RU2778407C2 - Compositions of non-viral non-capsid dna vectors based on lipid nanoparticles - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions is described, including a lipid nanoparticle for the delivery of a non-capsid non-viral DNA vector to a target site, and a pharmaceutical composition for the delivery of heterologous nucleic acid (options). In one option of the implementation, the lipid nanoparticle contains an ionized lipid and a non-viral non-capsid DNA vector with covalently closed ends (hereinafter – ceDNA vector), where the specified ceDNA vector contains at least one heterologous nucleotide sequence functionally located between asymmetrical inverted terminal repeats (hereinafter – asymmetrical ITR), where ITR are asymmetrical relatively to each other, and where one or more of asymmetrical ITR is modified by deletion, insertion, and/or replacement in at least one of areas selected from A, A’, B, B’, C, C’, D, and D’, wherein at least one of asymmetrical ITR contains a functional site of terminal resolution and Rep binding site.

EFFECT: invention expands the arsenal of means for the delivery of a non-capsid non-viral DNA vector to a target site.

37 cl, 37 dwg, 5 tbl, 13 ex

Description

CROSS REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. 62/556381, filed September 9, 2017, #62/675317, filed May 23, 2018, #62/675322, filed May 23, 2018, #62/675324, filed May 23, 2018, and #62 /675327 filed May 23, 2018, the contents of which are hereby incorporated by reference in their entirety.

SEQUENCE LIST

[0002] This application includes a sequence listing that was filed electronically in ASCII format and incorporated herein in its entirety by reference. The specified ASCII copy, created on September 7, 2018, is named 080170-090660WOPT_SL.txt and is 63790 bytes in size.

FIELD OF TECHNOLOGY

[0003] The present invention relates to non-viral capsidless lipid nanoparticle (LNP) vector formulations and their use for delivering exogenous DNA sequences to a target cell, tissue, target organ, or organism.

BACKGROUND OF THE INVENTION

[0004] Recently, non-viral capsidless DNA vectors with covalently closed ends have been described that contain transgenes flanked by AAV2 ITRs. However, the targeted delivery of these DNA vectors into cells, in vitro and in vivo, remains a challenge. Accordingly, there remains a need in the art for formulations to overcome these difficulties.

BRIEF DESCRIPTION OF THE INVENTION

[0005] According to one aspect of the present invention, novel lipid formulations are provided comprising an ionizable lipid and a non-capsid non-viral vector (ccDNA). The scDNA vectors described herein are capsidless linear duplex DNA molecules formed by a continuous strand of complementary DNA with covalently closed ends (linear, continuous and non-capsid structure) that contain a 5' inverted terminal repeat (ITR) sequence and 3 '-sequence of ITRs that are different from each other, or asymmetric with respect to each other. In one aspect, non-viral capsidless DNA vectors with covalently closed ends are preferably linear duplex molecules, and can be derived from a vector polynucleotide that encodes a heterologous nucleic acid that is functionally (i.e., operably) located between two different sequences of inverted terminal repeats (ITRs) (e.g., AAV ITRs), wherein at least one of the ITRs contains an end resolution site and a replication protein (RPS) binding site (sometimes referred to as a replicative protein binding site), e.g., a Rep binding site, and one of the ITRs contains a deletion, insertion and/or substitution with respect to another ITR, i.e. one of the ITRs is asymmetric with respect to the other ITR. In one embodiment, at least one of the ITRs is an AAV ITR, such as a wild-type AAV ITR or a modified AAV ITR. In one embodiment, at least one of the ITRs is an ITR modified from another ITR—that is, said scDNA contains ITRs that are asymmetric with respect to each other.

[0006] In one embodiment, at least one of the ITRs is a non-functional ITR.

[0007] In some embodiments, one or more ITRs are not wild-type ITRs.

[0008] In some embodiments, said scDNA vector, when digested with a single recognition site restriction enzyme on the scDNA vector and analyzed by both native and denaturing gel electrophoresis, exhibits characteristic bands of linear and continuous DNA, compared to linear and discontinuous DNA controls.

[0009] In some embodiments, one or more of the asymmetric ITR sequences of the ccDNA vector are from a virus selected from parvovirus, dependdovirus, and adeno-associated virus (AAV). In some embodiments, said asymmetric ITRs are from different viral serotypes. For example, one or more asymmetric ITRs may be from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.

[0010] In some embodiments, one or more of the asymmetric ITR sequences of the ccDNA vector are synthetic.

[0011] In some embodiments, at least one (e.g., one or both) of the asymmetric ITRs is modified by deletion, insertion, and/or substitution in at least one of the ITR regions selected from A, A', B, B', C, C', D and D'.

