker (e.g., 10, 15, 20, 25 amino acids) that connects the N-terminus of the VH to the C-terminus of the VL or the C-terminus of the VH to the N-terminus of the VL. The linker is usually rich in glycine for flexibility and rich in serine or threonine for solubility. Despite the removal of the constant regions and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies can be expressed from nucleic acids containing VH and VL coding sequences as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See also U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Application Publication Nos. 20050196754 and 20050196754. According to some embodiments, scFvs derived from Fab' may be used (e.g., obtained from a Fab library instead of from an antibody). In one embodiment, the scFv binds to human epidermal growth factor receptor 2 (HER2).

[0091] As used herein, the term "antigen" is meant to refer to a molecule that elicits an immune response. This immune response may include either antibody production or activation of specific immunologically competent cells, or both. Those skilled in the art will appreciate that any macromolecule, including virtually any protein or peptide, can function as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will appreciate that any DNA that includes a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response thus encodes an "antigen" as that term is used herein. Furthermore, those skilled in the art will appreciate that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. Furthermore, those skilled in the art will appreciate that an antigen need not be encoded by a "gene" at all. Antigens can be generated, synthesized, or derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids. In one embodiment, the antigen is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA).In one embodiment, the TAA or TSA is a glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha fetoprotein (AFP), lectin-reactive AFP, thyroglubilin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, HER2 / neu, survivin and telomerase, prostate carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF), IGF-II, IGF-I receptor and mesothelin, EphA2, HER2, GD2, glypican-3, 5T4, 8H9, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, kappa light chain, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, ERBB3, ERBB4, ErbB3 / 4, FAP, FAR, FBP, fetal AchR, folate receptor a, GD2, GD3, HLA-AI Selected from the group consisting of MAGE A1, HLA-A2, IL11Ra, IL13Ra2, KDR, Lambda, Lewis-Y, MCSP, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSC1, PSMA, ROR1, SURVIVIN, TAG72, TEM1, TEM8, VEGRR2, HMW-MAA, and VEGF receptor. In some embodiments, the TAA or TSA is an antigen present in the extracellular matrix of a tumor, such as fibronectin, tenascin, or an oncofetal variant of a necrotic region of a tumor.In some embodiments, the TAA or TSA is any membrane protein or biomarker expressed or overexpressed in tumor cells, including, but not limited to, integrins (e.g., integrin αvβ3, α5β1), EGF receptor family (e.g., EGFR2, Erbb2 / HER2 / neu, Erbb3, Erbb4), proteoglycans (e.g., heparan sulfate proteoglycans), disialogangliosides (e.g., GD2, GD3), B7-H3 (aka CD276), cancer antigen 125 (CA-125), epithelial cell adhesion molecule (EpCAM), vascular endothelial growth factor receptor 1 and 2 (VEGFR-1, VEGFR-2), CD52, carcinoembryonic antigen (CEA), tumor-associated glycoprotein (e.g., TAG-72), cluster of differentiation 19 (CD19), CD20, CD22, CD30, CD33, CD40, CD44, CD74, CD152, mucin 1 (MUC1), tumor necrosis factor receptor (e.g., TRAIL-R2), insulin-like growth factor receptor, folate receptor a, transmembrane glycoprotein NMB (GPNMB), CC chemokine antigen receptor (e.g., CCR4), prostate-specific membrane receptor (PSMA), receptor d'origin nantais (RON) receptor, cytotoxic T-lymphocyte antigen 4 (CTLA4), as well as other tumor-specific receptors or antigens, are any membrane proteins or biomarkers that are expressed or overexpressed in tumor cells.

[0092] In one embodiment, the antigen is human epidermal growth factor receptor 2 (HER2).

[0093] As used herein, phrases such as "anti-therapeutic nucleic acid immune response", "anti-transfer vector immune response", "immune response to therapeutic nucleic acid", "immune response to transfer vector" are meant to refer to any unwanted immune response to a therapeutic nucleic acid, whether viral or non-viral in origin. In some embodiments, the unwanted immune response is an antigen-specific immune response to the viral transfer vector itself. In some embodiments, the immune response is specific to the transfer vector, which may be double-stranded DNA, single-stranded RNA, or double-stranded RNA. In other embodiments, the immune response is specific to the sequence of the transfer vector. In other embodiments, the immune response is specific to the CpG content of the transfer vector.

[0094] As used herein, the term "aqueous solution" is meant to refer to a composition that comprises, in whole or in part, water.

[0095] As used herein, "base" includes purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogues, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications that place new reactive groups, such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0096] As used herein, the terms "carrier" and "excipient" are meant to include any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharma-ceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the composition. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce toxic, allergic, or similar untoward reactions when administered to a host.

[0097] As used herein, the term "ceDNA" is meant to refer to capsid-free, closed-ended, linear, double stranded (ds) duplex DNA for synthetic or other non-viral gene transfer. According to some embodiments, the ceDNA is a closed-ended linear duplex (CELiD) CELiD DNA. According to some embodiments, the ceDNA is a DNA-based minicircle. According to some embodiments, the ceDNA is a minimalistic immunologically-defined gene expression (MIDGE) vector. According to some embodiments, the ceDNA is a ministering DNA. According to some embodiments, the ceDNA is a dumbbell-shaped, linear, double stranded, closed-ended DNA that contains two hairpin structures of ITRs at the 5' and 3' ends of the expression cassette. According to some embodiments, the ceDNA is doggybone™ DNA. A detailed description of ceDNA is described in International Patent Application No. PCT / US2017 / 020828, filed March 3, 2017, the entire contents of which are expressly incorporated herein by reference. Particular methods for the production of ceDNA containing various inverted terminal repeat (ITR) sequences and configurations using cell-based methods are described in Example 1 of International Patent Application Nos. US18 / 49996, filed September 7, 2018, and US2018 / 064242, filed December 6, 2018, each of which is incorporated herein by reference in its entirety. Particular methods for the production of synthetic ceDNA vectors containing various ITR sequences and configurations are described, for example, in International Application No. PCT / US2019 / 14122, filed January 18, 2019, the entire contents of which are expressly incorporated herein by reference.

[0098] As used herein, the term "closed-ended DNA vector" refers to a capsid-free DNA vector having at least one covalently closed end and at least a portion of the vector having an intramolecular double-stranded structure.

[0099] As used herein, the terms "ceDNA vector" and "ceDNA" are used interchangeably and refer to a closed-end DNA vector that contains at least one terminal palindrome. In some embodiments, the ceDNA contains two covalently closed ends.

[0100] As used herein, the term "ceDNA-bacmid" is meant to refer to an infectious baculovirus genome that contains a ceDNA genome as an intermolecular duplex that can be propagated as a plasmid in E. coli, thereby acting as a shuttle vector for baculovirus.

[0101] As used herein, the term "ceDNA-baculovirus" is meant to refer to a baculovirus that contains a ceDNA genome as an intermolecular duplex within the baculovirus genome.

[0102] As used herein, the terms "ceDNA-baculovirus-infected insect cells" and "ceDNA-BIIC" are used interchangeably and are meant to refer to invertebrate host cells (including, but not limited to, insect cells (e.g., Sf9 cells)) infected with a ceDNA-baculovirus.

[0103] As used herein, the term "ceDNA genome" is meant to refer to an expression cassette that further incorporates at least one inverted terminal repeat (ITR) region. The ceDNA genome may further comprise one or more spacer regions. In some embodiments, the ceDNA genome is incorporated into a plasmid or viral genome as an intermolecular double-stranded polynucleotide of DNA.

[0104] As used herein, the terms "DNA regulatory sequence," "control element," and "regulatory element" are used interchangeably herein and are meant to refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, proteolytic signals, and the like, which provide and / or regulate the transcription of a non-coding sequence (e.g., a DNA-targeting RNA) or a coding sequence (e.g., a site-directed modifying polypeptide or a Cas9 / Csn1 polypeptide) and / or regulate the translation of the encoded polypeptide.

[0105] As used herein, the term "terminal repeat" or "TR" includes any viral or non-viral terminal or synthetic sequence that includes at least one minimally necessary origin of replication and a region that includes a palindromic hairpin structure. The Rep binding sequence (also referred to as "RBS" or Rep binding element (RBE)) and the terminal resolution site ("TRS") together constitute the "minimally necessary origin of replication" of AAV, and thus, a TR includes at least one RBS and at least one TRS. TRs that are reverse complements of each other within a given stretch of polynucleotide sequence are typically referred to as "inverted terminal repeats" or "ITRs", respectively. In the context of viruses, ITRs play an important role in mediating replication, packaging of viral particles and DNA, integration of DNA, and rescue of genomes and proviruses. TRs that are not reverse complements (palindromes) over their entire length can still perform the traditional functions of ITRs, and thus the term ITR is used to refer to TRs in viral or non-viral AAV vectors that can mediate replication in host cells. It will be understood by those skilled in the art that there may be more than two ITRs or asymmetric ITR pairs in a compound AAV vector construct.

[0106] "ITR" can be artificially synthesized using a set of oligonucleotides containing one or more desired functional sequences (e.g., palindromic sequences, RBS). The ITR sequence can be an AAV ITR, an artificial non-AAV ITR, or an ITR physically derived from a viral AAV ITR (e.g., an ITR fragment removed from the viral genome). For example, the ITR can be derived from the Parvoviridae family, which includes parvoviruses and dependoviruses (e.g., canine parvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus, human parvovirus B-19), or the SV40 hairpin, which serves as the origin of SV40 replication, can be used as an ITR, which can be further modified by truncation, substitution, deletion, insertion, and / or addition. The Parvoviridae family of viruses consists of two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect invertebrates. Dependoparvoviruses include the viral family of adeno-associated viruses (AAV), which are capable of replication in vertebrate hosts, including but not limited to human, primate, bovine, canine, equine, and ovine species. Typically, ITR sequences can be derived from AAV, as well as parvoviruses, lentiviruses, goose viruses, B19, in the configurations of wild type, "doggy bone" and "dumbbell", symmetric or asymmetric ITR orientation. ITRs are typically present at both the 5' and 3' ends of AAV vectors, but ITRs can be present at only one end of a linear vector. For example, ITRs can be present only at the 5' end. In some other cases, ITRs can be present only at the 3' end of a synthetic AAV vector. For convenience herein, an ITR that is located 5' to (upstream of) an expression cassette in a synthetic AAV vector is referred to as the "5' ITR" or "left ITR", and an ITR that is located 3' to (downstream of) an expression cassette in a vector or synthetic AAV is referred to as the "3' ITR" or "right ITR".

[0107] As used herein, "wild-type ITR" or "WT-ITR" refers to a sequence of a naturally occurring ITR sequence in an AAV genome or other dependant virus that maintains, for example, Rep binding activity and Rep nicking ability. The nucleotide sequence of a WT-ITR from any AAV serotype may differ slightly from the naturally occurring canonical sequence due to degeneracy of the genetic code or drift, and thus WT-ITR sequences encompassed for use herein include WT-ITR sequences as a result of naturally occurring variations (e.g., replication errors).

[0108] As used herein, the term "substantially symmetric WT-ITR" or "substantially symmetric WT-ITR pair" refers to a pair of WT-ITRs in a synthetic AAV vector, both wild-type ITRs having reverse-complementary sequences over their entire length. For example, an ITR can be considered to be a wild-type sequence even if it has one or more nucleotides that deviate from the naturally occurring canonical sequence, as long as the changes do not affect the physical and functional properties and overall three-dimensional structure (secondary and tertiary structures) of the sequence. In some embodiments, the deviating nucleotides represent conservative sequence changes. As a non-limiting example, the sequence has at least 95%, 96%, 97%, 98%, or 99% sequence identity (e.g., as measured using BLAST with default settings) to the canonical sequence, and has a symmetric three-dimensional spatial organization with respect to the other WT-ITR, such that their three-dimensional structures have the same shape in geometric space. A substantially symmetric WT-ITR has the same A, C-C', and B-B' loops in three-dimensional space. A substantially symmetric WT-ITR can be functionally confirmed as WT by determining that it has an operable Rep binding site (RBE or RBE') and a terminal separation site (trs) that pairs with an appropriate Rep protein. Optionally, other functions can be tested, including transgene expression under permissive conditions.

[0109] As used herein, the phrases "modified ITR" or "mod-ITR" or "mutated ITR" are used interchangeably and refer to an ITR that has a mutation in at least one or more nucleotides compared to the WT-ITR from the same serotype. The mutation may result in a change in one or more of the A, C, C', B, B' regions of the ITR, and may result in a change in the three-dimensional spatial configuration (i.e., its three-dimensional structure in geometric space) compared to the three-dimensional spatial configuration of the WT-ITR of the same serotype.

[0110] As used herein, the term "asymmetric ITR", also referred to as "asymmetric ITR pair", refers to a pair of ITRs in a single-stranded synthetic AAV genome that are not reverse-complementary over the entire length. As a non-limiting example, an asymmetric ITR pair does not have a symmetric three-dimensional spatial configuration with respect to their cognate ITRs, such that their three-dimensional structures are different shapes in geometric space. In other words, an asymmetric ITR pair differs in overall geometric structure, i.e., the configuration of their A, C-C', and B-B' loops in three-dimensional space (e.g., compared to the cognate ITR, one ITR may have a shorter C-C' arm and / or a shorter B-B' arm). The sequence difference between the two ITRs may be due to one or more nucleotide additions, deletions, truncations, or point mutations. In one embodiment, one ITR of the asymmetric ITR pair may be a wild-type AAV ITR sequence, and the other ITR may be a modified ITR (e.g., a non-wild-type or synthetic ITR sequence) as defined herein. In another embodiment, neither ITR of the asymmetric ITR pair is a wild-type AAV sequence, and the two ITRs are modified ITRs that have different shapes in geometric space (i.e., different overall geometric structures). In some embodiments, one mod-ITR of the asymmetric ITR pair can have a short C-C' arm, and the other ITR can have a different modification (e.g., a single arm, or a short B-B' arm, etc.) such that they have a different three-dimensional spatial organization compared to their cognate asymmetric mod-ITR.

[0111] As used herein, the term "symmetric ITR" refers to a pair of ITRs in a single-stranded AAV genome that are wild-type or mutant (e.g., modified relative to wild-type) depend virus ITR sequences and are reverse-complementary over their entire length. In one non-limiting example, both ITRs are wild-type ITR sequences from AAV2. In another example, neither ITR is a wild-type ITR AAV2 sequence (i.e., they are modified ITRs, also referred to as mutant ITRs), and may differ in sequence from the wild-type ITR due to nucleotide additions, deletions, substitutions, truncations, or point mutations. For convenience herein, the ITR located 5' (upstream of) the expression cassette in a synthetic AAV vector is referred to as the "5'ITR" or "left ITR", and the ITR located 3' (downstream of) the expression cassette in a synthetic AAV vector is referred to as the "3'ITR" or "right ITR".

[0112] As used herein, the term "substantially symmetric modified ITR" or "substantially symmetric mod-ITR pair" refers to a pair of modified ITRs in a synthetic AAV that both have reverse-complementary sequences over their entire length. For example, modified ITRs can be considered substantially symmetric even if there are some nucleotide sequences that deviate from the reverse-complementary sequence, as long as the changes do not affect the properties and overall shape. As a non-limiting example, the sequences have at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity (measured using BLAST with default settings) to the canonical sequence and have a symmetric three-dimensional spatial organization with respect to their cognate modified ITRs, such that their three-dimensional structures have the same shape in geometric space. In other words, substantially symmetric modified ITR pairs have the same A, C-C', and B-B' loops organized in three-dimensional space. In some embodiments, ITRs from a mod-ITR pair may have different reverse-complementary nucleotide sequences but still have the same symmetric three-dimensional spatial organization. That is, both ITRs have mutations that result in the same overall three-dimensional shape. For example, one ITR (e.g., 5'ITR) of a mod-ITR pair may be from one serotype and the other ITR (e.g., 3'ITR) may be from a different serotype, but both may have the same corresponding mutations (e.g., if the 5'ITR has a deletion in the C region, the cognate modified 3'ITR of the different serotype has a deletion in the corresponding position in the C' region), so that the modified ITR pair has the same symmetrical three-dimensional spatial organization. In such an embodiment, each ITR of the modified ITR pair may be from a different serotype (e.g., AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12), such as a combination of AAV2 and AAV6, and the modification of one ITR is reflected in the corresponding position of the cognate ITR of the different serotype. In one embodiment, a substantially symmetrical modified ITR pair refers to a pair of modified ITRs (mod-ITRs), so long as the differences in nucleotide sequence between the ITRs do not affect the properties or overall shape, and they have substantially the same shape in three-dimensional space.As non-limiting examples, a mod-ITR has at least 95%, 96%, 97%, 98%, or 99% sequence identity to a canonical mod-ITR as determined by standard means well known in the art, such as BLAST (Basic Local Alignment Search Tool) or BLASTN with default settings, and has a symmetric three-dimensional spatial organization such that their three-dimensional structures have the same shape in geometric space. A substantially symmetric mod-ITR pair has the same A, C-C', and B-B' loops in three-dimensional space. For example, if a modified ITR of a substantially symmetric mod-ITR pair has a deletion of the C-C' arm, then the cognate mod-ITR has a corresponding deletion of the C-C' loop and has a similar three-dimensional structure of the remaining A and B-B' loops that are the same shape in geometric space as its cognate mod-ITR.

[0113] As used herein, the phrase "effective amount" or "therapeutically effective amount" of a therapeutic agent, such as an active agent or therapeutic nucleic acid, is an amount sufficient to produce a desired effect, e.g., inhibition of expression of a target sequence, as compared to expression levels detected in the absence of the therapeutic nucleic acid. Suitable assays for measuring expression of a target gene or target sequence include, for example, examination of protein or RNA levels using techniques known to those of skill in the art, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays known to those of skill in the art.

[0114] As used herein, the term "expression" is meant to refer to the cellular processes involved in the production of RNA and proteins, and optionally secreted proteins, including, for example, but not limited to, transcription, transcription processing, translation, and protein folding, modification, and processing. As used herein, the phrase "expression product" includes RNA transcribed from a gene (e.g., a transgene) and polypeptides obtained by translation of mRNA transcribed from a gene.

[0115] As used herein, the term "expression vector" is meant to refer to a vector that directs the expression of an RNA or polypeptide from a sequence linked to a transcriptional regulatory sequence on the vector. The sequence to be expressed is often, but not necessarily, heterologous to the host cell. An expression vector can contain additional elements, for example, an expression vector can have two replication systems, so that it can be maintained in two organisms, for example, in human cells for expression, and in prokaryotic hosts for cloning and amplification. An expression vector can be a recombinant vector.

[0116] As used herein, the term "adjacent" is meant to refer to the relative position of one nucleic acid sequence with respect to another nucleic acid sequence. Generally, in the sequence ABC, B is adjacent to A and C. Similarly for the sequence A x B x C. Thus, an adjacent sequence may precede or follow the adjacent sequence, but need not be contiguous or immediately adjacent to the adjacent sequence.

[0117] As used herein, the term "spacer region" is meant to refer to an intervening sequence that separates functional elements within a vector or genome. In some embodiments, a spacer region holds two functional elements at a desired distance for optimal functionality. In some embodiments, a spacer region provides or increases the genetic stability of a vector or genome. In some embodiments, a spacer region facilitates easy genetic manipulation of a genome by providing a convenient location for cloning sites and gaps of a designed number of base pairs.

[0118] As used herein, the terms "expression cassette" and "expression unit" are used interchangeably and are meant to refer to a heterologous DNA sequence operably linked to a promoter or other DNA regulatory sequence sufficient to direct transcription of a transgene in a DNA vector, e.g., a synthetic AAV vector. Suitable promoters include, for example, tissue-specific promoters. The promoter may also be of AAV origin.

[0119] As used herein, the phrase "genetic disease" or "genetic disorder" is meant to refer to a disease that is caused, directly or indirectly, in part or in whole, by one or more abnormalities in the genome, particularly a condition that is present from birth. The abnormality may be a mutation, insertion, or deletion in a gene. The abnormality may affect the coding sequence of the gene or its regulatory sequence.

[0120] As used herein, the term "polypeptide" is meant to refer to a repeated sequence of amino acids.

[0121] As used herein, the term "lipids" is meant to refer to a group of organic compounds, including but not limited to esters of fatty acids, characterized by being insoluble in water but soluble in many organic solvents. They are generally divided into at least three classes: (1) "simple lipids," which include fats and oils as well as waxes, (2) "complex lipids," which include phospholipids and glycolipids, and (3) "derived lipids," such as steroids.

[0122] Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine.Other compounds that lack phosphorus, such as sphingolipids, glycosphingolipid families, diacylglycerols, and β-acyloxyacids, are also included in the group called amphipathic lipids.In addition, the above amphipathic lipids can be mixed with other lipids, including triglycerides and sterols.

[0123] In one embodiment, the lipid composition comprises one or more tertiary amino groups, one or more phenyl ester linkages, and a disulfide linkage.

[0124] As used herein, the term "lipid conjugate" is meant to refer to a conjugated lipid that inhibits aggregation of lipid particles (e.g., lipid nanoparticles). Such lipid conjugates include, but are not limited to, PEGylated lipids, such as PEG coupled to dialkyloxypropyl (e.g., PEG-DAA conjugates), PEG coupled to diacylglycerol (e.g., PEG-DAG conjugates), PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamine, and PEG conjugated to ceramide (see, e.g., U.S. Pat. No. 5,885,613), cationic PEGylated lipids, polyoxazoline (POZ)-lipid conjugates (e.g., POZ-DAA conjugates, see, e.g., U.S. Provisional Application No. 61 / 294,828, filed Jan. 13, 2010, and U.S. Provisional Application No. 61 / 295,140, ​​filed Jan. 14, 2010), polyamide oligomers (e.g., ATTA-lipid conjugates), and mixtures thereof. Additional examples of POZ-lipid conjugates are described in International Patent Application Publication No. 2010 / 006282. PEG or POZ can be directly conjugated to lipid or can be linked to lipid via a linker moiety. Any linker moiety suitable for coupling PEG or POZ to lipid can be used, including, for example, non-ester-containing linker moieties and ester-containing linker moieties. In certain preferred embodiments, non-ester-containing linker moieties such as amides or carbamates are used. The disclosures of each of the above patent documents are incorporated herein by reference in their entirety for all purposes.

[0125] As used herein, the term "lipid encapsulated" is meant to refer to lipid particles that provide complete encapsulation, partial encapsulation, or both, of an active or therapeutic agent, such as a nucleic acid (e.g., ceDNA). In preferred embodiments, the nucleic acid is completely encapsulated within the lipid particle (e.g., to form a lipid particle containing the nucleic acid).

[0126] As used herein, the term "lipid particle" or "lipid nanoparticle" is meant to refer to a lipid formulation that can be used to deliver a therapeutic agent, such as a nucleic acid therapeutic, to a target site of interest (e.g., a cell, tissue, organ, etc.). In one embodiment, the lipid particle of the present disclosure is a nucleic acid-containing lipid particle, which is typically formed from a cationic lipid, a non-cationic lipid, and optionally a conjugated lipid that prevents particle aggregation. In other preferred embodiments, a therapeutic agent, such as a therapeutic nucleic acid, can be encapsulated in the lipid portion of the particle, thereby protecting it from enzymatic degradation. In one embodiment, the lipid particle comprises a nucleic acid (e.g., ceDNA) and a lipid that comprises one or more tertiary amino groups, one or more phenyl ester bonds, and a disulfide bond.

[0127] According to some embodiments, lipid particles of the present disclosure typically have an average diameter of about 20 nm to about 75 nm, about 20 nm to about 70 nm, about 25 nm to about 75 nm, about 25 nm to about 70 nm, about 30 nm to about 75 nm, about 30 nm to about 70 nm, about 35 nm to about 75 nm, about 35 nm to about 70 nm, about 40 nm to about 75 nm, about 40 nm to about 70 nm, about 45 nm to about 75 nm, about The size is 50 nm to about 75 nm, about 50 nm to about 70 nm, about 60 nm to about 75 nm, about 60 nm to about 70 nm, about 65 nm to about 75 nm, about 65 nm to about 70 nm, or about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 51 nm, about 52 nm, about 53 nm, about 54 nm, about 55 nm, about 56 nm, about 57 nm, about 58 nm, about 59 nm, about 60 nm, about 61 nm, about 62 nm, about 63 nm, about 64 nm, about 65 nm, about 66 nm, about 67 nm, about 68 nm, about 69 nm, about 70 nm, about 71 nm, about 72 nm, about 73 nm, about 74 nm, or about 75 nm (± 3 nm).

[0128] Generally, the lipid particles (eg, lipid nanoparticles) of the present disclosure have an average diameter selected to provide the intended therapeutic effect.

[0129] According to some embodiments, the lipid particles of the present disclosure are typically sized to have an average diameter of less than about 75 nm, less than about 70 nm, less than about 65 nm, less than about 60 nm, less than about 55 nm, less than about 50 nm, less than about 45 nm, less than about 40 nm, less than about 35 nm, less than about 30 nm, less than about 25 nm, or less than about 20 nm.

[0130] As used herein, the term "cationic lipid" refers to any lipid that is positively charged at physiological pH. The cationic lipid in the lipid particle may include one or more cationic lipids, such as, for example, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), "SS-cleavable lipid", or mixtures thereof. In some embodiments, the cationic lipid is also an ionizable lipid, i.e., an ionizable cationic lipid. The corresponding quaternary lipids of all cationic lipids described herein (i.e., those in which the nitrogen atom in the cationic moiety is protonated and has four substituents) are contemplated to be within the scope of the present disclosure. Any cationic lipid described herein can be converted to the corresponding quaternary lipid by, for example, treating with chloromethane (CH3Cl) in acetonitrile (CH3CN) and chloroform (CHCl3).

[0131] As used herein, the term "anionic lipid" refers to any lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleylphosphatidylglycerol (POPG), and other anionic modifying groups associated with neutral lipids.

[0132] As used herein, the term "hydrophobic lipid" refers to a compound having a non-polar group, including but not limited to long chain saturated and unsaturated aliphatic hydrocarbon groups, and groups optionally substituted with one or more aromatic, alicyclic, or heterocyclic groups.Suitable examples include, but are not limited to, diacylglycerol, dialkylglycerol, N-N-dialkylamino, 1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

[0133] As used herein, the term "ionizable lipid" is meant to refer to a lipid, e.g., a cationic lipid, that has at least one protonatable or deprotonatable group such that the lipid is positively charged at or below physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably above physiological pH. It will be understood by those skilled in the art that the addition or removal of protons as a function of pH is an equilibrium process, and reference to charged or neutral lipids refers to the nature of the predominant species, and not all lipids need to be present in a charged or neutral form. In general, ionizable lipids have a pKa of the protonatable group in the range of about 4 to about 7. In some embodiments, ionizable lipids may include "cleavable lipids" or "SS-cleavable lipids."

[0134] As used herein, the term "neutral lipid" is meant to refer to any of a number of lipid species that exist in either uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol.

[0135] As used herein, the term "non-cationic lipid" is meant to refer to any amphipathic lipid, and any other neutral or anionic lipid.

