ApoE and ApoB modified lipid nanoparticle compositions and uses thereof

Abstract

Provided herein is a pharmaceutical composition comprising a lipid nanoparticle (LNP) and a therapeutic nucleic acid (TNA), wherein the LNP comprises an ApoE polypeptide, or a fragment thereof, and / or an ApoB polypeptide, or a fragment thereof, bound to the LNP, and at least one pharma- ceutically acceptable excipient. The ApoE polypeptide, or a fragment thereof, and / or the ApoB polypeptide, or a fragment thereof, can bind to a low-density lipoprotein (LDL) receptor or an LDL receptor family member, providing an LNP composition that can be advantageously directed to any cell or tissue expressing the LDL receptor.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 63 / 197,881, filed June 7, 2021. The entire contents of the aforementioned application are expressly incorporated herein by reference. [Background technology]

[0002] Ionizable lipid nanoparticles (Lipid NanoParticles, LNPs) have been widely used for systemic delivery of gene therapeutics, e.g., RNA therapeutics. Various types of ionizable lipid materials, such as C12292-200, 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 specific ligands has been shown to enhance the delivery and therapeutic efficiency of mRNA LNPs, but it has been recognized that conjugating moieties to enhance delivery and therapeutic efficiency 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 specificity of some 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] The mechanism of cellular uptake has been investigated, and it has been shown that the adsorption of serum ApoE (apolipoprotein E) onto the surface of LNPs is the main effector in promoting the intracellular delivery of LNPs to hepatocytes via the low-density lipoprotein (LDL) receptor (Akinc et al., Mol. Ther. 18, 1357-1364 (2010)). However, although LNPs have been shown to be advantageous for in vivo delivery, systemic delivery of gene therapy drugs to liver hepatocytes and other cell types remains very challenging.

[0004] With respect to the delivery of gene therapy drugs to hepatocytes, 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 the target cells (hepatocytes); (2) larger LNPs cannot be efficiently internalized by hepatocytes via clathrin-dependent 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. In addition, 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 relative to 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.

[0005] With regard to the delivery of gene therapy drugs to other types of cells (e.g., retinal cells), optimal methods for delivering these therapies to retinal pigment epithelial (RPE) cells and / or photoreceptor cells remain to be improved in order to increase transduction efficiency and reduce complications associated with the highly invasive surgery required for subretinal injection of viral vector suspensions. To date, when LNPs have been used for retinal gene transduction, the majority of expression has been seen in the retinal pigment epithelium (RPE) in the eyecup (Patel et al., Journal of Controlled Release Volume 303, 10 June 2019, Pages 91-100), and delivery of gene therapy to the actual photoreceptors within the retina remains a significant challenge. Thus, to realize the full potential of nucleic acid therapeutics for other cell types, such as liver hepatocytes and retinal cells, an efficient in vivo delivery system for nucleic acids is needed. [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] Akinc et al.,Mol.Ther.18, 1357-1364(2010) [Non-Patent Document 5] Patel et al.,Journal of Controlled Release Volume 303,10 June 2019,Pages 91-100 Summary of the Invention

[0007] This disclosure describes, for the first time, the combination of lipid nanoparticles, such as those having lipid A as described herein as an ionizable or cationic lipid, with gene therapy cargo (e.g., mRNA or closed-end DNA) for retinal delivery. Using mouse, rat, and non-human primate (NHP) in vivo systems, this disclosure demonstrated that robust transgene expression can be achieved in the retinal pigment epithelium (RPE) and neural retina, where photoreceptors reside, using LNP-delivered mRNA cargo. Importantly, the data presented herein showed that saturation was achieved with low doses of LNP-delivered mRNA, thereby successfully achieving an excellent therapeutic index and tolerability of the potential therapy. Previously, when LNPs have been used for retinal gene transduction, the majority of expression has been seen in the retinal pigment epithelium (RPE) in the retina (Patel et al., Journal of Controlled Release Volume 303, 10 June 2019, Pages 91-100), but getting to the actual photoreceptors within the retina has remained a significant challenge. The results presented in this disclosure surprisingly demonstrate that not only can LNPs enter the optic cup with expression equivalent to that of the RPE, but they can do so at low doses.

[0008] In one aspect, the disclosure provides a pharmaceutical composition comprising a lipid nanoparticle (LNP) and a therapeutic nucleic acid (TNA), wherein the LNP comprises an apolipoprotein E (ApoE) polypeptide, or a fragment thereof, and / or an apolipoprotein B (ApoB) polypeptide, or a fragment thereof, bound to the LNP, and at least one pharma- ceutically acceptable excipient. According to some embodiments, the ApoE polypeptide, or a fragment thereof, and / or the ApoB polypeptide, or a fragment thereof, can bind to a low-density lipoprotein (LDL) receptor or an LDL receptor family member. According to further embodiments, the LNP comprises an ApoE polypeptide, or a fragment thereof. According to some embodiments of any of the embodiments herein, the LNP comprises an ApoB polypeptide, or a fragment thereof. According to some embodiments of any of the embodiments herein, the ApoE polypeptide comprises an amino acid sequence of EELRVRLASHLRKLRKRLLRDADDLQKGGC (SEQ ID NO: 1) or has at least 80% sequence similarity to the amino acid sequence set forth in SEQ ID NO: 1. According to some embodiments, the ApoE polypeptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity to the amino acid sequence set forth in SEQ ID NO: 1. According to some embodiments, the ApoE polypeptide has at least 85% sequence similarity to the amino acid sequence set forth in SEQ ID NO: 1. According to some embodiments, the ApoE polypeptide has at least 90% sequence similarity to the amino acid sequence set forth in SEQ ID NO: 1. According to some embodiments, the ApoE polypeptide has at least 95% sequence similarity to the amino acid sequence set forth in SEQ ID NO: 1. According to some embodiments, the ApoE polypeptide has at least 99% sequence similarity to the amino acid sequence set forth in SEQ ID NO:1.According to some embodiments, the ApoE polypeptide consists of SEQ ID NO: 1. According to some embodiments of any of the embodiments herein, the ApoE polypeptide has at least 80% sequence similarity to the amino acid sequence set forth in SEQ ID NO: 3. According to some embodiments, the ApoE polypeptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity to the amino acid sequence set forth in SEQ ID NO: 3. According to some embodiments, the ApoE polypeptide has at least 85% sequence similarity to the amino acid sequence set forth in SEQ ID NO: 3. According to some embodiments, the ApoE polypeptide has at least 90% sequence similarity to the amino acid sequence set forth in SEQ ID NO: 3. According to some embodiments, the ApoE polypeptide has at least 95% sequence similarity to the amino acid sequence set forth in SEQ ID NO: 3. According to some embodiments, the ApoE polypeptide has at least 99% sequence similarity to the amino acid sequence shown in SEQ ID NO:3. According to some embodiments, the ApoE polypeptide comprises SEQ ID NO:3. According to some embodiments, the ApoE polypeptide consists of SEQ ID NO:3. According to some embodiments of any of the embodiments herein, the ApoE polypeptide bound to the LNP is a fragment of EELRVRLASHLRKLRKRLLRDADDLQKGGC shown in SEQ ID NO:1, which fragment is capable of binding to the LDL receptor. According to some embodiments of any of the embodiments herein, the ApoE polypeptide bound to the LNP is a fragment of EELRVRLASHLRKLRKRLLRDADDLQKGGC shown in SEQ ID NO:3, which fragment is capable of binding to the LDL receptor. According to some embodiments, the LNP is internalized into the cell. According to some embodiments of any of the embodiments herein, the ApoB polypeptide comprises the amino acid sequence SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGSGGC (SEQ ID NO:2) or has at least 80% sequence similarity to SEQ ID NO:2.According to some embodiments, the ApoB polypeptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity to the amino acid sequence shown in SEQ ID NO:2. According to some embodiments, the ApoB polypeptide has at least 85% sequence similarity to the amino acid sequence shown in SEQ ID NO:2. According to some embodiments, the ApoB polypeptide has at least 90% sequence similarity to the amino acid sequence shown in SEQ ID NO:2. According to some embodiments, the ApoB polypeptide has at least 95% sequence similarity to the amino acid sequence shown in SEQ ID NO:2. According to some embodiments, the ApoB polypeptide has at least 99% sequence similarity to the amino acid sequence shown in SEQ ID NO:2. According to some embodiments, the ApoB polypeptide has an amino acid sequence consisting of SEQ ID NO:2. According to some embodiments, the ApoB polypeptide consists of SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGSGGC (SEQ ID NO: 4). According to some embodiments of the aspects and embodiments herein, the ApoB polypeptide comprises the amino acid sequence of SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGSGGC (SEQ ID NO: 4) or has at least 80% sequence similarity to the amino acid sequence set forth in SEQ ID NO: 4. According to some embodiments, the ApoB polypeptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity to the amino acid sequence set forth in SEQ ID NO: 4. According to some embodiments, the ApoB polypeptide has at least 85% sequence similarity to the amino acid sequence set forth in SEQ ID NO: 4. According to some embodiments, the ApoB polypeptide has at least 90% sequence similarity to the amino acid sequence set forth in SEQ ID NO:4.According to some embodiments, the ApoB polypeptide has at least 95% sequence similarity to the amino acid sequence set forth in SEQ ID NO: 4. According to some embodiments, the ApoB polypeptide has at least 99% sequence similarity to the amino acid sequence set forth in SEQ ID NO: 4. According to some embodiments, the ApoB polypeptide consists of SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGSGGC (SEQ ID NO: 4). According to some embodiments of the aspects and embodiments herein, the ApoB polypeptide bound to the LNP is a fragment of EELRVRLASHLRKLRKRLLRDADDLQKGGC set forth in SEQ ID NO: 2, which fragment is capable of binding to the LDL receptor. According to some embodiments of the aspects and embodiments herein, the ApoB polypeptide bound to the LNP is a fragment of EELRVRLASHLRKLRKRLLRDADDLQKGG set forth in SEQ ID NO: 4, which fragment is capable of binding to the LDL receptor. According to some embodiments, the LNP is internalized into the cell. According to some embodiments of the aspects and embodiments herein, the LNP comprises a lipid selected from the group consisting of a cationic lipid, a sterol or a derivative thereof, a non-cationic lipid, and at least one PEGylated lipid.

[0009] According to some embodiments of the aspects and embodiments herein, the TNA is encapsulated in the LNP. According to some embodiments of the aspects and embodiments herein, the TNA is selected from the group consisting of minigenes, plasmids, minicircles, small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotide (ASO), ribozymes, closed-ended (ceDNA), ministrings, doggybone™, protelomeric closed-end DNA or dumbbell linear DNA, dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, gRNA, DNA virus vector, viral RNA vector, non-viral vector, and any combination thereof. According to some embodiments, the TNA is ceDNA. According to some embodiments, the ceDNA is linear double-stranded DNA. According to some embodiments, the TNA is mRNA. According to some embodiments, the TNA is an siRNA. According to some embodiments, the TNA is a plasmid.

[0010] According to some embodiments of the aspects and embodiments herein, the LNP comprises a PEGylated lipid, and the PEGylated lipid is attached to an ApoE polypeptide, or a fragment thereof, or the PEGylated lipid is attached to an ApoB polypeptide, or a fragment thereof. According to some embodiments, the ApoE polypeptide, or a fragment thereof, or the ApoB polypeptide, or a fragment thereof, is chemically conjugated to the PEGylated lipid.

[0011] According to some aspects and embodiments herein, the pharmaceutical composition is administered to a subject. According to some embodiments, the subject is a human patient in need of treatment with TNA-encapsulated LNPs.

[0012] According to some embodiments of the aspects and embodiments herein, the composition is delivered to LDLR expressing tissues via binding of ApoE and / or ApoB polypeptides present in the LNP to the LDLR receptor. According to some embodiments of the aspects and embodiments herein, the composition is delivered to retinal cells in the eye. According to some embodiments of the aspects and embodiments herein, the composition is delivered to photoreceptor (PR) cells. According to some embodiments of the aspects and embodiments herein, the composition is delivered to retinal pigment epithelium (RPE) cells. According to some embodiments of the aspects and embodiments herein, the composition is delivered to photoreceptor (PR) cells and retinal pigment epithelium (RPE) cells, and the expression of TNA in PR cells and the expression of TNA in RPE cells are uniformly distributed. According to some embodiments of the aspects and embodiments herein, the composition is delivered to hepatocytes in the liver. According to some embodiments of the aspects and embodiments herein, the composition is internalized in photoreceptor (PR) cells. According to some embodiments of the aspects and embodiments herein, the composition is internalized into retinal pigment epithelial (RPE) cells. According to some embodiments of the aspects and embodiments herein, the composition is internalized into hepatocytes.

[0013] According to some embodiments, the cationic lipid is represented by formula (I):

[0014] [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 C1~6 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 is an 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’ is an 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’ are each independently -CR a , -C(R a ) 2 CR a , or -[C(R a ) 2 ] 2 CR a and R a is independently H or C for each occurrence 1~3 Is it an alkyl group? or alternatively, R 4 But -C(R a ) 2 CR a , or -[C(R a ) 2 ] 2 CR 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 ) 2 CR a, or -[C(R a ) 2 ] 2 CR 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 each independently hydrogen, C 1~20 Alkylene or C 2~20 alkenylene, R 6 and R 6’ For each occurrence, 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.

[0015] According to some embodiments, the cationic lipid is represented by formula (II):

[0016] [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 (C 2 ~C 20 ) alkenyl, -C(O)O(C 2 ~C 20 ) alkyl, and (C 2 ~C 20 ) cyclopropyl substituted with alkyl; R 2 is (C 2 ~C 20 ) alkyl.

[0017] According to some embodiments, the cationic lipid has the following structural formula:

[0018] [ka] The compound is 1-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-(oleoyloxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanyl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl) 9-(tridecan-5-yl)nonanediate (Lipid 58), represented by the formula:

[0019] According to some embodiments, the lipid is represented by formula (V):

[0020] [ka] or a pharma- ceutically acceptable salt thereof, wherein: R 1 and R 1’ are each independently R a (C 1 ~C 6 ) alkylene; R 2 and R 2’ are each independently 1 ~C 2 ) alkylene; R 3 and R 3’ are each independently R b (C 1 ~C 6 ) alkyl, 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' are interrupted by -C(O)O- (C 2 ~C 6 ) alkylene; R 5 and R 5 ' are each independently 2 ~C 30 ) alkyl or (C 2 ~C 30 )alkenyl, each of which is optionally -C(O)O- or (C 3 ~C 6 ) interrupted by cycloalkyl, R a and R b are each halo or cyano.

[0021] According to some embodiments, the cationic lipid is represented by formula (XV):

[0022] [ka] or a pharma- ceutically acceptable salt thereof, wherein: R' is absent, hydrogen, or C 1 ~C 6 alkyl, where R' is hydrogen or C 1 ~C 6 When R', R 1 , and R 2 The nitrogen atom to which all are attached is protonated, R 1 and R 2 are each independently hydrogen, C 1 ~C 6 Alkyl or C 2 ~C 6 alkenyl, R 3 is C 1 ~C 12 Alkylene or C 2 ~C 12 alkenylene, R 4 is C 1 ~C 16 Unbranched alkyl, C2 ~C 16 unbranched alkenyl, or

[0023] [ka] where: R 4a and R 4b are each independently 1 ~C 16 Unbranched alkyl or C 2 ~C 16 is an unbranched alkenyl; R 5 does not exist or C 1 ~C 8 Alkylene or C 2 ~C 8 alkenylene, R 6a and R 6b are each independently 7 ~C 16 Alkyl or C 7 ~C 16 alkenyl, where R 6a and R 6b the total number of carbon atoms in the X 1 and X 2 each independently represents -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 ) 2 O-, -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 C 1 ~C 6 is alkyl, n is an integer selected from 1, 2, 3, 4, 5, and 6.

[0024] According to some embodiments, the cationic lipid is represented by formula (XX):

[0025] [ka] or a pharma- ceutically acceptable salt thereof, wherein: R' is absent, hydrogen, or C 1 ~C 3 alkyl, where R' is hydrogen or C 1 ~C 3 When R', R 1 , and R 2 The nitrogen atom to which all are attached is protonated, R 1 and R 2 are each independently hydrogen or C 1 ~C 3 is alkyl, R 3 is C 3 ~C 10 Alkylene or C 3 ~C 10 alkenylene, R 4 is C 1 ~C 16 Unbranched alkyl, C 2 ~C 16 unbranched alkenyl, or

[0026] [ka] where: R 4a and R 4b are each independently 1 ~C 16Unbranched alkyl or C 2 ~C 16 is an unbranched alkenyl; R 5 does not exist or C 1 ~C 6 Alkylene or C 2 ~C 6 alkenylene, R 6a and R 6b are each independently 7 ~C 14 Alkyl or C 7 ~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 ) 2 O-, -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 C 1 ~C 6 is alkyl, n is an integer selected from 1, 2, 3, 4, 5, and 6.

[0027] According to some embodiments, the cationic lipid is selected from any of the lipids in Table 2, Table 5, Table 6, Table 7, or Table 8.

[0028] According to some embodiments, the lipid is lipid A, represented by the structure:

[0029] [ka] or a pharma- ceutically acceptable salt thereof.

[0030] According to 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 structure:

[0031] [ka]

[0032] or a pharma- ceutically acceptable salt thereof.

[0033] According to some embodiments, the sterol or derivative thereof is cholesterol.

[0034] According to some embodiments, the sterol or derivative thereof is beta-sitosterol.

[0035] 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 ( 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-transPE, 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).

[0036] According to some embodiments, the PEGylated lipids are PEG-dilauryloxypropyl; PEG-dimyristyloxypropyl; PEG-dipalmityloxypropyl; PEG-distearyloxypropyl; 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (DMG-PEG); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azide(polyethylene glycol)], and distearoyl-rac-glycerol-poly(ethylene glycol). glycol), DSG-PEG;PEG-dilaurylglycerol;PEG-dipalmitoylglycerol;PEG-disterylglycerol;PEG-dilaurylglycamide;PEG-dimyristylglycamide;PEG-dipalmitoylglycamide;PEG-disterylglycamide;(l-[8'-(cholest-5-ene-3[beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol)(PEG-cholesterol);3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether(3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) glycol)ether, PEG-DMB; and l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol), DSPE-PEG; 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-hydroxyl, DSPE-PEG-OH;and l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-azide (DSPE-PEG-azide). According to some embodiments, the PEGylated lipid is DMG-PEG, DSPE-PEG, DSPE-PEG-OH, DSPE-PEG-azide, DSG-PEG, or a combination thereof. According to some embodiments, at least one PEGylated lipid is DMG-PEG2000, DSPE-PEG2000, DSPE-PEG2000-OH, DSPE-PEG2000-azide, DSG-PEG2000, or a combination thereof;

[0037] According to some embodiments of the aspects and embodiments herein, the ApoE polypeptide, or fragment thereof, or the ApoB polypeptide, or fragment thereof, is covalently attached to a PEGylated lipid of the LNP to form a PEGylated lipid conjugate. According to some embodiments, the PEGylated lipid to which the ApoE polypeptide, or fragment thereof, and / or the ApoB polypeptide, or fragment thereof, is covalently attached is DSPE-PEG or DSPE-PEG-azide.

[0038] According to some embodiments of the aspects and embodiments herein, the ApoE polypeptide, or fragment thereof, and / or the ApoB polypeptide, or fragment thereof, is covalently attached to the LNP via a non-cleavable linker. According to some embodiments, the non-cleavable linker is a maleimide-containing linker.

[0039] According to some embodiments of the aspects and embodiments herein, the ApoE polypeptide, or fragment thereof, and / or the ApoB polypeptide, or fragment thereof, is covalently attached to the LNP via a cleavable linker.

[0040] According to some embodiments of the aspects and embodiments herein, the ApoE polypeptide, or fragment thereof, and / or the ApoB polypeptide, or fragment thereof, is covalently attached to the LNP via a pyridyldisulfide (PDS)-containing linker.