[0012] In some embodiments, said scDNA vector contains at least two asymmetric ITRs selected from: (a) SEQ ID NO: 1 and SEQ ID NO: 52; and (b) SEQ ID NO: 2 and SEQ ID NO: 51.

[0013] In some embodiments, said scDNA vector is produced by a method comprising the steps of: (a) incubating a population of insect cells bearing an scDNA expression construct in the presence of at least one Rep protein, wherein said scDNA expression construct encodes said scDNA a vector, under conditions effective for, and for a period of time sufficient to induce the production of the scDNA vector in said insect cells; and (b) isolating said ccDNA vector from said insect cells. Without limitation, said scDNA expression construct may be a scDNA plasmid, scDNA bacmid, or scDNA baculovirus.

[0014] Typically, an insect cell expresses at least one Rep protein. The at least one Rep protein may be from a virus selected from parvovirus, dependdovirus, and adeno-associated virus (AAV). For example, said at least one Rep protein may be from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.

[0015] In some embodiments, said ionizable lipid is the lipid described in the publication from those listed in Table 1.

[0016] In some embodiments of the various aspects described herein, the presence of scDNA can be confirmed by digestion with a single recognition site restriction enzyme on an scDNA vector and analysis of the cleaved DNA material on denaturing and non-denaturing gels to confirm the presence of characteristic linear and continuous DNA versus linear and discontinuous DNA.

[0017] In some embodiments, said DNA vector is derived from a vector polynucleotide, wherein said vector polynucleotide encodes a heterologous nucleic acid operably located between two inverted terminal repeat (ITR) sequences, wherein said two ITSs are different from each other (are asymmetric) , and at least one of the ITRs is a functional ITR containing a functional terminal clearance site and a Rep binding site, and one of the ITRs contains a division, insertion, or substitution relative to the functional ITR; the presence of the Rep protein induces replication of the vector polynucleotide.

[0018] In some embodiments, the production of a DNA vector in an insect cell is provided. For example, said DNA vector may be a method comprising the steps of: (a) incubating a population of insect cells carrying said vector polynucleotide, which does not contain viral capsid coding sequences, in the presence of a Rep protein, under conditions effective for, and for a period of time sufficient to induce production of a capsid-free non-viral DNA vector in insect cells, characterized in that the capsid-free non-viral DNA produced is not contained in said insect cells in the absence of said vector; and (b) harvesting and isolating capsid-free, non-viral DNA from insect cells. In some additional embodiments, the presence of capsid-free, non-viral DNA isolated from insect cells can be confirmed. For example, the presence of non-capsid non-viral DNA isolated from insect cells can be confirmed by cleaving the DNA isolated from insect cells with a restriction enzyme with a single recognition site on said DNA vector and analyzing the digested DNA material on a non-denaturing gel to confirm the presence of characteristic linear bands. and continuous DNA, compared to linear and discontinuous DNA.

[0019] In some embodiments, said DNA vector is derived from a vector polynucleotide. For example, said DNA vector is derived from a vector polynucleotide encoding a heterologous nucleic acid operably located between the AAV2 first and second inverted terminal repeat (ITR) DNA polynucleotide sequences, wherein at least one of the ITRs contains at least one polynucleotide deletion, insertion or substitution relative to the corresponding ITR AAV2 wild-type SEQ ID NO: 1 or SEQ ID NO: 51 to induce replication of the specified DNA vector in the insect cell in the presence of Rep protein. In some additional embodiments of said DNA vector may be a method comprising the steps of: (a) incubating a population of insect cells carrying said vector polynucleotide that does not contain viral capsid coding sequences in the presence of a Rep protein, under conditions effective to, and for a period of time sufficient to induce production of capsid-free non-viral DNA in insect cells, said insect cells not containing viral capsid-coding sequences; and (b) harvesting and isolating capsid-free, non-viral DNA from insect cells. In some additional embodiments, the presence of capsid-free, non-viral DNA isolated from insect cells can be confirmed. For example, the presence of non-capsid non-viral DNA isolated from insect cells can be confirmed by cleaving the DNA isolated from insect cells with a restriction enzyme with a single recognition site on said DNA vector and analyzing the digested DNA material on a non-denaturing gel to confirm the presence of characteristic linear bands. and continuous DNA, compared to linear and discontinuous DNA.

[0020] In some embodiments, said lipid nanoparticle may further comprise a non-cationic lipid, a PEG-conjugated lipid, a sterol, or any combination thereof.