[0136] As used herein, the term "cleavable lipid" or "SS-cleavable lipid" refers to a lipid that contains a disulfide bond cleavable unit. The cleavable lipid may include a cleavable disulfide bond ("ss") containing lipid-like material that includes a pH-sensitive tertiary amine and an autolytic phenyl ester. For example, the SS-cleavable lipid can be ss-OP lipid (COATSOME® SS-OP), ss-M lipid (COATSOME® SS-M), ss-E lipid (COATSOME® SS-E), ss-EC lipid (COATSOME® SS-EC), ss-LC lipid (COATSOME® SS-LC), ss-OC lipid (COATSOME® SS-OC), and ss-PalmE lipid (see, e.g., Formulas I-IV), or lipids described by Togashi et al., (2018) Journal of Controlled Release "A hepatic pDNA delivery system based on an intracellular environment sensitive vitamin E -scaffold lipid-like material with the aid of an anti-inflammatory drug" 279:262-270. Additional examples of cleavable lipids are described in U.S. Patent No. 9,708,628 and U.S. Patent No. 10,385,030, the entire contents of which are incorporated herein by reference. In one embodiment, the cleavable lipid comprises a tertiary amine that responds to disulfide bonds that can be cleaved in acidic compartments, such as endosomes or lysosomes for membrane destabilization, and reducing environments such as the cytoplasm. In one embodiment, the cleavable lipid is a cationic lipid. In one embodiment, the cleavable lipid is an ionizable cationic lipid. Cleavable lipids are described in more detail herein.

[0137] As used herein, the term "organic lipid solution" is meant to refer to a composition that comprises an organic solvent having, in whole or in part, a lipid.

[0138] As used herein, the term "liposome" is meant to refer to lipid molecules assembled in a spherical configuration that encapsulates an internal aqueous volume separated from an aqueous exterior. Liposomes are vesicles with at least one lipid bilayer. Liposomes are typically used as carriers for drug / therapeutic drug delivery in the context of formulation development. They act by fusing with cell membranes and repositioning their lipid structures to deliver drugs or active formulation components. Liposome compositions for such delivery are typically composed of phospholipids, particularly compounds with phosphatidylcholine groups, although these compositions may also contain other lipids.

[0139] As used herein, the term "localized delivery" refers to the direct delivery of an active agent, such as an interfering RNA (e.g., siRNA), to a target site within an organism. For example, an agent can be locally delivered by direct injection into a disease site, such as a tumor, or other target site, such as an inflammation site, or into a target organ, such as the liver, heart, pancreas, or kidney.

[0140] As used herein, the term "nucleic acid" is meant to refer to a polymer containing at least two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in either single-stranded or double-stranded form, including DNA, RNA, and hybrids thereof. The DNA can be in the form of, for example, antisense molecules, plasmid DNA, DNA-DNA duplexes, pre-condensed DNA, PCR products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. The DNA can be in the form of minicircles, plasmids, bacmids, minigenes, ministring DNA (linear covalently closed DNA vectors), closed-end linear duplex DNA (CELiD or ceDNA), doggybone™ DNA, dumbbell DNA, minimal immunologically defined gene expression (MIDGE)-vectors, viral vectors, or non-viral vectors. RNA can be in the form of small interfering RNA (siRNA), dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and have similar binding properties as the reference nucleic acid. Examples of such analogs and / or modified residues include phosphorothioates, phosphorodiamidate morpholino oligomers (morpholinos), phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2'-O-methyl ribonucleotides, locked nucleic acids (LNA™), and peptide nucleic acids (PNAs). Unless otherwise limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses not only the sequence explicitly indicated, but also conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences.

[0141] As used herein, the phrases "nucleic acid therapy", "therapeutic nucleic acid" and "TNA" are used interchangeably and refer to any modality of therapy that uses nucleic acids as the active ingredient of a therapeutic agent to treat a disease or disorder. As used herein, these terms refer to RNA-based therapeutic agents and DNA-based therapeutic agents. Non-limiting examples of RNA-based therapeutic agents include mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), and microRNA (miRNA). Non-limiting examples of DNA-based therapeutics include minicircle DNA, minigenes, viral DNA (e.g., lentivirus or AAV genomes) or non-viral synthetic DNA vectors, closed-end linear double-stranded DNA (ceDNA / CELiD), plasmids, bacmids, DOGGYBONE™ DNA vectors, minimalistic immunologically-defined gene expression (MIDGE) vectors, non-viral ministring DNA vectors (linear covalently closed DNA vectors), or dumbbell-shaped DNA minimal vectors ("dumbbell DNA").

[0142] As used herein, a "nucleotide" comprises the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate group.

[0143] As used herein, the term "pharmaceutical acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate buffered saline, water, emulsions such as oil / water or water / oil, and various types of wetting agents. The term also encompasses any agent approved by a regulatory agency of the U.S. Federal government or listed in the U.S. Pharmacopeia for use in animals, including humans, as well as any carrier or diluent that does not cause significant irritation to the subject and does not destroy the biological activity and properties of the compound administered.

[0144] As used herein, the term "gap" is meant to refer to an interrupted portion of the synthetic DNA vector of the present disclosure, where a stretch of single-stranded DNA is created in the otherwise double-stranded ceDNA. A gap can be as long as 1 base pair to 100 base pairs in length for a single strand of the double-stranded DNA. Exemplary gaps designed and created by the methods described herein, and synthetic vectors generated thereby, can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 bp in length. Exemplary gaps in the present disclosure can be 1 bp to 10 bp in length, 1 to 20 bp in length, or 1 to 30 bp in length.

[0145] As used herein, the term "nick" refers to a discontinuity in a double-stranded DNA molecule in which there is no phosphodiester bond between adjacent nucleotides in one strand, typically due to damage or enzymatic action. It is understood that one or more nicks allow the strand to untwist during DNA replication, and that nicks are also believed to play a role in facilitating the binding of the transcription machinery.

[0146] "Receptor" refers to a polypeptide present on a cell membrane that selectively binds to one or more ligands, or a portion thereof. The term "receptor" as used herein is intended to encompass the entire receptor or a ligand-binding portion thereof. These portions of the receptor specifically include a region sufficient for specific binding of the ligand to occur.

[0147] As used herein, the term "cancer" refers to a physiological condition in multicellular eukaryotic organisms that is typically characterized by unregulated cell proliferation and malignant tumors. Thus, the term broadly encompasses solid tumors, blood cancers (e.g., leukemia), as well as myelofibrosis and multiple myeloma.

[0148] As used herein, the term "subject" is meant to refer to a human or animal to which treatment, including prophylactic treatment, with a therapeutic nucleic acid according to the present disclosure is provided. Typically, the animal is a vertebrate, such as, but not limited to, a primate, a rodent, a domestic animal, or a game animal. Primates include, but are not limited to, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domestic and game animals include, but are not limited to, cattle, horses, pigs, deer, bison, buffalo, feline species, such as domestic cats, canine species, such as dogs, foxes, wolves, avian species, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon. In certain embodiments of the aspects described herein, the subject is a mammal, such as a primate or a human. The subject can be male or female. Additionally, the subject may be an infant or child. In some embodiments, the subject may be a neonatal or fetal subject, e.g., the subject is present in utero. Preferably, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Non-human mammals may be advantageously used as subjects that represent animal models of diseases and disorders. In addition, the methods and compositions described herein may be used with domestic animals and / or pets. Human subjects may be of any age, sex, race, or ethnic group, e.g., Caucasian (white), Asian, African, Black, African American, African European, Latin American, Middle Eastern, etc. In some embodiments, the subject may be a patient or other subject in a clinical setting. In some embodiments, the subject has already undergone treatment. In some embodiments, the subject is an embryo, fetus, neonatal, infant, child, adolescent, or adult. In some embodiments, the subject is a human fetus, a human neonatal, a human infant, a human child, a human adolescent, or a human adult. In some embodiments, the subject is an animal embryo, or a non-human embryo or a non-human primate embryo, hi some embodiments, the subject is a human embryo.

[0149] As used herein, the phrase "subject in need," unless the context and usage of the phrase dictates otherwise, refers to a subject who (i) is to be administered ceDNA lipid particles (or a pharmaceutical composition comprising ceDNA lipid particles) in accordance with the disclosed subject matter set forth herein, (ii) has received ceDNA lipid particles (or a pharmaceutical composition comprising ceDNA lipid particles) in accordance with the disclosed subject matter set forth herein, or (iii) has received ceDNA lipid particles (or a pharmaceutical composition comprising ceDNA lipid particles) in accordance with the disclosed subject matter set forth herein.

[0150] As used herein, the terms "suppress," "reduce," "interfere," "inhibit," and / or "reduce" (and similar terms) generally refer to the act of directly or indirectly decreasing a concentration, level, function, activity, or behavior relative to natural, expected or average, or relative to a control condition.

[0151] As used herein, the term "systemic delivery" is meant to refer to delivery of lipid particles that results in widespread biodistribution of active agents, such as interfering RNA (e.g., siRNA) in an organism. Some administration techniques may lead to systemic delivery of a particular agent, while others may not. Systemic delivery means that a useful amount, preferably a therapeutic amount, of the agent is exposed to most parts of the body. To obtain widespread biodistribution, a blood lifetime is generally required such that the agent is not rapidly degraded or cleared (by first-pass organs (liver, lung, etc.) or by rapid non-specific cellular binding) before reaching disease sites distal to the administration site. Systemic delivery of lipid particles (e.g., lipid nanoparticles) can be by any means known in the art, including, for example, intravenous, subcutaneous, and intraperitoneal. In a preferred embodiment, systemic delivery of lipid particles (e.g., lipid nanoparticles) is by intravenous delivery.

[0152] As used herein, the terms "therapeutic amount", "therapeutically effective amount", "effective amount", or "pharmaceutical effective amount" of an active agent (e.g., ceDNA lipid particles described herein) are used interchangeably and refer to an amount sufficient to provide the intended benefit of treatment. However, dosage levels are based on a variety of factors, including the type of injury, age, weight, sex, medical condition of the patient, the severity of the medical condition, route of administration, and the specific active agent used. Thus, dosage regimens may vary widely, but can be routinely determined by a physician using standard methods. Additionally, the terms "therapeutic amount", "therapeutically effective amount", and "pharmaceutical effective amount" include prophylactic or preventative amounts of the compositions of the present disclosure described. In the prophylactic or preventative applications of the present disclosure described, the pharmaceutical composition or agent is administered to a patient susceptible to or otherwise at risk of a disease, disorder or condition, including the biochemical, histological and / or behavioral symptoms of the disease, disorder or condition, its complications, and intermediate pathological phenotypes manifested during the development of the disease, disorder or condition, in an amount sufficient to eliminate or reduce the risk, reduce the severity, or delay the onset of the disease, disorder or condition. It is generally preferred to use the maximum dose, i.e., the highest safe dose, according to some medical judgment. The terms "dose" and "administration" are used interchangeably herein.

[0153] As used herein, the term "therapeutic effect" refers to the outcome of treatment, which outcome is deemed desirable and beneficial. Therapeutic effect can include, directly or indirectly, the prevention, reduction, or elimination of disease symptoms. Therapeutic effect can also include, directly or indirectly, the prevention, reduction, or elimination of the progression of disease symptoms.

[0154] For any therapeutic agent described herein, the therapeutically effective amount can be determined first from preliminary in vitro studies and / or animal models. The therapeutically effective dose can also be determined from human data. The applied dose can be adjusted based on the relative bioavailability and efficacy of the administered compound. It is within the ability of a person skilled in the art to adjust the dose to achieve maximum efficacy based on the above methods and other known methods. The following summarizes the general principles for determining therapeutic efficacy, which can be found in Chapter 1 of Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th Edition, McGraw-Hill (New York) (2001), which is incorporated herein by reference.

[0155] Pharmacokinetic principles provide the basis for modifying dosing regimens to obtain the desired degree of therapeutic effect while minimizing unacceptable side effects. In situations where plasma concentrations of a drug can be measured and are related to the therapeutic window, additional guidance regarding dosage modifications can be obtained.

[0156] As used herein, the terms "treat", "treating" and / or "treatment" include inhibiting, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating the clinical symptoms of a condition, or substantially preventing the appearance of clinical symptoms of a condition, to obtain beneficial or desired clinical results. Treating further refers to achieving one or more of the following: (a) reducing the severity of the disorder, (b) limiting the onset of symptoms characteristic of the disorder being treated, (c) limiting the worsening of symptoms characteristic of the disorder being treated, (d) limiting the recurrence of the disorder in patients who previously had the disorder, and (e) limiting the recurrence of symptoms in patients who were previously asymptomatic for the disorder.

[0157] Beneficial or desired clinical results, such as pharmacological and / or physiological effects, include, but are not limited to, preventing the occurrence of a disease, disorder or condition in a subject who may have a predisposition to the disease, disorder or condition, but who has not yet experienced or exhibited symptoms of the disease (prophylactic treatment), alleviating the symptoms of the disease, disorder or condition, reducing the severity of the disease, disorder or condition, stabilizing (i.e., not worsening) the disease, disorder or condition, preventing the spread of the disease, disorder or condition, slowing or retarding the progression of the disease, disorder or condition, ameliorating or alleviating the disease, disorder or condition, and combinations thereof, as well as prolonging survival as compared to expected survival if not receiving treatment.

[0158] Beneficial or desired clinical results, such as pharmacological and / or physiological effects, include, but are not limited to, preventing the occurrence of a disease, disorder or condition in a subject who may have a predisposition to the disease, disorder or condition, but who has not yet experienced or exhibited symptoms of the disease (prophylactic treatment), alleviating the symptoms of the disease, disorder or condition, reducing the severity of the disease, disorder or condition, stabilizing (i.e., not worsening) the disease, disorder or condition, preventing the spread of the disease, disorder or condition, slowing or retarding the progression of the disease, disorder or condition, ameliorating or alleviating the disease, disorder or condition, and combinations thereof, as well as prolonging survival as compared to expected survival if not receiving treatment.

[0159] As used herein, the term "combination therapy" refers to a treatment regimen for clinical indications that includes two or more therapeutic agents. Thus, the term refers to a treatment regimen in which a first therapy, including a first composition (e.g., an active ingredient), is administered to a patient in conjunction with a second therapy, including a second composition (active ingredient), intended to treat the same or overlapping disease or clinical condition. Both the first and second compositions may act on the same cellular target, or on separate cellular targets. The phrase "in conjunction with" in the context of combination therapy means that in a subject receiving the combination therapy, the therapeutic effect of the first therapy overlaps temporally and / or spatially with the therapeutic effect of the second therapy. Thus, the combination therapy can be formulated as a single formulation for simultaneous administration or as separate formulations for sequential administration of the therapies.

[0160] As used herein, the term “alkyl” refers to a saturated monovalent hydrocarbon radical of 1 to 20 carbon atoms (i.e., C 1~20 "Monovalent" means that the alkyl has one point of attachment to the rest of the molecule. In one embodiment, the alkyl has 1 to 12 carbon atoms (i.e., C 1~12 alkyl), or 1 to 10 carbon atoms (i.e., C 1~10 In one embodiment, the alkyl has 1 to 8 carbon atoms (i.e., C 1~8 alkyl), 1 to 7 carbon atoms (i.e., C 1~7 alkyl), 1 to 6 carbon atoms (i.e., C 1~6 alkyl), 1 to 4 carbon atoms (i.e., C 1~4 alkyl), or 1 to 3 carbon atoms (i.e., C 1~3Examples include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl, 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, and the like. 1~6 Alkyl," "Straight or branched chain C 1~4 Alkyl" or "Straight or branched chain C 1~3 Straight chain or branched chain alkyl, such as "alkyl," means that the saturated monovalent hydrocarbon radical is straight chain or branched chain. As used herein, the term "straight chain" referring to an aliphatic hydrocarbon chain means that the chain is not branched.

[0161] As used herein, the term "alkylene" refers to a saturated divalent hydrocarbon radical of 1 to 20 carbon atoms (i.e., C 1~20 "Divalent" refers to an alkylene having 1 to 12 carbon atoms (i.e., C 1~12 alkylene), or 1 to 10 carbon atoms (i.e., C 1~10 In one embodiment, the alkylene has 1 to 8 carbon atoms (i.e., C 1~8 alkylene), 1 to 7 carbon atoms (i.e., C 1~7 alkylene), 1 to 6 carbon atoms (i.e., C 1~6 alkylene), 1 to 4 carbon atoms (i.e., C 1~4 alkylene), or 1 to 3 carbon atoms (i.e., C 1~3 Alkylene) and is ethylene or methylene. "Linear or branched C1~6 Alkylene," "straight or branched chain C 1~4 Alkylene" or "Straight or branched chain C 1~3 Linear or branched alkylene, such as "alkylene," means that the saturated divalent hydrocarbon radical is a linear or branched chain.

[0162] The term "alkenyl" refers to a straight-chain or branched-chain aliphatic hydrocarbon group having one or more (e.g., 1 or 2) carbon-carbon double bonds, and alkenyl groups include groups having "cis" and "trans" orientations, or, alternatively, "E" and "Z" orientations.

[0163] As used herein, "alkenylene" refers to an aliphatic divalent hydrocarbon group of 2 to 20 carbon atoms having one or two carbon-carbon double bonds (i.e., C 2~20 "Alkenylene" refers to an alkylene group having 2 to 12 carbon atoms (i.e., C 2~16 alkenylene), or 2 to 10 carbon atoms (i.e., C 2~10 In one embodiment, the alkenylene has 2 to 4 carbon atoms (C 2~4 Examples include, but are not limited to, ethylenylene or vinylene (-CH=CH-), allyl (-CHCH=CH-), etc. 2~6 Alkenylene," "straight or branched chain C 2~4 Alkenylene" or "Straight or branched chain C 2~3 Linear or branched alkenylene, such as "alkenylene," means that the unsaturated divalent hydrocarbon radical is linear or branched.

[0164] As used herein, "cycloalkylene" refers to a divalent saturated carbocyclic ring group having 3 to 12 carbon atoms as a monocyclic ring or 7 to 12 carbon atoms as a bicyclic ring. "Divalent" means that the cycloalkylene has two points of attachment to the rest of the molecule. In one embodiment, the cycloalkylene is a 3- to 7-membered monocyclic or a 3- to 6-membered monocyclic. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, cyclononylene, cyclodecylene, cycloundecylene, cyclododecylene, and the like. In one embodiment, the cycloalkylene is cyclopropylene.

[0165] The terms "heterocycle", "heterocyclyl", heterocyclic and "heterocyclic ring" are used interchangeably herein and refer to a cyclic group that contains at least one N atom, a heteroatom and optionally 1-3 additional heteroatoms selected from N and S, and is non-aromatic (i.e., partially or fully saturated). It can be monocyclic or bicyclic (bridged or fused). Examples of heterocyclic rings include, but are not limited to, aziridinyl, diaziridinyl, thiaaziridinyl, azetidinyl, diazetidinyl, triazetidinyl, thiadiazetidinyl, thiazetidinyl, pyrrolidinyl, pyrazolidinyl, imidazolinyl, isothiazolidinyl, thiazolidinyl, piperidinyl, piperazinyl, hexahydropyrimidinyl, azepanyl, azocanyl, and the like. The heterocycle contains 1 to 4 heteroatoms, which are selected from N and S and may be the same or different. In one embodiment, the heterocycle contains 1 to 3 N atoms. In another embodiment, the heterocycle contains 1 or 2 N atoms. In another embodiment, the heterocycle contains 1 N atom. "4-membered to 8-membered heterocyclyl" refers to a group having 4 to 8 atoms (including 1 to 4 heteroatoms selected from N and S, or 1 to 3 N atoms, or 1 or 2 N atoms, or 1 N atom) arranged in a monocyclic ring. "5- or 6-membered heterocyclyl" refers to a group having 5 or 6 atoms (including 1 to 4 heteroatoms selected from N and S, or 1 to 3 N atoms, or 1 or 2 N atoms, or 1 N atom) arranged in a monocyclic ring. The term "heterocycle" is intended to include all possible isomeric forms.Heterocycles are described in Paquette, Leo A., Principles of Modern Heterocyclic Chemistry (WA Benjamin, New York, 1968), especially Chapters 1, 3, 4, 6, 7, and 9, The Chemistry of Heterocyclic Compounds, A Series of Monographs (John Wiley & Sons, New York, 1950 to present), especially Volumes 13, 14, 16, 19, and 28, and J. Am. Chem. Soc. (1960) 82:5566. Heterocyclyl groups may be carbon (carbon-linked) or nitrogen (nitrogen-linked) attached to the remainder of the molecule, where such is possible.

[0166] When a group is described as "optionally substituted," the group can be (1) unsubstituted or (2) substituted. When a carbon of a group is described as optionally substituted with one or more of a list of substituents, one or more of the hydrogen atoms on the carbon (to the extent present) can be replaced separately and / or together with any independently selected substituents.

[0167] Suitable substituents for alkyl, alkylene, alkenylene, cycloalkylene, and heterocyclyl are those that do not significantly adversely affect the biological activity of the bifunctional compound. Unless otherwise specified, exemplary substituents for these groups include linear, branched, or cyclic alkyl, alkenyl, or alkynyl having 1 to 10 carbon atoms, aryl, heteroaryl, heterocyclyl, halogen, guanidinium [-NH(C=NH)NH2], -OR 100 , N.R. 101 R 102 , -NO2, -NR 101 COR 102 , -SR 100 , -SOR 101 Sulfoxides, represented by -SO2R 101 Sulfones, sulfonates -SO3M, sulfates -OSO3M, -SO2NR 101 R102 Sulfonamide, cyano, azido, -COR 101 , -OCOR 101 , -OCONR 101 R 102 , and polyethylene glycol units (-OCH2CH2) n R 101 where M is H or a cation (e.g., Na + Or K + ) is mentioned, and R 101 , R 102 and R 103 are each independently H, a linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms, a polyethylene glycol unit (-OCH2CH2) n -R 104 (n is an integer from 1 to 24), an aryl having 6 to 10 carbon atoms, a heterocyclic ring having 3 to 10 carbon atoms, and a heteroaryl having 5 to 10 carbon atoms; R 104 is H, a linear or branched cyclic alkyl having 1 to 4 carbon atoms, R 100 , R 101 , R 102 , R 103 and R 104 The alkyl, alkenyl, alkynyl, aryl, heteroaryl and heterocyclcyl of the group represented by the formula (I) are optionally substituted with one or more (e.g., 2, 3, 4, 5, 6, or more) substituents independently selected from halogen, -OH, -CN, -NO2, and unsubstituted straight or branched chain alkyl having 1 to 4 carbon atoms. Preferably, the substituents for the above optionally substituted alkyl, alkylene, alkenylene, cycloalkylene, and heterocyclyl are halogen, -CN, -NR 101 R 102 , -CF3, -OR 100 , aryl, heteroaryl, heterocyclyl, -SR 101 , -SOR 101 , -SO2R 101Alternatively, suitable substituents are selected from the group consisting of halogen, -OH, -NO2, -CN, C 1~4 Alkyl, -OR 100 , N.R. 101 R 102 , -NR 101 COR 102 , -SR 100 , -SO2R 101 , -SO2NR 101 R 102 , -COR 101 , -OCOR 101 , and -OCONR 101 R 102 wherein R 100 , R 101 , and R 102 are each independently -H or C 1~4 It is an alkyl.

[0168] As used herein, "halogen" refers to F, Cl, Br, or I. "Cyano" is --CN.

[0169] "Amine" or "amino," as used interchangeably herein, refers to a functional group containing a basic nitrogen atom bearing a lone pair of electrons.

[0170] As used herein, the term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of the ionizable lipids of the present disclosure. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, acid tartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate "mesylate", ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)), alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. A pharmaceutically acceptable salt may include the inclusion of another molecule, such as an acetate ion, a succinate ion, or other counter ion. A counter ion may be any organic or inorganic moiety that stabilizes the charge of the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. When multiple charged atoms are part of a pharmaceutically acceptable salt, it may have multiple counter ions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counter ions.

[0171] Grouping of alternative elements or embodiments of the disclosure disclosed herein should not be construed as limiting. Members of each group may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in or deleted from a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed herein to include the modified group, thus satisfying the description of all Markush groups used in the appended claims.

[0172] In some embodiments of any of the aspects, the disclosure described herein does not pertain to human cloning processes, processes for correcting the genetic identity of human germ lines, the use of human embryos for industrial or commercial purposes, or animals that are likely to cause suffering without providing any substantial medical benefit to humans or animals, and processes for correcting the genetic identity of animals resulting from such processes.

[0173] Other terms are defined herein within the description of various aspects of the disclosure.

[0174] All patents and other publications, including literature references, issued patents, published patent applications, and co-pending patent applications, cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that may be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicant and do not constitute any admission as to the accuracy of the dates or contents of these documents.

[0175] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. Although specific embodiments and examples of the present disclosure are described herein for illustrative purposes, those skilled in the art will recognize that various equivalent modifications are possible within the scope of the present disclosure. For example, while method steps or functions are presented in a given order, alternative embodiments may perform the functions in a different order, or the functions may be performed substantially simultaneously. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the present disclosure can be modified as appropriate to provide further embodiments of the present disclosure using the compositions, functions, and concepts of the above references and applications. Furthermore, some changes can be made to protein structures without affecting the type or amount of biological or chemical action, due to considerations of biological functional equivalence. These and other changes can be made to the present disclosure in light of the detailed description. All such modifications are intended to be within the scope of the appended claims.

[0176] Particular elements of any of the foregoing embodiments can be combined with or substituted for elements of other embodiments. Additionally, although advantages associated with particular embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily exhibit such advantages, to be within the scope of the present disclosure.

[0177] The techniques described herein are further illustrated by the following examples, which should not be construed as further limiting in any way. It should be understood that the present disclosure is not limited in any manner to the specific methodology, protocols, and reagents described herein, and as such may vary. The terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the scope of the present disclosure, which is defined solely by the claims.

[0178] II. Lipid Nanoparticle Compositions Provided herein is a pharmaceutical composition comprising a lipid nanoparticle (LNP) and a therapeutic nucleic acid (TNA), wherein the LNP comprises a single chain variable fragment (scFv) linked to the LNP. The scFv targets an antigen present on the surface of a cell. The term "linked" encompasses chemical conjugation, adsorption (physisorption and / or chemisorption). The types of bonds encompassed by the term "linked" are covalent and non-covalent interactions (e.g., hydrogen bonds, van der Waals bonds, ionic bonds, and hydrophobic bonds). According to some embodiments, the scFv is linked to the LNP via covalent conjugation. According to some embodiments, the scFv is linked to the LNP via maleimide linkage. It is the discovery of the present disclosure that maleimide conjugation of scFv to LNP resulted in a more robust conjugation to the LNP compared to other thiol-based crosslinking methods such as PDS conjugation, and importantly maintained the size and integrity of the LNP.

[0179] Thus, provided herein is a pharmaceutical composition comprising a lipid nanoparticle (LNP) and a therapeutic nucleic acid (TNA), wherein the LNP comprises a single chain variable fragment (scFv) linked to the LNP, the scFv being directed to an antigen present on the surface of a cell, and at least one pharma- ceutically acceptable excipient, wherein the scFv is covalently linked to the LNP via a non-cleavable linker. According to some embodiments, the non-cleavable linker is a maleimide-containing linker.