[0041] According to some embodiments of the aspects and embodiments herein, the ApoE polypeptide, or fragment thereof, and / or the ApoB polypeptide, or fragment thereof, are covalently attached to the LNP via strain promoted alkyne-azide cycloaddition (SPAAC) chemistry. According to some embodiments, the SPAAC chemistry comprises a reaction between a cyclooctyne or a derivative thereof and an azide compound. According to some embodiments, the cyclooctyne or a derivative thereof is dibenzocyclooctyne (DBCO) or a derivative thereof. According to some embodiments, the DBCO or a derivative thereof is a DBCO-functionalized ApoE polypeptide or a DBCO-functionalized ApoB polypeptide. According to some embodiments, the DBCO-functionalized ApoE polypeptide or a DBCO-functionalized ApoB polypeptide is represented by the following structure:

[0042] [ka]

[0043] According to some embodiments of the aspects and embodiments herein, the azide compound is DSPE-PEG2000-azide or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethylene glycol)-2000], or a salt thereof.

[0044] According to some embodiments of the aspects and embodiments herein, the ApoE polypeptide, or fragment thereof, and / or the ApoB polypeptide, or fragment thereof, is bound to the LNP via one or more non-covalent interactions selected from hydrogen bonds, van der Waals bonds, ionic bonds, and hydrophobic bonds.

[0045] According to some embodiments of the aspects and embodiments herein, the cationic lipid is present in a molar percentage of about 30% to about 80%.

[0046] According to some embodiments of the aspects and embodiments herein, the sterol is present in a molar percentage of about 20% to about 50%.

[0047] According to some embodiments of the aspects and embodiments herein, the non-cationic lipid is present in a molar percentage of about 2% to about 20%.

[0048] According to some embodiments of the aspects and embodiments herein, the at least one PEGylated lipid is present in a molar percentage of about 2.1% to about 10%, or the at least one PEGylated lipid is present in a molar percentage of about 1% to about 2%.

[0049] According to some embodiments of the aspects and embodiments herein, the ApoE and / or ApoB polypeptides are present in a total amount of about 0.02 μg / μg TNA to about 0.1 μg / μg TNA.

[0050] According to some embodiments of the aspects and embodiments herein, the pharmaceutical composition further comprises dexamethasone palmitate.

[0051] According to some embodiments of the aspects and embodiments herein, The LNPs include lipid A, DOPC, cholesterol, and DMG-PEG. According to some embodiments of the aspects and embodiments herein, the LNPs include lipid A, DOPC, cholesterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide. According to some embodiments of the aspects and embodiments herein, the LNPs include lipid A, DOPE, cholesterol, and DMG-PEG. According to some embodiments of the aspects and embodiments herein, the LNPs include lipid A, DOPE, cholesterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide. According to some embodiments of the aspects and embodiments herein, the LNPs include lipid A, DSPC, cholesterol, and DMG-PEG. According to some embodiments of the aspects and embodiments herein, the LNPs include lipid A, DSPC, cholesterol, and DMG-PEG. According to some embodiments of the aspects and embodiments herein, the LNPs include lipid A, DSPC, cholesterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide. According to some embodiments of the aspects and embodiments herein, the LNPs include lipid A, DOPC, beta-sitosterol, and DMG-PEG. According to some embodiments of the aspects and embodiments herein, the LNPs include lipid A, DOPC, beta-sitosterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide. According to some embodiments of the aspects and embodiments herein, the LNPs include lipid A, DOPE, beta-sitosterol, and DMG-PEG. According to some embodiments of the aspects and embodiments herein, the LNPs include lipid A, DOPE, beta-sitosterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide. According to some embodiments of the aspects and embodiments herein, the LNPs include lipid A, DSPC, beta-sitosterol, and DMG-PEG. According to some embodiments of the aspects and embodiments herein, the LNPs include lipid A, DSPC, beta-sitosterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide.

[0052] According to some embodiments of the aspects and embodiments herein, the DMG-PEG is DMG-PEG2000. According to some embodiments of the aspects and embodiments herein, the DSPE-PEG is DSPE-PEG2000 or DSPE-PEG5000. According to some embodiments of the aspects and embodiments herein, the DSPE-PEG-azide is DSPE-PEG2000-azide or DSPE-PEG5000-azide. According to some embodiments, the LNP comprises lipid A, DOPC, a sterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide in a molar ratio of about 51:7.3:38.3:2.9:0.5.

[0053] According to some embodiments of the aspects and embodiments herein, the LNP has the following structural formula:

[0054] [ka] The compound is 1-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-(oleoyloxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanyl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl) 9-(tridecan-5-yl)nonanediate (Lipid 58), represented by the formula:

[0055] According to some embodiments of the aspects and embodiments herein, the LNPs have a total lipid to TNA ratio of about 10:1 to about 40:1.

[0056] In another aspect, the present disclosure provides a pharmaceutical composition comprising a lipid nanoparticle (LNP), a therapeutic messenger RNA (mRNA), and at least one pharma- ceutically acceptable excipient, wherein the LNP comprises: an ApoE polypeptide or a fragment thereof and / or an ApoB polypeptide or a fragment thereof bound to an LNP; The following structural formula:

[0057] [ka] and a cationic lipid having the formula: (Lipid A) A pharmaceutical composition is provided in which the LNPs are capable of delivering mRNA to retinal cells.

[0058] According to some embodiments, the LNPs can deliver mRNA to photoreceptor (PR) cells. According to some embodiments of the aspects and embodiments herein, the LNPs can deliver mRNA to retinal pigment epithelium (RPE) cells. According to some embodiments of the aspects and embodiments herein, the LNPs can be internalized into PR cells and / or RPE cells. According to some embodiments of the aspects and embodiments herein, e mRNA expression is uniformly distributed in PR cells and RPE cells. According to some embodiments of the aspects and embodiments herein, the LNPs can deliver mRNA to retinal cells without causing retinal degeneration or thinning of the outer nuclear layer (ONL). According to some embodiments of the aspects and embodiments herein, the ApoE polypeptide, or a fragment thereof, and / or the ApoB polypeptide, or a fragment thereof, can bind to a low density lipoprotein (LDL) receptor or an LDL receptor family member. According to some embodiments of the aspects and embodiments herein, the LNPs include an ApoE polypeptide, or a fragment thereof. According to some embodiments of the aspects and embodiments herein, the LNP comprises an ApoB polypeptide, or a fragment thereof. According to some embodiments, the ApoE polypeptide comprises an amino acid sequence of EELRVRLASHLRKLRKRLLRDADDLQKGG (SEQ ID NO: 3), or has at least 80% sequence similarity to the amino acid sequence set forth in SEQ ID NO: 3. According to some embodiments, the ApoE polypeptide has at least 85%, at least 90%, at least 95%, or at least 99% sequence similarity to the amino acid sequence set forth in SEQ ID NO: 3. According to some embodiments, the ApoE polypeptide consists of EELRVRLASHLRKLRKRLLRDADDLQKGG (SEQ ID NO: 3). According to some embodiments of the aspects and embodiments herein, the ApoE polypeptide bound to the LNP is a fragment of EELRVRLASHLRKLRKRLLRDADDLQKGGC set forth in SEQ ID NO: 3, where the fragment is capable of binding to the LDL receptor.According to some embodiments, the ApoB polypeptide comprises an amino acid sequence of SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGSGGC (SEQ ID NO: 4) or has at least 80% sequence similarity to the amino acid sequence set forth in SEQ ID NO: 4. According to some embodiments, the ApoB polypeptide has at least 85%, at least 90%, at least 95%, or at least 99% sequence similarity to the amino acid sequence set forth in SEQ ID NO: 4. According to some embodiments, the ApoB polypeptide consists of SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGSGGC (SEQ ID NO: 4). According to some embodiments of the aspects and embodiments herein, the ApoB polypeptide bound to the LNP is a fragment of EELRVRLASHLRKLRKRLLRDADDLQKGG set forth in SEQ ID NO: 4, wherein the fragment is capable of binding to the LDL receptor.

[0059] According to some embodiments of the aspects and embodiments herein, the mRNA is encapsulated in the LNP.

[0060] According to some embodiments of the aspects and embodiments herein, the LNP further comprises a lipid selected from the group consisting of a sterol or a derivative thereof, a non-cationic lipid, and at least one PEGylated lipid. According to some embodiments, the sterol or a derivative thereof is cholesterol. According to some embodiments, the sterol or a derivative thereof is beta-sitosterol.

[0061] According to some embodiments of the aspects and embodiments herein, the non-cationic lipid is selected from the group consisting of dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), and dioleoyl-phosphatidylethanolamine (DOPE).

[0062] According to some embodiments of the aspects and embodiments herein, the PEGylated lipid is DMG-PEG, DSPE-PEG, DSPE-PEG-OH, DSPE-PEG-azide, DSG-PEG, or a combination thereof. According to some embodiments, at least one PEGylated lipid is DMG-PEG2000, DSPE-PEG2000, DSPE-PEG2000-OH, DSPE-PEG-azide, DSG-PEG, or a combination thereof. According to some embodiments, the LNPs are comprised of lipid A, DOPC, cholesterol, and DMG-PEG; or lipid A, DOPC, cholesterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide; or lipid A, DOPE, cholesterol, and DMG-PEG; lipid A, DOPE, cholesterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide; or lipid A, DSPC, cholesterol, and DMG-PEG; or lipid A, DOPC, cholesterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide; lipid A, DOPE, cholesterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide; OPC, beta-sitosterol and DMG-PEG; or lipid A, DOPC, beta-sitosterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide; or lipid A, DOPE, beta-sitosterol and DMG-PEG; or lipid A, DOPE, beta-sitosterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide; or lipid A, DSPC, beta-sitosterol and DMG-PEG; or lipid A, DOPC, beta-sitosterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide. According to some embodiments, the DMG-PEG is DMG-PEG2000. According to some embodiments of the aspects and embodiments herein, the DSPE-PEG is DSPE-PEG2000 or DSPE-PEG5000. According to some embodiments of the aspects and embodiments herein, the DSPE-PEG-azide is a DSPE-PEG2000-azide or a DSPE-PEG5000-azide.According to some embodiments, the LNPs comprise lipid A, DOPC, a sterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide in a molar ratio of about 51:7.3:38.3:2.9:0.5.

[0063] According to some embodiments of the aspects and embodiments herein, the LNP comprises a PEGylated lipid, and the PEGylated lipid is conjugated to an ApoE polypeptide, or a fragment thereof, or the PEGylated lipid is conjugated to an ApoB polypeptide, or a fragment thereof. According to some embodiments, the ApoE polypeptide, or a fragment thereof, or the ApoB polypeptide, or a fragment thereof, is covalently attached to the PEGylated lipid of the LNP to form a PEGylated lipid conjugate. According to some embodiments, the PEGylated lipid to which the ApoE polypeptide, or a fragment thereof, and / or the ApoB polypeptide, or a fragment thereof, is covalently attached is DSPE-PEG or DSPE-PEG-azide.

[0064] According to some embodiments of the aspects and embodiments herein, the ApoE polypeptide, or fragment thereof, and / or the ApoB polypeptide, or fragment thereof, is covalently attached 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 aspects and embodiments herein, the ApoE polypeptide, or fragment thereof, and / or the ApoB polypeptide, or fragment thereof, is covalently attached to the LNP via a cleavable linker.

[0066] According to some embodiments of the aspects and embodiments herein, the ApoE polypeptide, or fragment thereof, and / or the ApoB polypeptide, or fragment thereof, is covalently attached to the LNP via a pyridyl disulfide (PDS)-containing linker.

[0067] According to some embodiments of the aspects and embodiments herein, the ApoE polypeptide, or fragment thereof, and / or the ApoB polypeptide, or fragment thereof, are covalently attached to the LNP via strain-promoted alkyne-azide cycloaddition (SPAAC) chemistry.

[0068] According to some embodiments of the aspects and embodiments herein, the pharmaceutical composition is administered to the subject via subretinal, suprachoroidal, or intravitreal injection. According to some embodiments, the pharmaceutical composition is administered to the subject via subretinal injection.

[0069] According to another aspect, the present disclosure provides a dibenzocyclooctyne (DBCO)-functionalized ApoE or ApoB polypeptide represented by the structure:

[0070] [ka] During the ceremony, the ApoE polypeptide comprises an amino acid sequence of EELRVRLASHLRKLRKRLLRDADDLQKGG (SEQ ID NO:3) or has at least 80% sequence similarity to the amino acid sequence set forth in SEQ ID NO:3; A DBCO-functionalized ApoE or ApoB polypeptide is provided, wherein the ApoB polypeptide comprises an amino acid sequence of SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGSGGC (SEQ ID NO:4) or has at least 80% sequence similarity to the amino acid sequence set forth in SEQ ID NO:4.

[0071] According to some embodiments, pharmaceutical compositions are prepared using DBCO-functionalized ApoE or ApoB polypeptides in combination with an azide compound.

[0072] According to another aspect, the present disclosure provides lipid nanoparticle compositions prepared using the DBCO-functionalized ApoE or ApoB polypeptides of the aspects and embodiments herein in combination with an azide compound. According to some embodiments, the azide compound is DSPE-PEG2000-azide or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethylene glycol)-2000], or a salt thereof.

[0073] According to some embodiments of the aspects and embodiments herein, the LNPs have a diameter in the range of about 40 nm to about 120 nm.

[0074] According to some embodiments of the aspects and embodiments herein, the nanoparticles have a diameter of less than about 100 nm.

[0075] According to some embodiments of the aspects and embodiments herein, the nanoparticles have a diameter of about 60 nm to about 80 nm.

[0076] According to another aspect, the present disclosure provides a method for treating a genetic disorder in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition or lipid composition according to any one of the aspects or embodiments herein. According to some embodiments, the subject is a human. According to some embodiments of the present invention and its embodiments herein, the disorder is an eye disorder. According to some embodiments of the invention and embodiments thereof herein, the genetic disorder is sickle cell anemia, melanoma, hemophilia A (clotting factor VIII (FVIII) deficiency) and hemophilia B (clotting factor IX (FIX) deficiency), cystic fibrosis (CFTR), familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, phenylketonuria (PKU), congenital hepatic porphyria, inherited liver metabolic disorders, Lesch-Nyhan syndrome, sickle cell anemia, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom's syndrome, retinoblastoma, mucopolysaccharidosis (e.g., Hurler syndrome (MPS I), Scheie syndrome (MPS IS), Hurler-Scheie syndrome (MPS I), HS), Hunter syndrome (MPS II), Sanfilippo types A, B, C, and D (MPS III A, B, C, and D), Morquio types A and B (MPS IVA and MPS IVB), Maroteaux-Lamy syndrome (MPS VI), Sly syndrome (MPS VII), Hyaluronidase deficiency (MPS IX), Niemann-Pick disease A / B, C1 and C2, Fabry disease, Schindler disease, GM2-gangliosidosis type II (Sandhoff disease), Tay-Sachs disease, Metachromatic leukodystrophy, Krabbe disease, Mucolipidosis types I, II / III and IV, Sialidosis types I and II, Glycogen storage disease types I and II (Pompe disease), Gaucher disease types I, II and III, Cystinosis, Batten disease, Aspartylglucosaminuria, Salla disease, Danon disease (LAMP-2 deficiency), Lysosomal Acid LipaseLipase (LAL) deficiency, neuronal ceroid lipofuscinosis (CLN1-8, INCL, and LINCL), sphingolipidosis, galactosialidosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy, Friedreich's ataxia, Duchenne muscular dystrophy (DMD), Becker muscular dystrophies (BMD), dystrophic epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, generalized arterial calcification of infancy infancy (GACI), Leber congenital amaurosis, Stargardt macular dystrophy (ABCA4), ornithine transcarbamylase (OTC) deficiency, Usher syndrome, age-related macular degeneration (AMD), alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis (PFIC) type I (ATP8B1 deficiency), type II (ABCB11), type III (ABCB4), or type IV (TJP2), and cathepsin A deficiency.

[0077] According to some embodiments, the genetic disorder is hemophilia A. According to some embodiments, the genetic disorder is hemophilia B. According to some embodiments, the genetic disorder is phenylketonuria (PKU). According to some embodiments, the genetic disorder is Wilson's disease. According to some embodiments, the genetic disorder is Gaucher disease type I, II or III. According to some embodiments, the genetic disorder is Stargardt's macular dystrophy. According to some embodiments, the genetic disorder is LCA10. According to some embodiments, the genetic disorder is Usher syndrome. According to some embodiments, the genetic disorder is wet AMD.

[0078] According to another aspect, the present disclosure provides a method of delivering a therapeutic nucleic acid (TNA) to the retina of a subject or increasing the concentration of a TNA in the retina of a subject, comprising administering to the subject an effective amount of a pharmaceutical composition or lipid nanoparticle composition of any one of the aspects or embodiments herein.

[0079] According to another aspect, 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 or lipid nanoparticle composition of any one of the aspects or embodiments herein. [Brief description of the drawings]

[0080]