[0021] In some embodiments, said lipid nanoparticle further comprises a non-cationic lipid, wherein said non-ionic lipid is selected from the group consisting of distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylglycerol ( DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine ( DMPE), distearoylphosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine (such as 16-O-monomethyl-PE), dimethylphosphatidylethanolamine (such as 16-O-dimethyl-PE), 18-1-trans-PE, 1-stearoyl-2-oleoylphosphatidylethanolamine ( SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine ( EPC), dioleylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierukoylphosphatidylcholine (DEPC), palmitoyloleoylphosphatidylglycerol (POPG), dielaidoylphosphatidylethanolamine (DEPE), lecithin, phosphatidylazodilecitamine , phosphatidylserine, phosphatidylinositol, sphingomyelin, ovarian sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine and non-cationic lipids, as described, for example, in WO 2017/099823 or US 2018/0028664.

[0022] In some embodiments, said lipid particle further comprises a conjugated lipid, wherein said conjugated lipid is selected from the group consisting of PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG- DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, 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 dialkoxypropylcarbam, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl sodium salt -sn-glycero-3-phosphoethanolamine, and those described in Table 2.

[0023] In some embodiments, said lipid particle further comprises cholesterol or a cholesterol derivative as described in PCT Publication WO 2009/127060 or US Patent Publication US 2010/0130588.

[0024] In some embodiments, said lipid particle comprises an ionizable lipid, a non-cationic lipid, a conjugated lipid that inhibits particle aggregation, and a sterol. The amount of ionizable lipid, non-cationic lipid, conjugated lipid that inhibits particle aggregation, and sterol can vary independently. According to some embodiments, said lipid nanoparticle contains an ionizable lipid in an amount of from about 20 mol.% to about 90 mol.% of the total amount of lipids present in the specified particle, a non-cationic lipid in an amount of from about 5 mol.% to about 30 mol.% from the total amount of lipids present in the specified particle, a conjugated lipid that inhibits the aggregation of particles, in an amount of from about 0.5 mol.% to about 20 mol.% of the total amount of lipids present in the specified particle, and sterol in an amount of from about 20 mol.% to about 50 mol.% of the total amount of lipids present in the specified particle.

[0025] The ratio of total lipids to vector DNA may vary as needed. For example, the ratio of total lipids to vector DNA (w/w) can be from about 10:1 to about 30:1.

[0026] The present invention also provides a composition comprising a first lipid nanoparticle and an additional compound. Said first lipid nanoparticle contains an ionizable lipid and a first non-capsid non-viral vector.

Said non-capsid non-viral vector, after digestion with a single recognition site restriction enzyme on said DNA vector, is distinguished by the presence of characteristic linear and continuous DNA bands, compared to linear and discontinuous DNA in a non-denaturing gel assay.

[0027] In some embodiments, said additional compound is included in a second lipid nanoparticle. Said first and said second lipid nanoparticles may be the same or different. In some embodiments, said first and second lipid nanoparticles are different. In some embodiments, said first and second lipid nanoparticles are the same, i. an additional compound is included in the first lipid nanoparticle.

[0028] Any desired molecule can be used as an additional compound. In some embodiments, said additional compound is a second non-capsid non-viral vector. Said first and second non-capsid non-viral vectors may be the same or different. In some embodiments, said first and second non-capsid non-viral vectors are different.

[0029] In some embodiments, said additional compound is a therapeutic agent.

[0030] These and other aspects of the present invention are described in more detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The materials of this patent or application contain at least one color drawing. Copies of this patent or published patent application with color drawing(s) will be made available to the Office upon request and upon payment of the required fee.

[0032] FIG. 1A shows an example of the structure of an scDNA vector. In this embodiment, an exemplary scDNA vector includes an expression cassette containing the CAG promoter, WPRE and BGHpA. An open reading frame (ORF) encoding a luciferase transgene is inserted into a cloning site (R3/R4) between the CAG promoter and WPRE. The expression cassette is flanked by two inverted terminal repeats (ITRs) - the wild-type AAV2 ITR at the start (at the 5' end) and the modified ITR at the end (at the 3' end) of the expression cassette, respectively, these two ITRs flanking the expression cassette are asymmetrical with respect to each other.

[0033] FIG. 1B depicts an exemplary structure of a scDNA vector with an expression cassette containing the CAG promoter, WPRE and BGHpA. An open reading frame (ORF) encoding a luciferase transgene is inserted into a cloning site between the CAG promoter and WPRE. The expression cassette is flanked by two inverted terminal repeats (ITRs), a modified ITR at the start (at the 5' end) and a wild-type ITR at the end (at the 3' end) of the expression cassette.

[0034] FIG. 1C depicts an exemplary structure of a scDNA vector with an expression cassette containing an enhancer/promoter, an open reading frame (ORF) for transgene insertion, a post-transcriptional element (WPRE), and a poly(A) signal. An open reading frame (ORF) allows insertion of a transgene into a cloning site between the CAG promoter and WPRE. The expression cassette is flanked by two inverted terminal repeats (ITRs) that are asymmetrical with respect to each other; modified ITR at the beginning (at the 5'-end) and modified ITR at the end (at the 3'-end) of the expression cassette, while both the 5'-ITR and 3'ITR are modified but contain different modifications (i.e., do not contain the same modifications).