[0180] Also provided herein is a pharmaceutical composition comprising a lipid nanoparticle (LNP) and a therapeutic nucleic acid (TNA), wherein the LNP comprises a single chain variable fragment (scFv) linked to the LNP, where the scFv is directed to an antigen present on the surface of a cell, and at least one pharma- ceutical acceptable excipient, wherein the scFv is covalently linked to the LNP via a cleavable linker.

[0181] The LNPs described herein offer a number of therapeutic advantages, including a smaller size that allows for the encapsulation of large therapeutic nucleic acid molecules. It is an advantageous feature of the present disclosure that the scFv LNPs described herein are useful for targeting any cell or tissue that actively expresses an antigen present on the surface of the cell to which the scFv is directed. In some embodiments, the cell is a tumor cell. In some embodiments, the cell is a liver cell (hepatocyte).

[0182] According to some embodiments, the antigen is a tumor-associated antigen (TAA) or a tumor-selective antigen (TSA). A "tumor-associated antigen" or TAA is an antigen that is expressed on a tumor. A "tumor-selective antigen" or TSA is an antigen that is selectively expressed on a tumor. In one embodiment, the antigen is human epidermal growth factor receptor 2 (HER2).

[0183] In one embodiment, TAA expression may be restricted to tumor cell populations, expressed by all tumor cells, and expressed on the tumor cell surface. Other antigens may be overexpressed on tumor cells, but found at lower expression levels on normal cells, and thus are tumor-selective antigens (TSAs). Furthermore, some tumor antigens arise as "passenger mutations", i.e., non-essential antigens expressed by tumor cells that have defective control over DNA repair, and thus accumulate mutations in various proteins. Some tumor antigens are proteins produced by tumor cells that induce immune responses, particularly T cell-mediated immune responses.

[0184] According to some embodiments, the TAA or TSA is selected from the group consisting of glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha fetoprotein (AFP), lectin-reactive AFP, thyroglubilin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate cancer specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, HER2 / neu, survivin and telomerase, prostate carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin, EphA2, HER2, GD2, glypican-3, 5T4, 8H9, α vβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CEA, CD19, CD20, CD22, kappa light chain, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, ERBB3, ERBB4, ErbB3 / 4, FAP, FAR, FBP, fetal AchR, folate receptor a, GD2, GD3, HLA-AI The antigen is selected from the group consisting of MAGE A1, HLA-A2, IL11Ra, IL13Ra2, KDR, lambda, Lewis-Y, MCSP, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSC1, PSMA, ROR1, SURVIVIN, TAG72, TEM1, TEM8, VEGRR2, carcinoembryonic antigen, HMW-MAA, and VEGF receptor. Other exemplary antigens that can be used are antigens present in the extracellular matrix of tumors, such as fibronectin, tenascin, or carcinoembryonic variants of necrotic areas of tumors.

[0185] Additional tumor-selective molecules that can be used include any membrane protein or biomarker that is expressed or overexpressed in tumor cells, including, but not limited to, integrins (e.g., integrin αvβ3, α5β1), the EGF receptor family (e.g., EGFR2, Erbb2 / HER2 / neu, Erbb3, Erbb4), proteoglycans (e.g., heparan sulfate proteoglycans), disialogangliosides (e.g., GD2, GD3), B7-H3 (aka These include CD276), cancer antigen 125 (CA-125), epithelial cell adhesion molecule (EpCAM), vascular endothelial growth factor receptor 1 and 2 (VEGFR-1, VEGFR-2), CD52, carcinoembryonic antigen (CEA), tumor-associated glycoprotein (e.g., TAG-72), cluster of differentiation 19 (CD19), CD20, CD22, CD30, CD33, CD40, CD44, CD74, CD152, mucin 1 (MUC1), tumor necrosis factor receptor (e.g., TRAIL-R2), insulin-like growth factor receptor, folate receptor a, transmembrane glycoprotein NMB (GPNMB), CC chemokine receptor (e.g., CCR4), prostate-specific membrane antigen (PSMA), receptor d'origin nantais (RON) receptor, cytotoxic T lymphocyte antigen 4 (CTLA4), and other tumor-specific receptors or antigens.

[0186] The Cancer Antigenic Peptide Database is a publicly available database (caped.icp.ucl.ac.) that compiles information on human tumor antigens, including their peptide sequences and their locations in protein sequences. According to some embodiments, the scFv is directed to a tumor-associated antigen represented in the Cancer Antigenic Peptide Database.

[0187] According to some embodiments, the scFv binds to a tumor antigen associated with a hematological malignancy. In some embodiments, the scFv binds to a tumor antigen associated with a solid tumor.

[0188] The majority of antibody fragments currently in clinical development are for oncology applications. In addition to the general features of antibody fragments that make them attractive as immunotherapeutics, such as their small size and the lack of an Fc domain that reduces non-specific activation of innate immune cells, which gives them superior tissue and tumor penetration compared to traditional mAbs, there are a number of mechanisms of action that are unique to certain formats.

[0189] While oncology is the primary area in which antibody fragments have become a prominent class of therapeutic molecule, there are several other disease areas in which antibody fragments are being evaluated.

[0190] Autoimmune diseases are chronic and potentially life-threatening, and antibody therapy is usually very expensive, as it requires intensive, life-long treatment.The lower production cost of antibody fragments and the possibility of reduced immunogenicity due to their small size make the use of antibody fragments with half-life extension moieties a viable alternative to full-length antibodies.Furthermore, the development of antibody fragments for the treatment of autoimmune diseases, as with cancer immunotherapy, is growing at a fast pace, and there are many possibilities for bispecific targeting.

[0191] One of the first antibody fragments marketed for an autoimmune disease indication was certolizumab pegol (CIMZIA®), a pegylated Fab targeting TNF developed by UCB (Belgium), which was approved by the FDA for the treatment of Crohn's disease in 2008. It was subsequently approved for rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis. Two other Fabs are in clinical trials: FR104 (OSE / Janssen) against CD28 in Phase II for RA, and an anti-CD40L Fab developed by UCB, namely dapirolizumab, in Phase II for SLE.

[0192] One scFv format currently being evaluated in clinical trials for the treatment of RA is Dekavil or F8IL10 (Philogen), a fully human fusion protein consisting of the vascular-targeting scFv antibody F8 fused to the cytokine interleukin-10. Several other immunocytokines fused to scFvs are also in preclinical development.

[0193] It has been shown that antibody fragments such as Fab and scFv can penetrate the cornea, enter the eye, and achieve clinically useful concentrations in the anterior chamber over a reasonable period of time following topical administration (Thiel et al. Clin. Exp. Immunol. 2002.). The most common eye disorder treated with antibodies or antibody fragments is age-related macular degeneration (AMD), which is the leading cause of irreversible blindness in people over the age of 50 in developed countries. In the case of AMD, antibody fragments are applied directly to the eye via the intravitreal route. Ranibizumab (LUCENTIS®) is an anti-angiogenic monoclonal antibody fragment that targets VEGF-A, derived from the same parent mouse antibody as bevacizumab. It was approved for wet AMD in 2006, and then for diabetic macular edema and diabetic retinopathy in 2012 and 2015, respectively. Brolucizumab (Alcon / Novartis) is a VEGF-targeting scFv in phase III for wet AMD.

[0194] According to some embodiments, the scFv comprises SEQ ID NO:1. EVQLVESGGGLVQPGGSLRLSCAASGFNIDDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSADFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 1)

[0195] According to some embodiments, the scFv comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:1. According to some embodiments, the scFv comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1. According to some embodiments, the scFv comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:1. According to some embodiments, the scFv comprises an amino acid sequence that is at least 96% identical to SEQ ID NO:1. According to some embodiments, the scFv comprises an amino acid sequence that is at least 97% identical to SEQ ID NO:1. According to some embodiments, the scFv comprises an amino acid sequence that is at least 98% identical to SEQ ID NO:1. According to some embodiments, the scFv comprises an amino acid sequence that is at least 99% identical to SEQ ID NO:1. According to some embodiments, the scFv consists of SEQ ID NO:1.

[0196] According to some embodiments, the scFv comprises SEQ ID NO: 2, which contains a myc (bold underlined) tag and a His (italicized) tag, along with a c-terminal cysteine ​​required for maleimide conjugation.

[0197] [ka]

[0198] According to some embodiments, the scFv comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:2. According to some embodiments, the scFv comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:2. According to some embodiments, the scFv comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:2. According to some embodiments, the scFv comprises an amino acid sequence that is at least 96% identical to SEQ ID NO:2. According to some embodiments, the scFv comprises an amino acid sequence that is at least 97% identical to SEQ ID NO:2. According to some embodiments, the scFv comprises an amino acid sequence that is at least 98% identical to SEQ ID NO:2. According to some embodiments, the scFv comprises an amino acid sequence that is at least 99% identical to SEQ ID NO:2. According to some embodiments, the scFv consists of SEQ ID NO:2.

[0199] According to some embodiments, the scFv comprises SEQ ID NO: 3, which contains the same scFV core sequence as SEQ ID NO: 1, but with an N-terminal His (italics) tag and a c-terminal LLQGA polypeptide (bold and underlined) to facilitate transglutaminase-mediated conjugation.

[0200] [ka]

[0201] According to some embodiments, the scFv comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:3. According to some embodiments, the scFv comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:3. According to some embodiments, the scFv comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:3. According to some embodiments, the scFv comprises an amino acid sequence that is at least 96% identical to SEQ ID NO:3. According to some embodiments, the scFv comprises an amino acid sequence that is at least 97% identical to SEQ ID NO:3. According to some embodiments, the scFv comprises an amino acid sequence that is at least 98% identical to SEQ ID NO:3. According to some embodiments, the scFv comprises an amino acid sequence that is at least 99% identical to SEQ ID NO:3. According to some embodiments, the scFv consists of SEQ ID NO:3.

[0202] According to some embodiments, the LNP comprises a cationic lipid, a sterol or a derivative thereof, a non-cationic lipid, or a PEGylated lipid.

[0203] A. Cationic lipids In some embodiments, the lipid nanoparticles having an average diameter of 20-74 nm comprise a cationic lipid. In some embodiments, the cationic lipid is, for example, a non-fusogenic cationic lipid. By "non-fusogenic cationic lipid" is meant a cationic lipid that can condense and / or encapsulate a nucleic acid cargo such as ceDNA, but has little or no fusogenic activity.

[0204] In some embodiments, the cationic lipids are described in the International and U.S. Patent Application Publications listed in Table 1 below, and are determined to be non-fusogenic, for example, as measured by a membrane impermeable fluorescent dye exclusion assay (e.g., an assay described in the Examples section herein). The entire contents of these International and U.S. Patent Application Publications listed in Table 1 below are incorporated herein by reference in their entirety.

[0205] [Table 1-1]

[0206]

Table 1-2

[0207] In some embodiments, the cationic lipid is N-[1-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA); N-[1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA); chloride, DOTAP); 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC); 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DLEPC); 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC) ;1,2-Dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]ol butylcarboxamide ethyl 1-3,4-di[oleyloxy]-benzamide (MVL5);Dioctadecylamido-glycylspermine (DOGS);3b-[N-(N',N'-dimethylaminoethyl)carb amoyl]cholesterol (DC-Chol);Dioctadecyldimethylammonium Bromide (DDAB);Saint lipids (e.g., SAINT-2, N-methyl-4-(dioleyl)methylpyridinium);1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE); 1,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE); 1,2-dioleoyloxypropyl-3-dimethylhydroxyethyl ammonium chloride (DORI); Di-alkylated Amino Acid (DILA2) (e.g., C18:1-norArg-C16); Dioleyldimethylammonium chloride (DODAC); 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (1-palmitoyl-2-oleoyl-sn-glycero-3-ethylpho sphocholine, POEPC);and 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (MOEPC). In some variations, the condensing agent, e.g., cationic lipid, is selected from the group consisting of, for example, dioctadecyldimethylammonium bromide (DDAB), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,31-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), 1,2-dioleoyloxy-3- These lipids include dimethylaminopropane (DODAP), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), morpholinocholesterol (Mo-CHOL), (R)-5-(dimethylamino)pentane-1,2-diyl dioleate hydrochloride (DODAPen-C1), (R)-5-guanidinopentane-1,2-diyl dioleate hydrochloride (DOPen-G), and (R)-N,N,N-trimethyl-4,5-bis(oleoyloxy)pentan-1-aminium chloride (DOTAPen);

[0208] In some embodiments, the condensed lipid is DOTAP.

[0209] Ionizable lipids Also provided herein, according to some embodiments, are pharmaceutical compositions containing LNPs comprising an ionizable lipid and a therapeutic nucleic acid, such as a non-viral vector (e.g., ceDNA). Such LNPs can be used to deliver, for example, a pharmaceutical composition comprising a lipid nanoparticle (LNP) and a therapeutic nucleic acid (TNA), where the LNP comprises an scFv linked to the LNP, as described herein, to a target site of interest (e.g., a cell, tissue, organ, etc.).

[0210] Exemplary ionizable lipids are described in International Patent Application Publication Nos. 2015 / 095340, 2015 / 199952, 2018 / 011633, 2017 / 049245, 2015 / 061467, 2012 / 040184, 2012 / 000104, 2015 / 074085, 2016 / 081029, 2017 / 004143, 2017 / 075531, 2017 / 117528, 2011 / 022460, 2013 / 148541, 2013 / 116126, 201 1 / 153120, 2012 / 044638, 2012 / 054365, 2011 / 090965, 2013 / No. 016058, No. 2012 / 162210, No. 2008 / 042973, No. 2010 / 129709, No. 2010 / 14 No. 4740, No. 2012 / 099755, No. 2013 / 049328, No. 2013 / 086322, No. 2013 / 0863 No. 73, No. 2011 / 071860, No. 2009 / 132131, No. 2010 / 048536, No. 2010 / 088537 , 2010 / 054401, 2010 / 054406, 2010 / 054405, 2010 / 054384, No. 2012 / 016184, No. 2009 / 086558, No. 2010 / 042877, No. 2011 / 000106, No. 2 011 / 000107, 2005 / 120152, 2011 / 141705, 2013 / 126803, 200 6 / 007712, 2011 / 038160, 2005 / 121348, 2011 / 066651, 2009 / 1 Nos. 2011 / 141704, 2006 / 069782, 2012 / 031043, 2013 / 006825, 2013 / 033563, 2013 / 089151, 2017 / 099823, 2015 / 095346, and 2013 / 086354, and U.S. Patent Application Publication Nos. 2016 / 0311759, 2015 / 0376115, 2016 / 0151284, 2017 / 0210697, 2015 / 0140070, and 2013 / 0178541;Same No. 2013 / 0303587, No. 2015 / 0141678, No. 2015 / 0239926, No. 2016 / 0376224, No. 2017 / 0119904, No. 2012 / 014989 No. 4, No. 2015 / 0057373, No. 2013 / 0090372, No. 2013 / 0274523, No. 2013 / 0274504, No. 2013 / 0274504, No. 2009 / 002 No. 3673, No. 2012 / 0128760, No. 2010 / 0324120, No. 2014 / 0200257, No. 2015 / 0203446, No. 2018 / 0005363, No. 2014 / 0308304, 2013 / 0338210, 2012 / 0101148, 2012 / 0027796, 2012 / 0058144, 2013 / 0323269, 201 1 / 0117125, 2011 / 0256175, 2012 / 0202871, 2011 / 0076335, 2006 / 0083780, 2013 / 0123338, 2015 / 0064242, 2006 / 0051405, 2013 / 0065939, 2006 / 0008910, 2003 / 0022649, 2010 / 0130588 Nos. 2013 / 0116307, 2010 / 0062967, 2013 / 0202684, 2014 / 0141070, 2014 / 0255472, 2014 / 0039032, 2018 / 0028664, 2016 / 0317458, and 2013 / 0195920, the contents of all of which are incorporated herein by reference in their entireties.

[0211] In some embodiments, the ionizable lipid is MC3(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA or MC3), having the following structure:

[0212] [ka]

[0213] The lipid DLin-MC3-DMA is described in Jayaraman et al., Angew. Chem. Int. Ed Engl. (2012), 51(34):8529-8533, the contents of which are incorporated herein by reference in their entirety.

[0214] In some embodiments, the ionizable lipid is the lipid ATX-002, described in WO 2015 / 074085, the contents of which are incorporated herein by reference in their entirety.

[0215] In some embodiments, the ionizable lipid is (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine (compound 32), described in WO 2012 / 040184, the contents of which are incorporated herein by reference in their entirety.

[0216] In some embodiments, the ionizable lipid is compound 6 or compound 22, described in WO 2015 / 199952, the contents of which are incorporated herein by reference in their entirety.

[0217] Formula (I) According to some embodiments, the cationic lipid is represented by formula (I):

[0218] [ka] or a pharma- ceutically acceptable salt thereof, wherein: R 1 and R 1’ are each independently 1~3 is alkylene, R 2 and R 2’ are each independently a straight-chain or branched-chain C 1~6 Alkylene or C 3~6 is cycloalkylene, R3 and R 3’ each independently represents an optionally substituted C 1~6 Alkyl or optionally substituted C 3~6 cycloalkyl, or or alternatively, R 2 But branched chain C 1~6 When R is an alkylene, 3 But, C 1~6 If it is alkyl, R 2 and R 3 together with the intervening N atom form a 4- to 8-membered heterocyclyl, or or alternatively, R 2’ But branched chain C 1~6 When R is an alkylene, 3’ But, C 1~6 If it is alkyl, R 2’ and R 3’ together with the intervening N atom form a 4- to 8-membered heterocyclyl; R 4 and R 4’ are each independently -CH, -CHCH, or -(CH)CH; R 5 and R 5’ are independently hydrogen, C 1~20 Alkylene or C 2~20 alkenylene, R 6 and R 6’ But for each occurrence, independently, C 1~20 Alkylene, C 3~20 Cycloalkylene or C 2~20 alkenylene, m and n are each independently an integer selected from 1, 2, 3, 4, and 5.

[0219] According to some embodiments of any of the aspects or embodiments herein, R 2 and R 2’ are each independently C 1~3 It is alkylene.

[0220] According to some embodiments of any of the aspects or embodiments herein, R 1 or R 1’ A linear or branched chain C represented by 1~3 Alkylene, R 2 or R 2’ A linear or branched chain C represented by 1~6 Alkylene, and optionally substituted straight or branched chain C 1~6 Each alkyl is optionally substituted with one or more halo and cyano groups.

[0221] According to some embodiments of any of the aspects or embodiments herein, R 1 and R 2 Together, C 1~3 alkylene, R 1’ and R 2’ Together, C 1~3 Alkylene, for example, ethylene.

[0222] According to some embodiments of any of the aspects or embodiments herein, R 3 and R 3’ each independently represents an optionally substituted C 1~3 Alkyl, for example methyl.

[0223] According to some embodiments of any of the aspects or embodiments herein, R 4 and R 4’ are -CH, respectively.

[0224] According to some embodiments of any of the aspects or embodiments herein, R 2 is an optionally substituted branched chain C 1~6 alkylene, R 2 and R 3 together with their intervening N atoms form a 5- or 6-membered heterocyclyl. According to some embodiments of any of the aspects or embodiments herein, R 2’ is an optionally substituted branched chain C 1~6 R is an alkylene.2’ and R 3’ together with their intervening N atom form a 5- or 6-membered heterocyclyl, such as pyrrolidinyl or piperidinyl.

[0225] According to some embodiments of any of the aspects or embodiments herein, R 4 is -C(R a )2CR a , or -[C(R a )2]2CR a and R a is C 1~3 is alkyl, R 3 and R 4 together with their intervening N atoms form a 5- or 6-membered heterocyclyl. According to some embodiments of any of the aspects or embodiments herein, R 4’ is -C(R a )2CR a , or -[C(R a )2]2CR a and R a is C 1~3 is alkyl, R 3’ and R 4’ together with their intervening N atom form a 5- or 6-membered heterocyclyl, such as pyrrolidinyl or piperidinyl.

[0226] According to some embodiments of any of the aspects or embodiments herein, R 5 and R 5’ are each independently 1~10 Alkylene or C 2~10 In one embodiment, R is alkenylene. 5 and R 5’ are each independently 1~8 Alkylene or C 1~6 It is alkylene.

[0227] According to some embodiments of any of the aspects or embodiments herein, R 6 and R 6’ For each occurrence, independently, C1~10 Alkylene, C 3~10 Cycloalkylene or C 2~10 In one embodiment, C is an alkenylene. 1~6 Alkylene, C 3~6 Cycloalkylene or C 2~6 In one embodiment, C 3~10 Cycloalkylene or C 3~6 According to certain embodiments of any of the aspects or embodiments herein, m and n are each 3.

[0228] According to some embodiments of any of the aspects or embodiments herein, the cationic lipid is selected from any one of the lipids in Table 2, or a pharma- ceutically acceptable salt thereof.

[0229] [Table 2-1]

[0230] [Table 2-2]

[0231] [Table 2-3]

[0232] [Table 2-4]

[0233] [Table 2-5]

[0234] [Table 2-6]

[0235] [Table 2-7]

[0236] [Table 2-8]

[0237] [Table 2-9]

[0238] [Table 2-10]

[0239] [Table 2-11]

[0240] Formula (II) In some embodiments, the cationic lipid is of formula (II):

[0241] [ka] or a pharma- ceutically acceptable salt thereof, wherein: a is an integer ranging from 1 to 20 (e.g., a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); b is an integer ranging from 2 to 10 (e.g., b is 2, 3, 4, 5, 6, 7, 8, 9, or 10); R 1 does not exist or (C2~C 20 ) alkenyl, -C(O)O(C2-C 20 ) alkyl, and (C2-C 20 ) cyclopropyl substituted with alkyl; R 2 However, (C2~C 20 ) alkyl.

[0242] In a second chemical embodiment, the cationic lipid of formula (II) is of formula (XIII):

[0243] [ka] or a pharma- ceutically acceptable salt thereof, wherein c and d are each independently an integer in the range of 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and the remainder of the variables are as described for formula (XII).

[0244] In a third chemical embodiment, c and d in the cationic lipid of formula (II) or (III) are each independently an integer ranging from 2 to 8, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 8, 4 to 7, 4 to 6, 5 to 8, 5 to 7, or 6 to 8, and the remaining variables are as described for formula (XII).

[0245] In a fourth chemical embodiment, c in the cationic lipid of formula (II) or (III) is 2, 3, 4, 5, 6, 7, or 8, and the remaining variables are as described for formula (XII) or the second or third chemical embodiment. Alternatively, as part of the fourth chemical embodiment, c and d in the cationic lipid of formula (XII) or (XIII), or a pharma- ceutically acceptable salt thereof, are each independently 1, 3, 5, or 7, and the remaining variables are as described for formula (XII) or the second or third chemical embodiment.

[0246] In a fifth chemical embodiment, d in the cationic lipid of formula (II) or (III) is 2, 3, 4, 5, 6, 7, or 8, and the remaining variables are as described for formula (II) or the second or third or fourth chemical embodiment. Alternatively, as part of the fourth chemical embodiment, at least one of c and d in the cationic lipid of formula (II) or (III) or a pharma- ceutically acceptable salt thereof is 7, and the remaining variables are as described for formula (II) or the second or third or fourth chemical embodiment.

[0247] In a sixth chemical embodiment, the cationic lipid of formula (II) or (III) is of formula (IV):

[0248] [ka] or a pharma- ceutically acceptable salt thereof, wherein the remainder of the variables are as described for formula (I).

[0249] In a seventh chemical embodiment, b in the cationic lipid of formula (II), (III), or (IV) is an integer ranging from 3 to 9, and the remaining variables are as described for formula (II), or the second, third, fourth, or fifth chemical embodiment. Alternatively, as part of the seventh chemical embodiment, b in the cationic lipid of formula (II), (III), or (IV) is an integer ranging from 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 5 to 9, 5 to 8, 5 to 7, 6 to 9, 6 to 8, or 7 to 9, and the remaining variables are as described for formula (II), or the second, third, fourth, or fifth chemical embodiment. In another alternative, as part of the seventh chemical embodiment, b in the cationic lipid of formula (II), (III), or (IV) is 3, 4, 5, 6, 7, 8, or 9, and the remaining variables are as described for formula (XII), or the second, third, fourth, or fifth chemical embodiments.

[0250] In an eighth chemical embodiment, a in the cationic lipid of formula (II), (III), or (IV) is an integer ranging from 2 to 18, and the remaining variables are as described for formula (II), or the second, third, fourth, fifth, or seventh chemical embodiment. Alternatively, as part of the eighth embodiment, a in the cationic lipid of formula (II), (III), or (IV) is an integer ranging from 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 3 to 18, 3 to 17, 3 to 16, 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3~5, 4~18, 4~17, 4~16, 4~15, 4~14, 4~13, 4~12, 4~11, 4~10, 4~9, 4~8, 4~7, 4~6, 5~18, 5~17, 5~16, 5~15, 5~14, 5~13, 5~12, 5~11, 5~10, 5~9, 25~8, 5~7, 6~18, 6~17, 6~16, 6~15, 6~14, 6~13, 6~12, 6~11, 6~10, 6~9, 6~8, 7~18, 7~17, 7~16, 7~15, 7~14, 7~13, 7~12, 7~11, 7~10, 7~9, 8~18, 8~17, 8~16, 8~15, 8~14, 8~13, 8~12, 8~11, 8~10, 9~18, 9~17, 9~16, 9~15, 9~14, 9~13, 9~12, 9~11, 10~18, 10~17, 10~16, 10~15, 10~14, 10~13, 11~18, 11~17, 11~ 16, 11-15, 11-14, 11-13, 12-18, 12-17, 12-16, 12-15, 12-14, 13-18, 13-17, 13-16, 13-15, 14-18, 14-17, 14-16, 15-18, 15-17, or 16-18, and the remainder of the variables are as described for formula (II), or the second, third, fourth, fifth, or seventh chemical embodiment. In another alternative, as part of the eighth embodiment, a in the cationic lipid of formula (II), (III), or (IV) is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18, and the remaining variables are as described for formula (II), or the second, third, fourth, fifth, or seventh chemical embodiment.

[0251] In a ninth chemical embodiment, R in a cationic lipid of formula (II), (III), or (IV) or a pharma- ceutically acceptable salt thereof is 1 does not exist or (C5~C 15 )Alkenyl, -C(O)O(C4-C 18 ) alkyl, and (C4-C 16 ) cyclopropyl substituted with alkyl, and the remaining variables are as described for formula (II), (III), or (IV), or the second, third, fourth, fifth, seventh, or eighth chemical embodiment. Alternatively, as part of a ninth chemical embodiment, R in the cationic lipid of formula (II), (III), or (IV), or a pharma- ceutically acceptable salt thereof, is selected from the group consisting of cyclopropyl, ... 1 does not exist or (C5~C 15 )Alkenyl, -C(O)O(C4-C 16 ) alkyl, and (C4-C 16 ) cyclopropyl substituted with alkyl, and the remaining variables are as described for formula (II), (III), or (IV), or the second, third, fourth, fifth, seventh, or eighth chemical embodiment. Alternatively, as part of a ninth chemical embodiment, R in the cationic lipid of formula (II), (III), or (IV), or a pharma- ceutically acceptable salt thereof, is selected from the group consisting of cyclopropyl, ... 1 does not exist or (C5~C 12 )Alkenyl, -C(O)O(C4-C 12 ) alkyl, and (C4-C 12 ) cyclopropyl substituted with alkyl, and the remaining variables are as described for formula (II), (III), or (IV), or the second, third, fourth, fifth, seventh, or eighth chemical embodiment. In another alternative, as part of the ninth chemical embodiment, R in the cationic lipid of formula (II), (III), or (IV), or a pharma- ceutically acceptable salt thereof, is selected from the group consisting of cyclopropyl, ... 1 does not exist or (C5~C 10 )Alkenyl, -C(O)O(C4-C 10 ) alkyl, and (C4-C 10) cyclopropyl substituted with alkyl, and the remainder of the variables are as described for formula (II), (III), or (IV), or the second, third, fourth, fifth, seventh, or eighth chemical embodiment.