[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 1A]Graphs are presented showing that LNP-delivered mRNA and LNP-delivered ceDNA were tolerated in both mice (lower panel, FIG. 1B and FIG. 1D) and rats (upper panel, FIG. 1A and FIG. 1C). Degeneration scores are shown at 21 days. [Figure 1B] Graphs are presented showing that LNP-delivered mRNA and LNP-delivered ceDNA were tolerated in both mice (lower panel, FIG. 1B and FIG. 1D) and rats (upper panel, FIG. 1A and FIG. 1C). Degeneration scores are shown at 21 days. [Figure 1C] Graphs are presented showing that LNP-delivered mRNA and LNP-delivered ceDNA were tolerated in both mice (lower panel, FIG. 1B and FIG. 1D) and rats (upper panel, FIG. 1A and FIG. 1C). Degeneration scores are shown at 21 days. [Figure 1D] Graphs are presented showing that LNP-delivered mRNA and LNP-delivered ceDNA were tolerated in both mice (lower panel, FIG. 1B and FIG. 1D) and rats (upper panel, FIG. 1A and FIG. 1C). Degeneration scores are shown at 21 days. [Figure 2A] The results of fundus imaging for GFP expression in rats and mice at 24 hours are shown, as described in Example 2. Figures 2A-2I show fundus imaging of lipid A LNP / GFP mRNA treated mice (0.4 μg) (Group 2 described in Table 9, Figures 2A-2E) compared to untreated control mice (Group 1 described in Table 9, Figures 2F-2I). [Figure 2B] The results of fundus imaging for GFP expression in rats and mice at 24 hours are shown, as described in Example 2. Figures 2A-2I show fundus imaging of lipid A LNP / GFP mRNA treated mice (0.4 μg) (Group 2 described in Table 9, Figures 2A-2E) compared to untreated control mice (Group 1 described in Table 9, Figures 2F-2I). [Figure 2C]The results of fundus imaging for GFP expression in rats and mice at 24 hours are shown, as described in Example 2. Figures 2A-2I show fundus imaging of lipid A LNP / GFP mRNA treated mice (0.4 μg) (Group 2 described in Table 9, Figures 2A-2E) compared to untreated control mice (Group 1 described in Table 9, Figures 2F-2I). [Figure 2D] The results of fundus imaging for GFP expression in rats and mice at 24 hours are shown, as described in Example 2. Figures 2A-2I show fundus imaging of lipid A LNP / GFP mRNA treated mice (0.4 μg) (Group 2 described in Table 9, Figures 2A-2E) compared to untreated control mice (Group 1 described in Table 9, Figures 2F-2I). [Figure 2E] The results of fundus imaging for GFP expression in rats and mice at 24 hours are shown, as described in Example 2. Figures 2A-2I show fundus imaging of lipid A LNP / GFP mRNA treated mice (0.4 μg) (Group 2 described in Table 9, Figures 2A-2E) compared to untreated control mice (Group 1 described in Table 9, Figures 2F-2I). [Figure 2F] The results of fundus imaging for GFP expression in rats and mice at 24 hours are shown, as described in Example 2. Figures 2A-2I show fundus imaging of lipid A LNP / GFP mRNA treated mice (0.4 μg) (Group 2 described in Table 9, Figures 2A-2E) compared to untreated control mice (Group 1 described in Table 9, Figures 2F-2I). [Figure 2G] The results of fundus imaging for GFP expression in rats and mice at 24 hours are shown, as described in Example 2. Figures 2A-2I show fundus imaging of lipid A LNP / GFP mRNA treated mice (0.4 μg) (Group 2 described in Table 9, Figures 2A-2E) compared to untreated control mice (Group 1 described in Table 9, Figures 2F-2I). [Figure 2H]The results of fundus imaging for GFP expression in rats and mice at 24 hours are shown, as described in Example 2. Figures 2A-2I show fundus imaging of lipid A LNP / GFP mRNA treated mice (0.4 μg) (Group 2 described in Table 9, Figures 2A-2E) compared to untreated control mice (Group 1 described in Table 9, Figures 2F-2I). [Figure 2I] The results of fundus imaging for GFP expression in rats and mice at 24 hours are shown, as described in Example 2. Figures 2A-2I show fundus imaging of lipid A LNP / GFP mRNA treated mice (0.4 μg) (Group 2 described in Table 9, Figures 2A-2E) compared to untreated control mice (Group 1 described in Table 9, Figures 2F-2I). [Diagram 3] FIG. 13 is a graph showing the amount of GFP in the neural retina and RPE / eye cup as determined by ELISA at 12 and 24 hours post-treatment following administration of lipid A LNP / GFP mRNA (0.4 μg) to wild-type mice (Group 2 in Table 9). [Figure 4A] A comparison of GFP expression patterns in lipid A LNPs / GFP mRNA (0.4 μg) (FIG. 4B) compared to GFP transgenic mice (FIG. 4A). [Figure 4B] A comparison of GFP expression patterns in lipid A LNPs / GFP mRNA (0.4 μg) (FIG. 4B) compared to GFP transgenic mice (FIG. 4A). [Figure 5A] A comparison of GFP expression patterns in lipid A LNPs / GFP mRNA (0.4 μg) (FIG. 5B) compared to untreated vehicle control mice (FIG. 5A). [Figure 5B] A comparison of GFP expression patterns in lipid A LNPs / GFP mRNA (0.4 μg) (FIG. 5B) compared to untreated vehicle control mice (FIG. 5A). [Figure 6] 6 is a graph quantifying GFP expression.FIG 6 shows that lipid A LNP / GFP mRNA delivery in mice resulted in uniform distribution of GFP expression within the neural retina and optic cup. [Figure 7A] Figures 7A-7D show GFP expression in the neural retina and RPE in mice treated with lipid A LNP / GFP mRNA (0.4 μg) and AAV.GFP (AAV5-CAG-GFP) as described in Example 2. Figures 7A-7D are images showing results from immunohistochemistry (IHC). Figure 7E is a graph quantifying the results. [Figure 7B] Figures 7A-7D show GFP expression in the neural retina and RPE in mice treated with lipid A LNP / GFP mRNA (0.4 μg) and AAV.GFP (AAV5-CAG-GFP) as described in Example 2. Figures 7A-7D are images showing results from immunohistochemistry (IHC). Figure 7E is a graph quantifying the results. [Figure 7C] Figures 7A-7D show GFP expression in the neural retina and RPE in mice treated with lipid A LNP / GFP mRNA (0.4 μg) and AAV.GFP (AAV5-CAG-GFP) as described in Example 2. Figures 7A-7D are images showing results from immunohistochemistry (IHC). Figure 7E is a graph quantifying the results. [Figure 7D] Figures 7A-7D show GFP expression in the neural retina and RPE in mice treated with lipid A LNP / GFP mRNA (0.4 μg) and AAV.GFP (AAV5-CAG-GFP) as described in Example 2. Figures 7A-7D are images showing results from immunohistochemistry (IHC). Figure 7E is a graph quantifying the results. [Figure 7E] Figures 7A-7D show GFP expression in the neural retina and RPE in mice treated with lipid A LNP / GFP mRNA (0.4 μg) and AAV.GFP (AAV5-CAG-GFP) as described in Example 2. Figures 7A-7D are images showing results from immunohistochemistry (IHC). Figure 7E is a graph quantifying the results. [Figure 8A]Graphs quantifying GFP expression by ELISA in the neural retina (with photoreceptors or PRs) and eye cup (with retinal pigment epithelium or RPE cells) at increasing doses (0.2 μg, 0.4 μg, 1.0 μg) at 12 and 24 hours, with GFP concentration expressed as ng / eye (FIG. 8A) and ng / μg cargo (FIG. 8B). [Figure 8B] Graphs quantifying GFP expression by ELISA in the neural retina (with photoreceptors or PRs) and eye cup (with retinal pigment epithelium or RPE cells) at increasing doses (0.2 μg, 0.4 μg, 1.0 μg) at 12 and 24 hours, with GFP concentration expressed as ng / eye (FIG. 8A) and ng / μg cargo (FIG. 8B). [Figure 9A] 9 shows the results of fundus imaging in mouse and rat models as described in Example 4. When LNP-delivered mRNA such as lipid A LNP / GFP mRNA was dose-matched in mouse and rat models, GFP expression by the fundus in rats was found to be comparable to that by the fundus in mice. As shown in Figures 9E and 9F, lipid A LNP / GFP mRNA given to rats at medium and high doses (0.3 μg and 1.2 μg, respectively) achieved expression levels in rats that were comparable to that of lipid A LNP / GFP mRNA given to mice at medium and high doses (0.1 μg and 0.4 μg, respectively, see Figures 9B and 9C). [Figure 9B] 9 shows the results of fundus imaging in mouse and rat models as described in Example 4. When LNP-delivered mRNA such as lipid A LNP / GFP mRNA was dose-matched in mouse and rat models, GFP expression by the fundus in rats was found to be comparable to that by the fundus in mice. As shown in Figures 9E and 9F, lipid A LNP / GFP mRNA given to rats at medium and high doses (0.3 μg and 1.2 μg, respectively) achieved expression levels in rats that were comparable to that of lipid A LNP / GFP mRNA given to mice at medium and high doses (0.1 μg and 0.4 μg, respectively, see Figures 9B and 9C). [Figure 9C] 9 shows the results of fundus imaging in mouse and rat models as described in Example 4. When LNP-delivered mRNA such as lipid A LNP / GFP mRNA was dose-matched in mouse and rat models, GFP expression by the fundus in rats was found to be comparable to that by the fundus in mice. As shown in Figures 9E and 9F, lipid A LNP / GFP mRNA given to rats at medium and high doses (0.3 μg and 1.2 μg, respectively) achieved expression levels in rats that were comparable to that of lipid A LNP / GFP mRNA given to mice at medium and high doses (0.1 μg and 0.4 μg, respectively, see Figures 9B and 9C). [Figure 9D] 9 shows the results of fundus imaging in mouse and rat models as described in Example 4. When LNP-delivered mRNA such as lipid A LNP / GFP mRNA was dose-matched in mouse and rat models, GFP expression by the fundus in rats was found to be comparable to that by the fundus in mice. As shown in Figures 9E and 9F, lipid A LNP / GFP mRNA given to rats at medium and high doses (0.3 μg and 1.2 μg, respectively) achieved expression levels in rats that were comparable to that of lipid A LNP / GFP mRNA given to mice at medium and high doses (0.1 μg and 0.4 μg, respectively, see Figures 9B and 9C). [Figure 9E]9 shows the results of fundus imaging in mouse and rat models as described in Example 4. When LNP-delivered mRNA such as lipid A LNP / GFP mRNA was dose-matched in mouse and rat models, GFP expression by the fundus in rats was found to be comparable to that by the fundus in mice. As shown in Figures 9E and 9F, lipid A LNP / GFP mRNA given to rats at medium and high doses (0.3 μg and 1.2 μg, respectively) achieved expression levels in rats that were comparable to that of lipid A LNP / GFP mRNA given to mice at medium and high doses (0.1 μg and 0.4 μg, respectively, see Figures 9B and 9C). [Figure 9F] 9 shows the results of fundus imaging in mouse and rat models as described in Example 4. When LNP-delivered mRNA such as lipid A LNP / GFP mRNA was dose-matched in mouse and rat models, GFP expression by the fundus in rats was found to be comparable to that by the fundus in mice. As shown in Figures 9E and 9F, lipid A LNP / GFP mRNA given to rats at medium and high doses (0.3 μg and 1.2 μg, respectively) achieved expression levels in rats that were comparable to that of lipid A LNP / GFP mRNA given to mice at medium and high doses (0.1 μg and 0.4 μg, respectively, see Figures 9B and 9C). [Figure 10A] 10A-10D are images showing retinal degeneration in mice treated as described in Example 4. FIG. 10A shows vehicle treatment for reference. The images in FIG. 10B-10D show that one day after mice were administered increasing doses of LNP / GFP mRNA of lipid A at 0.03 μg, 0.1 μg, and 0.4 μg, no retinal degeneration occurred, thereby indicating a large tolerance window for LNP-delivered mRNA. [Figure 10B]10A-10D are images showing retinal degeneration in mice treated as described in Example 4. FIG. 10A shows vehicle treatment for reference. The images in FIG. 10B-10D show that one day after mice were administered increasing doses of LNP / GFP mRNA of lipid A at 0.03 μg, 0.1 μg, and 0.4 μg, no retinal degeneration occurred, thereby indicating a large tolerance window for LNP-delivered mRNA. [Figure 10C] 10A-10D are images showing retinal degeneration in mice treated as described in Example 4. FIG. 10A shows vehicle treatment for reference. The images in FIG. 10B-10D show that one day after mice were administered increasing doses of LNP / GFP mRNA of lipid A at 0.03 μg, 0.1 μg, and 0.4 μg, no retinal degeneration occurred, thereby indicating a large tolerance window for LNP-delivered mRNA. [Figure 10D] 10A-10D are images showing retinal degeneration in mice treated as described in Example 4. FIG. 10A shows vehicle treatment for reference. The images in FIG. 10B-10D show that one day after mice were administered increasing doses of LNP / GFP mRNA of lipid A at 0.03 μg, 0.1 μg, and 0.4 μg, no retinal degeneration occurred, thereby indicating a large tolerance window for LNP-delivered mRNA. [Figure 11A] FIG. 13 shows color fundus imaging of mouse eyes 2 days after treatment via subretinal injection of various LNP compositions formulated with GFP mRNA and different ionizable lipids listed in Table 12 (all at 0.2 μg dose). [Figure 11B] FIG. 13 shows color fundus imaging of mouse eyes 2 days after treatment via subretinal injection of various LNP compositions formulated with GFP mRNA and different ionizable lipids listed in Table 12 (all at 0.2 μg dose). [Figure 11C] FIG. 13 shows color fundus imaging of mouse eyes 2 days after treatment via subretinal injection of various LNP compositions formulated with GFP mRNA and different ionizable lipids listed in Table 12 (all at 0.2 μg dose). [Figure 11D]FIG. 13 shows color fundus imaging of mouse eyes 2 days after treatment via subretinal injection of various LNP compositions formulated with GFP mRNA and different ionizable lipids listed in Table 12 (all at 0.2 μg dose). [Figure 11E] FIG. 13 shows color fundus imaging of mouse eyes 2 days after treatment via subretinal injection of various LNP compositions formulated with GFP mRNA and different ionizable lipids listed in Table 12 (all at 0.2 μg dose). [Figure 11F] shows the corresponding cobalt blue fundus imaging (for GFP expression) of the same mouse eye sample. [Figure 11G] shows the corresponding cobalt blue fundus imaging (for GFP expression) of the same mouse eye sample. [Figure 11H] shows the corresponding cobalt blue fundus imaging (for GFP expression) of the same mouse eye sample. [Figure 11I] shows the corresponding cobalt blue fundus imaging (for GFP expression) of the same mouse eye sample. [Figure 11J] shows the corresponding cobalt blue fundus imaging (for GFP expression) of the same mouse eye sample. [Figure 12] 13 is a graph quantifying GFP expression in both the neural retina and the optic cup from the experiments performed in Example 5. [Figure 13A] 13A-13C are graphs showing inflammation and degeneration scores on day 1 in mouse eyes following treatment via subretinal injection with various LNP compositions formulated with GFP mRNA and different ionizable lipids (all at 0.2 μg doses), as described in this example. [Figure 13B] 13A-13C are graphs showing inflammation and degeneration scores on day 1 in mouse eyes following treatment via subretinal injection with various LNP compositions formulated with GFP mRNA and different ionizable lipids (all at 0.2 μg doses), as described in this example. [Figure 13C] These are graphs showing the inflammation score and degeneration score, respectively, of the same samples on day 1. [Figure 13D] These are graphs showing the inflammation score and degeneration score, respectively, of the same samples on day 1. [Figure 14A] 14A-14E are panels showing the results of OCT imaging on day 1 as described in Example 5. FIG. 14A shows a vehicle control. FIG. 14B shows lipid A LNP / GFP mRNA, FIG. 14C shows MC3 LNP / GFP mRNA, FIG. 14D and FIG. 14E show control (CTRL) lipid Z LNP 1 / GFP mRNA and control (CTRL) lipid Z LNP 2 / GFP mRNA, respectively, and FIG. 14F shows lipid 58 LNP / GFP mRNA. [Figure 14B] 14A-14E are panels showing the results of OCT imaging on day 1 as described in Example 5. FIG. 14A shows a vehicle control. FIG. 14B shows lipid A LNP / GFP mRNA, FIG. 14C shows MC3 LNP / GFP mRNA, FIG. 14D and FIG. 14E show control (CTRL) lipid Z LNP 1 / GFP mRNA and control (CTRL) lipid Z LNP 2 / GFP mRNA, respectively, and FIG. 14F shows lipid 58 LNP / GFP mRNA. [Figure 14C] 14A-14E are panels showing the results of OCT imaging on day 1 as described in Example 5. FIG. 14A shows a vehicle control. FIG. 14B shows lipid A LNP / GFP mRNA, FIG. 14C shows MC3 LNP / GFP mRNA, FIG. 14D and FIG. 14E show control (CTRL) lipid Z LNP 1 / GFP mRNA and control (CTRL) lipid Z LNP 2 / GFP mRNA, respectively, and FIG. 14F shows lipid 58 LNP / GFP mRNA. [Figure 14D] 14A-14E are panels showing the results of OCT imaging on day 1 as described in Example 5. FIG. 14A shows a vehicle control. FIG. 14B shows lipid A LNP / GFP mRNA, FIG. 14C shows MC3 LNP / GFP mRNA, FIG. 14D and FIG. 14E show control (CTRL) lipid Z LNP 1 / GFP mRNA and control (CTRL) lipid Z LNP 2 / GFP mRNA, respectively, and FIG. 14F shows lipid 58 LNP / GFP mRNA. [Figure 14E] 14A-14E are panels showing the results of OCT imaging on day 1 as described in Example 5. FIG. 14A shows a vehicle control. FIG. 14B shows lipid A LNP / GFP mRNA, FIG. 14C shows MC3 LNP / GFP mRNA, FIG. 14D and FIG. 14E show control (CTRL) lipid Z LNP 1 / GFP mRNA and control (CTRL) lipid Z LNP 2 / GFP mRNA, respectively, and FIG. 14F shows lipid 58 LNP / GFP mRNA. [Figure 14F] 14A-14E are panels showing the results of OCT imaging on day 1 as described in Example 5. FIG. 14A shows a vehicle control. FIG. 14B shows lipid A LNP / GFP mRNA, FIG. 14C shows MC3 LNP / GFP mRNA, FIG. 14D and FIG. 14E show control (CTRL) lipid Z LNP 1 / GFP mRNA and control (CTRL) lipid Z LNP 2 / GFP mRNA, respectively, and FIG. 14F shows lipid 58 LNP / GFP mRNA. [Figure 15A] 15A-15E are panels showing the results of OCT imaging on day 28 as described in Example 5. FIG. 15A shows the vehicle reference. On day 28, high degeneration scores of about 2.0 were observed in LNP compositions formulated with either MC3 or control (CTRL) lipid Z. Such high degeneration scores were supported by thinning of the outer nuclear layer (ONL) or retinal degeneration seen in OCT images taken on day 28 (see FIGS. 15C-15E). In contrast, on day 28, LNP / GFP mRNA with lipid A and LNP / GFP mRNA with lipid 58 recorded degeneration scores of less than 0.5 and about 1.0, respectively, and their corresponding OCT images in FIG. 15B and FIG. 15F (using the vehicle in FIG. 15A as a reference) demonstrate that the ONL layer maintained a healthy thickness. On day 1, none of the samples showed retinal degeneration, using the vehicle in FIG. 14A as a reference (see FIGS. 14A-14F). [Figure 15B]15A-15E are panels showing the results of OCT imaging on day 28 as described in Example 5. FIG. 15A shows the vehicle reference. On day 28, high degeneration scores of about 2.0 were observed in LNP compositions formulated with either MC3 or control (CTRL) lipid Z. Such high degeneration scores were supported by thinning of the outer nuclear layer (ONL) or retinal degeneration seen in OCT images taken on day 28 (see FIGS. 15C-15E). In contrast, on day 28, LNP / GFP mRNA with lipid A and LNP / GFP mRNA with lipid 58 recorded degeneration scores of less than 0.5 and about 1.0, respectively, and their corresponding OCT images in FIG. 15B and FIG. 15F (using the vehicle in FIG. 15A as a reference) demonstrate that the ONL layer maintained a healthy thickness. On day 1, none of the samples showed retinal degeneration, using the vehicle in FIG. 14A as a reference (see FIGS. 14A-14F). [Figure 15C] 15A-15E are panels showing the results of OCT imaging on day 28 as described in Example 5. FIG. 15A shows the vehicle reference. On day 28, high degeneration scores of about 2.0 were observed in LNP compositions formulated with either MC3 or control (CTRL) lipid Z. Such high degeneration scores were supported by thinning of the outer nuclear layer (ONL) or retinal degeneration seen in OCT images taken on day 28 (see FIGS. 15C-15E). In contrast, on day 28, LNP / GFP mRNA with lipid A and LNP / GFP mRNA with lipid 58 recorded degeneration scores of less than 0.5 and about 1.0, respectively, and their corresponding OCT images in FIG. 15B and FIG. 15F (using the vehicle in FIG. 15A as a reference) demonstrate that the ONL layer maintained a healthy thickness. On day 1, none of the samples showed retinal degeneration, using the vehicle in FIG. 14A as a reference (see FIGS. 14A-14F). [Figure 15D]15A-15E are panels showing the results of OCT imaging on day 28 as described in Example 5. FIG. 15A shows the vehicle reference. On day 28, high degeneration scores of about 2.0 were observed in LNP compositions formulated with either MC3 or control (CTRL) lipid Z. Such high degeneration scores were supported by thinning of the outer nuclear layer (ONL) or retinal degeneration seen in OCT images taken on day 28 (see FIGS. 15C-15E). In contrast, on day 28, LNP / GFP mRNA with lipid A and LNP / GFP mRNA with lipid 58 recorded degeneration scores of less than 0.5 and about 1.0, respectively, and their corresponding OCT images in FIG. 15B and FIG. 15F (using the vehicle in FIG. 15A as a reference) demonstrate that the ONL layer maintained a healthy thickness. On day 1, none of the samples showed retinal degeneration, using the vehicle in FIG. 14A as a reference (see FIGS. 14A-14F). [Figure 15E] 15A-15E are panels showing the results of OCT imaging on day 28 as described in Example 5. FIG. 15A shows the vehicle reference. On day 28, high degeneration scores of about 2.0 were observed in LNP compositions formulated with either MC3 or control (CTRL) lipid Z. Such high degeneration scores were supported by thinning of the outer nuclear layer (ONL) or retinal degeneration seen in OCT images taken on day 28 (see FIGS. 15C-15E). In contrast, on day 28, LNP / GFP mRNA with lipid A and LNP / GFP mRNA with lipid 58 recorded degeneration scores of less than 0.5 and about 1.0, respectively, and their corresponding OCT images in FIG. 15B and FIG. 15F (using the vehicle in FIG. 15A as a reference) demonstrate that the ONL layer maintained a healthy thickness. On day 1, none of the samples showed retinal degeneration, using the vehicle in FIG. 14A as a reference (see FIGS. 14A-14F). [Figure 15F]15A-15E are panels showing the results of OCT imaging on day 28 as described in Example 5. FIG. 15A shows the vehicle reference. On day 28, high degeneration scores of about 2.0 were observed in LNP compositions formulated with either MC3 or control (CTRL) lipid Z. Such high degeneration scores were supported by thinning of the outer nuclear layer (ONL) or retinal degeneration seen in OCT images taken on day 28 (see FIGS. 15C-15E). In contrast, on day 28, LNP / GFP mRNA with lipid A and LNP / GFP mRNA with lipid 58 recorded degeneration scores of less than 0.5 and about 1.0, respectively, and their corresponding OCT images in FIG. 15B and FIG. 15F (using the vehicle in FIG. 15A as a reference) demonstrate that the ONL layer maintained a healthy thickness. On day 1, none of the samples showed retinal degeneration, using the vehicle in FIG. 14A as a reference (see FIGS. 14A-14F). [Figure 16A] Shown are OCT images (taken on day 22) and hematoxylin and eosin (H&E) qualitative analysis images (taken on day 28) for the vehicle control and the 6 μg low dose. [Figure 16B] Shown are OCT images (taken on day 22) and hematoxylin and eosin (H&E) qualitative analysis images (taken on day 28) for the vehicle control and the 6 μg low dose. [Figure 16C] Shown are OCT images (taken on day 22) and hematoxylin and eosin (H&E) qualitative analysis images (taken on day 28) for the vehicle control and the 6 μg low dose. [Figure 16D] Shown are OCT images (taken on day 22) and hematoxylin and eosin (H&E) qualitative analysis images (taken on day 28) for the vehicle control and the 6 μg low dose. [Figure 17A] IHC images taken from untreated areas serving as negative controls (FIG. 17A), 6 μg low dose treatment (FIG. 17B), and 30 μg high dose (FIG. 17C) 24 hours after treatment. [Figure 17B]IHC images taken from untreated areas serving as negative controls (FIG. 17A), 6 μg low dose treatment (FIG. 17B), and 30 μg high dose (FIG. 17C) 24 hours after treatment. [Figure 17C] IHC images taken from untreated areas serving as negative controls (FIG. 17A), 6 μg low dose treatment (FIG. 17B), and 30 μg high dose (FIG. 17C) 24 hours after treatment. [Figure 18] FIG. 1 is a schematic diagram showing the association of an LDL receptor peptide with a polypeptide-based LNP described herein. [Figure 19A] FIG. 1 is a schematic showing a timeline of days 1-6 of the experiment used to determine LDL uptake via LDLR-mediated endocytosis by imaging in the ARPE-19 human retinal pigment epithelial (RPE) cell line. [Figure 19B] Western blot confirming LDLR knockdown. GAPDH was used as a loading control. [Figure 20] Figure 1 shows that ApoE and EpoB ligands enhanced cellular uptake of LNPs via cell surface receptors in ARPE-19 cells. Immunofluorescence was used to show the uptake of DiD-labeled LNPs or LDL. The left panel shows cells with (+) LDL receptor expression, and the right panel shows cells in which the LDL receptor was knocked down. [Figure 21] Figure 1 shows that ApoE and ApoB ligands enhanced cellular expression of LNP via cell surface receptors in ARPE-19 cells. Immunofluorescence was used to show ApoE / DiD-labeled LNP mRNA expression. The left panel shows cells with (+) LDL receptor expression, and the right panel shows cells in which the LDL receptor was knocked down. [Figure 22A]Figure 22A shows that ApoE and ApoB polypeptides enhanced LNP expression via cell surface receptors, but not their respective full proteins. Figure 22A shows the results of affinity chromatography used to confirm ligand binding. Figure 22B shows the results of affinity chromatography and in vitro uptake assays, which were used to confirm association with ApoB and ApoE polypeptides, but not their respective full proteins. [Figure 22B] Figure 22A shows that ApoE and ApoB polypeptides enhanced LNP expression via cell surface receptors, but not their respective full proteins. Figure 22A shows the results of affinity chromatography used to confirm ligand binding. Figure 22B shows the results of affinity chromatography and in vitro uptake assays, which were used to confirm association with ApoB and ApoE polypeptides, but not their respective full proteins. [Figure 23A] Figure 23 shows that ApoE and ApoB ligands increased GFP mRNA expression in both photoreceptors and RPE cells compared to basal levels in vivo. Figure 23A shows live imaging results that demonstrated an increase in total GFP mRNA expression by ApoE and ApoB ligands. Figure 23B is a graph showing the results of an ELISA assay that confirmed that ApoE and ApoB ligands boosted GFP expression (ng GFP / eye) in PR and RPE cells. [Figure 23B] Figure 23 shows that ApoE and ApoB ligands increased GFP mRNA expression in both photoreceptors and RPE cells compared to basal levels in vivo. Figure 23A shows live imaging results that demonstrated an increase in total GFP mRNA expression by ApoE and ApoB ligands. Figure 23B is a graph showing the results of an ELISA assay that confirmed that ApoE and ApoB ligands boosted GFP expression (ng GFP / eye) in PR and RPE cells. [Figure 24-1]1 shows ApoE (SEQ ID NO:3) and ApoB (SEQ ID NO:4) peptide sequences and physiochemical properties. [Figure 24-2] 1 shows ApoE (SEQ ID NO:3) and ApoB (SEQ ID NO:4) peptide sequences and physiochemical properties. [Diagram 25] 1 shows the results of SDS-PAGE used to determine the stability of ApoE and ApoB polypeptides in solution. [Figure 26A] The major pathways of conjugation using thiol-based crosslinking are illustrated and shown in Figure 26A. The maleimide (non-cleavable) linkage is shown in Figure 26A. The PDS (cleavable) linkage is shown in Figure 26B. [Figure 26B] The major pathways of conjugation using thiol-based crosslinking are illustrated and shown in Figure 26A. The maleimide (non-cleavable) linkage is shown in Figure 26A. The PDS (cleavable) linkage is shown in Figure 26B. [Figure 27] FIG. 1 shows a schematic of the conjugation protocol for maleimide chemistry. [Figure 28] The results show that lipid A / mCherry mRNA uptake was mediated by LDLR and blocked by treatment with 25-hydroxycholesterol (see Figure 28, A1 and A2), regardless of whether it was associated with ApoE via non-covalent (i.e., lipid A LNPs incubated with ApoE) or covalent (i.e., lipid A LNPs containing 0.5% DSPE-PEG2000-maleimide and reacted with ApoE) interactions. Furthermore, Figure 28 shows that lipid A / mCherry mRNA uptake was also mediated by LDLR when directly conjugated to ApoE via 0.5% DSPE-PEG2k-maleimide (see Figure 28, A4). [Figure 29A]The results of the AKTA binding assay are shown, which demonstrated binding of ApoE to LNPs in lipid A LNPs incubated with ApoE (Figure 29A, i.e., non-specific association), lipid A containing 0.1% DSPE-PEG5000 and incubated with ApoE (Figure 29B, i.e., also non-specific association), and lipid A containing 0.1% DSPE-PEG5k-OPDS + ApoE (Figure 29C, i.e., direct conjugation). [Figure 29B] The results of the AKTA binding assay are shown, which demonstrated binding of ApoE to LNPs in lipid A LNPs incubated with ApoE (Figure 29A, i.e., non-specific association), lipid A containing 0.1% DSPE-PEG5000 and incubated with ApoE (Figure 29B, i.e., also non-specific association), and lipid A containing 0.1% DSPE-PEG5k-OPDS + ApoE (Figure 29C, i.e., direct conjugation). [Figure 29C] The results of the AKTA binding assay are shown, which demonstrated binding of ApoE to LNPs in lipid A LNPs incubated with ApoE (Figure 29A, i.e., non-specific association), lipid A containing 0.1% DSPE-PEG5000 and incubated with ApoE (Figure 29B, i.e., also non-specific association), and lipid A containing 0.1% DSPE-PEG5k-OPDS + ApoE (Figure 29C, i.e., direct conjugation). [Diagram 30] 1 shows an SDS-PAGE gel analysis of various CTRL Lipid Z LNP formulations. [Diagram 31] 1 shows an SDS-PAGE gel analysis of various CTRL Lipid Z LNP formulations. [Figure 32-1] FIG. 1 shows a schematic for ApoE / ApoB polypeptide conjugation to LNPs using SPAAC chemistry. [Figure 32-2] FIG. 1 shows a schematic for ApoE / ApoB polypeptide conjugation to LNPs using SPAAC chemistry. [Figure 33A]The results of the experiment carried out in Example 11 are shown. Figure 33B confirms the cell viability in all samples at 48 hours. The results shown in Figure 33A and Figure 33C both show that as the molar ratio of DBCO-ApoE reacted with lipid A LNP (formulated with DSPE-PEG2k-N3) increases from 0.2 mol% to 1.0 mol%, GFP expression also increases gradually, thereby indicating LDLR-mediated uptake of lipid A / GFP mRNA. [Figure 33B] The results of the experiment carried out in Example 11 are shown. Figure 33B confirms the cell viability in all samples at 48 hours. The results shown in Figure 33A and Figure 33C both show that as the molar ratio of DBCO-ApoE reacted with lipid A LNP (formulated with DSPE-PEG2k-N3) increases from 0.2 mol% to 1.0 mol%, GFP expression also increases gradually, thereby indicating LDLR-mediated uptake of lipid A / GFP mRNA. [Figure 33C] The results of the experiment carried out in Example 11 are shown. Figure 33B confirms the cell viability in all samples at 48 hours. The results shown in Figure 33A and Figure 33C both show that as the molar ratio of DBCO-ApoE reacted with lipid A LNP (formulated with DSPE-PEG2k-N3) increases from 0.2 mol% to 1.0 mol%, GFP expression also increases gradually, thereby indicating LDLR-mediated uptake of lipid A / GFP mRNA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0081] AAV vectors are currently the viral vector of choice for retinal gene transfer. However, optimal methods for delivering these therapies to retinal pigment epithelial (RPE) cells and / or photoreceptor cells remain to be improved in order to increase transduction efficiency and reduce complications associated with the highly invasive surgery required for subretinal injection of viral vector suspensions. This disclosure describes, for the first time, the combination of lipid nanoparticles, such as those having lipid A as described herein as the ionizable or cationic lipid, with mRNA cargo for retinal delivery. Using mouse, rat, and non-human primate (NHP) in vivo systems, this disclosure has demonstrated that robust transgene expression can be achieved in the eye cup (RPE) and neural retina, where photoreceptors reside, using LNP-delivered mRNA cargo. Importantly, the data presented herein showed that saturation is achieved with low doses of LNP-delivered mRNA, thereby successfully achieving a superior therapeutic index and tolerability of the potential therapy. Previously, when LNPs have been used for retinal gene transduction, the majority of expression has been seen in the retinal pigment epithelium (RPE) in the optic cup (Patel et al., Journal of Controlled Release Volume 303, 10 June 2019, Pages 91-100), but getting into the actual photoreceptors in the retina has remained a significant challenge. The results presented in this disclosure surprisingly demonstrate that LNPs can not only get into the optic cup with expression equal to that of the RPE, but can do so at low doses.