[0035] FIG. 2A shows the T-shaped stalk-loop structure of one AAV2 wild-type ITR (SEQ ID NO: 538) with identified A-A' arm, B-B' arm, C-C' arm, two Rep binding sites (RBE and RBE') and end resolution site (trs). RBE contains a series of 4 duplex tetramers that are believed to interact with either Rep 78 or Rep 68. In addition, RBE' is also expected to interact with the Rep complex assembled on wild-type ITR or mutated ITR in this construct. The D and D' regions contain transcription factor binding sites and other conserved structures. In FIG. 2B shows the putative Rep-catalyzed nicking and ligating activity in the wild-type ITR of FIG. 2A, including a stem-loop T-shaped AAV2 wild-type ITR with identified A-A' arm, B-B' arm, C-C' arm, two Rep binding sites (RBE and RBE'), a terminal resolution site (trs); and a D and D' region containing several transcription factor binding sites and other conserved structures.

[0036] FIG. 3A shows the primary structure (polynucleotide sequence) (left) and the secondary structure (right) of the RBE-containing portions of the A-A' arm, and the C-C' arm and the B-B' arm of the left AAV2 wild-type ITR (SEQ ID NO: 540 ). In FIG. 3B shows an example sequence of a mutated ITR (also referred to as modified ITR) for a left ITR. Shown are the primary structure (left) and the predicted secondary structure (right) of the RBE portion of arm A-A', arm C, and arm B-B' from the mutated left ITR example (ITR-1, left) (SEQ ID NO: 113). Shown are the primary structure (left) and the predicted secondary structure (right) of the RBE-containing portion of the A-A' arm, C arm, and B-B' arm of an example mutated left ITR (ITR-1, left) (SEQ ID NO: 113). In FIG. 3C shows the primary structure (left) and secondary structure (right) of the RBE-containing portion of the A-A' loop, and arms B-B' and C-C' of the wild-type right AAV2 ITR (SEQ ID NO: 541). In FIG. 3D shows an example of a right modified ITR. Shown are the primary structure (left) and the predicted secondary structure (right) of the RBE-containing portion of the A-A', B-B', and C arms of an example mutant right ITR (ITR-1, right) (SEQ ID NO: 114). Any combination of left and right ITRs (eg, AAV2 or other viral serotype ITRs, or synthetic ITRs) may be used, provided that the left ITR is asymmetric to or different from the right ITR. On each of the FIGS. 3A-3D, the polynucleotide sequences refer to the sequence used in the plasmid or baculovirus baculovirus genome used to generate scDNA as described herein. Also in each of Figs. 3A-3D show the respective secondary structures of scDNA derived from the configurations of the scDNA vector in the plasmid or baculovirus baculovirus genome and predicted Gibbs free energies.

[0037] FIG. 4A is a schematic of an upstream process for producing baculovirus-infected insect cells (BVs) that are suitable for producing scDNA by the method schematically shown in FIG. 4B. In FIG. 4C shows the biochemical method and process for confirming the production of the scDNA vector. In FIG. 4D and FIG. 4E are schematic diagrams describing the process for identifying the presence of scDNA in DNA collected from cell pellets obtained during the scDNA preparation processes of FIG. 4B. In FIG. 4E shows DNA with a discontinuous pattern. The zcDNA can be cut with a restriction endonuclease having a single recognition site on the scDNA vector to produce two DNA fragments of different sizes (1 kb and 2 kb) under both neutral and denaturing conditions. In FIG. 4E also shows scDNA having a linear and continuous structure. Said scDNA vector can be cut with a restriction endonuclease to produce two DNA fragments migrating as 1 kb stretches. and 2 kb. under neutral conditions, however, under denaturing conditions, the chains remain connected and produce single chains migrating as 2 kb stretches. and 4 kb. In FIG. 4D is a schematic representation of expected bands for an example of scDNA uncleaved or restriction endonuclease cleaved and then subjected to electrophoresis on either native or denaturing gel. The leftmost schematic image shows a native gel with several bands suggesting that in duplex and uncut form, ccDNA exists in at least monomeric and dimeric states, which are seen as a faster migrating smaller monomer and a slower migrating dimer that is 2 times the size of the monomer. The second schematic from the left shows that when scDNA is cut with a restriction endonuclease, the original bands disappear and bands of more rapidly migrating fragments (eg, smaller) appear, corresponding to the expected sizes of the fragments remaining after cleavage. Under denaturing conditions, the original duplex DNA is single stranded and migrates as a compound twice as large as that seen on native gel, since the complementary strands are covalently linked. Accordingly, in the second diagram on the right, the cleaved scDNA shows a similar distribution of bands compared to that seen on the native gel, however the bands correspond to the migration of fragments that are twice the size of their native gel equivalents. The rightmost diagram shows that uncut scDNA under denaturing conditions migrates as a single-stranded open ring, and, accordingly, the size of the observed bands is twice the size of those observed under native conditions with an unopened ring. In the specified drawing "t.p.o." refers to the relative size of nucleotide molecules based, depending on context, on either the length of the nucleotide chain (eg, for single-stranded molecules observed under denaturing conditions) or the number of base pairs (eg, for double-stranded molecules observed under native conditions).