[0252] In a tenth chemical embodiment, R 1 is C 10 alkenyl, and the remainder of the variables are as described in any one of the preceding embodiments.

[0253] In an eleventh chemical embodiment, R in a cationic lipid of formula (II), (III), or (IV) or a pharma- ceutically acceptable salt thereof is 1 C(O)O(C2-C 20 ) alkyl, -C(O)O(C4-C 18 )Alkyl, -C(O)O(C4-C 12 ) alkyl, or -C(O)O(C4-C 10 ) alkyl is an unbranched alkyl, and the remainder of the variables are as described in any one of the preceding embodiments. In one chemical embodiment, R 1 is -C(O)O(C alkyl). Alternatively, in an eleventh chemical embodiment, R in the cationic lipid of formula (II), (III), or (IV) or a pharma- ceutically acceptable salt thereof is 1 -C(O)O(C4~C 18 )Alkyl, -C(O)O(C4-C 12 ) alkyl, or -C(O)O(C4-C 10 ) alkyl is a branched alkyl, and the remainder of the variables are as described in any one of the preceding chemical embodiments. In one chemical embodiment, R 1 is -C(O)O(C 17 alkyl), and the remainder of the variables are as described in any one of the preceding chemical embodiments.

[0254] In a twelfth chemical embodiment, R in a cationic lipid of formula (II), (III), or (IV) or a pharma- ceutically acceptable salt thereof is1 is selected from any of the groups listed in Table 3 below, where the wavy bond in each of the groups indicates the point of attachment of the group to the remainder of the lipid molecule, and the remaining variables are as described for formula (II), (III), or (IV), or the second, third, fourth, fifth, seventh, or eighth chemical embodiment. 1 Any one of the groups and R in Table 5 2 Further contemplated are combinations with any one of the groups, and the remaining variables are as described for formula (II), (III) or (IV), or the second, third, fourth, fifth, seventh or eighth chemical embodiments.

[0255] [Table 3]

[0256] In a thirteenth chemical embodiment, R in the cationic lipid of formula (II) or a pharma- ceutically acceptable salt thereof is 2 is selected from any of the groups listed in Table 4 below, where the wavy bond in each of the groups indicates the point of attachment of the group to the remainder of the lipid molecule, and the remaining variables are as described for formula (II), or the seventh, eighth, ninth, tenth, or eleventh chemical embodiment.

[0257] [Table 4]

[0258] Specific examples are provided in Table 5, the Exemplification section below, and are included as part of the 14th chemical embodiment herein of the cationic lipid of formula (II). Pharmaceutically acceptable salts and ionized and neutral forms are also included.

[0259] [Table 5-1]

[0260] [Table 5-2]

[0261] [Table 5-3]

[0262] [Table 5-4]

[0263] [Table 5-5]

[0264] Formula (V) In some embodiments, the cationic lipid is of formula (V):

[0265] [ka] or a pharma- ceutically acceptable salt thereof, wherein: R 1 and R 1’ However, each independently, R a is a (C1-C6) alkylene optionally substituted with one or more groups selected from R 2 and R 2’ are each independently (C1-C2) alkylene; R 3 and R 3’ However, each independently, R b is a (C1-C6) alkyl optionally substituted with one or more groups selected from or alternatively, R 2 and R 3 and / or R 2’ and R 3’ together with the intervening N atom form a 4- to 7-membered heterocyclyl; R 4 and R 4' is a (C2-C6) alkylene interrupted by -C(O)O-, R 5 and R 5 ' are each independently, (C2~C 30 ) alkyl or (C2-C 30 ) alkenyl, each of which is optionally interrupted by -C(O)O- or (C3-C6)cycloalkyl; R a and R b are each halo or cyano.

[0266] In a second chemical embodiment, R in the cationic lipid of formula (V) 1 and R 1’ are each independently (C1-C6) alkylene, and the remaining variables are as described above for formula (V). Alternatively, as part of a second chemical embodiment, R in the cationic lipid of formula (V) 1 and R 1’ is each independently a (C1-C3) alkylene, and the remainder of the variables are as described above for formula (V).

[0267] In a third chemical embodiment, the cationic lipid of formula (V) is Formula (VI):

[0268] [ka] or a pharma- ceutically acceptable salt thereof, wherein the remainder of the variables are as described above for formula (V).

[0269] In a fourth chemical embodiment, the cationic lipid of formula (V) is represented by formula (VII) or (VIII):

[0270] [ka] or a pharma- ceutically acceptable salt thereof, wherein the remainder of the variables are as described above for formula (V).

[0271] In a fifth chemical embodiment, the cationic lipid of formula (V) is represented by formula (IX) or (VI):

[0272] [ka] or a pharma- ceutically acceptable salt thereof, wherein the remainder of the variables are as described above for formula (V).

[0273] In a sixth chemical embodiment, the cationic lipid of formula (V) is represented by formula (XI), (XII), (XIII), or (XIV):

[0274] [ka] or a pharma- ceutically acceptable salt thereof, wherein the remainder of the variables are as described above for formula (XV).

[0275] In a seventh chemical embodiment, R in a cationic lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) 5 and R 5’ is a branched alkyl or branched alkenyl (wherein the number of carbon atoms is as described above for formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV)). In another alternative, as part of the seventh chemical aspect, at least one of R in the cationic lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) is 5 and R 5’ In another alternative, as part of the seventh chemical aspect, one of R in the cationic lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) is a branched alkyl or branched alkenyl. 5is a branched alkyl or branched alkenyl. In another alternative, as part of the seventh chemical aspect, R in the cationic lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) 5 is a branched alkyl or branched alkenyl.

[0276] In an eighth chemical embodiment, R in a cationic lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) 5 (C6~C 26 ) alkyl or (C6-C 26 ) alkenyl, each of which is optionally interrupted by -C(O)O- or (C-C)cycloalkyl, and the remaining variables are as described above for formula (I). Alternatively, as part of a seventh chemical aspect, R in a cationic lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) is 5 (C6~C 26 ) alkyl or (C6-C 26 ) alkenyl, each of which is optionally interrupted with -C(O)O- or (C-C)cycloalkyl, and the remaining variables are as described above for formula (V). In another alternative, as part of the eighth chemical aspect, R in the cationic lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) is 5 (C7~C 26 ) alkyl or (C7-C 26 ) alkenyl, each of which is optionally interrupted with -C(O)O- or (C-C)cycloalkyl, and the remaining variables are as described above for formula (V). In another alternative, as part of the eighth chemical aspect, R in the cationic lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) is 5 (C8~C 26) alkyl or (C8-C 26 ) alkenyl, each of which is optionally interrupted with -C(O)O- or (C-C)cycloalkyl, and the remaining variables are as described above for formula (V). In another alternative, as part of the eighth chemical aspect, R in the cationic lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) is 5 (C6~C 24 ) alkyl or (C6-C 24 ) alkenyl, each of which is optionally interrupted by -C(O)O- or cyclopropyl, and the remaining variables are as described above for formula (V). In another alternative, as part of the eighth chemical aspect, R in the cationic lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) is 5 (C8~C 24 ) alkyl or (C8-C 24 ) alkenyl, 24 ) alkyl is optionally interrupted with -C(O)O- or cyclopropyl, and the remaining variables are as described above for formula (V). In another alternative, as part of the eighth chemical aspect, R in the cationic lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) 5 (C8~C 10 ) alkyl, and the remaining variables are as described above for formula (V). In another alternative, as part of the eighth chemical aspect, R in the cationic lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) is 5 is interrupted by cyclopropyl (C 14 ~C 16) alkyl, and the remaining variables are as described above for formula (V). In another alternative, as part of the eighth chemical aspect, R in the cationic lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) is 5 is interrupted by -C(O)O- (C 10 ~C 24 ) alkyl, and the remaining variables are as described above for formula (V). In another alternative, as part of the eighth chemical aspect, R in the cationic lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) is 5 is (C 16 ~C 18 ) alkenyl, and the remaining variables are as described above for formula (V). In another alternative, as part of the eighth chemical aspect, R in the cationic lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) is 5 is -(CH2)3C(O)O(CH2)8CH3, -(CH2)5C(O)O(CH2)8CH 3、 -(CH2)7C(O)O(CH2)8CH3, -(CH2)7C(O)OCH[(CH2)7CH3]2, -(CH2)7-C3H6-(CH2)7CH3, -(CH2)7CH3, -(CH2)9CH 3、 -(CH2) 16 CH3, -(CH2)7CH=CH(CH2)7CH3, or -(CH2)7CH=CHCH2CH=CH(CH2)4CH3, and the remainder of the variables are as described above for formula (XV).

[0277] In a ninth chemical embodiment, R in a cationic lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) 5’ is interrupted by -C(O)O- (C 15 ~C 28) alkyl, and the remaining variables are as described above for formula (V) or the eighth chemical embodiment. Alternatively, as part of the ninth chemical embodiment, R in the cationic lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) is 5’ is interrupted by -C(O)O- (C 17 ~C 28 ) alkyl, and the remaining variables are as described above for formula (V) or the eighth chemical aspect. In another alternative, as part of the ninth embodiment, R in the cationic lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) is 5’ is interrupted by -C(O)O- (C 19 ~C 28 ) alkyl, and the remaining variables are as described above for formula (V) or the eighth chemical embodiment. In another alternative, as part of the ninth chemical embodiment, R in the cationic lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) is 5’ is interrupted by -C(O)O- (C 17 ~C 26 ) alkyl, and the remaining variables are as described above for formula (V) or the eighth chemical aspect. In another alternative, as part of the ninth embodiment, R in the cationic lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) is 5’ is interrupted by -C(O)O- (C 19 ~C 26 ) alkyl, and the remaining variables are as described above for formula (V) or the eighth chemical embodiment. In another alternative, as part of the ninth chemical embodiment, R in the cationic lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) is 5’ is interrupted by -C(O)O- (C 20 ~C 26) alkyl, and the remaining variables are as described above for formula (V) or the eighth chemical aspect. In another alternative, as part of the ninth embodiment, R 5 ' is interrupted by -C(O)O- (C 22 ~C 24 ) alkyl, and the remaining variables are as described above for formula (V) or the eighth chemical aspect. In another alternative, as part of the ninth embodiment, R 5’ is —(CH2)5C(O)OCH[(CH2)7CH3]2, —(CH2)7C(O)OCH[(CH2)7CH3]2, —(CH2)5C(O)OCH(CH2)2[(CH2)7CH3]2, or —(CH2)7C(O)OCH(CH2)2[(CH2)7CH3]2, and the remainder of the variables are as described above for formula (V) or the eighth chemical embodiment.

[0278] In another embodiment, the cationic lipid of formula (V), (VI), (VIII), (VIII), (IX), (X), (XII), (XIII), or (XIV) may be selected from any of the following lipids in Table 6, or a pharma- ceutically acceptable salt thereof.

[0279] [Table 6]

[0280] Formula (XV) In some embodiments, the cationic lipid is of formula (XV):

[0281] [ka] or a pharma- ceutically acceptable salt thereof, wherein: R' is absent, hydrogen, or C1-C6 alkyl, provided that when R' is hydrogen or C1-C6 alkyl, R', R 1 , and R 2 The nitrogen atoms to which all are attached are protonated, R 1 and R 2are each independently hydrogen, C1-C6 alkyl, or C2-C6 alkenyl; R 3 But, C1~C 12 Alkylene or C2-C 12 alkenylene, R 4 But, C1~C 16 Unbranched alkyl, C2-C 16 unbranched alkenyl, or

[0282] [ka] where: R 4a and R 4b However, each is independent, C1~C 16 Unbranched alkyl or C2-C 16 is an unbranched alkenyl; R 5 is absent, C1-C8 alkylene, or C2-C8 alkenylene; R 6a and R 6b However, each is independent, C7~C 16 Alkyl or C7-C 16 alkenyl, where R 6a and R 6b the total number of carbon atoms in the X 1 and X 2 are each independently -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -SS-, -C(R a )=N-, -N=C(R a )-, -C(R a )=NO-, -ON=C(R a )-, -C(=O)NR a -, -NR a C(=O)-, -NR a C(=O)NR a -, -OC(=O)O-, -OSi(R a )2O-, -C(=O)(CR a2) C(=O)O- or OC(=O)(CR a 2) C(=O)-, wherein R a is, for each occurrence, independently, hydrogen or C1-C6 alkyl; n is an integer selected from 1, 2, 3, 4, 5, and 6.

[0283] In a second embodiment, in the cationic lipid according to the first embodiment, or a pharma- ceutically acceptable salt thereof, X 1 and X 2 is the same and all other remaining variables are as described for formula (V) or the first embodiment.

[0284] In a third embodiment, the cationic lipid according to the first or second embodiment, or a pharma- ceutically acceptable salt thereof, is 1 and X 2 are each independently -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, or -SS-; or X 1 and X 2 are each independently -C(=O)O-, -C(=O)S-, or -SS-; or X 1 and X 2 is each independently -C(=O)O or -SS-, and all other remaining variables are as described for formula V or any one of the preceding embodiments.

[0285] In a fourth embodiment, the cationic lipid of the present disclosure is represented by formula (XVI):

[0286] [ka] or a pharma- ceutically acceptable salt thereof, where n is an integer selected from 1, 2, 3, and 4, and all other remaining variables are as described for formula (XV) or any one of the preceding embodiments.

[0287] In a fifth embodiment, the cationic lipid of the present disclosure is represented by formula (XVII):

[0288] [ka] or a pharma- ceutically acceptable salt thereof, where n is an integer selected from 1, 2, and 3, and all other remaining variables are as described for Formula (XV), Formula (XVI), or any one of the preceding embodiments.

[0289] In a sixth embodiment, the cationic lipid of the present disclosure is represented by formula (XVIII):

[0290] [ka] or a pharma- ceutically acceptable salt thereof, and all other remaining variables are as described for Formula (XV), Formula (XVI), Formula (XVII), or any one of the preceding embodiments.

[0291] In a seventh embodiment, in the cationic lipid according to Formula (XV), Formula (XVI), Formula (XVII), Formula (XVIII), or any one of the preceding embodiments, or a pharma- ceutically acceptable salt thereof, R 1 and R 2 are each independently hydrogen, C1-C6 alkyl or C2-C6 alkenyl, or C1-C5 alkyl or C2-C5 alkenyl, or C1-C4 alkyl or C2-C4 alkenyl, or C6 alkyl, or C5 alkyl, or C4 alkyl, or C3 alkyl, or C2 alkyl, or C1 alkyl, or C6 alkenyl, or C5 alkenyl, or C4 alkenyl, or C3 alkenyl, or C2 alkenyl, and all other remaining variables are as described for Formula (XV), Formula (XVI), Formula (XVII), Formula (XVIII), or any one of the preceding embodiments.

[0292] In an eighth embodiment, the cationic lipid of the present disclosure is represented by formula (XIX):

[0293] [ka] or a pharma- ceutically acceptable salt thereof, and all other remaining variables are as described for Formula (XV), Formula (XVI), Formula (XVII), Formula (XVIII), or any one of the preceding embodiments.

[0294] In a ninth embodiment, the cationic lipid according to formula (XV), formula (XVI), formula (XVII), formula (XVIII), formula (XIX), or any one of the preceding embodiments, or a pharma- ceutically acceptable salt thereof, is selected from the group consisting of R 3 is C1-C9 alkylene or C2-C9 alkenylene, C1-C7 alkylene or C2-C7 alkenylene, C1-C5 alkylene or C2-C5 alkenylene, C2-C8 alkylene or C2-C8 alkenylene, C3-C7 alkylene or C3-C7 alkenylene, C5-C7 alkylene or C5-C7 alkenylene, or R 3 But, C 12 Alkylene, C 11 Alkylene, C 10 Alkylene, C9 alkylene, or C8 alkylene, or C7 alkylene, or C6 alkylene, or C5 alkylene, or C4 alkylene, or C3 alkylene, or C2 alkylene, or C1 alkylene, or C 12 Alkenylene, C 11 Alkenylene, C 10 alkenylene, C9 alkenylene, or C8 alkenylene, or C7 alkenylene, or C6 alkenylene, or C5 alkenylene, or C4 alkenylene, or C3 alkenylene, or C2 alkenylene, and all other remaining variables are as described for Formula (XV), Formula (XVI), Formula (XVII), Formula (XVIII), Formula (XIX), or any one of the preceding embodiments.

[0295] In a tenth embodiment, in the cationic lipid according to formula (XV), formula (XVI), formula (XVII), formula (XVIII), formula (XIX), or any one of the preceding embodiments, or a pharma- ceutically acceptable salt thereof, R 5 is absent, C1-C6 alkylene, or C2-C6 alkenylene, or R 5 is absent, C1-C4 alkylene, or C2-C4 alkenylene, or R 5 does not exist or R 5 is C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkenylene, C alkenylene, C alkenylene, C alkenylene, C alkenylene, C alkenylene, or C alkenylene, and all other remaining variables are as described for Formula (XV), Formula (XVI), Formula (XVII), Formula (XVIII), Formula (XIX), or any one of the preceding embodiments.

[0296] In an eleventh embodiment, in the cationic lipid according to formula (XV), formula (XVI), formula (XVII), formula (XVIII), formula (XIX), or any one of the preceding embodiments, or a pharma- ceutically acceptable salt thereof, R 4 But, C1~C 14 Unbranched alkyl, C2-C 14 unbranched alkenyl, or

[0297] [ka] and R 4a and R 4b However, each is independent, C1~C 12 Unbranched alkyl or C2-C 12 unbranched alkenyl or R 4 But, C2~C 12 Unbranched alkyl or C2-C 12 unbranched alkenyl or R 4is a C5-C7 unbranched alkyl or a C5-C7 unbranched alkenyl, or R 4 But, C 16 Unbranched alkyl, C 15 Unbranched alkyl, C 14 Unbranched alkyl, C 13 Unbranched alkyl, C 12 Unbranched alkyl, C 11 Unbranched alkyl, C 10 Unbranched alkyl, C9 unbranched alkyl, C8 unbranched alkyl, C7 unbranched alkyl, C6 unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 unbranched alkyl, C1 unbranched alkyl, C 16 Unbranched alkenyl, C 15 Unbranched alkenyl, C 14 Unbranched alkenyl, C 13 Unbranched alkenyl, C 12 Unbranched alkenyl, C 11 Unbranched alkenyl, C 10 unbranched alkenyl, C9 unbranched alkenyl, C8 unbranched alkenyl, C7 unbranched alkenyl, C6 unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl, or C2 alkenyl, or R 4 but,

[0298] [ka] and R 4a and R 4b However, C2 to C 10 Unbranched alkyl or C2-C 10 unbranched alkenyl or R 4 but,

[0299] [ka] where R 4a and R 4b However, each independently, C 16 Unbranched alkyl, C 15 Unbranched alkyl, C 14 Unbranched alkyl, C13 Unbranched alkyl, C 12 Unbranched alkyl, C 11 Unbranched alkyl, C 10 Unbranched alkyl, C9 unbranched alkyl, C8 unbranched alkyl, C7 unbranched alkyl, C6 unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 alkyl, C1 alkyl, C 16 Unbranched alkenyl, C 15 Unbranched alkenyl, C 14 Unbranched alkenyl, C 13 Unbranched alkenyl, C 12 Unbranched alkenyl, C 11 Unbranched alkenyl, C 10 unbranched alkenyl, C9 unbranched alkenyl, C8 unbranched alkenyl, C7 unbranched alkenyl, C6 unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl, or C2 alkenyl, and all other remaining variables are as described for Formula (XV), Formula (XVI), Formula (XVII), Formula (XVIII), Formula (XIX), or any one of the preceding embodiments.

[0300] In a twelfth embodiment, in the cationic lipid according to formula (XV), formula (XVI), formula (XVII), formula (XVIII), formula (XIX), or any one of the preceding embodiments or a pharma- ceutically acceptable salt thereof, R 6a and R 6b However, independently, C6~C 14 Alkyl or C6~C 14 alkenyl or R 6a and R 6b are independently C8 to C 12 Alkyl or C8~C 12 alkenyl or R 6a and R 6b However, each independently, C 16 Alkyl, C 15 Alkyl, C 14 Alkyl, C 13 Alkyl, C 12 Alkyl, C 11 Alkyl, C 10Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 16 Alkenyl, C 15 Alkenyl, C 14 Alkenyl, C 13 Alkenyl, C 12 Alkenyl, C 11 Alkenyl, C 10 alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl, where R 6a and R 6b and all other remaining variables are as described for Formula (XV), Formula (XVI), Formula (XVII), Formula (XVIII), Formula (XIX), or any one of the preceding embodiments.

[0301] In a thirteenth embodiment, in the cationic lipid according to formula (XV), formula (XVI), formula (XVII), formula (XVIII), formula (XIX), or any one of the preceding embodiments or a pharma- ceutically acceptable salt thereof, R 6a and R 6b contain an equal number of carbon atoms, or R 6a and R 6b are the same or R 6a and R 6b Both are C 16 Alkyl, C 15 Alkyl, C 14 Alkyl, C 13 Alkyl, C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 16 Alkenyl, C 15 Alkenyl, C 14 Alkenyl, C 13 Alkenyl, C 12 Alkenyl, C 11 Alkenyl, C 10 alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl, where R 6a and R 6band all other remaining variables are as described for Formula (XV), Formula (XVI), Formula (XVII), Formula (XVIII), Formula (XIX), or any one of the preceding embodiments.

[0302] In a fourteenth embodiment, in the cationic lipid according to formula (XV), formula (XVI), formula (XVII), formula (XVIII), formula (XIX), or any one of the preceding embodiments, or a pharma- ceutically acceptable salt thereof, R is as defined in any one of the preceding embodiments. 6a and R 6b each contain a different number of carbon atoms, or R 6a and R 6b The number of carbon atoms in R differs by one or two carbon atoms, or 6a and R 6b The number of carbon atoms in R differs by one carbon atom, or 6a is C7 alkyl, and R 6a is C8 alkyl or R 6a is C8 alkyl, and R 6a is C7 alkyl or R 6a is C8 alkyl, and R 6a is C9 alkyl or R 6a is C9 alkyl, and R 6a is C8 alkyl or R 6a is C9 alkyl, and R 6a C 10 Alkyl or R 6a C 10 alkyl, and R 6a is C9 alkyl or R 6a C 10 alkyl, and R 6a C 11 Alkyl or R 6a C 11 alkyl, and R 6a C 10 Alkyl or R 6a C 11 alkyl, and R 6a C12 Alkyl or R 6a C 12 alkyl, and R 6a C 11 Alkyl or R 6a is C7 alkyl, and R 6a is C9 alkyl or R 6a is C9 alkyl, and R 6a is C7 alkyl or R 6a is C8 alkyl, and R 6a C 10 Alkyl or R 6a C 10 alkyl, and R 6a is C8 alkyl or R 6a is C9 alkyl, and R 6a C 11 Alkyl or R 6a C 11 alkyl, and R 6a is C9 alkyl or R 6a C 10 alkyl, and R 6a C 12 Alkyl or R 6a C 12 alkyl, and R 6a C 10 Alkyl or R 6a C 11 alkyl, and R 6a C 13 alkyl or R 6a C 13 alkyl, and R 6a C 11 alkyl, etc., and all other remaining variables are as described for Formula I, Formula II, Formula III, Formula IV, Formula V, or any one of the preceding embodiments.

[0303] In one embodiment, the cationic lipid of the present disclosure, or the cationic lipid of formula (XV), formula (XVI), formula (XVII), formula (XVIII) or formula (XIX), is any one of the lipids selected from the lipids in Table 7, or a pharma- ceutically acceptable salt thereof.

[0304] [Table 7-1]

[0305] [Table 7-2]

[0306] Formula (XX) In some embodiments, the cationic lipid is of formula (XX):

[0307] [ka] (XX) or a pharma- ceutically acceptable salt thereof, wherein: R' is absent, hydrogen, or C1-C3 alkyl, provided that when R' is hydrogen or C1-C3 alkyl, R', R 1 , and R 2 The nitrogen atoms to which all are attached are protonated, R 1 and R 2 are each independently hydrogen or C1-C3 alkyl; R 3 But C3~C 10 Alkylene or C3-C 10 alkenylene, R 4 But, C1~C 16 Unbranched alkyl, C2-C 16 unbranched alkenyl, or

[0308] [ka] where: R 4a and R 4b However, each is independent, C1~C 16 Unbranched alkyl or C2-C 16 is an unbranched alkenyl; R5 is absent, C1-C6 alkylene, or C2-C6 alkenylene; R 6a and R 6b However, each is independent, C7~C 14 Alkyl or C7-C 14 alkenyl, X is -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -SS-, -C(R a )=N-, -N=C(R a )-, -C(R a )=NO-, -ON=C(R a )-, -C(=O)NR a -, -NR a C(=O)-, -NR a C(=O)NR a -, -OC(=O)O-, -OSi(R a )2O-, -C(=O)(CR a 2) C(=O)O- or OC(=O)(CR a 2) C(=O)-, wherein R a is, for each occurrence, independently, hydrogen or C1-C6 alkyl; n is an integer selected from 1, 2, 3, 4, 5, and 6.

[0309] In a second embodiment, in the cationic lipid according to the first embodiment, or a pharma- ceutically acceptable salt thereof, X is -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, or -SS-, and all other remaining variables are as described for Formula I or the first embodiment.

[0310] In a third embodiment, the cationic lipid of the present disclosure is represented by formula (XXI):

[0311] [ka] or a pharma- ceutically acceptable salt thereof, where n is an integer selected from 1, 2, 3, and 4, and all other remaining variables are as described for formula (XX), or any one of the preceding embodiments. In an alternative third embodiment, n is an integer selected from 1, 2, and 3, and all other remaining variables are as described for formula (XX), or any one of the preceding embodiments.

[0312] In a fourth embodiment, the cationic lipid of the present disclosure is represented by formula (XXII):

[0313] [ka] or a pharma- ceutically acceptable salt thereof, and all other remaining variables are as described for Formula (XX), Formula (XXI), or any one of the preceding embodiments.

[0314] In a fifth embodiment, in the cationic lipid according to the first embodiment or a pharma- ceutically acceptable salt thereof, R 1 and R 2 are each independently hydrogen, C1-C2 alkyl, or C2-C3 alkenyl, or R', R 1 , and R 2 are each independently hydrogen, C1-C2 alkyl, and all other remaining variables are as described for Formula (XX), Formula (XXI), or any one of the preceding embodiments.