[0082] In some embodiments, the present disclosure provides lipid nanoparticle (LNP) compositions (e.g., pharmaceutical compositions) comprising a therapeutic nucleic acid (TNA), where the LNP comprises an ApoE polypeptide and / or an ApoB polypeptide linked to the LNP. It is an advantageous feature of the present disclosure that the ApoE- or ApoB-bound LNPs described herein are useful for delivery of TNA to any cell or tissue expressing an LDL receptor (LDLR), and are not limited to a particular cell or tissue type. The present disclosure has surprisingly found that the physiochemical properties of the LNP compositions described herein depend in part on the lipids used in the LNP compositions comprising a therapeutic nucleic acid (TNA), where the LNP comprises an ApoE polypeptide and / or an ApoB polypeptide linked to the LNP. For example, it has been found by the present disclosure that the LNP size of lipid 2 increases as the ApoB loading / LNP increases, and the LNP yield of lipid 2 decreases as the ApoB loading / LNP increases. It was also found that the LNP size of lipid 1 was more stable after ApoB addition, and the LNP yield of lipid 1 decreased with increasing ApoB loading / LNP.

[0083] As an additional advantage, LNPs comprising ApoE and / or ApoB polypeptides linked to the LNPs described herein provide more efficient delivery of therapeutic nucleic acids, better tolerability, and improved safety profiles. 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.

[0084] One of the most difficult hurdles in developing therapies, especially in rare diseases, is the large number of individual conditions. Approximately 350 million people on the planet live with rare disorders, and the National Institutes of Health defines a rare disorder as a disorder or condition with fewer than 200,000 people diagnosed. Approximately 80% of these rare disorders are genetic in origin, and approximately 95% of them do not have FDA-approved treatments (rarediseases.info.nih.gov / diseases / pages / 31 / faqs-about-rare-diseases). The advantage of the ceDNA lipid particles (e.g., lipid nanoparticles) described herein is that they provide an approach that can be rapidly adapted to multiple diseases, especially rare monogenic diseases, which can meaningfully change the current state of treatment for many genetic disorders or diseases.

[0085] 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 understood that the present invention 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 present invention, 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 (eds.), 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.

[0086] 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.

[0087] 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."

[0088] The use of the alternative (eg, "or") should be understood to mean either one, both, or any combination thereof.

[0089] 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.

[0090] 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).

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] As used herein, the term "aqueous solution" is meant to refer to a composition that comprises, in whole or in part, water.

[0099] As used herein, the term "azide compound" is meant to refer to any compound, synthetic or natural, that contains an azide (N3) moiety.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] As used herein, the term "cyclooctyne or a derivative thereof" is meant to refer to any synthetic or natural compound having a cyclooctyne moiety. According to some embodiments, cyclooctyne is a C cyclooctyne having at least one C≡C triple bond.8 It is an alkyne.

[0110] 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.

[0111] "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".

[0112] 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).

[0113] 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.

[0114] As used herein, the phrases "modified ITR" or "mod-ITR" or "mutant 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.

[0115] 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.

[0116] 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".

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] As used herein, the term "polypeptide" is meant to refer to a repeated sequence of amino acids. According to some embodiments, the polypeptide of the present disclosure is an ApoE or ApoB polypeptide. According to some embodiments, the ApoE polypeptide is a functional fragment (or functional portion) of a full-length ApoE polypeptide. According to some embodiments, the ApoE polypeptide is a functional fragment (or functional portion) of a full-length ApoB polypeptide. According to some embodiments, the ApoE polypeptide is 30 amino acids or less in length. According to some embodiments, the ApoB polypeptide is 30 amino acids or less in length.

[0126] As used herein, the term "LDL" refers to low density lipoprotein particles.

[0127] As used herein, the terms "LDL-R" and "LDL receptor" are used interchangeably and refer to low density lipoprotein particle receptors. According to some embodiments, LDL-R expression can be determined, for example, by mRNA or protein assays. Non-limiting examples of LDLR family members include LDLR, very low-density lipoprotein (VLDL) receptor, ApoE receptor, LDL receptor-related protein 1 (LRP-1), LRP-1b, and LRP-2 / megalin (see Strickland et al., 2002, TRENDS in Endocrinol. & Metab. 13:66-74). In some instances, the LDLR or ligand of an LDLR family member may bind to multiple members of the LDLR family.

[0128] As used herein, the term "LDLR ligand" is meant to refer to a ligand capable of binding to the LDLR and / or one or more members of the LDLR family of receptors.

[0129] 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.

[0130] 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.

[0131] In one embodiment, the lipid composition comprises one or more tertiary amino groups, one or more phenyl ester linkages, and a disulfide linkage.

[0132] 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 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 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.

[0133] 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).

[0134] 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.

[0135] 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 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 7 ... The size is about 5 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).

[0136] Generally, the lipid particles (eg, lipid nanoparticles) of the present disclosure have an average diameter selected to provide the intended therapeutic effect.

[0137] 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.

[0138] 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 of the cationic lipids described herein can be dissolved in, for example, acetonitrile (CH 3 CN) and chloroform (CHCl 3 ) in chloromethane (CH 3 Cl) can be converted to the corresponding quaternary lipids.

[0139] 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, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic modification groups associated with neutral lipids.

[0140] 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.

[0141] 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."

[0142] 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.

[0143] As used herein, the term "non-cationic lipid" is meant to refer to any amphipathic lipid, and any other neutral or anionic lipid.

[0144] 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.

[0145] As used herein, the term "organic lipid solution" is meant to refer to a composition comprising an organic solvent having, in whole or in part, a lipid.

[0146] 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.

[0147] 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 desired 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.

[0148] 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, gRNA, 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.

[0149] 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 genome) 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").

[0150] 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.

[0151] 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.

[0152] As used herein, the term "gap" is meant to refer to an interrupted portion of the synthetic DNA vector of the invention, 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.

[0153] 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.

[0154] The term "receptor" as used herein is intended to encompass the entire receptor or ligand-binding portions thereof. These portions of the receptor particularly include the region sufficient for specific binding of the ligand to occur.

[0155] As used herein, the term "ocular disorder" is meant to include ocular diseases associated with elevated intraocular pressure (IOP), such as conditions associated with ocular neovascularization, dry eye, inflammatory conditions, ocular hypertension, and glaucoma.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] 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 is available.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] 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 1~6alkyl), 1 to 4 carbon atoms (i.e., C 1~4 alkyl), or 1 to 3 carbon atoms (i.e., C 1~3 Examples 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 or branched. As used herein, the term "straight chain" referring to an aliphatic hydrocarbon chain means that the chain is not branched.

[0169] 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 1~4alkylene), or 1 to 3 carbon atoms (i.e., C 1~3 Alkylene) and is ethylene or methylene. "Straight or branched chain C 1~6 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 straight or branched chain.

[0170] 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.

[0171] 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 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 ethylenylene or vinylene (-CH=CH-), allyl (-CH 2 "Linear or branched chain C 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.

[0172] 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.

[0173] 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.

[0174] 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.

[0175] 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)NH 2 ], -OR 100 , N.R. 101 R 102 , -NO 2 , -NR 101 COR 102 , -SR 100 , -SOR 101 Sulfoxides, represented by -SO 2 R 101Sulfones, sulfonates represented by -SO 3 M, sulfate-OSO 3 M, -SO 2 NR 101 R 102 Sulfonamide, cyano, azido, -COR 101 , -OCOR 101 , -OCONR 101 R 102 , and polyethylene glycol units (-OCH 2 CH 2 ) 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 each independently represents H, a linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms, a polyethylene glycol unit (-OCH 2 CH 2 ) 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 groups represented by the formula (I) are halogen, -OH, -CN, -NO 2 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 R102 , -CF 3 , -OR 100 , aryl, heteroaryl, heterocyclyl, -SR 101 , -SOR 101 , -SO 2 R 101 , and -SO 3 M. Alternatively, suitable substituents are halogen, -OH, -NO 2 , -CN,C 1~4 Alkyl, -OR 100 , N.R. 101 R 102 , -NR 101 COR 102 , -SR 100 , -SO 2 R 101 , -SO 2 NR 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.

[0176] As used herein, "halogen" refers to F, Cl, Br, or I. "Cyano" is --CN.

[0177] "Amine" or "amino," as used interchangeably herein, refers to a functional group containing a basic nitrogen atom bearing a lone pair of electrons.

[0178] 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.

[0179] Grouping of alternative elements or embodiments of the invention 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 to include the modified group herein, thus satisfying the description of all Markush groups used in the appended claims.

[0180] 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.

[0181] Other terms are defined herein within the description of various aspects of the invention.

[0182] 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 invention 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.

[0183] 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.

[0184] 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.

[0185] The techniques described herein are further illustrated by the following examples, which should not be construed as further limiting in any way. It is to be understood that the present invention is not limited in any manner 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 specific embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0186] 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 an ApoE polypeptide, or a fragment thereof, bound to the LNP. The term "bound" 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). Also provided herein is a pharmaceutical composition comprising a lipid nanoparticle (LNP) and a therapeutic nucleic acid (TNA), wherein the LNP comprises an ApoB polypeptide, or a fragment thereof, bound to the LNP. According to some embodiments, the ApoE polypeptide, or a fragment thereof, and / or the ApoB polypeptide, or a fragment thereof, can bind to a low density lipoprotein (LDL) receptor or an LDL receptor family member. A number of ligands that bind to members of the LDLR family of receptors are provided, for example, in Strickland et al., 2002, TRENDS in Endocrinol. & Metab. 13:66-74, the disclosure of which is incorporated herein by reference. According to some embodiments, a particularly preferred family of ligands includes peptides that include the LDLR-binding domain of apolipoprotein B (ApoB, Spencer and Verma, 2007, Proc. Natl. Acad. Sci. USA 104:7594-7599) or apolipoprotein E (ApoE, Lalazar et al., 1988, J. Biol. Chem. 263:3542-3545), which are the nominal LDL receptor ligands. Three major isoforms of ApoE have been identified, including apoE2, apoE3, and apoE4, and a number of ApoE variants have been described (see, e.g., de Knijff et al., 1994, Hum. Mutat. 4:178-194).