[0038] FIG. 5 is an exemplary denaturing gel run of examples of scDNA vectors with (+) or without (-) endonuclease digestion (EcoRI for scDNA constructs 1 and 2; BamH1 for scDNA constructs 3 and 4; SpeI for scDNA constructs 5 and 6 ; and XhoI for scDNA constructs 7 and 8). Dimensions for bands marked with an asterisk are defined and shown at the bottom of the drawing.

[0039] FIG. 6A shows an example of a Rep bacmid in plasmid pFBDLSR containing the nucleic acid sequences for the Rep proteins Rep52 and Rep78. The specified example Rep-bakmida contains: fragment of the IE1 promoter (SEQ ID NO.66); the Rep78 nucleotide sequence, including the Kozak sequence (SEQ ID NO:67), the polyhedrin promoter sequence for Rep52 (SEQ ID NO:68), and the Rep58 nucleotide sequence starting with the Kozak sequence gccgccacc) (SEQ ID NO:69). In FIG. 8B is a schematic representation of an example zcDNA plasmid-1, with wt-L ITR, CAG promoter, luciferase transgene, WPRE, and polyadenylation sequence; and mod-R (modified right) ITR.

[0040] FIG. 7A shows the results of an in vitro protein expression assay measuring luciferase activity (Y-axis, RQ (Luc)) in HEK293 cells 48 hours after transfection for 400 ng (black), 200 ng (grey), or 100 ng (white). constructs identified on the x-axis (construct-1, construct-3, construct-5, construct-7 (Table 5 in Example 1). Figure 7B shows luciferase activity (Y-axis, RQ(Luc)) measured in cells HEK293 48 hours after transfection of 400 ng (black), 200 ng (grey) or 100 ng (white) constructs identified on the x-axis (construct-2, construct-4, construct-6, construct-8) ( Table 5) Also shown is luciferase activity measured in Fugene-treated HEK293 cells without any plasmids ("Fugene"), or untreated HEK293 cells ("untreated").

[0041] In FIG. 8A shows the viability of HEK293 cells (Y-axis) 48 hours after transfection with 400 ng (black), 200 ng (grey) or 100 ng (white) of the constructs identified on the X-axis (construct-1, construct-3, construct -5, construction-7). In FIG. 8B shows the viability of HEK293 cells (Y-axis) 48 hours after transfection with 400 ng (black), 200 ng (grey) or 100 ng (white) of the constructs identified on the x-axis (construct-2, construct-4, construct -6, construction-8).

[0042] FIG. 9-11 are bar graphs showing the average lipid nanoparticle size and scDNA encapsulation of some examples of lipid nanoparticles prepared with buffers containing different salts at a constant N/P ratio (FIG. 9) or varying N/P ratios (FIGS. 10 and 11 ).

[0043] FIG. 12 is a bar graph showing the effect of serum/BSA on encapsulation in examples of lipid nanoparticles.

[0044] FIG. 13 is a bar graph showing the release of ccDNA from examples of lipid nanoparticles in the presence of dioleylphosphatidylserine (DOPS) liposomes.

[0045] FIG. 14 is a bar graph showing the effect of serum/BSA on encapsulation in examples of lipid nanoparticles.

[0046] FIG. 15 is a bar graph showing the release of ccDNA from examples of lipid nanoparticles in the presence of dioleylphosphatidylserine (DOPS) liposomes.

[0047] FIG. 16 shows ApoE binding for some examples of lipid nanoparticles.

[0048] FIG. 16 is a bar graph showing expression in HEK293 of scDNA examples.

[0049] FIG. 18 are gel electrophoresis photographs showing expression in HEK293 of scDNA examples.

[0050] FIG. 19 is a bar graph showing expression in HEK293 of scDNA examples.

[0051] FIG. 20 shows some examples of compounds of formula (I) and formula (II) described in example 10.

[0052] FIG. 21 shows the general scheme for the synthesis of compounds of formula (I) and (II) described in example 10.

[0053] FIG. 22 is a general scheme for the synthesis of compounds of formulas (I) and (II) described in example 10.

[0054] FIG. 23 shows some examples of the compounds of formula (III) described in example 11.