[0315] In a sixth embodiment, the cationic lipid of the present disclosure is represented by formula (XXII):

[0316] [ka] or a pharma- ceutically acceptable salt thereof, and all other remaining variables are as described for Formula (XX), Formula (XXI), Formula (XXII), or any one of the preceding embodiments.

[0317] In a seventh embodiment, in the cationic lipid according to formula (XX), formula (XXI), formula (XXII), formula (XXIII), or any one of the preceding embodiments, or a pharma- ceutically acceptable salt thereof, R 5 is absent or is a C1-C8 alkylene, or R 5 is absent, C1-C6 alkylene, or C2-C6 alkenylene, or R 5 is absent, C1-C4 alkylene, or C2-C4 alkenylene, or R 5 does not exist or R 5 is C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkenylene, C alkenylene, C alkenylene, C alkenylene, C alkenylene, C alkenylene, or C alkenylene, and all other remaining variables are as described for Formula (XX), Formula (XXI), Formula (XXII), Formula (XXIII), or any one of the preceding embodiments.

[0318] In an eighth embodiment, the cationic lipid of the present disclosure is represented by formula (XXIV):

[0319] [ka] or a pharma- ceutically acceptable salt thereof, and all other remaining variables are as described for Formula (XX), Formula (XXI), Formula (XXII), Formula (XXIII), or any one of the preceding embodiments.

[0320] In a ninth embodiment, in the cationic lipid according to formula (XX), formula (XXI), formula (XXII), formula (XXIII), formula (XXIV), or any one of the preceding embodiments, or a pharma- ceutically acceptable salt thereof, R 4 But, C1~C 14 Unbranched alkyl, C2-C 14 unbranched alkenyl, or

[0321] [ka] and R 4a and R 4b However, each is independent, C1~C 12 Unbranched alkyl or C2-C 12 unbranched alkenyl or R 4 But, C2~C 12 Unbranched alkyl or C2-C 12 unbranched alkenyl or R 4 But C5~C 12 Unbranched alkyl or C5-C 12 unbranched alkenyl or R 4 But, C 16 Unbranched alkyl, C 15 Unbranched alkyl, C 14 Unbranched alkyl, C 13 Unbranched alkyl, C 12 Unbranched alkyl, C 11 Unbranched alkyl, C 10 Unbranched alkyl, C9 unbranched alkyl, C8 unbranched alkyl, C7 unbranched alkyl, C6 unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 unbranched alkyl, C1 unbranched alkyl, C 16 Unbranched alkenyl, C 15 Unbranched alkenyl, C 14 Unbranched alkenyl, C 13 Unbranched alkenyl, C 12 Unbranched alkenyl, C 11 Unbranched alkenyl, C 10 unbranched alkenyl, C9 unbranched alkenyl, C8 unbranched alkenyl, C7 unbranched alkenyl, C6 unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl, or C2 alkenyl, or R 4 but,

[0322] [ka] and R 4a and R4b However, C2 to C 10 Unbranched alkyl or C2-C 10 unbranched alkenyl or R 4 but,

[0323] [ka] where R 4a and R 4b However, each independently, C 16 Unbranched alkyl, C 15 Unbranched alkyl, C 14 Unbranched alkyl, C 13 Unbranched alkyl, C 12 Unbranched alkyl, C 11 Unbranched alkyl, C 10 Unbranched alkyl, C9 unbranched alkyl, C8 unbranched alkyl, C7 unbranched alkyl, C6 unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 alkyl, C1 alkyl, C 16 Unbranched alkenyl, C 15 Unbranched alkenyl, C 14 Unbranched alkenyl, C 13 Unbranched alkenyl, C 12 Unbranched alkenyl, C 11 Unbranched alkenyl, C 10 unbranched alkenyl, C9 unbranched alkenyl, C8 unbranched alkenyl, C7 unbranched alkenyl, C6 unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl, or C2 alkenyl, and all other remaining variables are as described for Formula (XX), Formula (XXI), Formula (XXII), Formula (XXIII), Formula (XXIV), or any one of the preceding embodiments.

[0324] In a tenth embodiment, in the cationic lipid according to formula (XX), formula (XXI), formula (XXII), formula (XXIII), formula (XXIV), or any one of the preceding embodiments, or a pharma- ceutically acceptable salt thereof, R 3is C3-C8 alkylene or C3-C8 alkenylene, C3-C7 alkylene or C3-C7 alkenylene, or C3-C5 alkylene or C3-C5 alkenylene, or R 3 is C alkylene, or C alkylene, or C6 alkylene, or C5 alkylene, or C4 alkylene, or C3 alkylene, or C1 alkylene, or C8 alkenylene, or C7 alkenylene, or C6 alkenylene, or C5 alkenylene, or C4 alkenylene, or C3 alkenylene, and all other remaining variables are as described for Formula (XX), Formula (XXI), Formula (XXII), Formula (XXIII), Formula (XXIV), or any one of the preceding embodiments.

[0325] In an eleventh embodiment, in the cationic lipid according to formula (XX), formula (XXI), formula (XXII), formula (XXIII), formula (XXIV), or any one of the preceding embodiments or a pharma- ceutically acceptable salt thereof, R 6a and R 6b However, each is independent, C7~C 12 Alkyl or C7~C 12 alkenyl or R 6a and R 6b are independently C8 to C 10 Alkyl or C8~C 10 alkenyl or R 6a and R 6b are each independently 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 12 Alkenyl, C 11 Alkenyl, C 10 alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl, and all other remaining variables are as described for Formula (XX), Formula (XXI), Formula (XXII), Formula (XXIII), Formula (XXIV), or any one of the preceding embodiments.

[0326] In a twelfth embodiment, in the cationic lipid according to formula (XX), formula (XXI), formula (XXII), formula (XXIII), formula (XXIV), or any one of the preceding embodiments or a pharma- ceutically acceptable salt thereof, R 6a and R 6b contain an equal number of carbon atoms, or R 6a and R 6b are the same or R 6a and R 6b Both are C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 12 Alkenyl, C 11 Alkenyl, C 10 alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl, and all other remaining variables are as described for Formula (XX), Formula (XXI), Formula (XXII), Formula (XXIII), Formula (XXIV), or any one of the preceding embodiments.

[0327] In a thirteenth embodiment, in the cationic lipid according to formula (XX), formula (XXI), formula (XXII), formula (XXIII), formula (XXIV), or any one of the preceding embodiments or a pharma- ceutically acceptable salt thereof, R is as defined in any one of the preceding embodiments. 6a and R 6b each contain a different number of carbon atoms, or R 6a and R 6b The number of carbon atoms in R differs by one or two carbon atoms, or 6a and R 6b The number of carbon atoms in R differs by one carbon atom, or 6a is C7 alkyl, and R 6a is C8 alkyl or R 6a is C8 alkyl, and R 6a is C7 alkyl or R 6a is C8 alkyl, and R 6a is C9 alkyl or R 6ais C9 alkyl, and R 6a is C8 alkyl or R 6a is C9 alkyl, and R 6a C 10 Alkyl or R 6a C 10 alkyl, and R 6a is C9 alkyl or R 6a C 10 alkyl, and R 6a C 11 Alkyl or R 6a C 11 alkyl, and R 6a C 10 Alkyl or R 6a C 11 alkyl, and R 6a C 12 Alkyl or R 6a C 12 alkyl, and R 6a C 11 Alkyl or R 6a is C7 alkyl, and R 6a is C9 alkyl or R 6a is C9 alkyl, and R 6a is C7 alkyl or R 6a is C8 alkyl, and R 6a C 10 Alkyl or R 6a C 10 alkyl, and R 6a is C8 alkyl or R 6a is C9 alkyl, and R 6a C 11 Alkyl or R 6a C 11 alkyl, and R 6a is C9 alkyl or R 6a C 10 alkyl, and R 6a C 12 Alkyl or R 6a C 12 alkyl, and R 6a C 10alkyl, etc., and all other remaining variables are as described for Formula (XX), Formula (XXI), Formula (XXII), Formula (XXIII), Formula (XXIV), or any one of the preceding embodiments.

[0328] In a fourteenth embodiment, in the cationic lipid according to formula (XX), formula (XXI), formula (XXII), formula (XXIII), formula (XXIV), or any one of the preceding embodiments, or a pharma- ceutically acceptable salt thereof, R' is absent, and all other remaining variables are as described for formula (XX), formula (XXI), formula (XXII), formula (XXIII), formula (XXIV), or any one of the preceding embodiments.

[0329] In one embodiment, the cationic lipid of the present disclosure, or the cationic lipid of formula (XX), formula (XXI), formula (XXII), formula (XXIII), formula (XXIV), is any one of the lipids selected from the lipids in Table 8 or a pharma- ceutically acceptable salt thereof.

[0330] [Table 8-1]

[0331] [Table 8-2]

[0332] Specific examples are provided in the Exemplification section below and are included as part of the cationic or ionizable lipids described herein. Pharmaceutically acceptable salts and neutral forms are also included.

[0333] Cleavable lipids According to some embodiments, provided herein is a pharmaceutical composition comprising a lipid nanoparticle (LNP) and a therapeutic nucleic acid (TNA), wherein the LNP comprises an scFv (e.g., the scFv is directed to an antigen present on the surface of a cell) linked to the LNP via a cleavable lipid that can be used to deliver a capsid-free non-viral DNA vector to a target site of interest (e.g., a cell, tissue, organ, etc.). As used herein, the term "cleavable lipid" refers to a cationic lipid that comprises a disulfide bond ("SS") cleavable unit. In one embodiment, the SS-cleavable lipid comprises a tertiary amine that responds to disulfide bonds that can be cleaved in acidic compartments (e.g., endosomes or lysosomes) and reducing environments (e.g., the cytoplasm) for membrane destabilization. The SS-cleavable lipid may comprise SS-cleavable and pH-activated lipid-like substances such as ss-OP lipids, ssPalm lipids, ss-M lipids, ss-E lipids, ss-EC lipids, ss-LC lipids, and ss-OC lipids.

[0334] According to some embodiments, the SS-cleavable lipids are described in International Patent Application Publication No. 2019188867, which is incorporated by reference in its entirety.

[0335] According to some embodiments, the LNPs described herein have a size range of about 20 to about 70 nm in average diameter, e.g., about 20 nm to about 70 nm, about 25 nm to about 70 nm, about 30 nm to about 70 nm, about 35 nm to about 70 nm, about 40 nm to about 70 nm, about 45 nm to about 80 nm, about 50 nm to about 70 nm, about 60 nm to about 70 nm, about 65 nm to about 70 nm, or about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm. According to some embodiments, the average diameter of the LNPs is about 50 nm to about 70 nm, which is significantly smaller and therefore advantageous for targeting and evading immune responses. Furthermore, the LNPs described herein can encapsulate greater than about 60% up to about 90% of double-stranded DNA, such as ceDNA. According to some embodiments, the LNPs described herein can encapsulate greater than about 60% double stranded DNA, such as ceDNA, greater than about 65% double stranded DNA, such as ceDNA, greater than about 70% double stranded DNA, such as ceDNA, greater than about 75% double stranded DNA, such as ceDNA, greater than about 80% double stranded DNA, such as ceDNA, greater than about 85% double stranded DNA, such as ceDNA, or greater than about 90% double stranded DNA, such as ceDNA.

[0336] The lipid particles described herein (e.g., LNPs comprising an scFv (e.g., an scFv directed to an antigen present on the surface of a cell) linked to the LNP) can be advantageously used to increase the delivery of nucleic acids (e.g., ceDNA, mRNA) to target cells / tissues compared to LNPs produced by other processes and compared to other lipids, e.g., ionizable cationic lipids. Thus, the lipid particles described herein (e.g., LNPs comprising an scFv (e.g., an scFv directed to an antigen present on the surface of a cell) linked to the LNP) provided maximum nucleic acid delivery compared to lipid particles prepared by processes and methods known in the art. Although the mechanism has not yet been determined and without being bound by theory, it is believed that lipid particles (e.g., LNPs comprising an scFv (e.g., an scFv directed to an antigen present on the surface of a cell) linked to the LNP) to hepatocytes avoid phagocytosis from the nucleus and transport to the nucleus more efficiently. Another advantage of the lipid particles described herein (e.g., LNPs comprising an scFv (e.g., the scFv is directed to an antigen present on the surface of a cell) linked to the LNP) is their better tolerability compared to other lipids, e.g., ionizable cationic lipids, e.g., MC3.

[0337] In one embodiment, the cleavable lipid may comprise three components: an amine head group, a linker group, and a hydrophobic tail. In one embodiment, the cleavable lipid comprises one or more phenyl ester bonds, one of many tertiary amino groups, and a disulfide bond. The tertiary amine group provides pH responsiveness and induces endosomal escape, the phenyl ester bond enhances the degradability (autolysis) of the structure, and the disulfide bond cleaves in a reducing environment.

[0338] In one embodiment, the cleavable lipid is a ss-OP lipid. In one embodiment, the ss-OP lipid comprises the structure shown in Formula A below:

[0339] [ka]

[0340] In one embodiment, the SS-cleavable lipid is SS-cleavable and pH-activated lipid mimic (ssPalm). ssPalm lipid is well known in the art. For example, see Togashi et al., Journal of Controlled Release, 279 (2018) 262-270, the entire contents of which are incorporated herein by reference. In one embodiment, the ssPalm is ssPalmM lipid comprising the structure of lipid B.

[0341] [ka]

[0342] In one embodiment, the ssPalmE lipid is a ssPalmE-P4-C2 lipid comprising the structure of lipid C.

[0343] [ka]

[0344] In one embodiment, the ssPalmE lipid is a ssPalmE-Paz4-C2 lipid comprising the structure of lipid D.

[0345] [ka]

[0346] In one embodiment, the cleavable lipid is a ss-M lipid. In one embodiment, the ss-M lipid comprises the structure shown in lipid E ​​below.

[0347] [ka]

[0348] In one embodiment, the cleavable lipid is a ss-E lipid. In one embodiment, the ss-E lipid comprises the structure shown in lipid F below.

[0349] [ka]

[0350] In one embodiment, the cleavable lipid is a ss-EC lipid. In one embodiment, the ss-EC lipid comprises the structure shown in lipid G below.

[0351] [ka]

[0352] In one embodiment, the cleavable lipid is a ss-LC lipid. In one embodiment, the ss-LC lipid comprises the structure shown in lipid H below.

[0353] [ka]

[0354] In one embodiment, the cleavable lipid is a ss-OC lipid. In one embodiment, the ss-OC lipid comprises the structure shown in lipid J below.

[0355] [ka]

[0356] In one embodiment, lipid particle (e.g., LNPs comprising an scFv (e.g., the scFv is directed to an antigen present on the surface of a cell) linked to the LNP) formulation is made and loaded with ceDNA obtained by the process disclosed in International Patent Application No. PCT / US2018 / 050042, filed September 7, 2018, which is incorporated by reference in its entirety. This can be achieved by high energy mixing of ethanol lipids with aqueous ceDNA at low pH, which protonates the lipids and provides favorable energetics for ceDNA / lipid association and nucleation of the particles. The particles can be further stabilized by aqueous dilution and removal of the organic solvent. The particles can be concentrated to a desired level. In one embodiment, the present disclosure provides ceDNA-lipid particles comprising lipids of formula I prepared by the process described in Example 2 of U.S. Provisional Application No. 63 / 194,620.

[0357] Generally, lipid particles (e.g., LNPs comprising an scFv (e.g., an scFv directed to an antigen present on the surface of a cell) linked to an LNP) are prepared with a total lipid to ceDNA (mass or weight) ratio of about 10:1 to 60:1. In some embodiments, the lipid to ceDNA ratio (mass / mass ratio, w / w ratio) can be within the range of about 1:1 to about 60:1, about 1:1 to about 55:1, about 1:1 to about 50:1, about 1:1 to about 45:1, about 1:1 to about 40:1, about 1:1 to about 35:1, about 1:1 to about 30:1, about 1:1 to about 25:1, about 10:1 to about 14:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, about 6:1 to about 9:1, about 30:1 to about 60:1. According to some embodiments, lipid particles (e.g., LNPs comprising an scFv (e.g., an scFv directed to an antigen present on the surface of a cell) linked to an LNP) are prepared with a ceDNA (mass or weight) to total lipid ratio of about 60:1. According to some embodiments, lipid particles (e.g., LNPs comprising an scFv (e.g., an scFv directed to an antigen present on the surface of a cell) linked to an LNP) are prepared with a ceDNA (mass or weight) to total lipid ratio of about 30:1. The amount of lipid and ceDNA can be adjusted to provide a desired N / P ratio, e.g., an N / P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or more. In general, the total lipid content of lipid particle formulations can range from about 5 mg / ml to about 30 mg / mL.

[0358] In some embodiments, the lipid nanoparticles include an agent for condensing and / or encapsulating nucleic acid cargo, such as ceDNA. Such agents are also referred to herein as condensing agents or encapsulating agents. Without limitation, any compound known in the art for condensing and / or encapsulating nucleic acid can be used as long as it is non-fusogenic. In other words, the agent can condense and / or encapsulate nucleic acid cargo, such as ceDNA, but has little or no fusogenic activity. Without wishing to be bound by theory, the condensing agent may have some fusogenic activity when it does not condense / encapsulate nucleic acid, such as ceDNA, but the nucleic acid encapsulated in the lipid nanoparticle formed with the condensing agent may be non-fusogenic.

[0359] According to some embodiments, LNPs comprising an scFv (e.g., an scFv directed to an antigen present on the surface of a cell) linked to a LNP described herein can encapsulate greater than about 60% of rigid double-stranded DNA such as ceDNA, greater than about 65% of rigid double-stranded DNA such as ceDNA, greater than about 70% of rigid double-stranded DNA such as ceDNA, greater than about 75% of rigid double-stranded DNA such as ceDNA, greater than about 80% of rigid double-stranded DNA such as ceDNA, greater than about 85% of rigid double-stranded DNA such as ceDNA, or greater than about 90% of rigid double-stranded DNA such as ceDNA.

[0360] Cationic lipids are typically used to condense nucleic acid cargo, such as ceDNA, at low pH and to drive membrane association and membrane fusogenicity.Generally, cationic lipids are lipids that are positively charged or contain at least one amino group that is protonated under acidic conditions, such as pH 6.5 or less.Cationic lipids can also be ionizable lipids, such as ionizable cationic lipids.By "non-fusogenic cationic lipid" is meant cationic lipids that can condense and / or encapsulate nucleic acid cargo, such as ceDNA, but have little or no fusogenic activity.

[0361] In one embodiment, the cationic lipid may comprise 20-90% (mol) of the total lipid present in the lipid particle (e.g., lipid nanoparticle). For example, the cationic lipid molar content may be 20-70% (mol), 30-60% (mol), 40-60% (mol), 40-55% (mol), or 45-55% (mol) of the total lipid present in the lipid particle (e.g., lipid nanoparticle). In some embodiments, the cationic lipid comprises about 50 mol% to about 90 mol% of the total lipid present in the lipid particle (e.g., an LNP that includes an scFv (e.g., an scFv directed to an antigen present on the surface of a cell) linked to the LNP).

[0362] In one embodiment, the SS-cleavable lipid is not MC3(6Z,9Z,28Z,3lZ)-heptatriaconta-6,9,28,3 l-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA or MC3). DLin-MC3-DMA is described in Jayaraman et al., Angew.Chem.Int.Ed Engl.(2012),51(34):8529-8533, the contents of which are incorporated herein by reference in their entirety. The structure of D-Lin-MC3-DMA (MC3) is shown below as lipid K.

[0363] [ka]

[0364] In one embodiment, the cleavable lipid is not lipid ATX-002. Lipid ATX-002 is described in WO2015 / 074085, the contents of which are incorporated herein by reference in their entirety. In one embodiment, the cleavable lipid is not (13Z.16Z)- / V, / V-dimethyl-3-nonyldocosa-13,16-dien-1-amine (compound 32). Compound 32 is described in WO2012 / 040184, the contents of which are incorporated herein by reference in their entirety. In one embodiment, the cleavable lipid is not compound 6 or compound 22. Compounds 6 and 22 are described in WO2015 / 199952, the contents of which are incorporated herein by reference in their entirety.

[0365] Non-limiting examples of cationic lipids include SS-cleavable and pH-activated lipid mimic-OP (ss-OP; Formula I), SS-cleavable and pH-activated lipid mimic-M (SS-M; Formula V), SS-cleavable and pH-activated lipid mimic-E (SS-E; Formula VI), SS-cleavable and pH-activated lipid mimic-EC (SS-EC; Formula VII), SS-cleavable and pH-activated lipid mimic-LC (SS-LC; Formula VIII), SS-cleavable and pH-activated lipid mimic-OC (SS-OC; Formula IX), polyethyleneimine, polyamidoamine (PAMAM) star dendrimers, lipofectin (a combination of DOTMA and DOPE), lipofectase, LIPOFECTAMINE™ (e.g., LIPOFECTAMINE™ 2000), DOPE, Cytofectin (Gilead Sciences, Foster City, Calif.), and Eufectin (JBL, San Luis Exemplary cationic liposomes include N-[1-(2,3-dioloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3-dioloxy)-propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP), 3b-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), 2,3-dioleyloxy-N[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide, and dimethyldioctadecylammonium bromide (DDAB). The nucleic acid (e.g., ceDNA or CELiD) may also be complexed, for example, with poly(L-lysine) or avidin, and a lipid may or may not be included in the mixture, e.g., steryl-poly(L-lysine).

[0366] In one embodiment, the cationic lipid is ss-OP of formula I. In another embodiment, the cationic lipid is SS-PAZ of formula II.

[0367] In one embodiment, the ceDNA vectors disclosed herein are delivered using cationic lipids as described in U.S. Pat. No. 8,158,601, or polyamine compounds or lipids as described in U.S. Pat. No. 8,034,376.

[0368] B. Noncationic lipids In one embodiment, the lipid particle (e.g., an LNP comprising an scFv (e.g., the scFv is directed to an antigen present on the surface of a cell) linked to the LNP) can further comprise a non-cationic lipid. The non-cationic lipid can serve to increase fusogenicity and increase the stability of the LNP during formation. Non-cationic lipids include amphipathic lipids, neutral lipids, and anionic lipids. Thus, the non-cationic lipid can be a neutral uncharged, zwitterionic, or anionic lipid. Non-cationic lipids are typically used to enhance membrane fusogenicity.

[0369] Exemplary non-cationic lipids include distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (e.g. 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (e.g. 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), dierucoyl phosphatidylcholine (DEPC), palmitoyl oleyl phosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE); 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE);The phospholipids include, but are not limited to, lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof.It should be understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used.The acyl group in these lipids is preferably C; 10 ~C 24 The acyl group is derived from a fatty acid having a carbon chain, for example, lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0370] Other examples of non-cationic lipids suitable for use in lipid particles (e.g., LNPs comprising an scFv (e.g., the scFv is directed to an antigen present on the surface of a cell) linked to the LNP) include non-phospholipids such as, for example, stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramide, sphingomyelin, and the like.

[0371] In one embodiment, the non-cationic lipid is a phospholipid. In one embodiment, the non-cationic lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments, the non-cationic lipid is DSPC. In other embodiments, the non-cationic lipid is DOPC. In other embodiments, the non-cationic lipid is DOPE.

[0372] In some embodiments, the non-cationic lipid may comprise 0-20% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, the non-cationic lipid content is 0.5-15% (mol) of the total lipid present in the lipid particle (e.g., lipid nanoparticle). In some embodiments, the non-cationic lipid content is 5-12% (mol) of the total lipid present in the lipid particle (e.g., lipid nanoparticle). In some embodiments, the non-cationic lipid content is 5-10% (mol) of the total lipid present in the lipid particle (e.g., lipid nanoparticle). In one embodiment, the non-cationic lipid content is about 6% (mol) of the total lipid present in the lipid particle (e.g., lipid nanoparticle). In one embodiment, the non-cationic lipid content is about 7.0% (mol) of the total lipid present in the lipid particle (e.g., lipid nanoparticle). In one embodiment, the non-cationic lipid content is about 7.5% (mol) of the total lipid present in the lipid particle (e.g., lipid nanoparticle). In one embodiment, the non-cationic lipid content is about 8.0% (mol) of the total lipid present in the lipid particle (e.g., lipid nanoparticle). In one embodiment, the non-cationic lipid content is about 9.0% (mol) of the total lipid present in the lipid particle (e.g., lipid nanoparticle). In some embodiments, the non-cationic lipid content is about 10% (mol) of the total lipid present in the lipid particle (e.g., lipid nanoparticle). In one embodiment, the non-cationic lipid content is about 11% (mol) of the total lipid present in the lipid particle (e.g., lipid nanoparticle).

[0373] Exemplary non-cationic lipids are described in International Patent Application Publication No. 2017 / 099823 and U.S. Patent Application Publication No. 2018 / 0028664, the contents of both of which are incorporated by reference in their entireties.

[0374] In one embodiment, the lipid particles (e.g., lipid nanoparticles) may further comprise components such as sterols to provide membrane integrity and stability of the lipid particles. In one embodiment, an exemplary sterol that can be used in the lipid particles is cholesterol or a derivative thereof. Non-limiting examples of cholesterol derivatives include polar analogs such as 5α-cholestanol, 5β-coprostanol, cholesteryl-(2′-hydroxy)-ethyl ether, cholesteryl-(4′-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogs such as 5α-cholestane, cholestenone, 5α-cholestanone, 5β-cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analog such as cholesteryl-(4′-hydroxy)-butyl ether. In some embodiments, the cholesterol derivative is cholestyryl hemisuccinate (CHEMS).

[0375] Exemplary cholesterol derivatives are described in International Patent Application Publication No. WO 2009 / 127060 and US Patent Application Publication No. 2010 / 0130588, the contents of both of which are incorporated herein by reference in their entireties.

[0376] In one embodiment, components that provide membrane integrity, such as sterols, may comprise 0-50% (mol) of the total lipids present in the lipid particle (e.g., lipid nanoparticle). In some embodiments, such components are 20-50% (mol) of the total lipid content of the lipid particle (e.g., lipid nanoparticle). In some embodiments, such components are 30-40% (mol) of the total lipid content of the lipid particle (e.g., lipid nanoparticle). In some embodiments, such components are 35-45% (mol) of the total lipid content of the lipid particle (e.g., lipid nanoparticle). In some embodiments, such components are 38-42% (mol) of the total lipid content of the lipid particle (e.g., lipid nanoparticle).

[0377] In one embodiment, the lipid particles (e.g., lipid nanoparticles) may further comprise polyethylene glycol (PEG) or conjugated lipid molecules. These are generally used to inhibit aggregation and / or provide steric stabilization of the lipid particles (e.g., lipid nanoparticles). Exemplary conjugated lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic-polymer lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the conjugated lipid molecule is a PEGylated lipid, e.g., a (methoxypolyethylene glycol)-conjugated lipid. In some other embodiments, the PEGylated lipid is a PEGylated lipid, e.g., a (methoxypolyethylene glycol)-conjugated lipid. 2000 -DMG (dimyristoylglycerol).