[0187] In some embodiments, the LNP comprises an ApoE polypeptide, or a fragment thereof. In some embodiments, the ApoE polypeptide is 30 amino acids in length. In some embodiments, the ApoE polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 below. EELRVRLASHLRKLRKRLLRDADDLQKGGC (SEQ ID NO: 1) EELRVRLASHLRKLRKRLLRDADDLQKGG (SEQ ID NO: 3)

[0188] According to some embodiments, the ApoE polypeptide has at least 80% sequence similarity to the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3. According to some embodiments, the ApoE polypeptide has at least 80% sequence similarity to the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3. According to some embodiments, the ApoE polypeptide has at least 81% sequence similarity to SEQ ID NO:1 or SEQ ID NO:3. According to some embodiments, the ApoE polypeptide has at least 82% sequence similarity to SEQ ID NO:1 or SEQ ID NO:3. According to some embodiments, the ApoE polypeptide has at least 83% sequence similarity to SEQ ID NO:1 or SEQ ID NO:3. According to some embodiments, the ApoE polypeptide has at least 84% sequence similarity to SEQ ID NO:1 or SEQ ID NO:3. According to some embodiments, the ApoE polypeptide has at least 85% sequence similarity to SEQ ID NO:1 or SEQ ID NO:3. According to some embodiments, the ApoE polypeptide has at least 86% sequence similarity to SEQ ID NO:1 or SEQ ID NO:3. According to some embodiments, the ApoE polypeptide has at least 87% sequence similarity to SEQ ID NO:1 or SEQ ID NO:3. According to some embodiments, the ApoE polypeptide has at least 88% sequence similarity to SEQ ID NO:1 or SEQ ID NO:3. According to some embodiments, the ApoE polypeptide has at least 89% sequence similarity to SEQ ID NO:1 or SEQ ID NO:3. According to some embodiments, the ApoE polypeptide has at least 90% sequence similarity to SEQ ID NO:1 or SEQ ID NO:3. According to some embodiments, the ApoE polypeptide has at least 91% sequence similarity to SEQ ID NO:1 or SEQ ID NO:3. According to some embodiments, the ApoE polypeptide has at least 92% sequence similarity to SEQ ID NO:1 or SEQ ID NO:3. According to some embodiments, the ApoE polypeptide has at least 93% sequence similarity to SEQ ID NO:1 or SEQ ID NO:3. According to some embodiments, the ApoE polypeptide has at least 94% sequence similarity to SEQ ID NO:1 or SEQ ID NO:3.According to some embodiments, the ApoE polypeptide has at least 95% sequence similarity to SEQ ID NO:1 or SEQ ID NO:3. According to some embodiments, the ApoE polypeptide has at least 96% sequence similarity to SEQ ID NO:1 or SEQ ID NO:3. According to some embodiments, the ApoE polypeptide has at least 97% sequence similarity to SEQ ID NO:1 or SEQ ID NO:3. According to some embodiments, the ApoE polypeptide has at least 98% sequence similarity to SEQ ID NO:1 or SEQ ID NO:3. According to some embodiments, the ApoE polypeptide has at least 99% sequence similarity to SEQ ID NO:1 or SEQ ID NO:3. According to some embodiments, the ApoE polypeptide consists of SEQ ID NO:1 or SEQ ID NO:3. According to some embodiments, the ApoE polypeptide comprises SEQ ID NO:1 or SEQ ID NO:3, and the ApoE polypeptide is capable of binding to the LDL receptor. According to some embodiments, the ApoE polypeptide comprises SEQ ID NO:1 or SEQ ID NO:3, and the ApoE polypeptide is capable of binding to the LDL receptor and is capable of internalizing the LNP into the cell. According to some embodiments, the ApoE polypeptide bound to the LNP is a fragment of EELRVRLASHLRKLRKRLLRDADDLQKGGC as set forth in SEQ ID NO: 1 or a fragment of EELRVRLASHLRKLRKRLLRDADDLQKGG as set forth in SEQ ID NO: 3, which fragment is capable of binding to the LDL receptor. According to some embodiments, the ApoE polypeptide bound to the LNP is a fragment of EELRVRLASHLRKLRKRLLRDADDLQKGGC as set forth in SEQ ID NO: 1 or a fragment of EELRVRLASHLRKLRKRLLRDADDLQKGG as set forth in SEQ ID NO: 3, which fragment is capable of binding to the LDL receptor and internalizing the LNP into the cell.

[0189] In some embodiments, the LNP comprises an ApoB polypeptide, or a fragment thereof. In some embodiments, the ApoB polypeptide is 30 amino acids in length. In some embodiments, the ApoB polypeptide comprises the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:4 below. SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGSGGC (SEQ ID NO: 2) SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGSGG (SEQ ID NO: 4)

[0190] According to some embodiments, the ApoB polypeptide has at least 80% sequence similarity to the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:4. According to some embodiments, the ApoB polypeptide has at least 80% sequence similarity to the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:4. According to some embodiments, the ApoB polypeptide has at least 81% sequence similarity to SEQ ID NO:2 or SEQ ID NO:4. According to some embodiments, the ApoB polypeptide has at least 82% sequence similarity to SEQ ID NO:2 or SEQ ID NO:4. According to some embodiments, the ApoB polypeptide has at least 83% sequence similarity to SEQ ID NO:2 or SEQ ID NO:4. According to some embodiments, the ApoB polypeptide has at least 84% sequence similarity to SEQ ID NO:2 or SEQ ID NO:4. According to some embodiments, the ApoB polypeptide has at least 85% sequence similarity to SEQ ID NO:2 or SEQ ID NO:4. According to some embodiments, the ApoB polypeptide has at least 86% sequence similarity to SEQ ID NO:2 or SEQ ID NO:4. According to some embodiments, the ApoB polypeptide has at least 87% sequence similarity to SEQ ID NO:2 or SEQ ID NO:4. According to some embodiments, the ApoB polypeptide has at least 88% sequence similarity to SEQ ID NO:2 or SEQ ID NO:4. According to some embodiments, the ApoB polypeptide has at least 89% sequence similarity to SEQ ID NO:2 or SEQ ID NO:4. According to some embodiments, the ApoB polypeptide has at least 90% sequence similarity to SEQ ID NO:2 or SEQ ID NO:4. According to some embodiments, the ApoB polypeptide has at least 91% sequence similarity to SEQ ID NO:2 or SEQ ID NO:4. According to some embodiments, the ApoB polypeptide has at least 92% sequence similarity to SEQ ID NO:2 or SEQ ID NO:4. According to some embodiments, the ApoB polypeptide has at least 93% sequence similarity to SEQ ID NO:2 or SEQ ID NO:4. According to some embodiments, the ApoB polypeptide has at least 94% sequence similarity to SEQ ID NO:2 or SEQ ID NO:4.According to some embodiments, the ApoB polypeptide has at least 95% sequence similarity to SEQ ID NO:2 or SEQ ID NO:4. According to some embodiments, the ApoB polypeptide has at least 96% sequence similarity to SEQ ID NO:2 or SEQ ID NO:4. According to some embodiments, the ApoB polypeptide has at least 97% sequence similarity to SEQ ID NO:2 or SEQ ID NO:4. According to some embodiments, the ApoB polypeptide has at least 98% sequence similarity to SEQ ID NO:2 or SEQ ID NO:4. According to some embodiments, the ApoB polypeptide has at least 99% sequence similarity to SEQ ID NO:2 or SEQ ID NO:4. According to some embodiments, the ApoB polypeptide consists of SEQ ID NO:2 or SEQ ID NO:4. According to some embodiments, the ApoB polypeptide comprises SEQ ID NO:2, and the ApoB polypeptide is capable of binding to the LDL receptor. According to some embodiments, the ApoB polypeptide comprises SEQ ID NO:2 or SEQ ID NO:4, and the ApoB polypeptide is capable of binding to the LDL receptor and is capable of internalizing the LNP into the cell. According to some embodiments, the ApoB polypeptide bound to the LNP is a fragment of SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGSGGC as set forth in SEQ ID NO: 2, or a fragment of SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGSGG as set forth in SEQ ID NO: 4, which fragment is capable of binding to the LDL receptor, and which fragment is capable of binding to the LDL receptor. According to some embodiments, the ApoB polypeptide bound to the LNP is a fragment of SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGSGGC as set forth in SEQ ID NO: 2, or a fragment of SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGSGGC as set forth in SEQ ID NO: 4, which fragment is capable of binding to the LDL receptor, and which fragment is capable of internalizing the LNP into the cell.

[0191] 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 ApoE- or ApoB-binding LNPs described herein are useful for delivering the LNP to any cell or tissue that actively expresses the LDLR.

[0192] According to some embodiments, the LNP comprises a cationic lipid, a sterol or a derivative thereof, a non-cationic lipid, or a PEGylated lipid.

[0193] 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.

[0194] 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.

[0195] [Table 1]

[0196] In some embodiments, the cationic lipid is selected from the group consisting of N-[1-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA); N-[1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium 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-aminopropyl)amino]ol butylcarboxamide ethyl 1-3,4-di[oleyloxy]-benzamide (MVL5); Dioctadecylamido-glycylspermine (DOGS); 3b-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol ( ... 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);

[0197] In some embodiments, the condensed lipid is DOTAP.

[0198] 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 ApoE polypeptide, or fragment thereof, and / or an ApoB polypeptide, or fragment thereof, conjugated to the LNP as described herein, to a site of interest (e.g., a cell, tissue, organ, etc.).

[0199] Exemplary ionizable lipids are those described in International PCT Patent 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, 2014 / 022460, 2015 ... 2011 / 153120, 2012 / 044638, 2012 / 054365, 2011 / 090965, 20 13 / 016058, 2012 / 162210, 2008 / 042973, 2010 / 129709, 2010 / No. 144740, No. 2012 / 099755, No. 2013 / 049328, No. 2013 / 086322, No. 2013 / 08 No. 6373, No. 2011 / 071860, No. 2009 / 132131, No. 2010 / 048536, No. 2010 / 08853 No. 7, No. 2010 / 054401, No. 2010 / 054406, No. 2010 / 054405, No. 2010 / 054384 , 2012 / 016184, 2009 / 086558, 2010 / 042877, 2011 / 000106, 2011 / 000106, No. 2011 / 000107, No. 2005 / 120152, No. 2011 / 141705, No. 2013 / 126803, No. 2 006 / 007712, 2011 / 038160, 2005 / 121348, 2011 / 066651, 2009 Nos. 2011 / 141704, 2006 / 069782, 2012 / 031043, 2013 / 006825, 2013 / 033563, 2013 / 089151, 2017 / 099823, 2015 / 095346, and 2013 / 086354, as well as U.S. Patent 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 / 0023 No. 673, No. 2012 / 0128760, No. 2010 / 0324120, No. 2014 / 0200257, No. 2015 / 0203446, No. 2018 / 0005363, No. 2014 / 0 308304, 2013 / 0338210, 2012 / 0101148, 2012 / 0027796, 2012 / 0058144, 2013 / 0323269, 2011 / 0117125, 2011 / 0256175, 2012 / 0202871, 2011 / 0076335, 2006 / 0083780, 2013 / 0123338, 2 015 / 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.

[0200] 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:

[0201] [ka]

[0202] 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.

[0203] 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.

[0204] 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.

[0205] 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.

[0206] Formula (I) According to some embodiments, the ionizable lipid is represented by formula (I):

[0207] [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, R 3 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, -CH 2 CH, or -(CH 2 ) 2 CH, R 5 and R 5’ are each independently hydrogen, C 1~20 Alkylene or C 2~20 alkenylene, R 6 and R 6’ For each occurrence, 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.

[0208] According to some embodiments of any of the aspects or embodiments herein, R 2 and R 2’ are each independently C 1~3It is alkylene.

[0209] 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.

[0210] 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.

[0211] 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.

[0212] According to some embodiments of any of the aspects or embodiments herein, R 4 and R 4’ are -CH, respectively.

[0213] 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 certain 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.

[0214] According to some embodiments of any of the aspects or embodiments herein, R 4 is -C(R a ) 2 CR a , or -[C(R a ) 2 ] 2 CR a and R a is C2 1~3 is alkyl, R 3 and R 4 together with their intervening N atoms form a 5- or 6-membered heterocyclyl. According to certain embodiments of any of the aspects or embodiments herein, R 4’ is -C(R a ) 2 CR a , or -[C(R a ) 2 ] 2 CR 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.

[0215] 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.

[0216] According to some embodiments of any of the aspects or embodiments herein, R 6 and R 6’ For each occurrence, independently, C 1~10 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.

[0217] According to some embodiments of any of the aspects or embodiments herein, the ionizable lipid is selected from any one of the lipids in Table 2, or a pharma- ceutically acceptable salt thereof.

[0218] [Table 2-1]

[0219] [Table 2-2]

[0220] [Table 2-3]

[0221] [Table 2-4]

[0222] [Table 2-5]

[0223] [Table 2-6]

[0224] [Table 2-7]

[0225] [Table 2-8]

[0226] [Table 2-9]

[0227] [Table 2-10]

[0228] [Table 2-11]

[0229] Formula (II) In some embodiments, the ionizable lipid is of formula (II):

[0230] [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 (C 2 ~C 20 ) alkenyl, -C(O)O(C2 ~C 20 ) alkyl, and (C 2 ~C 20 ) cyclopropyl substituted with alkyl; R 2 is (C 2 ~C 20 ) alkyl.

[0231] In a second chemical embodiment, the ionizable lipid of formula (II) is of formula (XIII):

[0232] [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).

[0233] In a third chemical embodiment, c and d in the ionizable 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).

[0234] In a fourth chemical embodiment, c in the ionizable 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 ionizable 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.

[0235] In a fifth chemical embodiment, d in the ionizable 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 ionizable 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.

[0236] In a sixth chemical embodiment, the ionizable lipid of formula (II) or (III) is of formula (IV):

[0237] [ka] or a pharma- ceutically acceptable salt thereof, wherein the remainder of the variables are as described for formula (I).

[0238] In a seventh chemical embodiment, b in the ionizable 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 ionizable 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 ionizable 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 embodiment.

[0239] In an eighth chemical embodiment, a in the ionizable 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 ionizable 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 ionizable 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.

[0240] In a ninth chemical embodiment, R in an ionizable lipid of formula (II), (III) or (IV) or a pharma- ceutically acceptable salt thereof is 1 does not exist or (C 5 ~C 15 ) alkenyl, -C(O)O(C 4 ~C 18 ) alkyl, and (C 4 ~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 ionizable lipid of formula (II), (III) or (IV), or a pharma- ceutically acceptable salt thereof, is selected from the group consisting of cyclopropyl substituted with alkyl, ... 1 does not exist or (C 5 ~C 15 ) alkenyl, -C(O)O(C 4 ~C 16 ) alkyl, and (C 4 ~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 ionizable lipid of formula (II), (III) or (IV), or a pharma- ceutically acceptable salt thereof, is selected from the group consisting of cyclopropyl substituted with alkyl, ... 1 does not exist or (C 5 ~C 12 ) alkenyl, -C(O)O(C 4 ~C 12 ) alkyl, and (C 4 ~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 an ionizable lipid of formula (II), (III) or (IV), or a pharma- ceutically acceptable salt thereof, is selected from 1 does not exist or (C 5 ~C 10 ) alkenyl, -C(O)O(C 4 ~C 10 ) alkyl, and (C 4 ~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.

[0241] 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.

[0242] In an eleventh chemical embodiment, R in an ionizable lipid of formula (II), (III) or (IV) or a pharma- ceutically acceptable salt thereof is 1 C(O)O(C 2 ~C 20 ) alkyl, -C(O)O(C 4 ~C 18 ) alkyl, -C(O)O(C 4 ~C 12 ) alkyl or -C(O)O(C 4 ~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 9Alternatively, in an eleventh chemical embodiment, R in an ionizable lipid of formula (II), (III), or (IV), or a pharma- ceutically acceptable salt thereof, is 1 -C(O)O(C 4 ~C 18 ) alkyl, -C(O)O(C 4 ~C 12 ) alkyl or -C(O)O(C 4 ~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 teeth, -C(O)O(C 17 alkyl), and the remainder of the variables are as described in any one of the preceding chemical embodiments.

[0243] In a twelfth chemical embodiment, R in an ionizable lipid of formula (II), (III), or (IV), or a pharma- ceutically acceptable salt thereof, 1 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.

[0244] [Table 3]

[0245] In a thirteenth chemical embodiment, R in an ionizable lipid of formula (II) or a pharma- ceutically acceptable salt thereof is 2is 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.

[0246] [Table 4]

[0247] Specific examples are provided in Table 5 and in the Exemplification section below, and are included as part of the fourteenth chemical embodiment herein of the ionizable lipid of formula (I). Pharmaceutically acceptable salts and ionized and neutral forms are also included.

[0248] [Table 5-1]

[0249] [Table 5-2]

[0250] [Table 5-3]

[0251] [Table 5-4]

[0252] [Table 5-5]

[0253] In some embodiments, lipid nanoparticles of the present disclosure comprise 1-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-(oleoyloxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanyl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl)9-(tridecan-5-yl)nonanediate, listed above as lipid 58.

[0254] [ka]

[0255] Formula (V) In some embodiments, the ionizable lipid is of formula (V):

[0256] [ka] or a pharma- ceutically acceptable salt thereof, wherein: R 1 and R 1’ are each independently R a (C 1 ~C 6 ) alkylene; R 2 and R 2’ are each independently 1 ~C 2 ) alkylene; R 3 and R 3’ are each independently R b (C 1 ~C 6 ) alkyl, 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' are interrupted by -C(O)O- (C 2 ~C 6 ) alkylene; R 5 and R 5 ' are each independently 2 ~C 30 ) alkyl or (C 2 ~C 30 )alkenyl, each of which is optionally -C(O)O- or (C 3 ~C 6 ) interrupted by cycloalkyl, R a and R b are each halo or cyano.

[0257] In a second chemical embodiment, R in an ionizable lipid of formula (V) 1 and R 1’ are each independently (C 1 ~C 6 ) alkylene, and the remaining variables are as described above for formula (V). Alternatively, as part of the second chemical embodiment, R in the ionizable lipid of formula (V) 1 and R 1’ are each independently (C 1 ~C 3 ) alkylene, and the remainder of the variables are as described above for formula (V).

[0258] In a third chemical embodiment, the ionizable lipid of formula (V) is Formula (VI):

[0259] [ka] or a pharma- ceutically acceptable salt thereof, wherein the remainder of the variables are as described above for formula (V).

[0260] In a fourth chemical embodiment, the ionizable lipid of formula (V) is represented by formula (VII) or (VIII):

[0261] [ka] or a pharma- ceutically acceptable salt thereof, wherein the remainder of the variables are as described above for formula (V).

[0262] In a fifth chemical embodiment, the ionizable lipid of formula (V) is represented by formula (IX) or (VI):

[0263] [ka] or a pharma- ceutically acceptable salt thereof, wherein the remainder of the variables are as described above for formula (V).

[0264] In a sixth chemical aspect, the ionizable lipid of formula (V) is of formula (XI), (XII), (XIII), or (XIV):

[0265] [ka] or a pharma- ceutically acceptable salt thereof, wherein the remainder of the variables are as described above for formula (XV).

[0266] In a seventh chemical embodiment, R in an ionizable 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 (the number of carbon atoms being 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, R in the ionizable 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 ionizable lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) is a branched alkyl or branched alkenyl. 5 is a branched alkyl or branched alkenyl. In another alternative, as part of the seventh chemical aspect, R in the ionizable lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) 5’ is a branched alkyl or branched alkenyl.