[0055] FIG. 24 shows general schemes for the synthesis of compounds of formula (III), formula (IV) and formula (V) described in example 11.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0056] Unless otherwise indicated herein, scientific and technical terms used in connection with the present invention have the meanings commonly implied by those skilled in the art to which the present invention pertains. It should be understood that the present invention is not limited to the specific methodology, protocols, and reagents, and the like described herein, and therefore, such methods, protocols, and reagents 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. Definitions of common immunology and molecular biology terms can be found in the following sources: The Merck Manual of Diagnosis and Therapy, 19th ed., published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), Fields Virology, 6th ed., edition Lippincott Williams & Wilkins, Philadelphia, PA, USA (2013), ^Knipe , DM and Howley, PM (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, edition Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (eds.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, Elsevier edition, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, ^{4th ed} ., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), content which are incorporated by reference in this document in their entirety.

[0057] As used herein, the term "lipid nanoparticle" refers to a vesicle formed by one or more lipid components. Lipid nanoparticles are commonly used as nucleic acid delivery vehicles in the context of pharmaceutical development. They work by fusing with the cell membrane and rearranging its lipid structure to deliver a drug or active pharmaceutical ingredient (API). Typically, the composition of lipid nanoparticles for such delivery includes synthetic ionizable or cationic lipids, phospholipids (particularly compounds containing a phosphatidylcholine group), cholesterol, and polyethylene glycol lipid (PEG lipid); however, these compositions may also include other lipids. The resulting lipid composition generally determines the surface characteristics and thus the protein content (opsonization) in biological systems, and thus the control of biodistribution and cellular uptake characteristics.

[0058] As used herein, the term "liposome" refers to lipid molecules assembled in a spherical configuration encapsulating an inner volume of an aqueous solution separated from an outer volume of an aqueous solution. Liposomes are vesicles having at least one lipid bilayer. Liposomes are commonly used as drug/therapeutic delivery vehicles in the context of pharmaceutical development. They work by fusing with the cell membrane and rearranging its lipid structure to deliver a drug or active pharmaceutical ingredient. Liposomal formulations for such delivery typically consist of phospholipids, in particular compounds containing a phosphatidylcholine group, however, these formulations may also include other lipids.

[0059] As used herein, the term "ionizable lipid" refers to lipids containing at least one protonable or deprotonable group, such that said lipid is positively charged at a pH value equal to or less than the physiological pH value (e.g., pH 7.4 ), and is neutral at a second pH, preferably equal to or greater than the physiological pH. One skilled in the art will appreciate that pH dependent proton addition or removal is an equilibrium process, and that reference to a charged or neutral lipid refers to the nature of the predominant lipid species and not necessarily all lipids will be in the charged or neutral form. . Typically, ionizable lipids have a pK _a of the protonated group in the range of about 4 to about 7. Ionizable lipids are also referred to herein as cationic lipids.

[0060] As used herein, the term "non-cationic lipid" refers to any amphipathic lipid, as well as any other neutral lipid or anionic lipid. Suitably, said non-cationic lipid may be a neutral, uncharged, zwitterionic, or anionic lipid.

[0061] As used herein, the term "conjugated lipid" refers to a lipid molecule conjugated to a non-lipid molecule such as PEG, polyoxazoline, polyamide, or a polymer (eg, a cationic polymer).

[0062] As used herein, the term "adjuvant" refers to pharmacologically inactive ingredients that are formulated with an API, e.g., ccDNA, and/or lipid nanoparticles, to bulk up and/or stabilize the formulation during formulation. General categories of excipients include, for example, bulking agents, fillers, diluents, release agents, binders, coatings, leavening agents, flavoring agents, coloring agents, lubricants, lubricants, sorbents, preservatives, sweeteners, and products used to aid absorption or solubility. drug or other pharmacokinetic characteristics.

[0063] As used herein, the terms "heterologous nucleotide sequence" and "transgene" are used interchangeably and refer to a nucleic acid of interest (which is not a nucleic acid encoding a capsid polypeptide) that is included in a scDNA vector and can be delivered and expressed by scDNA -vector as described herein. Transgenes of interest include, but are not limited to, nucleic acids encoding polypeptides, preferably therapeutic (eg for medical, diagnostic or veterinary use) or immunogenic polypeptides (eg for vaccines). In some embodiments, nucleic acids of interest include nucleic acids transcribed into therapeutic RNA. Provided transgenes for use in ccDNA vectors according to the present invention include, but are not limited to, transgenes that express or encode one or more polypeptides, peptides, ribozymes, aptamers, peptide nucleic acids, siRNA, RNAi (RNA interference), siRNA, dncRNA , antisense oligo- or polynucleotides, antibodies, antigen-binding fragments, or any combination of the above.