[0378] Exemplary PEGylated lipids include PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG diacylglycerol succinate (PEGS-DAG) (such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-1-0-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbamate, N-(carbonyl-methoxypolyethyleneglycol), N-(carboxymethyl)-1,1-dimethylethyl)-2,3-dimethylethyl-1,1-dimethylethyl-2,2 ... Examples of PEG-lipid conjugates include, but are not limited to, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or mixtures thereof. Additional exemplary PEG-lipid conjugates are described, for example, in U.S. Patent Application Publication Nos. 5,885,613, 6,287,591, 2003 / 0077829, 2003 / 0077829, 2005 / 0175682, 2008 / 0020058, 2011 / 0117125, 2010 / 0130588, 2016 / 0376224, and 2017 / 0119904, the contents of all of which are incorporated herein by reference in their entirety.

[0379] In one embodiment, the PEG-DAA PEGylated lipid can be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can be, for example, PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol (1-[8'-(cholest-5-en-3[beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethyleneglycol). In one embodiment, the PEG lipid can be one or more of PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], PEG-DMB (3,4-ditetradecaoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000].

[0380] [ka] may be selected from the group consisting of:

[0381] In some embodiments, the PEGylated lipid is N-(carbonyl-methoxypolyethylene glycol n)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG n , where n is 350, 500, 750, 1000 or 2000), N-(carbonyl-methoxypolyethylene glycol n )-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG n, where n is 350, 500, 750, 1000 or 2000), DSPE-polyglycerin-cyclohexyl-carboxylic acid, DSPE-polyglycerin-2-methylglutar-carboxylic acid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugated polyethylene glycol (DSPE-PEG-OH), polyethylene glycol-dimyristol glycerol (PEG-DMG), polyethylene glycol-distearoyl glycerol (PEG-DSG), or N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)200011 (C8 PEG2000 ceramide). DMPE-PEG, where n is 350, 500, 750, 1000 or 2000. n In some examples, the PEG-lipid is N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG 2,000). n In some examples, the PEG-lipid is N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG 2,000). In some embodiments, the PEG-lipid is DSPE-PEG-OH. In some preferred embodiments, the PEG-lipid is PEG-DMG.

[0382] In some embodiments, the conjugated lipid, e.g., the PEGylated lipid, comprises a tissue-specific targeting ligand, e.g., a first or second targeting ligand, e.g., PEG-DMG conjugated to a GalNAc ligand.

[0383] In one embodiment, lipids conjugated with molecules other than PEG can be used instead of PEG-lipids. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic-polymer lipid (CPL) conjugates can be used instead of or in addition to PEG-lipids. Exemplary conjugated lipids, i.e., PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates, and cationic polymer-lipids, are described in International Patent Application Publication Nos. 1996 / 010392, 1998 / 051278, 2002 / 087541, 2005 / 026372, 2008 / 147372, 2009 / 023113, 2010 / 022162, 2011 / 022162, 2012 / 022162, 2013 / 022162, 2014 / 022162, 2015 / 022162, 2016 / 022162, 2017 / 022162, 2018 / 022162, 2019 / 022162, 2013 ... 438, 2009 / 086558, 2012 / 000104, 2017 / 117528, 2017 / 099823, 2015 / 199952, 2017 / 004143, 2015 / 095346, 2012 / 000104, 2012 / 000104, and 2010 / 0062 82, U.S. Patent Application Publication Nos. 2003 / 0077829, 2005 / 0175682, 2008 / 0020058, 2011 / 0117125, 2013 / 0303587, 2018 / 0028664, 2015 / 0376115, 2016 / 0376224, and 2016 / 0317458 Nos. 2013 / 0303587, 2013 / 0303587, and 2011 / 0123453, as well as U.S. Pat. Nos. 5,885,613, 6,287,591, 6,320,017, and 6,586,559, the contents of all of which are incorporated herein by reference in their entireties.

[0384] In some embodiments, the PEGylated lipid may comprise 0-20% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, the PEGylated lipid content is 0.5-10% (mol). In some embodiments, the PEGylated lipid content is 1-5% (mol). In some embodiments, the PEGylated lipid content is 2-4% (mol). In some embodiments, the PEGylated lipid content is 2-3% (mol). In one embodiment, the PEGylated lipid content is about 2% (mol). In one embodiment, the PEGylated lipid content is about 2.5% (mol). In some embodiments, the PEGylated lipid content is about 3% (mol). In one embodiment, the PEGylated lipid content is about 3.5% (mol). In one embodiment, the PEGylated lipid content is about 4% (mol).

[0385] It is understood that the molar ratio of cationic lipid, e.g., ionizable cationic lipid, to non-cationic lipid, sterol, and PEGylated lipid can be varied as needed. For example, lipid particles (e.g., lipid nanoparticles) may contain 30-70% cationic lipid by molar or total weight of the composition, 0-60% cholesterol by molar or total weight of the composition, 0-30% non-cationic lipid by molar or total weight of the composition, and 2-5% PEGylated lipid by molar or total weight of the composition. In one embodiment, the composition contains 40-60% cationic lipid by molar or total weight of the composition, 30-50% cholesterol by molar or total weight of the composition, 5-15% non-cationic lipid by molar or total weight of the composition, and 2-5% PEG or conjugated lipid by molar or total weight of the composition. In one embodiment, the composition is 40-60% cationic lipid by molar or total weight of the composition, 30-40% cholesterol by molar or total weight of the composition, 5-10% non-cationic lipid by molar or total weight of the composition, and 2-5% PEGylated lipid by molar or total weight of the composition. The composition may contain 60-70% cationic lipid by molar or total weight of the composition, 25-35% cholesterol by molar or total weight of the composition, 5-10% non-cationic lipid by molar or total weight of the composition, and 2-5% PEGylated lipid by molar or total weight of the composition. The composition may also contain up to 45-55% cationic lipid by molar or total weight of the composition, 35-45% cholesterol by molar or total weight of the composition, 2-15% non-cationic lipid by molar or total weight of the composition, and 2-5% PEGylated lipid by molar or total weight of the composition.The formulation may also be a lipid nanoparticle formulation, for example, a composition comprising 8-30% cationic lipid by molar or total weight of the composition, 5-15% non-cationic lipid by molar or total weight of the composition, and 0-40% cholesterol by molar or total weight of the composition; 4-25% cationic lipid by molar or total weight of the composition, 4-25% non-cationic lipid by molar or total weight of the composition, 2-25% cholesterol by molar or total weight of the composition, 10-35% conjugated lipid by molar or total weight of the composition, and 5% cholesterol by molar or total weight of the composition. or 2-30% cationic lipid by molar or total weight of the composition, 2-30% non-cationic lipid by molar or total weight of the composition, 1-15% cholesterol by molar or total weight of the composition, 2-35% PEGylated lipid by molar or total weight of the composition, and 1-20% cholesterol by molar or total weight of the composition; or even up to 90% cationic lipid by molar or total weight of the composition, and 2-10% non-cationic lipid by molar or total weight of the composition, or even 100% cationic lipid by molar or total weight of the composition. In some embodiments, the lipid particle formulation comprises cationic lipid, non-cationic phospholipid, cholesterol, and PEGylated lipid (conjugated lipid) in a molar ratio of about 50:9:38.5:2.5.

[0386] In one embodiment, the lipid particle (e.g., lipid nanoparticle) formulation comprises a cationic lipid, a non-cationic phospholipid, cholesterol, and a PEGylated lipid (conjugated lipid) in a molar ratio of about 50:7:40:3.

[0387] In another aspect, the present disclosure provides a lipid nanoparticle formulation comprising a phospholipid, a lecithin, a phosphatidylcholine, and a phosphatidylethanolamine.

[0388] In one embodiment, the lipid particle (e.g., lipid nanoparticle) comprises a cationic lipid, a non-cationic lipid (e.g., a phospholipid), a sterol (e.g., cholesterol), and a PEGylated lipid (conjugated lipid), with the molar ratio of lipids ranging from 20-70 mole percent for the cationic lipid (target 30-60), the molar percent of the non-cationic lipid ranging from 0-30 (target 0-15), the molar percent of the sterol ranging from 20-70 (target 30-50), and the molar percent of the PEGylated lipid (conjugated lipid) ranging from 1-6 (target 2-5).

[0389] Lipid nanoparticles (LNPs) containing ceDNA are disclosed in International Patent Application No. US2018 / 050042, filed September 7, 2018, which is incorporated herein in its entirety and are contemplated for use in the methods and compositions disclosed herein.

[0390] The size of lipid particles (e.g., lipid nanoparticles) can be determined by quasi-elastic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK). According to some embodiments, the mean diameter of the LNPs as determined by light scattering is less than about 75 nm or less than about 70 nm. According to some embodiments, the mean diameter of the LNPs as determined by light scattering is between about 50 nm and about 75 nm or between about 50 nm and about 70 nm.

[0391] The pKa of the formulated cationic lipids can be correlated with the efficacy of the LNPs for delivery of nucleic acids (see Jayaraman et al., Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al., Nature Biotechnology 28, 172-176 (2010), both of which are incorporated by reference in their entirety). In one embodiment, the pKa of each cationic lipid is determined in the lipid nanoparticles using a fluorescence-based assay of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). Lipid nanoparticles consisting of cationic lipid / DSPC / cholesterol / PEG-lipid (50 / 10 / 38.5 / 1.5 mol%) in PBS at a concentration of 0.4 mM total lipid can be prepared using the in-line process described herein and elsewhere. TNS can be prepared as a 100 mM stock solution in distilled water. Vesicles can be diluted to 24 mM lipid in 2 mL of buffer solution containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, 130 mM NaCl, with a pH ranging from 2.5 to 11. An aliquot of TNS solution can be added to a final concentration of 1 mM, followed by vortex mixing. Emission intensity is measured at room temperature in an SLM Aminco Series 2 emission spectrophotometer using an excitation wavelength of 321 nm and an emission wavelength of 445 nm. A sigmoidal best-fit analysis can be applied to the fluorescence data, and the pKa is determined as the pH that produces half-optimal fluorescence intensity.

[0392] In one embodiment, relative activity can be determined by measuring luciferase expression in the liver 4 hours after administration via tail vein injection. Activity is compared at doses of 0.3 and 1.0 mg ceDNA / kg and expressed as ng luciferase / g liver measured 4 hours after administration.

[0393] Without being limited thereto, the lipid particles (e.g., lipid nanoparticles) of the present disclosure include lipid formulations that can be used to deliver capsid-free, non-viral DNA vectors to a desired target site (e.g., cells, tissues, organs, etc.). In general, the lipid particles (e.g., lipid nanoparticles) comprise a capsid-free, non-viral DNA vector and a cationic lipid or a salt thereof.

[0394] In one embodiment, the lipid particle (e.g., lipid nanoparticle) comprises a molar ratio of cationic lipid / non-cationic lipid / sterol / conjugated lipid of 50:10:38.5:1.5. In one embodiment, the present disclosure provides a lipid particle (e.g., lipid nanoparticle) formulation comprising phospholipid, lecithin, phosphatidylcholine and phosphatidylethanolamine.

[0395] III. Closed-end DNA (ceDNA) Vectors The embodiments of the present disclosure are based on methods and compositions comprising closed-end linear double-stranded (ceDNA) vectors capable of expressing transgenes (e.g., therapeutic nucleic acids (TNAs)). ceDNA vectors as described herein do not have the packaging constraints imposed by the limited space within the viral capsid. ceDNA vectors represent a versatile eukaryotically produced alternative to prokaryotically produced plasmid DNA vectors as opposed to encapsulated AAV genomes. This allows for the insertion of control elements, such as regulatory switches as disclosed herein, large transgenes, multiple transgenes, etc.

[0396] The ceDNA vector preferably has a linear, continuous structure rather than a discontinuous structure. A linear, continuous structure is believed to be more stable against attack by cellular endonucleases and at the same time less likely to recombine and cause mutagenesis. Thus, a linear, continuous structure ceDNA vector is a preferred embodiment. A continuous, linear, single-stranded intramolecular duplex ceDNA vector may be covalently linked at the termini without sequences encoding AAV capsid proteins. These ceDNA vectors are structurally different from plasmids (including the ceDNA plasmids described herein), which are circular, double-stranded nucleic acid molecules of bacterial origin. Complementary strands of a plasmid can be separated following denaturation to produce two nucleic acid molecules, whereas, conversely, a ceDNA vector has complementary strands but is a single DNA molecule and therefore likely to remain a single molecule even when denatured. In some embodiments, ceDNA vectors, unlike plasmids, may be produced without DNA base methylation of prokaryotic cell types. Thus, ceDNA vectors and ceDNA-plasmids differ both in terms of structure (in particular linear vs. circular) and in terms of the methods used to produce and purify these different objects, and also in terms of their DNA methylation, which in the case of ceDNA-plasmids is of prokaryotic cell type and in the case of ceDNA vectors is of eukaryotic cell type.

[0397] Provided herein are non-viral capsid-free ceDNA molecules (ceDNA) with covalently closed ends. These non-viral capsid-free ceDNA molecules can be produced in permissive host cells from expression constructs (e.g., ceDNA-plasmids, ceDNA-bacmids, ceDNA-baculoviruses, or integrative cell lines) that contain a heterologous gene (e.g., a transgene, particularly a therapeutic transgene) located between two different inverted terminal repeat (ITR) sequences, the ITRs being different with respect to each other. In some embodiments, one of the ITRs is modified by deletion, insertion, and / or substitution compared to the wild-type ITR sequence (e.g., AAV ITR), and at least one of the ITRs includes a functional terminal resolution site (trs) and a Rep binding site. The ceDNA vector is preferably double-stranded, e.g., self-complementary, over at least a portion of the molecule, such as an expression cassette (e.g., the ceDNA is not a double-stranded circular molecule). The ceDNA vectors have covalently closed ends and are therefore resistant to exonuclease digestion (e.g., exonuclease I or exonuclease III) at 37°C for, for example, one hour or more.

[0398] In one aspect, the ceDNA vector comprises, in the 5' to 3' direction, a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette as described herein), and a second AAV ITR. In one embodiment, the first ITR (5'ITR) and the second ITR (3'ITR) are asymmetric with respect to each other. That is, they have different three-dimensional spatial configurations from each other. As an exemplary embodiment, the first ITR can be a wild-type ITR and the second ITR can be a mutant or modified ITR, or vice versa, the first ITR can be a mutant or modified ITR and the second ITR can be a wild-type ITR. In one embodiment, the first ITR and the second ITR are both modified, but are of different sequences, or have different modifications, or are not the same modified ITR, and have different three-dimensional spatial configurations. In other words, a ceDNA vector with asymmetric ITRs may have ITRs in which any changes in one ITR relative to the WT-ITR are not reflected in the other ITR, or alternatively, the asymmetric ITRs may have a different sequence and a different three-dimensional shape relative to each other if they have a modified asymmetric ITR pair.

[0399] In one embodiment, the ceDNA vector comprises, in the 5' to 3' direction, a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette as described herein), and a second AAV ITR, where the first ITR (5'ITR) and the second ITR (3'ITR) are symmetrical or substantially symmetrical with respect to each other, i.e., the ceDNA vector may comprise ITR sequences that have a symmetrical three-dimensional spatial organization, whereby their structures are the same shape in geometric space or have the same A, C-C', B-B' loops in three-dimensional space. In such an embodiment, the symmetrical ITR pair or the substantially symmetrical ITR pair may be a modified ITR (e.g., mod-ITR) that is not a wild-type ITR. The mod-ITR pair may have the same sequence that has one or more modifications from the wild-type ITR and is the reverse complement (inversion) of each other. In one embodiment, the modified ITR pair is substantially symmetrical as defined herein, i.e., the modified ITR pair may have different sequences but may have corresponding or the same symmetrical three-dimensional shapes. In some embodiments, the symmetrical ITR or substantially symmetrical ITR may be wild-type (WT-ITR) as described herein. That is, both ITRs have wild-type sequences, but are not necessarily WT-ITRs of the same AAV serotype. In one embodiment, one WT-ITR may be derived from one AAV serotype and the other WT-ITR may be derived from a different AAV serotype. In such an embodiment, the WT-ITR pair is substantially symmetrical as defined herein, i.e., they may have one or more conservative nucleotide modifications while maintaining a symmetrical three-dimensional spatial configuration.

[0400] The wild-type or mutant or otherwise modified ITR sequences provided herein represent DNA sequences contained in an expression construct (e.g., ceDNA-plasmid, ceDNA-bacmid, ceDNA-baculovirus) for the production of a ceDNA vector. Thus, the ITR sequences actually contained in a ceDNA vector produced from a ceDNA-plasmid or other expression construct may or may not be identical to the ITR sequences provided herein as a result of naturally occurring variations (e.g., replication errors) that occur during the production process.

[0401] In one embodiment, the ceDNA vector described herein, which comprises an expression cassette having a transgene that is a therapeutic nucleic acid sequence, can be operably linked to one or more regulatory sequences that allow or control the expression of the transgene. In one embodiment, the polynucleotide comprises a first ITR sequence and a second ITR sequence, and the nucleotide sequence of interest is flanked by the first and second ITR sequences, and the first and second ITR sequences are asymmetric with respect to each other or symmetric with respect to each other.

[0402] In one embodiment, the expression cassette is located between two ITRs and includes, in that order, a promoter operably linked to a transgene, a post-transcriptional regulatory element, and one or more of a polyadenylation and termination signal. In one embodiment, the promoter is regulatable-inducible or repressible. The promoter can be any sequence that promotes transcription of the transgene. In one embodiment, the promoter is a CAG promoter or a variant thereof. The post-transcriptional regulatory element is a sequence that regulates the expression of the transgene, and as a non-limiting example, any sequence that creates a tertiary structure that enhances the expression of the transgene, which is a therapeutic nucleic acid sequence.

[0403] In one embodiment, the post-transcriptional regulatory element comprises a WPRE. In one embodiment, the polyadenylation and termination signal comprises a BGH polyA. Any cis-regulatory element known in the art, or combinations thereof, may additionally be used, such as, but not limited to, the SV40 late polyA signal upstream enhancer sequence (USE) or other post-transcriptional processing elements, including, but not limited to, the thymidine kinase gene of herpes simplex virus or hepatitis B virus (HBV). In one embodiment, the expression cassette length in the 5' to 3' direction exceeds the maximum length known to be encapsidated in AAV virions. In one embodiment, the length is greater than 4.6 kb, or greater than 5 kb, or greater than 6 kb, or greater than 7 kb. Various expression cassettes are exemplified herein.

[0404] In one embodiment, the expression cassette may comprise more than 4000 nucleotides, 5000 nucleotides, 10,000 nucleotides, or 20,000 nucleotides, or 30,000 nucleotides, or 40,000 nucleotides, or 50,000 nucleotides, or any range of about 4000-10,000 nucleotides, or 10,000-50,000 nucleotides, or more than 50,000 nucleotides. In some embodiments, the expression cassette may comprise a transgene that is a therapeutic nucleic acid sequence in the range of 500-50,000 nucleotides in length. In one embodiment, the expression cassette may comprise a transgene that is a therapeutic nucleic acid sequence in the range of 500-75,000 nucleotides in length. In one embodiment, the expression cassette may comprise a transgene that is a therapeutic nucleic acid sequence in the range of 500-10,000 nucleotides in length. In one embodiment, the expression cassette may comprise a transgene that is a therapeutic nucleic acid sequence in the range of 1000-10,000 nucleotides in length. In one embodiment, the expression cassette may contain a transgene that is a therapeutic nucleic acid sequence ranging from 500 to 5,000 nucleotides in length. The ceDNA vector is capable of delivering large sized expression cassettes to the host since it does not have the size limitations of the encapsidated AAV vector. In one embodiment, the ceDNA vector lacks prokaryotic cell specific methylation.

[0405] In one embodiment, the rigid therapeutic nucleic acid may be a plasmid.

[0406] In one embodiment, the ceDNA vectors disclosed herein are used for therapeutic purposes (e.g., medical, diagnostic, or veterinary uses) or immunogenic polypeptides.

[0407] The expression cassette can include any transgene that is a therapeutic nucleic acid sequence. In certain embodiments, the ceDNA vector includes any gene of interest in a subject, including one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNAs, RNAis, antisense oligonucleotides, antisense polynucleotides, antibodies, antigen-binding fragments, or any combination thereof.

[0408] In one embodiment, the ceDNA expression cassette may include an expressible exogenous sequence (e.g., an open reading frame) that encodes, for example, a protein that is absent, inactive, or insufficiently active in the recipient subject, or a gene that encodes a protein with a desired biological or therapeutic effect. In one embodiment, the exogenous sequence, such as a donor sequence, may encode a gene product that can function to correct the expression of a defective gene or transcript. In one embodiment, the expression cassette may also encode a corrective DNA strand, encode a polypeptide, a sense or antisense oligonucleotide, or an RNA (coding or non-coding; e.g., siRNA, shRNA, microRNA, and their antisense counterparts (e.g., antagoMiR)). In one embodiment, the expression cassette may include an exogenous sequence that encodes a reporter protein used for experimental or diagnostic purposes, such as b-lactamase, b-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others known in the art.

[0409] Thus, the expression cassette may include any gene that encodes a protein, polypeptide, or RNA that is reduced or absent due to a mutation, or that provides a therapeutic effect when overexpression is considered within the scope of this disclosure. The ceDNA vector may include a template or donor nucleotide sequence that is used as a corrective DNA strand to be inserted after a double-stranded break (or nick) provided by a nuclease. The ceDNA vector may include a template nucleotide sequence that is used as a corrective DNA strand to be inserted after a double-stranded break (or nick) provided by an inducible RNA nuclease, meganuclease, or zinc finger nuclease.

[0410] IV. Therapeutic Nucleic Acids Aspects of the disclosure generally provide compositions (e.g., pharmaceutical compositions) comprising a lipid nanoparticle (LNP) and a therapeutic nucleic acid (TNA), where the LNP comprises an scFv linked to the LNP. According to embodiments, the disclosure provides pharmaceutical compositions comprising a lipid nanoparticle (LNP) and a therapeutic nucleic acid (TNA), where the LNP comprises an scFv linked to the LNP, where the scFv is directed to an antigen present on the surface of a cell, and where the scFv is linked to the LNP by maleimide conjugation.

[0411] Exemplary therapeutic nucleic acids of the present disclosure can include, but are not limited to, minigenes, plasmids, minicircles, small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASO), ribozymes, closed-ended double-stranded DNA (e.g., ceDNA, CELiD, linear covalently closed DNA ("ministring"), doggybone™, protelomeric closed-ended DNA, or dumbbell linear DNA), dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, and DNA viral vectors, viral RNA vectors, and any combination thereof.

[0412] siRNA or miRNA, which can downregulate the intracellular level of a particular protein through a process called RNA interference (RNAi), are also contemplated by the present disclosure as nucleic acid therapeutics. After siRNA or miRNA is introduced into the cytoplasm of a host cell, these double-stranded RNA constructs can bind to a protein called RISC. The sense strand of siRNA or miRNA is removed by the RISC complex. When the RISC complex binds to complementary mRNA, it cleaves the mRNA and releases the cleaved strand. RNAi is by inducing specific destruction of mRNA, which leads to the downregulation of the corresponding protein.

[0413] Antisense oligonucleotides (ASOs) and ribozymes that inhibit mRNA translation into protein can be nucleic acid therapeutics. In the case of antisense constructs, these single-stranded deoxynucleic acids have sequences complementary to the sequences of target protein mRNA, and can bind to mRNA by Watson and Crick base pairing. This binding prevents the translation of target mRNA and / or induces RNaseH degradation of the mRNA transcript. As a result, antisense oligonucleotides have enhanced specificity of action (i.e., downregulation of specific disease-related proteins).

[0414] In any of the aspects and embodiments provided herein, the therapeutic nucleic acid can be a therapeutic RNA. The therapeutic RNA can be an inhibitor of mRNA translation, an RNA interference (RNAi) agent, a catalytically active RNA molecule (ribozyme), a transfer RNA (tRNA), or an mRNA transcript (ASO), an RNA that binds to a protein or other molecular ligand (aptamer). In any of the methods provided herein, the RNAi agent can be a double-stranded RNA, a single-stranded RNA, a microRNA, a short interfering RNA, a small hairpin RNA, or a triple-helix forming oligonucleotide.

[0415] Modified Therapeutic Nucleic Acids Embodiments of the present disclosure further provide a composition (e.g., a pharmaceutical composition) comprising a lipid particle (e.g., a lipid nanoparticle (LNP) and a therapeutic nucleic acid (TNA), where the LNP comprises an scFv (e.g., the scFv is directed to an antigen present on the surface of a cell) linked to the LNP) and a modified therapeutic nucleic acid (TNA), where the TNA is as defined above.

[0416] In one embodiment, the denatured TNA is closed-ended DNA (ceDNA). The term "denatured therapeutic nucleic acid" refers to a partial or complete TNA whose conformation has been altered from the standard B-form conformation. The conformational change may include a change in secondary structure (i.e., base pair interactions within a single nucleic acid molecule) and / or a change in tertiary structure (i.e., double helix structure). Without being bound by theory, it was believed that TNA treated with alcohol / water solutions or pure alcohol solvents results in denaturation of the nucleic acid into a conformation that enhances encapsulation efficiency by LNPs and produces LNP formulations with smaller diameter sizes (i.e., less than 75 nm, e.g., average size of about 68-74 nm in diameter). All LNP average diameter sizes and size ranges described herein apply to LNPs containing denatured TNA.

[0417] When DNA is in an aqueous environment, it has a B-form structure with 10.4 base pairs in each complete helical turn. Gradually changing this aqueous environment by adding a moderately less polar alcohol such as methanol untwists the helix, causing the DNA to smoothly change to a form with only 10.2 base pairs per helical turn, as visualized by circular dichroism (CD) spectroscopy. In one embodiment, the denatured TNA in the pharmaceutical composition provided herein has a 10.2-form structure.

[0418] In contrast to this behavior, when water is replaced with a slightly less polar alcohol such as ethanol, the same type of conformational change occurs only until about 65% of the water is replaced with ethanol. At this point, the DNA suddenly changes to A-form structure, which has a tighter twisted helix containing 11 base pairs per helix turn, as visualized by CD. In one embodiment, the denatured TNA in the pharmaceutical composition provided herein has A-form structure.

[0419] According to some embodiments, the denatured TNA in the pharmaceutical composition provided herein has a rod-like structure when visualized under a transmission electron microscope (TEM). According to some embodiments, the denatured TNA in the pharmaceutical composition provided herein has a ring-like structure when visualized under a transmission electron microscope (TEM). In comparison, undenatured TNA has a chain-like structure.

[0420] Production of V.ceDNA vector The embodiments of the present disclosure are based on compositions comprising lipid nanoparticles (LNPs) and therapeutic nucleic acids (TNAs). ceDNA vectors as described herein do not have the packaging constraints imposed by the limited space within the viral capsid. ceDNA vectors represent a versatile eukaryotically produced alternative to prokaryotically produced plasmid DNA vectors as opposed to the encapsulated AAV genome. This allows the insertion of control elements, such as regulatory switches as disclosed herein, large transgenes, multiple transgenes, etc.