[0267] In an eighth chemical embodiment, R in an ionizable lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) 5 is (C 6 ~C 26 ) alkyl or (C 6 ~C 26 )alkenyl, each of which is optionally -C(O)O- or (C 3 ~C 6 ) cycloalkyl, and the remaining variables are as described above for formula (I). Alternatively, as part of the seventh chemical aspect, R in the ionizable lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) 5 is (C 6 ~C 26 ) alkyl or (C 6 ~C 26 )alkenyl, each of which is optionally -C(O)O- or (C 3 ~C 5 ) cycloalkyl, and the remaining variables are as described above for formula (I). In another alternative, as part of the eighth chemical aspect, R in an ionizable lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) 5 is (C 7 ~C26 ) alkyl or (C 7 ~C 26 )alkenyl, each of which is optionally -C(O)O- or (C 3 ~C 5 ) cycloalkyl, and the remaining variables are as described above for formula (I). In another alternative, as part of the eighth chemical aspect, R in an ionizable lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) 5 is (C 8 ~C 26 ) alkyl or (C 8 ~C 26 )alkenyl, each of which is optionally -C(O)O- or (C 3 ~C 5 ) cycloalkyl, and the remaining variables are as described above for formula (V). In another alternative, as part of the eighth chemical aspect, R in an ionizable lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) 5 is (C 6 ~C 24 ) alkyl or (C 6 ~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 an ionizable lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) is 5 is (C 8 ~C 24 ) alkyl or (C 8 ~C 24 ) alkenyl, 8 ~C 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 an ionizable lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) 5 is (C 8 ~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 ionizable 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 ionizable 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 ionizable 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 ionizable lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) is 5 is -(CH 2 ) 3 C(O)O(CH 2 ) 8 CH 3 , -(CH 2 ) 5 C(O)O(CH 2 ) 8 CH 3、 -(CH 2 ) 7 C(O)O(CH 2 ) 8 CH 3 , -(CH 2 ) 7 C(O)OCH[(CH 2 ) 7 CH 3 ] 2 , -(CH 2 ) 7 -C 3 H 6 -(CH 2 ) 7 CH 3 , -(CH 2 ) 7 CH 3 , -(CH 2 ) 9 CH 3、 -(CH 2 ) 16 CH 3 , -(CH 2 ) 7 CH=CH(CH 2 ) 7 CH 3 or -(CH 2 ) 7 CH=CHCH 2 CH=CH(CH 2 ) 4 CH 3 and the remainder of the variables are as described above for formula (XV).

[0268] In a ninth chemical embodiment, R in an ionizable 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 ionizable lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) 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 ionizable 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 ionizable 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 ionizable 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 ionizable 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 -(CH 2 ) 5 C(O)OCH[(CH 2 ) 7 CH 3 ] 2 , -(CH 2 ) 7 C(O)OCH[(CH 2 ) 7 CH 3 ] 2 , -(CH 2 ) 5 C(O)OCH(CH 2 ) 2 [(CH 2 ) 7 CH 3 ] 2 , or -(CH 2 ) 7 C(O)OCH(CH 2 ) 2 [(CH 2 ) 7 CH 3 ] 2 and the remainder of the variables are as described above for formula (V) or the eighth chemical embodiment.

[0269] In another embodiment, the ionizable 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.

[0270] [Table 6]

[0271] Formula (XV) In some embodiments, the ionizable lipid is of formula (XV):

[0272] [ka] or a pharma- ceutically acceptable salt thereof, wherein: R' is absent, hydrogen, or C 1 ~C 6 alkyl, where R' is hydrogen or C 1 ~C 6 When R', R 1 , and R 2 The nitrogen atom to which all are attached is protonated, R 1 and R 2 are each independently hydrogen, C 1 ~C 6 Alkyl or C 2 ~C 6 alkenyl, R 3 is C 1 ~C 12 Alkylene or C 2 ~C 12 alkenylene, R 4 is C 1 ~C 16 Unbranched alkyl, C 2 ~C 16 unbranched alkenyl, or

[0273] [ka] where: R 4a and R 4b are each independently 1 ~C 16 Unbranched alkyl or C 2 ~C 16 is an unbranched alkenyl; R 5 does not exist or C 1 ~C 8 Alkylene or C2 ~C 8 alkenylene, R 6a and R 6b are each independently 7 ~C 16 Alkyl or C 7 ~C 16 alkenyl, where R 6a and R 6b the total number of carbon atoms in the X 1 and X 2 each independently represents -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 ) 2 O-, -C(=O)(CR a 2 )C(=O)O- or OC(=O)(CR a 2 )C(=O)-, where R a is, for each occurrence, independently, hydrogen or C 1 ~C 6 is alkyl, n is an integer selected from 1, 2, 3, 4, 5, and 6.

[0274] In a second embodiment, in the ionizable 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.

[0275] In a third embodiment, in the ionizable lipid according to the first or second embodiment, or a pharma- ceutically acceptable salt thereof, X 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.

[0276] In a fourth embodiment, the ionizable lipid of the present disclosure is represented by formula (XVI):

[0277] [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.

[0278] In a fifth embodiment, the ionizable lipid of the present disclosure is represented by formula (XVII):

[0279] [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.

[0280] In a sixth embodiment, the ionizable lipid of the present disclosure is represented by formula (XVIII):

[0281] [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.

[0282] In a seventh embodiment, in the ionizable 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, C 1 ~C 6 Alkyl or C 2 ~C 6 Alkenyl, or C 1 ~C 5 Alkyl or C 2 ~C 5 Alkenyl, or C 1 ~C 4 Alkyl or C 2 ~C 4 Alkenyl, or C 6 Alkyl or C 5 Alkyl or C 4 Alkyl or C 3 Alkyl or C 2 Alkyl or C 1 Alkyl or C 6 Alkenyl, or C 5 Alkenyl, or C 4 Alkenyl, or C 3 Alkenyl, or C 2 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.

[0283] In an eighth embodiment, the ionizable lipid of the present disclosure is represented by formula (XIX):

[0284] [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.

[0285] In a ninth embodiment, in the ionizable 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 3 is C 1 ~C 9 Alkylene or C 2 ~C 9 Alkenylene, C 1 ~C 7 Alkylene or C 2 ~C 7 Alkenylene, C 1 ~C 5 Alkylene or C 2 ~C 5 Alkenylene or C 2 ~C 8 Alkylene or C 2 ~C 8 Alkenylene or C 3 ~C 7 Alkylene or C 3 ~C 7 Alkenylene or C 5 ~C 7 Alkylene or C 5 ~C 7 alkenylene or R 3 But, C 12 Alkylene, C 11 Alkylene, C 10 Alkylene, C 9 Alkylene or C 8 Alkylene or C 7 Alkylene or C 6 Alkylene or C 5 Alkylene or C 4 Alkylene or C 3 Alkylene or C 2 Alkylene or C1 Alkylene or C 12 Alkenylene, C 11 Alkenylene, C 10 Alkenylene, C 9 Alkenylene or C 8 Alkenylene or C 7 Alkenylene or C 6 Alkenylene or C 5 Alkenylene or C 4 Alkenylene or C 3 Alkenylene or C 2 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.

[0286] 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 But it doesn't exist or C 1 ~C 6 Alkylene or C 2 ~C 6 alkenylene or R 5 But it doesn't exist or C 1 ~C 4 Alkylene or C 2 ~C 4 alkenylene or R 5 does not exist or R 5 But, C 8 Alkylene, C 7 Alkylene, C 6 Alkylene, C 5 Alkylene, C 4 Alkylene, C 3 Alkylene, C 2 Alkylene, C 1 Alkylene, C 8 Alkenylene, C 7 Alkenylene, C 6 Alkenylene, C 5 Alkenylene, C 4Alkenylene, C 3 Alkenylene or C 2 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.

[0287] 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, C 1 ~C 14 Unbranched alkyl, C 2 ~C 14 unbranched alkenyl, or

[0288] [ka] and R 4a and R 4b However, each independently, C 1 ~C 12 Unbranched alkyl or C 2 ~C 12 unbranched alkenyl or R 4 But, C 2 ~C 12 Unbranched alkyl or C 2 ~C 12 unbranched alkenyl or R 4 But, C 5 ~C 7 Unbranched alkyl or C 5 ~C 7 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, C 9 Unbranched alkyl, C 8Unbranched alkyl, C 7 Unbranched alkyl, C 6 Unbranched alkyl, C 5 Unbranched alkyl, C 4 Unbranched alkyl, C 3 Unbranched alkyl, C 2 Unbranched alkyl, C 1 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, C 9 Unbranched alkenyl, C 8 Unbranched alkenyl, C 7 Unbranched alkenyl, C 6 Unbranched alkenyl, C 5 Unbranched alkenyl, C 4 Unbranched alkenyl, C 3 Unbranched alkenyl, or C 2 alkenyl or R 4 but,

[0289] [ka] and R 4a and R 4b However, each independently, C 2 ~C 10 Unbranched alkyl or C 2 ~C 10 unbranched alkenyl or R 4 but,

[0290] [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, C12 Unbranched alkyl, C 11 Unbranched alkyl, C 10 Unbranched alkyl, C 9 Unbranched alkyl, C 8 Unbranched alkyl, C 7 Unbranched alkyl, C 6 Unbranched alkyl, C 5 Unbranched alkyl, C 4 Unbranched alkyl, C 3 Unbranched alkyl, C 2 Alkyl, C 1 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, C 9 Unbranched alkenyl, C 8 Unbranched alkenyl, C 7 Unbranched alkenyl, C 6 Unbranched alkenyl, C 5 Unbranched alkenyl, C 4 Unbranched alkenyl, C 3 Unbranched alkenyl, or C 2 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.

[0291] 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, each independently, C 6 ~C 14 Alkyl or C 6 ~C 14 alkenyl or R 6a and R 6b are each independently 8~C 12 Alkyl or C 8 ~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 10 Alkyl, C 9 Alkyl, C 8 Alkyl, C 7 Alkyl, C 16 Alkenyl, C 15 Alkenyl, C 14 Alkenyl, C 13 Alkenyl, C 12 Alkenyl, C 11 Alkenyl, C 10 Alkenyl, C 9 Alkenyl, C 8 Alkenyl or C 7 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.

[0292] 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 12Alkyl, C 11 Alkyl, C 10 Alkyl, C 9 Alkyl, C 8 Alkyl, C 7 Alkyl, C 16 Alkenyl, C 15 Alkenyl, C 14 Alkenyl, C 13 Alkenyl, C 12 Alkenyl, C 11 Alkenyl, C 10 Alkenyl, C 9 Alkenyl, C 8 Alkenyl or C 7 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.

[0293] 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 C 7 alkyl, and R 6a C 8 Alkyl or R 6a C 8 alkyl, and R 6a C 7 Alkyl or R 6a C 8 alkyl, and R 6a C9 Alkyl or R 6a C 9 alkyl, and R 6a C 8 Alkyl or R 6a C 9 alkyl, and R 6a C 10 Alkyl or R 6a C 10 alkyl, and R 6a C 9 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 C 7 alkyl, and R 6a C 9 Alkyl or R 6a C 9 alkyl, and R 6a C 7 Alkyl or R 6a C 8 alkyl, and R 6a C 10 Alkyl or R 6a C 10 alkyl, and R 6a C 8 Alkyl or R 6a C 9 alkyl, and R 6a C 11 Alkyl or R 6a C 11 alkyl, and R 6a 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.

[0294] 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.

[0295] [Table 7-1]

[0296] [Table 7-2]

[0297] [Table 7-3]

[0298] [Table 7-4]

[0299] Formula (XX) In some embodiments, the cationic lipid is of formula (XX):

[0300] [ka] or a pharma- ceutically acceptable salt thereof, wherein: R' is absent, hydrogen, or C 1 ~C 3 alkyl, where R' is hydrogen or C 1 ~C 3 When R', R 1 , and R 2 The nitrogen atom to which all are attached is protonated, R 1 and R 2 are each independently hydrogen or C 1 ~C 3 is alkyl, R 3 is C 3 ~C 10 Alkylene or C 3 ~C 10 alkenylene, R 4 is C 1 ~C 16 Unbranched alkyl, C 2 ~C 16 unbranched alkenyl, or

[0301] [ka] where: R 4a and R 4b are each independently 1 ~C 16 Unbranched alkyl or C 2 ~C 16 is an unbranched alkenyl; R 5 does not exist or C 1 ~C 6 Alkylene or C 2 ~C 6 alkenylene, R 6a and R6b are each independently 7 ~C 14 Alkyl or C 7 ~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 ) 2 O-, -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 C 1 ~C 6 is alkyl, n is an integer selected from 1, 2, 3, 4, 5, and 6.

[0302] 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 (XX) or the first embodiment.

[0303] In a third embodiment, the cationic lipid of the present disclosure is represented by formula (XXI):

[0304] [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.

[0305] In a fourth embodiment, the cationic lipid of the present disclosure is represented by formula (XXII):

[0306] [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.

[0307] 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 or C 1 ~C 2 Alkyl or C 2 ~C 3 alkenyl, or R′, R 1 , and R 2 are each independently hydrogen, C 1 ~C 2 and all other remaining variables are as described for Formula (XX), Formula (XXI), or any one of the preceding embodiments.

[0308] In a sixth embodiment, the cationic lipid of the present disclosure is represented by formula (XXII):

[0309] [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.

[0310] In a seventh embodiment, in the cationic lipid according to formula (XX), formula (XXI), formula (XXII), formula (XXIII), formula (XIX), or any one of the preceding embodiments, or a pharma- ceutically acceptable salt thereof, R 5 But it doesn't exist or C 1 ~C 8 alkylene or R 5 But it doesn't exist or C 1 ~C 6 Alkylene or C 2 ~C 6 alkenylene or R 5 But it doesn't exist or C 1 ~C 4 Alkylene or C 2 ~C 4 alkenylene or R 5 does not exist or R 5 is C 6 Alkylene, C 5 Alkylene, C 4 Alkylene, C 3 Alkylene, C 2 Alkylene, C 1 Alkylene, C 6 Alkenylene, C 5 Alkenylene, C 4 Alkenylene, C 3 Alkenylene or C 2 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.

[0311] In an eighth embodiment, the cationic lipid of the present disclosure is represented by formula (XXIV):

[0312] [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.

[0313] 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, C 1 ~C 14 Unbranched alkyl, C 2 ~C 14 unbranched alkenyl, or

[0314] [ka] and R 4a and R 4b However, each independently, C 1 ~C 12 Unbranched alkyl or C 2 ~C 12 unbranched alkenyl or R 4 But, C 2 ~C 12 Unbranched alkyl or C 2 ~C 12 unbranched alkenyl or R 4 But, C 5 ~C 12 Unbranched alkyl or C 5 ~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, C 9 Unbranched alkyl, C 8 Unbranched alkyl, C7 Unbranched alkyl, C 6 Unbranched alkyl, C 5 Unbranched alkyl, C 4 Unbranched alkyl, C 3 Unbranched alkyl, C 2 Unbranched alkyl, C 1 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, C 9 Unbranched alkenyl, C 8 Unbranched alkenyl, C 7 Unbranched alkenyl, C 6 Unbranched alkenyl, C 5 Unbranched alkenyl, C 4 Unbranched alkenyl, C 3 Unbranched alkenyl, or C 2 alkenyl or R 4 but,

[0315] [ka] and R 4a and R 4b However, each independently, C 2 ~C 10 Unbranched alkyl or C 2 ~C 10 unbranched alkenyl or R 4 but,

[0316] [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 12Unbranched alkyl, C 11 Unbranched alkyl, C 10 Unbranched alkyl, C 9 Unbranched alkyl, C 8 Unbranched alkyl, C 7 Unbranched alkyl, C 6 Unbranched alkyl, C 5 Unbranched alkyl, C 4 Unbranched alkyl, C 3 Unbranched alkyl, C 2 Alkyl, C 1 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, C 9 Unbranched alkenyl, C 8 Unbranched alkenyl, C 7 Unbranched alkenyl, C 6 Unbranched alkenyl, C 5 Unbranched alkenyl, C 4 Unbranched alkenyl, C 3 Unbranched alkenyl, or C 2 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.

[0317] 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 3 But, C 3 ~C 8 Alkylene or C 3 ~C 8 Alkenylene, C 3 ~C 7 Alkylene or C 3 ~C 7 Alkenylene or C 3~C 5 Alkylene or C 3 ~C 5 alkenylene or R 3 But, C 8 Alkylene or C 7 Alkylene or C 6 Alkylene or C 5 Alkylene or C 4 Alkylene or C 3 Alkylene or C 1 Alkylene or C 8 Alkylene, C 8 Alkenylene or C 7 Alkenylene or C 6 Alkenylene or C 5 Alkenylene or C 4 Alkenylene or C 3 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.

[0318] 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 independently, C 7 ~C 12 Alkyl or C 7 ~C 12 alkenyl or R 6a and R 6b are each independently 8 ~C 10 Alkyl or C 8 ~C 10 alkenyl or R 6a and R 6b are each independently 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C 9 Alkyl, C8 Alkyl, C 7 Alkyl, C 12 Alkenyl, C 11 Alkenyl, C 10 Alkenyl, C 9 Alkenyl, C 8 Alkenyl or C 7 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.

[0319] 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, C 9 Alkyl, C 8 Alkyl, C 7 Alkyl, C 12 Alkenyl, C 11 Alkenyl, C 10 Alkenyl, C 9 Alkenyl, C 8 Alkenyl or C 7 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.

[0320] 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 C 7 alkyl, and R 6a C 8 Alkyl or R 6a C 8 alkyl, and R 6a C 7 Alkyl or R 6a C 8 alkyl, and R 6a C 9 Alkyl or R 6a C 9 alkyl, and R 6a C 8 Alkyl or R 6a C 9 alkyl, and R 6a C 10 Alkyl or R 6a C 10 alkyl, and R 6a C 9 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 6aC 12 alkyl, and R 6a C 11 Alkyl or R 6a C 7 alkyl, and R 6a C 9 Alkyl or R 6a C 9 alkyl, and R 6a C 7 Alkyl or R 6a C 8 alkyl, and R 6a C 10 Alkyl or R 6a C 10 alkyl, and R 6a C 8 Alkyl or R 6a C 9 alkyl, and R 6a C 11 Alkyl or R 6a C 11 alkyl, and R 6a C 9 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, 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.

[0321] 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.

[0322] 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.

[0323] [Table 8-1]

[0324] [Table 8-2]

[0325] Specific examples are provided in the Exemplification section below and are included as part of the ionizable lipids described herein. Pharmaceutically acceptable salts and neutral forms are also included.

[0326] 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 ApoE polypeptide, or a fragment thereof, and / or an ApoB polypeptide, or a fragment thereof, bound to the LNP via a cleavable lipid that can be used to deliver a capsid-free non-viral DNA vector to a 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 include SS-cleavable and pH-activated lipid-like substances such as ss-OP lipid, ssPalm lipid, ss-M lipid, ss-E lipid, ss-EC lipid, ss-LC lipid, and ss-OC lipid.

[0327] 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.

[0328] 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.

[0329] The lipid particles described herein (e.g., LNPs comprising ApoE polypeptides, or fragments thereof, and / or ApoB polypeptides, or fragments thereof, conjugated to LNPs) can be advantageously used to increase the delivery of nucleic acids (e.g., ceDNA, mRNA) to 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 ApoE polypeptides, or fragments thereof, and / or ApoB polypeptides, or fragments thereof, conjugated to LNPs) 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 is not bound by theory, it is believed that lipid particles (e.g., LNPs comprising ApoE polypeptides, or fragments thereof, and / or ApoB polypeptides, or fragments thereof, conjugated to LNPs) 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 ApoE polypeptide, or fragment thereof, and / or an ApoB polypeptide, or fragment thereof, conjugated to the LNP) is their better tolerability compared to other lipids, e.g., ionizable cationic lipids, e.g., MC3.

[0330] 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.

[0331] In one embodiment, the cleavable lipid is a ss-cleavable lipid. In one embodiment, the ss-cleavable lipid comprises the structure shown below:

[0332] Lipid A

[0333] [ka]

[0334] 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.

[0335] lipid B

[0336] [ka]

[0337] In one embodiment, the ssPalmE lipid is a ssPalmE-P4-C2 lipid comprising the structure of lipid C.

[0338] lipid C

[0339] [ka]

[0340] In one embodiment, the ssPalmE lipid is a ssPalmE-Paz4-C2 lipid comprising the structure of lipid D.

[0341] lipid D

[0342] [ka]

[0343] 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.

[0344] Lipid E

[0345] [ka]

[0346] 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.

[0347] lipid F

[0348] [ka]

[0349] 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.

[0350] lipid G

[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] lipid H

[0354] [ka]

[0355] 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.

[0356] lipid J

[0357] [ka]

[0358] In some embodiments, the lipid nanoparticles of the present disclosure comprise lipid A, as listed above.

[0359] In some embodiments, the lipid nanoparticles of the present disclosure may comprise lipid A, DOPC, cholesterol, and PEG-DMG. In some embodiments, the lipid nanoparticles of the present disclosure may comprise lipid A, DOPC, cholesterol, and PEG2000-DMG.