[0064] As used herein, the terms "expression cassette" and "transcriptional cassette" are used interchangeably and refer to a linear stretch of nucleic acids that includes a transgene that is operably linked to one or more promoters or other regulatory sequences sufficient to direct transcription of said transgene, however, does not include capsid coding sequences, other sequences of the vector, or regions of inverted terminal repeats. The expression cassette may further comprise one or more cis-acting sequences (eg, promoters, enhancers, or repressors), one or more introns, and one or more post-transcriptional regulatory elements.

[0065] As used herein, the term "terminal repeat" or "TR" includes any viral terminal repeat or synthetic sequence that contains at least one minimum required origin of replication and a region containing a palindromic hairpin structure. The Rep binding sequence ("RBS") and the terminal resolution site ("TRS") together constitute the "minimum required origin of replication" and, accordingly, the terminal repeat (TR) contains at least one RBS and at least one TRS. All TRs that are reversely complementary to each other within a given stretch of polynucleotide sequence are generally referred to as "inverted terminal repeats" or "ITRs". In the context of a virus, ITRs mediate virus replication, packaging, integration, and provirus rescue. As was surprisingly discovered by the authors of the described invention, TRs that are not reverse complementary throughout can still perform the traditional functions of ITR, and accordingly, the term ITR in this document refers to TRs in the scDNA genome or scDNA vector, which is able to mediate replication of the scDNA vector. One skilled in the art will recognize that more than two ITRs or pairs of asymmetric ITRs may be present in complex ccDNA configurations. The ITR may be an AAV ITR or a non-AAV ITR, or may be derived from an AAV ITR or a non-AAV ITR. For example, ITR may be derived from a virus of the Parvoviridae family, which includes parvoviruses and dependoviruses (e.g., canine parvovirus, bovine parvovirus, murine 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. Viruses of the family Parvoviridae consist of two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect invertebrates. Dependoparvoviruses include the adeno-associated virus (AAV) family of viruses capable of replication in vertebrate hosts, including but not limited to humans, primate species, bovine, canine, equine, and ovine

[0066] As used herein, the term "asymmetric ITRs" refers to a pair of ITRs within the same scDNA genome or scDNA vector that are not reversely complementary throughout. The difference in the sequences of the two ITRs can be due to the addition of nucleotides, deletion, truncation or point mutation. In one embodiment, one ITR of a pair may be a wild-type AAV sequence and the other a non-wild-type or synthetic sequence. In another embodiment, neither ITR of the pair is a wild-type AAV sequence, and the sequences of the two ITRs are different. For convenience, the ITR located 5' to the expression cassette (above) in a scDNA vector is referred to herein as the "5'-ITR" or "left ITR", and the ITR located 3' to the expression cassette (below) in the scDNA vector is referred to as the "3'-ITR" or "right ITR".

[0067] As used herein, the term "ccDNA genome" refers to an expression cassette that further includes at least one inverted terminal repeat region. The scDNA genome may further comprise one or more spacer regions. In some embodiments, said scDNA genome is incorporated as a DNA polynucleotide with intermolecular duplex structures into a plasmid or viral genome.

[0068] As used herein, the term "scDNA spacer region" refers to an intermediate sequence that separates functional elements in said scDNA vector or scDNA genome. In some embodiments, the scDNA spacer regions hold the two functional elements at the required distance for optimal functionality. In some embodiments, spacer regions of the ccDNA provide or enhance the genetic stability of the ccDNA genome in, for example, a plasmid or baculovirus. In some embodiments, scDNA spacer regions facilitate prepared genetic manipulation of the scDNA genome by providing a convenient location for cloning sites and the like. For example, in certain aspects, an oligonucleotide "polylinker" containing multiple endonuclease restriction sites or a non-open reading frame sequence designed to contain no known protein binding sites (e.g., a transcription factor) can be placed in the zcDNA genome to separate cis -acting factors, for example, 6-mer, 12-mer, 18-mer, 24-mer, 48-mer, 86-mer, 176-mer, etc. can be inserted between the end clearance site and located in the direction 5' transcriptional regulatory element. Similarly, a spacer may be included between the polyadenylation signal sequence and the 3' clearance site.