[0421] Methods for the production of ceDNA vectors described herein comprising asymmetric or symmetric ITR pairs as defined herein are described in Section IV of International Application PCT / US18 / 49996, filed September 7, 2018, which is incorporated herein by reference in its entirety. As described herein, ceDNA vectors can be obtained, for example, by a process comprising: a) incubating a population of host cells (e.g., insect cells) harboring a polynucleotide expression construct template (e.g., ceDNA-plasmid, ceDNA-bacmid, and / or ceDNA-baculovirus), where the host cells lack viral capsid coding sequences under conditions effective to induce production of ceDNA vectors in the host cells in the presence of Rep proteins, and for a time sufficient therefor, and b) harvesting and isolating the ceDNA vectors from the host cells. The presence of Rep proteins induces replication of vector polynucleotides with modified ITRs to produce ceDNA vectors in the host cells.

[0422] The following are provided as non-limiting examples.

[0423] According to some embodiments, synthetic ceDNA is produced via excision from a double-stranded DNA molecule. Synthetic production of ceDNA vectors is described in Examples 2-6 of International Patent Application No. US19 / 14122, filed January 18, 2019, which is incorporated herein by reference in its entirety. One exemplary method of producing ceDNA vectors using a synthetic method involving excision of a double-stranded DNA molecule. Briefly, ceDNA vectors can be generated using a double-stranded DNA construct. See, for example, Figures 7A-8E of International Patent Application No. PCT / US19 / 14122. In some embodiments, the double-stranded DNA construct is a ceDNA plasmid, see, for example, Figure 6 of International Patent Application No. US2018 / 064242, filed December 6, 2018.

[0424] In some embodiments, the constructs for generating ceDNA vectors contain additional components for regulating expression of the transgene, such as a regulatory switch for regulating expression of the transgene, or a kill switch that can kill a cell containing the vector.

[0425] A molecular regulatory switch is one that responds to a signal and produces a measurable change in state. Such regulatory switches can be usefully combined with the ceDNA vectors described herein to control the output of transgene expression. In some embodiments, the ceDNA vector includes a regulatory switch that serves to fine-tune the expression of the transgene. For example, it can serve as a biological containment function for the ceDNA vector. In some embodiments, the switch is an "on / off" switch designed to start or stop (i.e., shut down) the controllable and regulatable expression of a gene of interest in the ceDNA vector. In some embodiments, the switch can include a "kill switch" that can instruct a cell containing the synthetic ceDNA vector to undergo programmed cell death once the switch is activated. Exemplary regulatory switches encompassed for use in ceDNA vectors can be used to regulate expression of a transgene and are discussed more fully in International Application No. US18 / 49996, which is incorporated by reference in its entirety and described herein.

[0426] Another exemplary method for producing a ceDNA vector using synthetic methods involving the assembly of various oligonucleotides is provided in Example 3 of International Application No. PCT / US19 / 14122, where the ceDNA vector is produced by synthesizing a 5' oligonucleotide and a 3' ITR oligonucleotide and ligating the ITR oligonucleotide to a double-stranded polynucleotide comprising an expression cassette. Figure 11B of PCT / US19 / 14122, which is incorporated herein by reference in its entirety, shows an exemplary method for ligating a 5' ITR oligonucleotide and a 3' ITR oligonucleotide to a double-stranded polynucleotide comprising an expression cassette.

[0427] An exemplary method for producing a ceDNA vector using synthetic methods is provided in Example 4 of PCT / US19 / 14122, which is incorporated herein by reference in its entirety, and uses a single-stranded linear DNA that includes two sense ITRs flanking the sense expression cassette sequence and covalently linked to two antisense ITRs flanking the antisense expression cassette, and then ligating the ends of this single-stranded linear DNA to form a closed-ended single-stranded molecule. A non-limiting example is to synthesize and / or produce a single-stranded DNA molecule, anneal portions of the molecule to form a single linear DNA molecule with one or more base-paired regions of secondary structure, and then ligate the free 5' and 3' ends together to form a closed single-stranded molecule.

[0428] In yet another aspect, the present disclosure provides host cell lines that stably integrate the DNA vector polynucleotide expression template (ceDNA template) described herein into their own genome for use in producing non-viral DNA vectors. Methods for producing such cell lines are described in Lee, L. et al. (2013) Plos One 8(8):e69879, incorporated herein by reference in its entirety. For example, Rep protein is added to the host cells at an MOI of 3. In one embodiment, the host cell line is an invertebrate cell line, preferably insect Sf9 cells. When the host cell line is a mammalian cell line, preferably 293 cells, the cell line may have a stably integrated polynucleotide vector template, and a second vector, such as a herpes virus, may be used to introduce Rep protein into the cells, allowing excision and amplification of ceDNA in the presence of Rep.

[0429] Any promoter can be operably linked to the heterologous nucleic acid (e.g., reporter nucleic acid or therapeutic transgene) of the vector polynucleotide. The expression cassette can contain a synthetic regulatory element, such as the CAG promoter. The CAG promoter includes (i) a cytomegalovirus (CMV) early enhancer element, (ii) a promoter, the first exon and the first intron of the chicken beta actin gene, and (ii) a splice acceptor of the rabbit beta globin gene. Alternatively, the expression cassette can include an alpha-1-antitrypsin (AAT) promoter, a liver-specific (LP1) promoter, or a human elongation factor-1 alpha (EF1-α) promoter. In some embodiments, the expression cassette includes one or more constitutive promoters, such as a retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), a cytomegalovirus (CMV) immediate early promoter (optionally with a CMV enhancer). Alternatively, inducible or repressible promoters, the native promoter of the transgene, tissue-specific promoters, or various promoters known in the art can be used. Suitable transgenes for gene therapy are well known to those skilled in the art.

[0430] Capsid-free ceDNA vectors can also be produced from vector polynucleotide expression constructs that further include a cis-regulatory element or combination of cis-regulatory elements, non-limiting examples include woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and BGH polyA, or, for example, beta-globin polyA. Other posttranscriptional processing elements include, for example, the thymidine kinase gene of herpes simplex virus or hepatitis B virus (HBV). The expression cassette can include any polyadenylation sequence known in the art, such as naturally isolated from bovine BGHpA or viral SV40pA, or synthetic, or variations thereof. Some expression cassettes can also include an SV40 late polyA signal upstream enhancer (USE) sequence. USE can be used in combination with SV40pA or a heterologous polyA signal.

[0431] The time for harvesting and collecting the DNA vectors described herein from the cells can be selected and optimized to achieve high yield production of ceDNA vectors. For example, the harvest time can be selected taking into consideration cell viability, cell morphology, cell growth, etc. In one embodiment, the cells are grown under sufficient conditions and harvested after sufficient time has passed since baculovirus infection to produce the DNA vector, but before the majority of the cells begin to die due to viral toxicity. The DNA-vector can be isolated using a plasmid purification kit, such as the Qiagen Endo-Free Plasmid Kit. Other methods developed for plasmid isolation can also be adapted for DNA vectors. In general, any nucleic acid purification method can be employed.

[0432] The DNA vector may be purified by any means known to those skilled in the art for the purification of DNA. In one embodiment, the ceDNA vector is purified as a DNA molecule. In another embodiment, the ceDNA vector is purified as an exosome or microparticle.

[0433] In one embodiment, the capsid-free non-viral DNA vector comprises or is derived from a plasmid comprising a polynucleotide template comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette of an exogenous DNA), and a modified AAV ITR, in that order, and the template nucleic acid molecule lacks an AAV capsid protein coding sequence. In a further embodiment, the nucleic acid template of the present disclosure lacks viral capsid protein coding sequences (i.e., lacks not only AAV capsid genes, but also capsid genes of other viruses). In addition, in certain embodiments, the template nucleic acid molecule also lacks an AAV Rep protein coding sequence. Thus, in a preferred embodiment, the nucleic acid molecule of the present disclosure lacks both functional AAV cap and AAV rep genes.

[0434] In one embodiment, the ceDNA vector may comprise an ITR structure that is mutated relative to the wild-type AAV2 ITRs disclosed herein, but still retains operational RBE, TRS, and RBE' portions.

[0435] ceDNA Plasmids ceDNA-plasmids are plasmids used for the later production of ceDNA vectors. In one embodiment, ceDNA-plasmids can be constructed using known techniques to provide at least (1) a modified 5'ITR sequence, (2) an expression cassette containing cis-regulatory elements, such as promoters, inducible promoters, regulatory switches, enhancers, etc., and (3) a modified 3'ITR sequence (the 3'ITR sequence is asymmetric with respect to the 5'ITR sequence) as operably linked components in the transcriptional direction. In some embodiments, the expression cassette flanked by ITRs contains cloning sites for introducing exogenous sequences. The expression cassette replaces the rep and cap coding regions of the AAV genome.

[0436] In one embodiment, the ceDNA vector is derived from a plasmid, referred to herein as a "ceDNA-plasmid," that encodes, in that order, a first adeno-associated virus (AAV) inverted terminal repeat (ITR), an expression cassette comprising a transgene, and a mutated or modified AAV ITR, the ceDNA-plasmid lacking an AAV capsid protein coding sequence. In an alternative embodiment, the ceDNA-plasmid encodes, in that order, a first (or 5') modified or mutated AAV ITR, an expression cassette comprising a transgene, and a second (or 3') modified AAV ITR, the ceDNA-plasmid lacking an AAV capsid protein coding sequence, the 5' and 3' ITRs being symmetrical with respect to each other. In an alternative embodiment, a ceDNA-plasmid encodes, in that order, a first (or 5') modified or mutated AAV ITR, an expression cassette containing a transgene, and a second (or 3') mutated or modified AAV ITR, wherein the ceDNA-plasmid lacks the AAV capsid protein coding sequence, and the 5' and 3' modified ITRs have the same modifications (i.e., they are reverse complements or symmetrical to each other).

[0437] In one embodiment, the ceDNA-plasmid system lacks viral capsid protein coding sequences (i.e., lacks not only AAV capsid genes but also capsid genes of other viruses). In one embodiment, the ceDNA-plasmid also lacks AAV Rep protein coding sequences. In one embodiment, the ceDNA-plasmid lacks functional AAV cap and AAV rep genes (GG-3' for AAV2) as well as variable palindromic sequences that allow hairpin formation. In one embodiment, the ceDNA-plasmid of the present disclosure can be generated using the native nucleotide sequence of the genome of any AAV serotype known in the art. In one embodiment, the ceDNA-plasmid backbone is derived from the AAV1, AAV2, AAV3, AAV4, AAV5, AAV 5, AAV7, AAV8, AAV9, AAV 10, AAV 11, AAV 12, AAVrh8, AAVrhlO, AAV-DJ, and AAV-DJ8 genomes, e.g., Kotin and Smith, The Springer Index of Viruses, available at NCBI: NC 002077; NC001401, NC001729, NC001829, NC006152, NC006260, NC006261, URLs maintained by Springer. In one embodiment, the ceDNA-plasmid backbone is derived from the AAV2 genome. In one embodiment, the ceDNA-plasmid backbone is a synthetic backbone engineered to include 5' and 3' ITRs from one of these AAV genomes.

[0438] In one embodiment, the ceDNA-plasmid may optionally include a selectable or selectable marker for use in establishing a ceDNA vector producing cell line. In one embodiment, the selectable marker may be inserted downstream (i.e., 3') of the 3'ITR sequence. In another embodiment, the selectable marker may be inserted upstream (i.e., 5') of the 5'ITR sequence. Suitable selectable markers include, for example, those that confer drug resistance. The selectable marker may be, for example, a blasticidin S resistance gene, kanamycin, geneticin, etc.

[0439] VI. Preparation of Lipid Particles Lipid particles (e.g., lipid nanoparticles) can form spontaneously upon mixing of ceDNA and lipids. Depending on the desired particle size distribution, the resulting nanoparticle mixture can be extruded through a membrane (e.g., 100 nrn cutoff) using, for example, a thermobarrel extruder such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be achieved, for example, by dialysis or tangential flow filtration. In one embodiment, lipid nanoparticles are formed as described in Example 3 described in U.S. Provisional Application No. 63 / 194,620.

[0440] In general, lipid particles (e.g., lipid nanoparticles) can be formed by any method known in the art. For example, lipid particles (e.g., lipid nanoparticles) can be prepared by methods described in, for example, US2013 / 0037977, US2010 / 0015218, US2013 / 0156845, US2013 / 0164400, US2012 / 0225129, and US2010 / 0130588, the contents of each of which are incorporated herein by reference in their entirety. In some embodiments, lipid particles (e.g., lipid nanoparticles) can be prepared using a continuous mixing method, a direct dilution process, or an in-line dilution process. Processes and apparatus for preparing lipid nanoparticles using direct dilution and in-line dilution processes are described in US2007 / 0042031, the contents of which are incorporated herein by reference in their entirety. A process and apparatus for preparing lipid nanoparticles using a serial dilution process is described in US 2004 / 0142025, the contents of which are incorporated herein by reference in their entirety.

[0441] According to some embodiments, the present disclosure provides LNPs comprising a DNA vector, including a ceDNA vector as described herein, and an ionizable lipid. For example, lipid nanoparticle formulations made and loaded with therapeutic nucleic acids, such as ceDNA, obtained by the process disclosed in International Patent Application No. PCT / US2018 / 050042, filed September 7, 2018, which is incorporated herein by reference in its entirety.

[0442] In one embodiment, lipid particles (e.g., lipid nanoparticles) can be prepared by an impinging jet process. Generally, the particles are formed by mixing lipid dissolved in alcohol (e.g., ethanol) with ceDNA dissolved in a buffer, such as citrate buffer, sodium acetate buffer, sodium acetate and magnesium chloride buffer, malic acid buffer, malic acid and sodium chloride buffer, or sodium citrate and sodium chloride buffer. The lipid to ceDNA mixture ratio can be about 45-55% lipid and about 65-45% ceDNA.

[0443] The lipid solution can contain cationic lipids (e.g., ionizable cationic lipids), non-cationic lipids (e.g., phospholipids such as DSPC, DOPE, and DOPC), PEG or PEG-conjugated molecules (e.g., PEG-lipids), and sterols (e.g., cholesterol) in a total lipid concentration of 5-30 mg / mL, more likely 5-15 mg / mL, and most likely 9-12 mg / mL in an alcohol, e.g., ethanol. In the lipid solution, the molar ratios of lipids can range from about 25-98%, preferably about 35-65%, for cationic lipids; about 0-15%, preferably about 0-12%, for non-ionic lipids; about 0-15%, preferably about 1-6%, for PEG or PEG-conjugated lipid molecules; and about 0-75%, preferably about 30-50%, for sterols.

[0444] The ceDNA solution can contain ceDNA in a buffer solution having a pH in the range of 3.5 to 5, at a concentration in the range of 0.3 to 1.0 mg / mL, preferably 0.3 to 0.9 mg / mL.

[0445] To form the LNPs, in one exemplary but non-limiting embodiment, the two liquids are heated to a temperature in the range of about 15-40°C, preferably about 30-40°C, and then mixed, for example, in an impinging jet mixer, to instantly form the LNPs. The mixing flow rate can be in the range of 10-600 mL / min. The tube ID can have a range of 0.25-1.0 mm, with a total flow rate of 10-600 mL / min. The combination of flow rate and tube ID can have the effect of controlling the particle size of the LNPs to 30-200 nm. The solution can then be mixed with a buffer solution at a higher pH, in a mixing ratio ranging from 1:1 to 1:3 volume:volume, preferably about 1:2 volume:volume. Optionally, this buffer solution can be at a temperature in the range of 15-40°C or 30-40°C. The mixed LNPs can then undergo an anion exchange filtration step. Prior to anion exchange, the mixed LNPs can be incubated for a period of time, for example 30 minutes to 2 hours. The temperature during incubation can be in the range of 15-40 °C or 30-40 °C. After incubation, filter the solution through a filter, such as a 0.8 µm filter, which includes an anion exchange separation step. Tube IDs ranging from 1 mm ID to 5 mm ID and flow rates from 10 to 2000 mL / min can be used in this process.

[0446] After formation, the LNPs can be concentrated and ultrafiltered via an ultrafiltration process in which the alcohol is removed and the buffer is exchanged for a final buffer solution, e.g., phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

[0447] The ultrafiltration process can use a tangential flow filtration format (TFF) with membranes having a nominal molecular weight cutoff range of 30-500 kD. The membrane formats are hollow fiber or flat sheet cassettes. In a TFF process with the appropriate molecular weight cutoff, the LNPs can be retained in the retentate, while the filtrate or permeate contains alcohol, citrate buffer, and waste of the final buffer. The TFF process is a multi-step process that results in an initial concentration of ceDNA between 1-3 mg / mL. After concentration, the LNP solution is ultrafiltered with 10-20 volumes against the final buffer to remove alcohol and perform a buffer exchange. The material can then be further concentrated 1-3 times. The concentrated LNP solution can be sterile filtered.

[0448] VII. Pharmaceutical Compositions and Formulations Provided herein is a pharmaceutical composition comprising a lipid nanoparticle (LNP) and a therapeutic nucleic acid (TNA), wherein the LNP comprises an scFv linked to the LNP, and at least one pharma- ceutically acceptable excipient. According to one aspect, provided herein is a pharmaceutical composition comprising a lipid nanoparticle (LNP) and a therapeutic nucleic acid (TNA), wherein the LNP comprises a single chain variable fragment (scFv) linked to the LNP, the scFv directed to an antigen present on the surface of a cell, and at least one pharma- ceutically acceptable excipient, wherein the scFv is covalently linked to the LNP via a non-cleavable linker. According to one aspect, provided herein is a pharmaceutical composition comprising a lipid nanoparticle (LNP) and a therapeutic nucleic acid (TNA), wherein the LNP comprises a single chain variable fragment (scFv) linked to the LNP, wherein the scFv is directed to an antigen present on the surface of a cell, and at least one pharma- ceutical acceptable excipient, wherein the scFv is covalently linked to the LNP via a cleavable linker. According to one aspect, provided herein is a pharmaceutical composition comprising a lipid nanoparticle (LNP) and a therapeutic nucleic acid (TNA), wherein the LNP comprises a single chain variable fragment (scFv) linked to the LNP, wherein the scFv is directed to an antigen present on the surface of a cell, and at least one pharma- ceutical acceptable excipient, wherein the scFv is non-covalently linked to the LNP.

[0449] According to some embodiments, the TNA (e.g., ceDNA) is encapsulated in lipid. In one embodiment, a TNA (e.g., ceDNA) lipid particle (e.g., lipid nanoparticle) is provided with complete or partial encapsulation of a therapeutic nucleic acid. In one embodiment, the nucleic acid therapeutic is completely encapsulated in a lipid particle (e.g., lipid nanoparticle) to form a nucleic acid containing lipid particle. In one embodiment, the nucleic acid is encapsulated within the lipid portion of the particle, thereby protecting it from enzymatic degradation.

[0450] Depending on the intended use of the lipid particle (e.g., lipid nanoparticle), the ratio of components may vary, and the delivery efficiency of a particular formulation may be measured using, for example, an endosomal release parameter (ERP) assay.

[0451] In one embodiment, the lipid particles (eg, lipid nanoparticles) may be conjugated to other moieties to prevent aggregation. Such lipid conjugates include, but are not limited to, PEG-lipid conjugates, such as PEG coupled to dialkyloxypropyl (e.g., PEG-DAA conjugates), PEG coupled to diacylglycerol (e.g., PEG-DAG conjugates), PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamine, and PEG conjugated to ceramide (see, e.g., U.S. Pat. No. 5,885,613), cationic PEG lipids, polyoxazoline (POZ)-lipid conjugates (e.g., POZ-DAA conjugates, see, e.g., U.S. Provisional Application No. 61 / 294,828, filed Jan. 13, 2010, and U.S. Provisional Application No. 61 / 295,140, ​​filed Jan. 14, 2010), polyamide oligomers (e.g., ATTA-lipid conjugates), and mixtures thereof. Additional examples of POZ-lipid conjugates are described in PCT Publication No. 2010 / 006282. PEG or POZ can be directly conjugated to lipid or can be linked to lipid via a linker moiety. Any linker moiety suitable for coupling PEG or POZ to lipid can be used, including, for example, non-ester-containing linker moieties and ester-containing linker moieties. In certain preferred embodiments, non-ester-containing linker moieties such as amides or carbamates are used. The disclosures of each of the above patent documents are incorporated herein in their entirety by reference for all purposes.

[0452] In one embodiment, the TNA (e.g., ceDNA) may be complexed with the lipid portion of the particle or encapsulated in the lipid locus of the lipid particle (e.g., lipid nanoparticle). In one embodiment, the TNA may be fully encapsulated in the lipid locus of the lipid particle (e.g., lipid nanoparticle), thereby protecting it from degradation by nucleases, for example, in aqueous solution. In one embodiment, the TNA in the lipid particle (e.g., lipid nanoparticle) is not substantially degraded after exposure of the lipid particle (e.g., lipid nanoparticle) to nucleases for at least about 20, 30, 45, or 60 minutes at 37° C. In some embodiments, the TNA in the lipid particle (e.g., lipid nanoparticle) is not substantially degraded after incubation of the particle in serum for at least about 30, 45, or 60 minutes at 37° C., or for at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours.

[0453] In one embodiment, the lipid particles (eg, lipid nanoparticles) are substantially non-toxic to a subject, eg, a mammal, such as a human.

[0454] In one embodiment, a pharmaceutical composition comprising a therapeutic nucleic acid of the present disclosure may be formulated into a lipid particle (e.g., lipid nanoparticle). In some embodiments, the lipid particle comprising a therapeutic nucleic acid may be formed from a cationic lipid. In some other embodiments, the lipid particle comprising a therapeutic nucleic acid may be formed from a non-cationic lipid. In a preferred embodiment, the lipid particles of the present disclosure are nucleic acid-containing lipid particles formed from cationic lipids comprising a therapeutic nucleic acid selected from the group consisting of mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), minicircle DNA, minigenes, viral DNA (e.g., lentivirus or AAV genome) or non-viral synthetic DNA vectors, closed-end linear double-stranded DNA (ceDNA / CELiD), plasmids, bacmids, doggybone™ DNA vectors, minimal immunologically defined gene expression (MIDGE) vectors, non-viral ministring DNA vectors (linear covalently closed DNA vectors), or dumbbell-shaped DNA minimal vectors ("dumbbell DNA").

[0455] In another preferred embodiment, the lipid particles of the present disclosure are nucleic acid-containing lipid particles, which are formed from non-cationic lipids, and optionally conjugated lipids that prevent aggregation of the particles.

[0456] In one embodiment, the lipid particle formulation is an aqueous solution. In one embodiment, the lipid particle (e.g., lipid nanoparticle) formulation is a lyophilized powder.

[0457] According to some aspects, the present disclosure provides lipid particle formulations further comprising one or more pharmaceutical excipients. In one embodiment, the lipid particle (e.g., lipid nanoparticle) formulation further comprises sucrose, tris, trehalose, and / or glycine.

[0458] In one embodiment, the lipid particles (e.g., lipid nanoparticles) disclosed herein can be incorporated into a pharmaceutical composition suitable for administration to a subject for in vivo delivery to a cell, tissue, or organ of the subject. Typically, the pharmaceutical composition comprises the TNA (e.g., ceDNA) lipid particles (e.g., lipid nanoparticles) disclosed herein and a pharma- ceutically acceptable carrier. In one embodiment, the TNA (e.g., ceDNA) lipid particles (e.g., lipid nanoparticles) of the present disclosure can be incorporated into a pharmaceutical composition suitable for the desired route of therapeutic administration (e.g., parenteral administration). Passive tissue transduction via high pressure intravenous or intra-arterial infusion is also contemplated, as well as intracellular injection, such as intranuclear microinjection or intracytoplasmic injection. Pharmaceutical compositions for therapeutic purposes can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high TNA (e.g., ceDNA) vector concentrations. Sterile injectable solutions can be prepared by incorporating the required amount of TNA (e.g., ceDNA) vector compound in an appropriate buffer with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.

[0459] The lipid particles disclosed herein can be incorporated into pharmaceutical compositions suitable for local, systemic, intra-amniotic, intra-subarachnoid, intracranial, intra-arterial, intravenous, intralymphatic, intraperitoneal, subcutaneous, tracheal, intra-tissue (e.g., intramuscular, intracardiac, intrahepatic, intrarenal, intracerebral), intra-subarachnoid, intravesical, conjunctival (e.g., extra-orbital, intra-orbital, retro-orbital, intra-retinal, sub-retinal, choroidal, subchoroidal, interstitial, intracameral and intravitreal), intracochlear, and mucosal (e.g., oral, rectal, nasal) administration. Passive tissue transduction via high pressure intravenous or intra-arterial infusion is also contemplated, as well as intracellular injections such as intranuclear microinjection or intracytoplasmic injection.

[0460] Pharmaceutically active compositions comprising TNA (e.g., ceDNA) lipid particles (e.g., lipid nanoparticles) can be formulated to deliver the transgene in the nucleic acid to recipient cells, resulting in therapeutic expression of the transgene therein. The compositions can also include a pharma- ceutically acceptable carrier.

[0461] Pharmaceutical compositions for therapeutic purposes must typically be sterile and stable under the conditions of manufacture and storage.The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for high TNA (e.g., ceDNA) vector concentration.A sterile injectable solution can be prepared by incorporating the required amount of ceDNA vector compound in a suitable buffer with one or a combination of the above-listed ingredients as needed, followed by filtration sterilization.

[0462] In one embodiment, the lipid particles (e.g., lipid nanoparticles) are solid core particles having at least one lipid bilayer. In one embodiment, the lipid particles (e.g., lipid nanoparticles) have a non-bilayer structure, i.e., a non-lamellar (i.e., non-bilayer) morphology. Non-bilayer morphology can include, but is not limited to, three-dimensional tube, rod, cubic symmetry, etc. The non-lamellar morphology (i.e., non-bilayer structure) of lipid particles (e.g., lipid nanoparticles) can be determined using analytical techniques known and used by those skilled in the art. Such techniques include, but are not limited to, cryo-transmission electron microscopy ("Cryo-TEM"), differential scanning calorimetry ("DSC"), X-ray diffraction, etc. For example, the morphology (lamellar vs. non-lamellar) of lipid particles can be easily assessed and characterized using Cryo-TEM analysis, for example, as described in US2010 / 0130588, the contents of which are incorporated herein by reference in their entirety.

[0463] In one embodiment, lipid particles (eg, lipid nanoparticles) having a non-lamellar morphology are electron dense.

[0464] In one embodiment, the present disclosure provides lipid particles (e.g., lipid nanoparticles) that are either unilamellar or multilamellar in structure. In some aspects, the present disclosure provides lipid particle (e.g., lipid nanoparticle) formulations that include multivesicular particles and / or effervescent particles. By controlling the composition and concentration of the lipid components, the rate at which lipid conjugates exchange out of the lipid particle (lipid nanoparticle) and then the rate at which the lipid nanoparticle becomes fusogenic can be controlled. In addition, other variables, including, for example, pH, temperature, or ionic strength, can be used to vary and / or control the rate at which the lipid particle (e.g., lipid nanoparticle) becomes fusogenic. Other methods that can be used to control the rate at which the lipid particle (e.g., lipid nanoparticle) becomes fusogenic will be apparent to those skilled in the art based on the present disclosure. It will also be apparent that the lipid particle size can be controlled by controlling the composition and concentration of the lipid conjugate.