[0360] In some embodiments, the lipid nanoparticles of the present disclosure comprise lipid A, DOPC, cholesterol, PEG, 2000 In a further embodiment, the lipid nanoparticles may comprise lipid A, DOPC, cholesterol, PEG, PEG-DMG, and GalNAc. 2000 -DMG and GalNAc may be contained in a molar ratio of 50%:10%:38%:1.5%:0.5%, respectively.

[0361] In one embodiment, lipid particle (e.g., LNPs comprising ApoE polypeptide or fragment thereof and / or ApoB polypeptide or fragment thereof linked to LNPs) formulations are 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 herein by reference in its entirety. This can be accomplished by high energy mixing of ethanolic 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.

[0362] In general, lipid particles (e.g., LNPs comprising an ApoE polypeptide, or fragment thereof, and / or an ApoB polypeptide, or fragment thereof bound to the 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 ApoE polypeptides, or fragments thereof, and / or ApoB polypeptides, or fragments thereof bound to the LNPs) are prepared with a ceDNA (mass or weight) to total lipid ratio of about 10:1 to 60:1. According to some embodiments, lipid particles (e.g., LNPs comprising ApoE polypeptides, or fragments thereof, and / or ApoB polypeptides, or fragments thereof bound to the LNPs) 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.

[0363] 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.

[0364] According to some embodiments, LNPs comprising an ApoE polypeptide, or fragment thereof, and / or an ApoB polypeptide, or fragment thereof, bound to the LNPs 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.

[0365] 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.

[0366] 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., LNP comprising an ApoE polypeptide, or fragment thereof, and / or an ApoB polypeptide, or fragment thereof bound to the LNP).

[0367] 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.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 formula X.

[0368] lipid K

[0369] [ka]

[0370] 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.

[0371] 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).

[0372] In one embodiment, the cationic lipid is ss-OP of formula I. In another embodiment, the cationic lipid is SS-PAZ of formula II.

[0373] 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.

[0374] B. Noncationic lipids In one embodiment, the lipid particle (e.g., an LNP comprising an ApoE polypeptide, or fragment thereof, and / or an ApoB polypeptide, or fragment thereof bound to the LNP) may further comprise a non-cationic lipid. The non-cationic lipid may serve to enhance fusogenicity and enhance the stability of the LNP during formation. Non-ionizable lipids include amphipathic lipids, neutral lipids, and anionic lipids. Thus, the non-cationic lipid may be a neutral uncharged, zwitterionic, or anionic lipid. The non-cationic lipids are typically used to enhance membrane fusogenicity.

[0375] 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 ... 16-O-dimethyl-1-phenylethanolamine (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 (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);It is to 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.

[0376] Other examples of non-cationic lipids suitable for use in lipid particles (e.g., LNPs comprising an ApoE polypeptide, or fragment thereof, and / or an ApoB polypeptide, or fragment thereof bound 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.

[0377] 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.

[0378] 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).

[0379] 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.

[0380] 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).

[0381] 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.

[0382] 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).

[0383] 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).

[0384] 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-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbamate, N-(carbonyl-methoxypolyethyleneglycol) 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.

[0385] 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). PEG lipids can be one or more of PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], PEG-DMB (3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In one embodiment, the PEG lipid is one or more of PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000],

[0386] [ka] The compound may be selected from the group consisting of:

[0387] In some embodiments, the PEGylated lipid is N-(carbonyl-methoxypolyethylene glycol n)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG n , where n (representing the average molecular weight of PEG) 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, 2000, or 5000), 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). DSPE-PEG, where n (representing the average molecular weight of PEG) is 350, 500, 750, 1000, 2000, or 5000. 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 PEGylated lipid is DSPE-PEG-OH. In some embodiments, the PEGylated lipid is DSPE-PEG-azide. In some embodiments, the PEGylated lipid is PEG-DMG. In some embodiments, the PEGylated lipid is PEG-DSG.

[0388] In some embodiments, the conjugated lipid, e.g., the PEGylated lipid, comprises a tissue-specific ligand, e.g., a first or second ligand, e.g., DSPE-PEG conjugated to a GalNAc ligand, DSG-PEG conjugated to a GalNAc ligand.

[0389] 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.

[0390] 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 about 0.5-10% (mol). In some embodiments, the PEGylated lipid content is about 1-5% (mol). In some embodiments, the PEGylated lipid content is about 2-4% (mol). In some embodiments, the PEGylated lipid content is about 2-3% (mol). In some embodiments, the PEGylated lipid content is about 1-3% (mol). In some embodiments, the PEGylated lipid content is about 0.75-2.5% (mol). In some embodiments, the PEGylated lipid content is about 0.75-2.0% (mol). In some embodiments, the PEGylated lipid content is about 0.75-1.8% (mol). In some embodiments, the PEGylated lipid content is about 1-2% (mol). In some embodiments, the PEGylated lipid content is about 0.75-1.5% (mol). In some embodiments, the PEGylated lipid content is about 1-1.8% (mol). In some embodiments, the PEGylated lipid content is about 1-1.5% (mol). In some embodiments, the PEGylated lipid content is about 1-1.3% (mol). In some embodiments, the PEGylated lipid content is about 1-1.2% (mol). In some embodiments, the PEGylated lipid content is about 0.75-1.5% (mol). In some embodiments, the PEGylated lipid content is about 0.75-1.25% (mol). In some embodiments, the PEGylated lipid content is about 1.5-1.8% (mol). In some embodiments, the PEGylated lipid content is about 1.2-1.5% (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).

[0391] 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) can comprise 30-70% 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 comprises 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.

[0392] 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.

[0393] In another aspect, the present disclosure provides a lipid nanoparticle formulation comprising a phospholipid, a lecithin, a phosphatidylcholine, and a phosphatidylethanolamine.

[0394] 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).

[0395] 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.

[0396] 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.

[0397] 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(201 0), 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.

[0398] 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.

[0399] 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 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.

[0400] 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.

[0401] III. Closed-end DNA (ceDNA) Vectors The embodiments of the present disclosure are based on methods and compositions comprising closed-end linear duplex (ceDNA) vectors capable of expressing a transgene (e.g., a therapeutic nucleic acid (TNA)). 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 for the insertion of control elements, such as regulatory switches as disclosed herein, large transgenes, multiple transgenes, etc.

[0402] 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.

[0403] 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.

[0404] 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.

[0405] 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.

[0406] 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.

[0407] 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.

[0408] 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.

[0409] 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.

[0410] 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.

[0411] In one embodiment, the rigid therapeutic nucleic acid may be a plasmid.

[0412] In one embodiment, the ceDNA vectors disclosed herein are used for therapeutic purposes (e.g., medical, diagnostic, or veterinary uses) or immunogenic polypeptides.

[0413] 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.

[0414] In one embodiment, the ceDNA expression cassette may include an expressible exogenous sequence (e.g., an open reading frame) encoding, for example, a protein that is absent, inactive, or insufficiently active in the recipient subject, or a gene encoding a protein having 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 also encodes a corrective DNA strand, and may be 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., 112inisteriR). In one embodiment, the expression cassette may include an exogenous sequence encoding 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.

[0415] 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.

[0416] IV. Therapeutic Nucleic Acids Aspects of the present disclosure generally provide compositions (e.g., pharmaceutical compositions) comprising a lipid nanoparticle (LNP) and a therapeutic nucleic acid (TNA), wherein the LNP comprises an ApoE polypeptide, or a fragment thereof, and / or an ApoB polypeptide, or a fragment thereof, bound to the LNP.

[0417] Exemplary therapeutic nucleic acids of the present disclosure include, but are not limited to, minigenes, plasmids, minicircles, small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASO), ribozymes, closed-end double-stranded DNA (e.g., ceDNA, CELId, linear covalently closed DNA (113inisteringg"), doggybone™, protelomeric closed-end RNA, or dumbbell linear RNA), dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, gRNA, DNA viral vectors, viral RNA vectors, and any combination thereof.

[0418] 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.

[0419] 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).

[0420] 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.

[0421] Modified Therapeutic Nucleic Acids Aspects of the present disclosure further provide a lipid particle comprising a lipid nanoparticle (LNP) and a therapeutic nucleic acid (TNA) (e.g., a composition (e.g., a pharmaceutical composition) comprising an ApoE polypeptide, or fragment thereof, and / or an ApoB polypeptide, or fragment thereof, bound to the LNP), and a pharmaceutical composition comprising a modified therapeutic nucleic acid (TNA), where TNA is as defined above.

[0422] In one embodiment, the DNA vector 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.

[0423] 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.

[0424] 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.

[0425] 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.

[0426] 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.

[0427] 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.

[0428] The following are provided as non-limiting examples.

[0429] 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 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 Application No. 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.

[0430] 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.

[0431] 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.

[0432] 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.

[0433] 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.

[0434] In yet another aspect, the present invention 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.

[0435] 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.

[0436] 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.

[0437] 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.

[0438] 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.

[0439] 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 invention 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 invention lacks both functional AAV cap and AAV rep genes.

[0440] 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.

[0441] ceDNA Plasmids ceDNA-plasmids are plasmids used for the later production of ceDNA vectors. In some embodiments, 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, and the like, 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.

[0442] 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).

[0443] 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.

[0444] 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.

[0445] 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.

[0446] 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.

[0447] 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.

[0448] 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.

[0449] 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.

[0450] 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.

[0451] 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.

[0452] 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.

[0453] 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.

[0454] VII. Pharmaceutical Compositions and Formulations Provided herein is a pharmaceutical composition comprising a lipid nanoparticle (LNP) and a therapeutic nucleic acid (TNA), the LNP comprising an ApoE polypeptide, or a fragment thereof, and / or an ApoB polypeptide, or a fragment thereof, bound to the LNP, and at least one pharma- ceutically acceptable excipient. According to some embodiments, the pharmaceutical composition is delivered to an LDLR-expressing tissue via binding of the ApoE polypeptide and / or ApoB polypeptide present in the LNP to an LDLR receptor. According to some embodiments, the composition is delivered to retinal cells in the eye. According to some embodiments, the composition is delivered to hepatic cells in the liver. According to some embodiments, the composition is internalized by retinal cells in the eye. According to some embodiments, the composition is internalized by hepatic cells in the liver.

[0455] In one embodiment, the lipid particles (eg, lipid nanoparticles) are substantially non-toxic to a subject, eg, a mammal, such as a human.

[0456] In some embodiments, the LNPs comprise a lipid selected from the group consisting of a cationic lipid described herein, a sterol or derivative thereof, a non-cationic lipid, and at least one PEGylated lipid. In some embodiments, the LNPs comprise a cationic lipid. In some embodiments, the LNPs comprise a sterol or derivative thereof. In some embodiments, the LNPs comprise a non-cationic lipid. In some embodiments, the LNPs comprise at least one PEGylated lipid.

[0457] 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.

[0458] According to some embodiments, the TNA (e.g., ceDNA) is encapsulated in lipids. In one embodiment, the TNA may be fully encapsulated in the lipid location 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.

[0459] 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 de...

Claims

1. A pharmaceutical composition comprising a lipid nanoparticle (Lipid NanoParticle, LNP) and a therapeutic nucleic acid (Therapeutic Nucleic Acid, TNA), wherein the LNP comprises an apolipoprotein E (Apolipoprotein E, ApoE) polypeptide or a fragment thereof, and / or an apolipoprotein B (Apolipoprotein B, ApoB) polypeptide or a fragment thereof, which are bound to the LNP, and at least one pharmaceutically acceptable excipient, the ApoE polypeptide or the fragment thereof, and / or the ApoB polypeptide or the fragment thereof, can bind to a low-density lipoprotein (Low-Density Lipoprotein, LDL) receptor on the cell or an LDL receptor family member, and the LNP can be internalized into the cell, a pharmaceutical composition.

2. The ApoE polypeptide bound to the LNP comprises an amino acid sequence having at least 85% identity to the amino acid sequence shown in EELRVRLASHLRKLKRLLRDADDLQKGGGC (SEQ ID NO: 1) or the amino acid sequence shown in SEQ ID NO: 1; the ApoE polypeptide bound to the LNP comprises an amino acid sequence having at least 85% identity to the amino acid sequence shown in EELRVRLASHLRKLKRLLRDADDLQKGG (SEQ ID NO: 3) or the amino acid sequence shown in SEQ ID NO: 3; the ApoE polypeptide bound to the LNP comprises an amino acid sequence comprising a fragment of EELRVRLASHLRKLKRLLRDADDLQKGGGC (SEQ ID NO: 1), and the fragment can bind to the LDL receptor; or the ApoE polypeptide bound to the LNP comprises an amino acid sequence comprising a fragment of EELRVRLASHLRKLKRLLRDADDLQKGG (SEQ ID NO: 3), and the fragment can bind to the LDL receptor, the pharmaceutical composition according to claim 1. **Claim 3**: The pharmaceutical composition according to claim 2, wherein the ApoE polypeptide bound to the LNP has an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO:

1. **Claim 4**: The pharmaceutical composition according to claim 2, wherein the ApoE polypeptide bound to the LNP has an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID NO:

1. **Claim 5**: The pharmaceutical composition according to claim 2, wherein the ApoE polypeptide bound to the LNP has an amino acid sequence having at least 99% identity to the amino acid sequence shown in SEQ ID NO:

1. **Claim 6**: The pharmaceutical composition according to claim 2, wherein the ApoE polypeptide bound to the LNP has an amino acid sequence consisting of SEQ ID NO:

1. **Claim 7**: The pharmaceutical composition according to claim 2, wherein the ApoE polypeptide bound to the LNP comprises SEQ ID NO:

1. **Claim 8**: The pharmaceutical composition according to claim 2, wherein the ApoE polypeptide bound to the LNP has an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO:

3. **Claim 9**: The pharmaceutical composition according to claim 8, wherein the ApoE polypeptide bound to the LNP has an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID NO:

3. **Claim 10**: The pharmaceutical composition according to claim 9, wherein the ApoE polypeptide bound to the LNP has an amino acid sequence having at least 99% identity to the amino acid sequence shown in SEQ ID NO:

3. **Claim 11**: The pharmaceutical composition according to claim 10, wherein the ApoE polypeptide bound to the LNP comprises SEQ ID NO:

3. **Claim 12**: The pharmaceutical composition according to claim 2, wherein the ApoE polypeptide bound to the LNP has an amino acid sequence consisting of SEQ ID NO:

3. **Claim 13**: The ApoB polypeptide bound to the LNP comprises an amino acid sequence shown in SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGSGGGC (SEQ ID NO: 2) or an amino acid sequence having at least 85% identity to the amino acid sequence shown in SEQ ID NO: 2; The ApoB polypeptide bound to the LNP comprises an amino acid sequence shown in SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGSGGG (SEQ ID NO: 4) or an amino acid sequence having at least 85% identity to the amino acid sequence shown in SEQ ID NO: 4; The ApoB polypeptide bound to the LNP comprises an amino acid sequence comprising a fragment of SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGSGGGC (SEQ ID NO: 2), and the fragment is capable of binding to the LDL receptor; or The pharmaceutical composition according to claim 1, wherein the ApoB polypeptide bound to the LNP comprises an amino acid sequence comprising a fragment of SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGSGGG (SEQ ID NO: 4), and the fragment is capable of binding to the LDL receptor. **Claim 14**: The pharmaceutical composition according to claim 13, wherein the ApoB polypeptide bound to the LNP has an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO:

2. **Claim 15**: The pharmaceutical composition according to claim 13, wherein the ApoB polypeptide bound to the LNP has an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID NO:

2. **Claim 16**: The pharmaceutical composition according to claim 13, wherein the ApoB polypeptide bound to the LNP has an amino acid sequence having at least 99% identity to the amino acid sequence shown in SEQ ID NO:

2. **Claim 17**: The pharmaceutical composition according to claim 13, wherein the ApoB polypeptide bound to the LNP has an amino acid sequence comprising SEQ ID NO:

2. **Claim 18**: The pharmaceutical composition according to claim 13, wherein the ApoB polypeptide bound to the LNP has an amino acid sequence consisting of SSVIDALQYKLEGTTRLTRKRGLKLA TALSLSNKFVEGSGG C (SEQ ID NO: 2). **Claim 19**: The pharmaceutical composition according to claim 13, wherein the ApoB polypeptide bound to the LNP has an amino acid sequence containing SEQ ID NO:

4. **Claim 20**: The pharmaceutical composition according to claim 13, wherein the ApoB polypeptide bound to the LNP has an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO:

4. **Claim 21**: The pharmaceutical composition according to claim 13, wherein the ApoB polypeptide bound to the LNP has an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID NO:

4. **Claim 22**: The pharmaceutical composition according to claim 13, wherein the ApoB polypeptide bound to the LNP has an amino acid sequence having at least 99% identity to the amino acid sequence shown in SEQ ID NO:

4. **Claim 23** The pharmaceutical composition according to claim 13, wherein the ApoB polypeptide has an amino acid sequence consisting of SSVIDALQYKLEGTTRLTRKRGLKLA TALSLSNKFVEGSGG (SEQ ID NO: 4). **Claim 24** The pharmaceutical composition according to claim 1, wherein the LNP contains at least one lipid selected from the group consisting of a cationic lipid, a sterol or its derivative, a non-cationic lipid, and at least one PEGylated lipid. **Claim 25** The pharmaceutical composition according to claim 1, wherein the TNA is encapsulated in the LNP. **Claim 26** The pharmaceutical composition according to claim 1, wherein the TNA is selected from the group consisting of a minigene, a plasmid, a minicircle, small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotide (ASO), ribozyme, closed-end (ceDNA), ministring, doggybone (trademark), telomere closed-end DNA, dumbbell linear DNA, dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), mRNA, tRNA, rRNA, gRNA, DNA virus vector, viral RNA vector, non-viral vector, and any combination thereof.

27. The LNP contains at least one PEGylated lipid, and the at least one PEGylated lipid is bound to the ApoE polypeptide or a fragment thereof; The LNP contains at least one PEGylated lipid, and the at least one PEGylated lipid is bound to the ApoB polypeptide or a fragment thereof; The LNP contains at least one PEGylated lipid, and the ApoE polypeptide or a fragment thereof and / or the ApoB polypeptide or a fragment thereof are chemically conjugated to the at least one PEGylated lipid; The LNP contains a cationic lipid selected from the group consisting of the lipids shown in Table 2, Table 5, Table 6, Table 7, and Table 8; The LNP 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), dieleoyl 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);Comprising a non-cationic lipid selected from the group consisting of lecithin, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, lysophosphatidylcholine, dilinoleoyl phosphatidylcholine, and mixtures thereof; The LNP is a sterol or a derivative thereof, and the sterol or a derivative thereof is cholesterol; The LNP is a sterol or a derivative thereof, and the sterol or a derivative thereof is beta-sitosterol; The pharmaceutical composition is for administration to a subject; The subject is a human patient in need of treatment with LNP encapsulated with TNA; The composition is delivered to LDLR-expressing tissue via the binding of the ApoE polypeptide and / or the ApoB polypeptide present in the LNP to the LDLR in the LNP; The composition is for delivery to retinal cells in the eye; The composition is for delivery to photoreceptor (PR) cells; The composition is for delivery to retinal pigment epithelium (RPE) cells; The composition is for delivery to photoreceptor (PR) cells and retinal pigment epithelium (RPE) cells, and the expression of the TNA in the PR cells and the expression of the TNA in the RPE cells are uniformly distributed; and / or The composition is for delivery to hepatocytes in the liver. The pharmaceutical composition according to claim 1.

28. The pharmaceutical composition according to claim 27, wherein the ApoE polypeptide, or a fragment thereof, or the ApoB polypeptide, or a fragment thereof, is chemically conjugated to the PEGylated lipid. **Claim 29** The cationic lipid has the following lipid structure: (i) 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, optionally substituted, R ２ and R ２’ are each independently a straight-chain or branched-chain C １～６ alkylene, optionally substituted, R ３ and R ３’ are each independently a straight-chain or branched-chain C １～６ alkyl, or alternatively, when R ２ is a branched-chain C １～６ alkylene, optionally substituted, R ２ and R ３ together with the intervening N atom form a 4- to 8-membered heterocyclyl, or alternatively, when R ２’ is a branched-chain C １～６ alkylene, optionally substituted, R ２’ and R ３’ together with the intervening N atom form a 4- to 8-membered heterocyclyl, and R ４ and R ４’ are each independently -CR ａ -, -C(R ａ ) ２ CR ａ -, or -[C(R ａ ) ２ ２ CR ａand R ａ is, for each occurrence, independently H or C １～３ alkyl, 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’ are, 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; (ii) 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; (iii) Formula (V): [Chemical Formula 64] 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, or 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; (iv) Formula (XV): [Chemical Formula 65] 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 66】 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)-, wherein, 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 (v) Formula (XX): [Chemical Formula 67] or a pharmaceutically acceptable salt thereof, wherein 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 Formula 68] wherein, 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, for each occurrence, independently hydrogen or C1-C6 alkyl, n is an integer selected from 1, 2, 3, 4, 5, and 6; (vi) any lipid structure in Table 2, Table 5, Table 6, Table 7, or Table 8; (vii) lipid A represented by the following structure: [Chemical formula 69] or a pharmaceutically acceptable salt thereof; (viii) 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, [Chemical formula 70] or a pharmaceutically acceptable salt thereof represented by one of; and / or (ix) 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-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (e.g., 16-O-monomethyl PE), dimethyl-phosphatidyl-ethanolamine (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, dipalmitoyl phosphate, lysophosphatidylcholine, dilinoleoyl phosphatidylcholine, and mixtures thereof; and / or; The pharmaceutical composition according to claim 24, selected from the group consisting of dioleoyl phosphatidylcholine (DOPC), distearoyl phosphatidylcholine (DSPC), and dioleoyl-phosphatidylethanolamine (DOPE).

30. (i) The cationic lipid has the following structural formula 【Chemical formula 63】 Represented by 1-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-(oleoyloxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanylethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl)9-(tridecan-5-yl)nonanedioate (lipid 58); (ii) The cationic lipid has the following structure: 【Chemical formula 69】 Is lipid A represented by or a pharmaceutically acceptable salt thereof; or (iii) The cationic lipid has the following structure: 【Chemical formula 70】 MC3(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA or MC3) having or a pharmaceutically acceptable salt thereof, the pharmaceutical composition according to claim 24.

31. The at least one PEGylated lipid is selected from the group consisting of PEG-dilauroxypropyl; PEG-dimyristyloxypropyl; PEG-dipalmityloxypropyl, PEG-distearyloxypropyl; l-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (DMG-PEG); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethylene glycol], and distearoyl-rac-glycerol-poly(ethylene glycol) (DSG-PEG); PEG-dilauryl glycerol; PEG-dipalmitoyl glycerol; PEG-disteryl glycerol; PEG-dilauryl glycamide; PEG-dimyristyl glycamide; PEG-dipalmitoyl glycamide; PEG-disteryl glycamide; (l-[8'-(cholesta-5-en-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), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-hydroxyl (DSPE-PEG-OH); and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-azide (DMPE-PEG-azide); The at least one PEGylated lipid is selected from the group consisting of DMG-PEG2000, DSPE-PEG2000, DSPE-PEG2000-OH, DSPE-PEG2000-azide, DSG-PEG2000, and combinations thereof; The ApoE polypeptide, or a fragment thereof, and / or the ApoB polypeptide, or a fragment thereof, is covalently attached to the at least one PEGylated lipid of the LNP to form a PEGylated lipid conjugate; and / or The pharmaceutical composition according to claim 27, wherein the ApoE polypeptide, or a fragment thereof, and / or the ApoB polypeptide, or a fragment thereof, is covalently bound to the at least one PEGylated lipid comprising DSPE-PEG or DSPE-PEG-azide.

32. The ApoE polypeptide, or a fragment thereof, and / or the ApoB polypeptide, or a fragment thereof, is covalently bound to the LNP via a non-cleavable linker; The ApoE polypeptide, or a fragment thereof, and / or the ApoB polypeptide, or a fragment thereof, is covalently bound to the LNP via a non-cleavable maleimide-containing linker; The ApoE polypeptide, or a fragment thereof, and / or the ApoB polypeptide, or a fragment thereof, is covalently bound to the LNP via a cleavable linker; The ApoE polypeptide, or a fragment thereof, and / or the ApoB polypeptide, or a fragment thereof, is covalently bound to the LNP via a pyridyldisulfide (PDS)-containing linker; The ApoE polypeptide, or a fragment thereof, and / or the ApoB polypeptide, or a fragment thereof, is bound to the LNP via one or more non-covalent binding interactions selected from the group consisting of hydrogen bonds, van der Waals bonds, ionic bonds, and hydrophobic bonds; and / or, The ApoE polypeptide, or a fragment thereof, and / or the ApoB polypeptide, or a fragment thereof, is covalently bound to the LNP via strain-promoted alkyne-azide cycloaddition (SPAAC) chemistry, and the SPAAC chemistry includes a reaction between a cyclooctyne or a derivative thereof and an azide compound, The cyclooctyne or a derivative thereof is dibenzocyclooctyne (DBCO) or a derivative thereof, The DBCO or a derivative thereof has the following structure: 【Chemical Formula 71】 a DBCO-functionalized ApoE polypeptide or a DBCO-functionalized ApoB polypeptide represented thereby; and / or The pharmaceutical composition according to claim 1, wherein the azide compound is DSPE-PEG2000-azide or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethylene glycol)-2000], or a salt thereof.

33. 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 PEGylated lipid is present in a molar percentage of about 2.1% to about 10%; and / or The pharmaceutical composition according to claim 24, wherein the PEGylated lipid is present in a molar percentage of about 1% to about 2%.

34. The pharmaceutical composition according to claim 1, wherein the ApoE polypeptide, or a fragment thereof, and / or the ApoB polypeptide, or a fragment thereof, is present in a total amount of about 0.02 μg / μg of TNA to about 0.1 μg / μg of TNA.

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

36. The LNP is Lipid A, DOPC, cholesterol and DMG-PEG; Lipid A, DOPC, cholesterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide; Lipid A, DOPE, cholesterol and DMG-PEG; Lipid A, DOPE, cholesterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide; Lipid A, DSPC, cholesterol and DMG-PEG; Lipid A, DSPC, cholesterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide; Lipid A, DOPC, beta-sitosterol and DMG-PEG; Lipid A, DOPC, beta-sitosterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide; Lipid A, DOPE, beta-sitosterol and DMG-PEG; Lipid A, DOPE, beta-sitosterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide; Lipid A, DSPC, beta-sitosterol and DMG-PEG; or Lipid A, DSPC, beta-sitosterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide The pharmaceutical composition according to claim 29, comprising:

37. The DMG-PEG is DMG-PEG2000; The DSPE-PEG is DSPE-PEG2000 or DSPE-PEG5000; and / or The DSPE-PEG-azide is DSPE-PEG2000-azide or DSPE-PEG5000-azide, the pharmaceutical composition according to claim 36.

38. The LNP contains lipid A, DOPC, sterol, DMG-PEG and DSPE-PEG or DSPE-PEG-azide in a molar ratio of about 51:7.3:38.3:2.9:0.5, the pharmaceutical composition according to claim 37.

39. The LNP has a ratio of total lipid TNA of about 10:1 to about 40:1, the pharmaceutical composition according to claim 1.

40. A pharmaceutical composition comprising lipid nanoparticles (Lipid NanoParticle, LNP), therapeutic messenger RNA (mRNA), and at least one pharmaceutically acceptable excipient, wherein the LNP is The ApoE polypeptide or a fragment thereof and / or the ApoB polypeptide or a fragment thereof bound to the LNP, and The following structural formula 【Chemical Formula 73】 (Lipid A) A cationic lipid having the formula, and A pharmaceutical composition, wherein the LNP can deliver the mRNA to retinal cells.

41. The pharmaceutical composition according to claim 40, wherein the LNP can deliver the mRNA to photoreceptor (PR) cells or retinal pigment epithelium (RPE) cells.

42. The pharmaceutical composition according to claim 41, wherein the LNP can be internalized into the PR cells and / or the RPE cells.

43. The pharmaceutical composition according to claim 42, wherein the mRNA expression is uniformly distributed in the PR cells and / or the RPE cells.

44. The pharmaceutical composition according to claim 40, wherein the LNP can deliver the mRNA to retinal cells without causing retinal degeneration or thinning of the outer nuclear layer (ONL).

45. The pharmaceutical composition according to claim 40, wherein the ApoE polypeptide or a fragment thereof and / or the ApoB polypeptide or a fragment thereof can bind to a low-density lipoprotein (LDL) receptor or an LDL receptor family member.

46. The pharmaceutical composition according to claim 40, wherein the LNP contains an ApoE polypeptide or a fragment thereof.

47. The pharmaceutical composition according to claim 40, wherein the LNP contains an ApoB polypeptide or a fragment thereof.

48. The pharmaceutical composition according to claim 40, wherein the ApoE polypeptide comprises the amino acid sequence shown in EELRVRLASHLRKLRKRLlRDADDLQKGG (SEQ ID NO: 3), or has at least 85% identity to the amino acid sequence shown in SEQ ID NO:

3.

49. The pharmaceutical composition according to claim 48, wherein the ApoE polypeptide has at least 85%, at least 90%, at least 95%, or at least 99% identity to the amino acid sequence shown in SEQ ID NO:

3.

50. The pharmaceutical composition according to claim 48, wherein the ApoE polypeptide has an amino acid sequence consisting of the amino acid sequence EELRVRLASHLRKLRKRLlRDADDLQKGG (SEQ ID NO: 3).

51. The pharmaceutical composition according to claim 40, wherein the ApoE polypeptide is a fragment of the amino acid sequence EELRVRLASHLRKLRKRLlRDADDLQKGG (SEQ ID NO: 3) shown in SEQ ID NO: 1, and the fragment can bind to the LDL receptor.

52. The pharmaceutical composition according to claim 40, wherein the ApoB polypeptide comprises the amino acid sequence SSVIDALQYKL EGTTRLTRKRGLKLATALSLSNKFVEGSGG (SEQ ID NO: 2), or has at least 80% identity to the amino acid sequence shown in SEQ ID NO:

2.

53. The pharmaceutical composition according to claim 52, wherein the ApoB polypeptide has at least 85%, at least 90%, at least 95%, or at least 99% identity to the amino acid sequence shown in SEQ ID NO:

2.

54. The pharmaceutical composition according to claim 52, wherein the ApoB polypeptide has an amino acid sequence consisting of SSVIDALQYKL EGTTRLTRKRGLKLATALSLSNKFVEGSGG (SEQ ID NO: 2). **Claim 55**: The pharmaceutical composition according to claim 40, wherein the ApoB polypeptide comprises an amino acid sequence that is a fragment of EELRVRLASHLRKLRKRLRRDADDLQKGG shown in SEQ ID NO: 3, and the fragment is capable of binding to the LDL receptor. **Claim 56** The pharmaceutical composition according to claim 40, wherein the mRNA is encapsulated in the LNP. **Claim 57** The pharmaceutical composition according to claim 40, wherein the LNP further comprises at least one lipid selected from the group consisting of a sterol or a derivative thereof, a non-cationic lipid, and at least one PEGylated lipid. **Claim 58** The pharmaceutical composition according to claim 57, wherein the sterol or a derivative thereof is cholesterol, or the sterol or a derivative thereof is beta-sitosterol. **Claim 59** The pharmaceutical composition according to claim 57, wherein the non-cationic lipid is selected from the group consisting of dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), and dioleoyl-phosphatidylethanolamine (DOPE). **Claim 60** The pharmaceutical composition according to claim 57, wherein the PEGylated lipid is DMG-PEG, DSPE-PEG, DSPE-PEG-OH, DSPE-PEG-azide, DSG-PEG, or a combination thereof. **Claim 61** The pharmaceutical composition according to claim 60, wherein the at least one PEGylated lipid is selected from the group consisting of DMG-PEG2000, DSPE-PEG2000, DSPE-PEG2000-OH, DSPE-PEG-azide, DSG-PEG, and combinations thereof. **Claim 62** The LNP is Lipid A, DOPC, cholesterol, and DMG-PEG; Lipid A, DOPC, cholesterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide; Lipid A, DOPE, cholesterol and DMG-PEG; Lipid A, DOPE, cholesterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide; Lipid A, DSPC, cholesterol and DMG-PEG; Lipid A, DSPC, cholesterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide; Lipid A, DOPC, beta-sitosterol and DMG-PEG; Lipid A, DOPC, beta-sitosterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide; Lipid A, DOPE, beta-sitosterol and DMG-PEG; Lipid A, DOPE, beta-sitosterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide; Lipid A, DSPC, beta-sitosterol and DMG-PEG; or The pharmaceutical composition according to claim 40, comprising Lipid A, DSPC, beta-sitosterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide.

63. The pharmaceutical composition according to claim 62, wherein the DMG-PEG is DMG-PEG2000.

64. The pharmaceutical composition according to claim 62, wherein the DSPE-PEG is DSPE-PEG2000 or DSPE-PEG5000.

65. The pharmaceutical composition according to claim 62, wherein the DSPE-PEG-azide is DSPE-PEG2000-azide or DSPE-PEG5000-azide.

66. The pharmaceutical composition according to claim 62, wherein the LNP comprises lipid A, DOPC, sterol, DMG-PEG and DSPE-PEG or DSPE-PEG-azide in a molar ratio of about 51:7.3:38.3:2.9:0.

5.

67. The pharmaceutical composition according to claim 40, wherein the LNP comprises a PEGylated lipid, and the PEGylated lipid is bound to the ApoE polypeptide or a fragment thereof, or the PEGylated lipid is bound to the ApoB polypeptide or a fragment thereof.

68. The pharmaceutical composition according to claim 67, wherein the ApoE polypeptide or a fragment thereof, or the ApoB polypeptide or a fragment thereof is covalently bound to the PEGylated lipid of the LNP to form a PEGylated lipid conjugate.

69. The pharmaceutical composition according to claim 68, wherein the PEGylated lipid to which the ApoE polypeptide or a fragment thereof, and / or the ApoB polypeptide or a fragment thereof is covalently bound is DSPE-PEG or DSPE-PEG-azide.

70. The pharmaceutical composition according to claim 40, wherein the ApoE polypeptide or a fragment thereof, and / or the ApoB polypeptide or a fragment thereof is covalently bound to the LNP via a non-cleavable linker.

71. The pharmaceutical composition according to claim 70, wherein the non-cleavable linker is a maleimide-containing linker.

72. The pharmaceutical composition according to claim 40, wherein the ApoE polypeptide or a fragment thereof, and / or the ApoB polypeptide or a fragment thereof is covalently bound to the LNP via a cleavable linker.

73. The pharmaceutical composition according to claim 40, wherein the ApoE polypeptide, or a fragment thereof, and / or the ApoB polypeptide, or a fragment thereof, is covalently bound to the LNP via a pyridyldisulfide (PDS)-containing linker.

74. The pharmaceutical composition according to claim 40, wherein the ApoE polypeptide, or a fragment thereof, and / or the ApoB polypeptide, or a fragment thereof, is covalently bound to the LNP via strain-promoted alkyne-azide cycloaddition (SPAAC) chemistry.

75. A dibenzocyclooctyne (DBCO)-functionalized ApoE polypeptide or ApoB polypeptide represented by the following structure: 【Chemical Formula 74】 wherein: the ApoE polypeptide comprises the amino acid sequence shown in EELRVRLASHLRKLRKRLRRDADDLQKGGG (SEQ ID NO: 3), or an amino acid sequence having at least 85% identity to the amino acid sequence shown in SEQ ID NO: 3; the ApoB polypeptide comprises the amino acid sequence shown in SSVIDALQYKL EGTTRLTRKRGLKLATALSLSNKFVEGSGGGC (SEQ ID NO: 2), or an amino acid sequence having at least 85% identity to the amino acid sequence shown in SEQ ID NO: 2, the DBCO-functionalized ApoE polypeptide or ApoB polypeptide.

76. A pharmaceutical composition prepared using the DBCO-functionalized ApoE polypeptide or ApoB polypeptide according to claim 75 as a reagent in combination with an azide compound.

77. A lipid nanoparticle composition prepared using the DBCO-functionalized ApoE polypeptide or ApoB polypeptide according to claim 75 in combination with an azide compound.

78. The pharmaceutical composition according to claim 76 or the lipid nanoparticle composition according to claim 77, wherein the azide compound is DSPE-PEG2000-azide or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethylene glycol)-2000], or a salt thereof.

79. The LNP has a diameter of about 40 nm to about 120 nm; The LNP has a diameter of less than about 100 nm; or The pharmaceutical composition according to claim 1, wherein the LNP has a diameter of about 60 nm to about 80 nm.

80. Use of the pharmaceutical composition according to claim 1 in the manufacture of a medicament for treating a genetic disorder in a subject.

81. The use according to claim 80, wherein the subject is a human.

82. The use according to claim 80, wherein the disorder is an eye disorder.

83. The genetic disorder is melanoma, hemophilia A (factor VIII (FVIII) deficiency), hemophilia B (factor IX (FIX) deficiency), cystic fibrosis (CFTR deficiency), familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, phenylketonuria (PKU), congenital hepatic porphyria, hereditary liver metabolic disorders, Lesch-Nyhan syndrome, sickle cell anemia, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom syndrome, retinoblastoma, mucopolysaccharidosis (e.g., Hurler syndrome (MPS I type), Scheie syndrome (MPS I S type), Hurler-Scheie syndrome (MPS I H-S type), Hunter syndrome (MPS II type), Sanfilippo A, B, C, and D types (MPS III A, B, C, and D types), Morquio A and B types (MPS IVA and MPS IVB), Maroteaux-Lamy syndrome (MPS VI type), Sly syndrome (MPS VII type), hyaluronidase deficiency (MPSType IX), Niemann-Pick disease A / B, C1 and C2 types, Fabry disease, Schindler disease, GM2-gangliosidosis type II (Sandhoff disease), Tay-Sachs disease, metachromatic leukodystrophy, Krabbe disease, mucolipidosis I, II / III and IV types, sialidosis I and II types, glycogen storage disease types I and II (Pompe disease), Gaucher disease types I, II and III, cystinosis, Batten disease, aspartylglucosaminuria, Salla disease, Danon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinosis (CLN1-8, INCL, and LINCL), sphingolipidosis, galactosialidosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy, Friedrich's ataxia, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), dystrophic epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, generalized arterial calcification of infancy (GACI), Leber congenital amaurosis, Stargardt macular dystrophy (ABCA4 deficiency), ornithine transcarbamylase (OTC) deficiency, Usher syndrome, age-related macular degeneration (AMD), alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis (PFIC) type I (ATP8B1 deficiency), type II (ABCB11 deficiency), type III (ABCB4 deficiency), or type IV (TJP2 deficiency), and cathepsin A deficiency, the use according to claim 80, selected from the group consisting of.

84. Use of the pharmaceutical composition according to claim 1 in the manufacture of a medicament for delivering a therapeutic nucleic acid (TNA) to the retina of a subject or increasing the concentration of said TNA in the retina of a subject.

85. Use of the pharmaceutical composition according to claim 1 in the manufacture of a medicament for delivering a therapeutic nucleic acid (TNA) to the liver of a subject or increasing the concentration of said TNA in the liver of a subject. **Claim 86**: The use according to claim 84, wherein the agent is for delivery to an LDLR-expressing tissue via binding of the ApoE polypeptide or a fragment thereof and / or the ApoB polypeptide or a fragment thereof present in the LNP to the LDLR receptor. **Claim 87**: The use according to claim 85, wherein the agent is for delivery to an LDLR-expressing tissue via binding of the ApoE polypeptide or a fragment thereof and / or the ApoB polypeptide or a fragment thereof present in the LNP to the LDLR receptor. **Claim 88**: The use according to claim 84, wherein the agent is for delivery to cells selected from the group consisting of retinal cells, photoreceptor (PR) cells, and retinal pigment epithelial (RPE) cells of the eye. **Claim 89**: The use according to claim 84, wherein the agent is for administration by one or more routes selected from the group consisting of subretinal injection, suprachoroidal injection, and intravitreal injection. **Claim 90**: The pharmaceutical composition according to claim 1, for treating a genetic disorder in a subject. **Claim 91**: The pharmaceutical composition according to claim 1, for delivering a therapeutic nucleic acid (TNA) to the retina of a subject or increasing the concentration of the TNA in the retina of the subject. **Claim 92**: The pharmaceutical composition according to claim 1, for delivering a therapeutic nucleic acid (TNA) to the liver of a subject or increasing the concentration of the TNA in the liver of the subject.

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