[0465] In one embodiment, the pKa of the formulated cationic lipid can be correlated with the efficacy of the LNP for delivery of nucleic acids (see Jayaraman et al., Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al., Nature Biotechnology 28, 172-176 (2010), both of which are incorporated by reference in their entireties). In one embodiment, the preferred range of pKa is from about 5 to about 7. In one embodiment, the pKa of the cationic lipid can be determined in lipid particles (e.g., lipid nanoparticles) using an assay based on the fluorescence of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS).

[0466] In one embodiment, the encapsulation of TNA (e.g., ceDNA) in lipid particles (e.g., lipid nanoparticles) can be determined by performing a membrane-impermeable fluorescent dye exclusion assay, such as the Oligreen® assay or the PicoGreen® assay, which uses a dye that has enhanced fluorescence when associated with nucleic acid. In general, encapsulation is determined by adding a dye to the lipid particle formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed upon addition of a small amount of non-ionic detergent. Detergent-mediated disruption of the lipid bilayer releases the encapsulated TNA (e.g., ceDNA) and allows it to interact with the membrane-impermeable dye. The encapsulation of ceDNA can be calculated as E=(Io-I) / Io, where I and Io refer to the fluorescence intensity before and after the addition of detergent.

[0467] According to some embodiments, for ophthalmic delivery, the interfering RNA-ligand conjugates and nanoparticle-ligand conjugates can be combined with ophthalmologically acceptable preservatives, co-solvents, surfactants, viscosity enhancers, penetration enhancers, buffers, sodium chloride, or water to form aqueous, sterile ophthalmic suspensions or solutions.

[0468] Unit Dose In one embodiment, the pharmaceutical composition may be presented in a unit dosage form. The unit dosage form will typically be adapted for one or more routes of administration of the pharmaceutical composition. In some embodiments, the unit dosage form is adapted for administration by inhalation. In some embodiments, the unit dosage form is adapted for administration by an inhaler. In some embodiments, the unit dosage form is adapted for administration by a nebulizer. In some embodiments, the unit dosage form is adapted for administration by an aerosolizer. In some embodiments, the unit dosage form is adapted for oral administration, buccal administration, or sublingual administration. In some embodiments, the unit dosage form is adapted for intravenous, intramuscular, or subcutaneous administration. In some embodiments, the unit dosage form is adapted for intrathecal or intraventricular administration. In some embodiments, the pharmaceutical composition is formulated for topical administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound that produces a therapeutic effect.

[0469] VIII. Treatment method A pharmaceutical composition comprising a lipid nanoparticle (LNP) and a therapeutic nucleic acid (TNA), where the LNP comprises an scFv (e.g., the scFv is directed to an antigen present on the surface of a cell) linked to the LNP as described herein, can be used to introduce a nucleic acid sequence (e.g., the TNA) into a cell to treat or prevent a disease or disorder. According to some embodiments, the pharmaceutical composition can be used in diagnostic methods.

[0470] Provided herein is a method of treating a disease or disorder in a subject, the method comprising introducing into a target cell in need thereof (e.g., a muscle cell or muscle tissue, or other diseased cell type) of the subject a therapeutically effective amount of a pharmaceutical composition comprising a lipid nanoparticle (LNP) and a therapeutic nucleic acid (TNA), wherein the LNP comprises an scFv linked to the LNP, the scFv being directed to an antigen present on the surface of the cell, and the cell being a tumor cell.

[0471] Thus, according to some aspects, the pharmaceutical compositions described herein may be used in methods of treating cancer, according to other aspects, the pharmaceutical compositions described herein may be used in methods of preventing cancer or preventing the recurrence of cancer.

[0472] As used herein, the term "cancer" refers to any of a variety of malignant neoplasms characterized by the proliferation of undifferentiated cells that tend to invade surrounding tissues and metastasize to new body sites, and to pathological conditions characterized by the proliferation of such malignant neoplasms. Cancer may be localized (e.g., solid tumors) or systemic. In the context of this disclosure, the term "localized" (as in "localized tumor") refers to an anatomically isolated or isolatable abnormality, such as a solid malignant tumor, as opposed to a systemic disease. For example, certain cancers, such as certain leukemias (e.g., myelofibrosis) and multiple myeloma, may have both a localized (e.g., bone marrow) and a systemic (e.g., circulating blood cells) component to the disease. In some embodiments, the cancer may be systemic, such as a hematological malignancy. Cancers that may be treated according to the present disclosure include, but are not limited to, all types of lymphoma / leukemia, carcinoma and sarcoma, such as cancers or tumors found in the anus, bladder, bile duct, bone, brain, breast, cervix, colon / rectum, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum (chest), mouth, ovaries, pancreas, penis, prostate, skin, small intestine, stomach, spinal cord, tailbone, testes, thyroid and uterus. Types of cancer that may be treated by the methods of the present disclosure include, but are not limited to, papilloma / carcinoma, choriocarcinoma, endodermal sinus tumor, teratoma, adenoma / adenocarcinoma, melanoma, fibroma, lipoma, leiomyoma, rhabdomyoma, mesothelioma, hemangioma, osteoma, chondroma, glioma, lymphoma / leukemia, squamous cell carcinoma, small cell carcinoma, large cell undifferentiated carcinoma, basal cell carcinoma and sinonasal undifferentiated carcinoma. Types of sarcomas include, but are not limited to, soft tissue sarcomas, such as alveolar soft part sarcoma, angiosarcoma, dermatofibrosarcoma, desmoid tumor, desmoplastic small round cell tumor, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma, angiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, and Askin's tumor, Ewing's sarcoma (primitive neuroectodermal tumor), malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and chondrosarcoma.

[0473] The TNA (e.g., ceDNA) lipid nanoparticles can be administered via any suitable route described herein and known in the art. In one embodiment, the target cells are in a human subject.

[0474] Provided herein is a method for providing a diagnostically or therapeutically effective amount of a pharmaceutical composition comprising LNPs and TNAs, the LNPs comprising scFvs, the scFvs being directed to an antigen present on the surface of a cell as described herein and linked to the LNPs, to a subject in need thereof, the method comprising providing to a cell, tissue or organ of the subject in need thereof an amount of the pharmaceutical composition comprising LNPs and TNAs, the LNPs comprising scFvs, the scFvs being directed to an antigen present on the surface of a cell as described herein and linked to the LNPs, for a time effective to allow expression of a transgene from a ceDNA vector, thereby providing to the subject a diagnostically or therapeutically effective amount of the pharmaceutical composition comprising LNPs and TNAs, the LNPs comprising scFvs, the scFvs being directed to an antigen present on the surface of a cell as described herein and linked to the LNPs. In one embodiment, the subject is a human.

[0475] Provided herein are methods that include using pharmaceutical compositions comprising LNPs and TNAs, the LNPs comprising scFvs, the scFvs being directed to antigens present on the surface of cells and linked to LNPs, as described herein, to treat or alleviate one or more symptoms of a disease or disease state. There are several genetic diseases with known defective genes, typically falling into two classes: enzyme deficiency states, which are usually inherited recessively, and imbalance states, which may involve regulatory or structural proteins, but are typically not always inherited dominantly. For deficiency state diseases, pharmaceutical compositions comprising LNPs and TNAs, the LNPs comprising scFvs, the scFvs being directed to antigens present on the surface of cells and linked to LNPs, as described herein, can be used to deliver transgenes that bring normal genes to affected tissues for replacement therapy, and in some embodiments, antisense mutations can be used to create animal models of the disease. As used herein, a disease state is treated by partially or fully repairing the deficiency or imbalance that causes the disease or makes it more severe.

[0476] In general, a pharmaceutical composition comprising an LNP and a TNA, where the LNP comprises an scFv, where the scFv is directed to an antigen present on the surface of a cell and linked to the LNP as described herein, can be used to deliver any transgene as described above to treat, prevent, or ameliorate symptoms associated with any disorder associated with gene expression. Exemplary disease states include, but are not limited to, cystic fibrosis (and other lung diseases), hemophilia A, hemophilia B, thalassemia, anemia and other blood disorders, AIDS, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, epilepsy and other neurological disorders, cancer, diabetes, muscular dystrophies (e.g., Duchenne, Becker), Hurler's disease, adenosine deaminase deficiency, metabolic disorders, retinal degenerative diseases (and other eye diseases), mitochondriopathy (e.g., Leber's hereditary optic neuropathy (LHON), Leigh's syndrome, and subacute sclerosing encephalitis), myopathy (e.g., facioscapulohumeral myopathy (FSHD) and cardiomyopathies), diseases of solid organs (e.g., brain, liver, kidney, heart), and the like. In some embodiments, the ceDNA vectors disclosed herein may be advantageously used in the treatment of individuals with metabolic disorders (e.g., omegaly transcarbamylase deficiency).

[0477] In one embodiment, a pharmaceutical composition comprising an LNP and a TNA, wherein the LNP comprises an scFv, and the scFv is directed to an antigen present on the surface of a cell (as described herein) and linked to the LNP, can be used to treat, ameliorate, and / or prevent a disease or disorder caused by a mutation in a gene or gene product. Exemplary diseases or disorders that can be treated with ceDNA vectors (e.g., pharmaceutical compositions comprising LNPs and TNAs, where the LNPs comprise scFvs, where the scFvs are directed to antigens present on the surface of a cell, as described herein, and linked to the LNPs) include cancers and tumors, metabolic diseases or disorders (e.g., Fabry disease, Gaucher disease, phenylketonuria (PKU), glycogen storage diseases); urea cycle diseases or disorders (e.g., ornithine transcarbamylase (OTC) deficiency); lysosomal storage diseases or disorders (e.g., metachromatic leukodystrophy (MLD), mucopolysaccharidosis type II (MPSII; Hunter syndrome)); liver diseases or disorders (e.g., progressive familial intrahepatic cholestasis (PFIC); blood diseases or disorders (e.g., hemophilia (A and B), thalassemia, and anemia); and genetic diseases or disorders (e.g., cystic fibrosis).

[0478] In one embodiment, a pharmaceutical composition comprising an LNP and a TNA, where the LNP comprises an scFv, where the scFv is directed to an antigen present on the surface of a cell as described herein and is linked to the LNP, may be used to deliver a heterologous nucleotide sequence in situations where it is desirable to modulate the level of transgene expression (e.g., a transgene encoding a hormone or growth factor as described herein).

[0479] In one embodiment, a pharmaceutical composition comprising an LNP and a TNA, the LNP comprising an scFv, the scFv being directed to an antigen present on the surface of a cell and linked to the LNP as described herein, can be used to correct the abnormal levels and / or function of a gene product (e.g., the absence of, or a defect in, a protein) that causes a disease or disorder. The ceDNA vectors in the lipid nanoparticles described herein can produce functional proteins and / or correct the levels of the protein to alleviate or reduce symptoms or confer a benefit resulting from a particular disease or disorder caused by the absence of, or a defect in, the protein. For example, treatment of OTC deficiency can be achieved by producing a functional OTC enzyme; treatment of hemophilia A and B can be achieved by modifying the levels of factors VIII, IX, and X; treatment of PKU can be achieved by modifying the levels of phenylalanine hydroxylase enzyme; treatment of Fabry disease or Gaucher disease can be achieved by producing functional alpha-galactosidase or beta-glucocerebrosidase, respectively; treatment of MFD or MPSII can be achieved by producing functional arylsulfatase A or iduronate-2-sulfatase, respectively; treatment of cystic fibrosis can be achieved by producing a functional cystic fibrosis transmembrane regulator; treatment of glycogen storage disease can be achieved by restoring functional G6Pase function; treatment of PFIC can be achieved by producing functional ATP8B1, ABCB11, ABCB4, or TJP2 genes.

[0480] In one embodiment, a pharmaceutical composition comprising an LNP and a TNA, the LNP comprising an scFv, the scFv being directed to an antigen present on the surface of the cell and linked to the LNP as described herein, can be used to provide an RNA-based therapeutic to a cell in vitro or in vivo. Examples of RNA-based therapeutics include, but are not limited to, mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA ...

Claims

**Claim 1**: A pharmaceutical composition comprising: Lipid nanoparticles (LNP), wherein the LNP comprises a cationic lipid, a sterol, a non-cationic lipid, and a PEG5000 PEGylated lipid; A therapeutic nucleic acid (TNA); and At least one pharmaceutically acceptable excipient wherein the LNP comprises a single-chain variable fragment (scFv) linked to the LNP, and the scFv binds to an antigen present on the surface of a cell. **Claim 2**: The pharmaceutical composition according to claim 1, wherein the scFv is covalently linked to the LNP or the scFv is chemically conjugated to the LNP. **Claim 3**: The pharmaceutical composition according to claim 2, wherein the scFv is chemically conjugated to the LNP via the PEG5000 PEGylated lipid. **Claim 4**: The pharmaceutical composition according to claim 3, wherein the PEG5000 PEGylated lipid to which the scFv is chemically conjugated is DSPE-PEG5000. **Claim 5**: The pharmaceutical composition according to claim 2, wherein the scFv is chemically conjugated to the LNP via a non-cleavable linker or a cleavable linker. **Claim 6**: The pharmaceutical composition according to claim 5, wherein the non-cleavable linker is a maleimide-containing linker or the cleavable linker is a pyridyldisulfide (PDS)-containing linker. **Claim 7**: The pharmaceutical composition according to claim 1, wherein the scFv is linked to the LNP via transglutaminase-mediated conjugation. **Claim 8**: The pharmaceutical composition according to claim 1, wherein the antigen is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). **Claim 9**: The pharmaceutical composition according to claim 8, wherein the TAA or TSA is human epidermal growth factor receptor 2 (HER2). **Claim 10**: The pharmaceutical composition according to claim 1, wherein the scFv is bivalent. **Claim 11**: The pharmaceutical composition according to claim 1, wherein the LNP is capable of being internalized into the cell. **Claim 12**: ​ ​ ​ ​ ​ ​ ​ ​ The scFv of the pharmaceutical composition according to claim 1 comprises the amino acid sequence shown in SEQ ID NO: 2, has at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 2, comprises the amino acid sequence shown in SEQ ID NO: 3, or has at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO:

3.

13. The pharmaceutical composition according to claim 1, wherein the TNA is encapsulated in the LNP.

14. The TNA of the pharmaceutical composition according to claim 1 is selected from the group consisting of minigenes, plasmids, minicircles, small interfering RNAs (siRNAs), microRNAs (miRNAs), antisense oligonucleotides (ASOs), ribozymes, closed-ended (ceDNAs), ministring DNAs, doggybone (trademark) DNAs, proteolomer closed-ended DNAs, dumbbell linear DNAs, dicer substrates dsRNAs, small hairpin RNAs (shRNAs), asymmetric interfering RNAs (aiRNAs), mRNAs, tRNAs, rRNAs, DNA virus vectors, viral RNA vectors, non-viral vectors, and any combination thereof.

15. The pharmaceutical composition according to claim 14, wherein the TNA is mRNA.

16. The pharmaceutical composition according to claim 14, wherein the TNA is siRNA.

17. The pharmaceutical composition according to claim 1, which is administered to a subject.

18. The pharmaceutical composition according to claim 17, wherein the subject is a human.

19. The pharmaceutical composition according to claim 1 targets cells that express the cell surface antigen to which the scFv binds.

20. Targeting tumor cells; Targeting liver cells; and / or Targeting hepatocytes in the liver, the pharmaceutical composition according to claim 1.

21. (a) The cationic lipid is represented by formula (I), 【Chemical Formula 61】 or a pharmaceutically acceptable salt thereof, wherein R １ and R １’ are each independently a straight-chain or branched-chain C １～３ alkylene which is optionally substituted R ２ and R ２’ are each independently a straight-chain or branched-chain C １～６ alkylene which is optionally substituted, R ３ and R ３’ are each independently, optionally substituted linear or branched C １～６ alkyl, or Or alternatively, R ２ is optionally substituted branched chain C １～６ alkylene, then R ２ and R ３ together with the intervening N atoms form a 4- to 8-membered heterocyclyl, or Or alternatively, R ２’ is, when optionally substituted branched-chain C １～６ alkylene, R ２’ and R ３’ together with their intervening N atoms form a 4- to 8-membered heterocyclyl, R ４ and R ４’ are each independently, -CR ａ , -C(R ａ ) ２ CR ａ , or -[C(R ａ ) ２ ２ CR ａ wherein R ａ is, for each occurrence, independently H or C １～３ alkyl, or Or alternatively, R ４ is -C(R ａ ), ２ CR ａ , or -[C(R ａ ), ２ ２ CR ａ and when R ａ is C １～３ alkyl, R ３ and R ４ together with the intervening N atom form a 4- to 8-membered heterocyclyl, Or alternatively, R ４’ is, -C(R ａ ), ２ CR ａ , or -[C(R ａ ), ２ ２ CR ａ and when R ａ is C １～３ alkyl, R ３’ and R ４’ together with the intervening N atom form a 4- to 8-membered heterocyclyl, R ５ and R ５’ are each independently hydrogen, C １～２０ alkylene or C ２～２０ alkenylene, R ６ and R ６’ is, for each occurrence, independently C １～２０ alkylene, C ３～２０ cycloalkylene, or C ２～２０ alkenylene, and m and n are each independently an integer selected from 1, 2, 3, 4, and 5; or (b) The cationic lipid is represented by formula (II), 【Chemical Formula 62】 or a pharmaceutically acceptable salt thereof, wherein a is an integer in the range of 1 to 20, b is an integer in the range of 2 to 10, R1 is absent or is selected from (C2-C20) alkenyl, -C(O)O(C2-C20) alkyl, and cyclopropyl substituted with (C2-C20) alkyl, R2 is (C2-C20) alkyl; or (c) the cationic lipid is represented by formula (V), 【Chemical Formula 63】 or a pharmaceutically acceptable salt thereof, wherein R1 and R1' are each independently (C1-C6) alkylene optionally substituted with one or more groups selected from Ra, R2 and R2' are each independently (C1-C2) alkylene, R3 and R3' are each independently (C1-C6) alkyl optionally substituted with one or more groups selected from Rb, alternatively, R2 and R3 and / or R2' and R3' together with the intervening N atom form a 4- to 7-membered heterocyclyl, R4 and R4' are each (C2-C6) alkylene interrupted by -C(O)O-, R5 and R5' are each independently (C2-C30) alkyl or (C2-C30) alkenyl, each of which is optionally interrupted by -C(O)O- or (C3-C6) cycloalkyl, Ra and Rb are each halo or cyano; or (d) the cationic lipid is represented by formula (XV), 【Chemical Formula 64】 or a pharmaceutically acceptable salt thereof, wherein R' is absent, hydrogen, or C1-C6 alkyl, provided that when R' is hydrogen or C1-C6 alkyl, the nitrogen atom to which R', R1, and R2 are all attached is protonated, R1 and R2 are each independently hydrogen, C1-C6 alkyl, or C2-C6 alkenyl, R3 is C1-C12 alkylene or C2-C12 alkenylene, R4 is C1-C16 unbranched alkyl, C2-C16 unbranched alkenyl, or 【Chemical Formula 65】 wherein R4a and R4b are each independently C1-C16 unbranched alkyl or C2-C16 unbranched alkenyl, R5 is absent, C1-C8 alkylene, or C2-C8 alkenylene, R6a and R6b are each independently C7-C16 alkyl or C7-C16 alkenyl, provided that the total number of carbon atoms in R6a and R6b combined is greater than 15, X1 and X2 are each independently -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -S-S-, -C(Ra)=N-, -N=C(Ra)-, -C(Ra)=NO-, -O-N=C(Ra)-, -C(=O)NRa-, -NRaC(=O)-, -NRaC(=O)NRa-, -OC(=O)O-, -OSi(Ra)2O-, -C(=O)(CRa2)C(=O)O-, or OC(=O)(CRa2)C(=O)-, where Ra is, for each occurrence, independently hydrogen or C1-C6 alkyl, n is an integer selected from 1, 2, 3, 4, 5, and 6; or (e) the cationic lipid is represented by formula (XX), 【Chemical Formula 66】 or a pharmaceutically acceptable salt thereof, where R' is absent, hydrogen, or C1-C3 alkyl, provided that when R' is hydrogen or C1-C3 alkyl, the nitrogen atom to which R', R1, and R2 are all attached is protonated, R1 and R2 are each independently hydrogen or C1-C3 alkyl, R3 is C3-C10 alkylene or C3-C10 alkenylene, R4 is C1-C16 unbranched alkyl, C2-C16 unbranched alkenyl, or 【Chemical 67】 where R4a and R4b are each independently C1-C16 unbranched alkyl or C2-C16 unbranched alkenyl, R5 is absent, C1-C6 alkylene, or C2-C6 alkenylene, R6a and R6b are each independently C7-C14 alkyl or C7-C14 alkenyl, X is -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -S-S-, -C(Ra)=N-, -N=C(Ra)-, -C(Ra)=NO-, -O-N=C(Ra)-, -C(=O)NRa-, -NRaC(=O)-, -NRaC(=O)NRa-, -OC(=O)O-, -OSi(Ra)2O-, -C(=O)(CRa2)C(=O)O-, or OC(=O)(CRa2)C(=O)-, wherein, Ra is, independently for each occurrence, hydrogen or C1-C6 alkyl, n is an integer selected from 1, 2, 3, 4, 5, and 6; or (f) the cationic lipid is selected from lipids comprising any of the lipids shown in Table 2, Table 5, Table 6, Table 7, and Table 8; (g) the cationic lipid is a lipid having the following structure, 【Chemical Formula 68】 or a pharmaceutically acceptable salt thereof; or (h) the cationic lipid is MC3(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA or MC3) having the following structure, or 【Chemical Formula 69】 a pharmaceutically acceptable salt thereof, the pharmaceutical composition according to claim 1.

22. the sterol or a derivative thereof is cholesterol or beta-sitosterol; The non-cationic lipid is distearoyl-sn-glycero-phosphoethanolamine (DSPE), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl phosphatidylethanolamine (POPE), dioleoyl phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), distearoyl phosphatidylethanolamine (DSPE), monomethyl phosphatidylethanolamine (e.g., 16-O-monomethyl PE), dimethyl phosphatidylethanolamine (e.g., 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoyl phosphatidylethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), dielaidoyl phosphatidylcholine (DEPC), palmitoyl oleoyl phosphatidylglycerol (POPG), dielaidoyl phosphatidylethanolamine (DEPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE); 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE);Selected from the group consisting of lecithin, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof; optionally, said non-cationic lipid is selected from the group consisting of dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), and dioleoylphosphatidylethanolamine (DOPE); and / or; The PEG5000 PEGylated lipid is selected from the group consisting of PEG-dilauroxypropyl; PEG-dimyristyloxypropyl; PEG-dipalmitoyloxypropyl, PEG-distearyloxypropyl; 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (DMG-PEG); PEG-dilauroyl glycerol; PEG-dipalmitoyl glycerol; PEG-distearyl glycerol; PEG-dilauroyl glycamide; PEG-dimyristyl glycamide; PEG-dipalmitoyl glycamide; PEG-distearyl glycamide; (1-[8'-(cholesta-5-en-3[beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol))(PEG-cholesterol); 3,4-ditetradecaoxyl benzyl-[omega]-methyl-poly(ethylene glycol) ether (PEG-DMB), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-hydroxyl (DSPE-PEG-OH); The pharmaceutical composition according to claim 1, wherein the at least one PEGylated lipid is DMG-PEG5000, DSPE-PEG5000, DSPE-PEG5000-OH, or a combination thereof.

23. The cationic lipid is present in a molar percentage of about 30% to about 80%; The sterol is present in a molar percentage of about 20% to about 50%; The non-cationic lipid is present in a molar percentage of about 2% to about 20%; The at least one PEGylated lipid is present in a molar percentage of about 2.1% to about 10%, The LNP has a ratio of total lipid to TNA of about 10:1 to about 40:1; and / or The scFv is present in a total amount of about 0.02 μg / μg of TNA to about 0.1 μg / μg of TNA. The pharmaceutical composition according to claim 21.

24. The pharmaceutical composition according to claim 1, further comprising dexamethasone palmitate.

25. The pharmaceutical composition according to claim 1, wherein the LNP has a diameter of about 40 nm to about 120 nm; a diameter of about 60 nm to about 80 nm; or a diameter of less than about 100 nm. **Claim 26** The TNA includes an expression cassette; The expression cassette includes a promoter sequence and a transgene; and / or The pharmaceutical composition according to claim 14, wherein the expression cassette includes a polyadenylation sequence. **Claim 27** The pharmaceutical composition according to claim 14, wherein the cDNA includes at least one inverted terminal repeat (ITR) adjacent to either the 5' or 3' end of the expression cassette. **Claim 28** The expression cassette is adjacent to two ITRs, and the two ITRs include one 5' ITR and one 3' ITR; The expression cassette is linked to an ITR (3' ITR) at the 3' end; The expression cassette is linked to an ITR (5' ITR) at the 5' end; The at least one ITR is selected from the group consisting of an ITR derived from an AAV serotype, an ITR derived from an ITR of avian virus, an ITR derived from a B19 virus ITR, and a wild-type ITR derived from parvovirus; At least one of the 5' ITR and the 3' ITR is a wild-type AAV ITR; At least one of the 5' ITR and the 3' ITR is a modified or mutant ITR; The 5' ITR and the 3' ITR are symmetric; The 5' ITR and the 3' ITR are asymmetric; The cDNA further includes a spacer sequence between the 5' ITR and the expression cassette; and / or The pharmaceutical composition according to claim 27, wherein the cDNA further includes a spacer sequence between the 3' ITR and the expression cassette. **Claim 29** The pharmaceutical composition according to claim 28, wherein the AAV serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. **Claim 30** The pharmaceutical composition according to claim 28, wherein the spacer sequence has a length of at least 5 base pairs. **Claim 31** The pharmaceutical composition according to claim 14, wherein the cDNA has a nick or a gap. **Claim 32** The pharmaceutical composition according to claim 14, wherein the cDNA is CELiD, a DNA-based minicircle, MIDGE, a ministring DNA, a dumbbell-shaped linear double-stranded closed-ended DNA containing two hairpin structures of ITR at the 5' and 3' ends of an expression cassette, or doggybone (trademark) DNA.

33. A pharmaceutical composition for use in the treatment of cancer in a subject, the pharmaceutical composition comprising an effective amount of the pharmaceutical composition according to any one of claims 1 to 32.

34. The pharmaceutical composition according to claim 33, wherein the subject is a human.

35. A pharmaceutical composition for use in delivering a therapeutic nucleic acid (TNA) to a tumor in a subject or increasing the concentration of the TNA, the pharmaceutical composition comprising an effective amount of the pharmaceutical composition according to any one of claims 1 to 32.

36. A pharmaceutical composition for use in delivering a therapeutic nucleic acid (TNA) to the liver of a subject or increasing the concentration of the TNA, the pharmaceutical composition comprising an effective amount of the pharmaceutical composition according to any one of claims 1 to 32.

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