[0069] As used herein, the terms "Rep binding site", "Rep binding element", "RBE", and "RBS" are used interchangeably and refer to a Rep protein binding site (e.g., Rep 78 AAV or Rep 68 AAV) that, when binding by the Rep protein allows said Rep protein to exhibit the appropriate site-specific endonuclease activity on the sequence comprising the RBS. The RBS sequence and its reverse complementary sequence together form one RBS. RBS sequences are known in the art and include, for example, 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 531), an RBS sequence identified in AAV2. Any known RBS sequence may be used in accordance with embodiments of the present invention, including other known AAV RBS sequences and other known natural or synthetic RBS sequences. Without wishing to be bound by theory, it is believed that the nuclease domain of the Rep protein binds to the GCTC duplex nucleotide sequence, and, accordingly, to the 5'-(GCGC)(GCTC)(GCTC)(GCTC)-3' duplex oligonucleotide (SEQ ID NO : 531) direct binding and stable assembly of two known Rep AAV proteins occurs. In addition, soluble aggregated conformers (i.e., an indeterminate number of interconnected Rep proteins) dissociate and bind to oligonucleotides that contain Rep binding sites. Each Rep protein interacts with both nitrogenous bases and a phosphodiester backbone on each strand. Interactions with nitrogenous bases provide sequence specificity, while interactions with the phosphodiester backbone are nonspecific or less sequence specific and stabilize the protein-DNA complex.

[0070] As used herein, the terms "terminal clearance site" and "TRS" are used interchangeably and refer to the region in which Rep forms via a tyrosine-phosphodiester bond with 5'-thymidine to form 3'-OH, which serves as a substrate to complete the DNA with a cellular DNA polymerase, such as delta DNA polymerase or epsilon DNA polymerase. Alternatively, the Rep-thymidine complex may be involved in a coordinated ligation reaction. In some embodiments, the TRS encompasses at least unpaired thymidine. In some embodiments, the nicking efficiency of the TRS can be controlled, at least in part, by the distance between it and the RBS within a single molecule. If the acceptor substrate is a complementary ITR, the final product is an intramolecular duplex. TRS sequences are known in the art and include, for example, 5'-GGTTGA-3' (SEQ ID NO: 45), a hexanucleotide sequence identified in AAV2. Any known TRS sequence may be used in accordance with embodiments of the present invention, including other known AAV TRS sequences and other known natural or synthetic TRS sequences such as AGTT (SEQ ID NO: 46), GGTTGG (SEQ ID NO: 47), AGTTGG (SEQ ID NO: 48), AGTTGA (SEQ ID NO: 49); and other motifs such as RRTTRR (SEQ ID NO: 50).

[0071] As used herein, the term "ccDNA plasmid" refers to a plasmid that contains the scDNA genome as an intermolecular duplex.

[0072] As used herein, the term “scDNA bacmida” refers to an infectious baculovirus genome comprising an intermolecular duplex scDNA genome capable of replicating in E. coli as a plasmid and thus functioning as a shuttle vector for baculovirus.

[0073] As used herein, the term "ccDNA baculovirus" refers to a baculovirus that contains the scDNA genome as an intermolecular duplex within the baculovirus genome.

[0074] As used herein, the terms “ccDNA-baculovirus-infected insect cell” and “ccDNA-VPS” are used interchangeably, and refer to an invertebrate host cell (including, but not limited to, an insect cell (e.g., an Sf9 cell). )) infected with scDNA-baculovirus.

[0075] As used herein, the terms "closed-ended DNA vector", "ccDNA vector", and "ccDNA" are used interchangeably and refer to a non-viral capsidless DNA vector with at least one covalently closed end (i.e., containing intramolecular duplex). In some embodiments, the scDNA contains two covalently closed ends.

[0076] As defined herein, "reporters" refers to proteins that can be used to provide detectable readings. Reporters typically produce a measurable signal, such as fluorescent, color, or luminescent. Reporter protein coding sequences encode proteins whose presence in a cell or organism is readily observable. For example, fluorescent proteins cause the cell to fluoresce when excited by light of a specific wavelength, luciferases cause the cell to catalyze a reaction that generates light, and enzymes such as β-galactosidase convert the substrate into a colored product. Examples of reporter polypeptides suitable for experimental or diagnostic purposes include, but are not limited to, β-lactamase, β-galactosidase (LacZ), alkaline phosphatase (AP), thymidine kinase (TK), green fluorescent protein (GFP) and other fluorescent proteins, chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art.

[0077] As used herein, the term "effector protein" refers to a polypeptide that provides a detectable indication, either, for example, as a reporter polypeptide, or, more preferably, as a cell-killing polypeptide, for example, a toxin, or an agent that renders the cell sensitive to killing (murder, destruction) by a certain agent or in the absence of a certain agent. Effector proteins include any protein or peptide that directly targets or damages the host cell's DNA and/or RNA. For example, effector proteins may include, but are not limited to, a restriction endonuclease that targets a host cell's DNA sequence (genomic or extrachromosomal element), a protease that degrades a polypeptide target essential for cell survival, a DNA gyrase inhibitor, and a ribonuclease-type toxin. In some embodiments, expression of an effector protein controlled by a synthetic biological circuit as described herein can be factored into another synthetic biological circuit, thereby expanding the range and complexity of the response of the biological circuit system.

[0078] The term "transcriptional regulators" refers to transcriptional activators and repressors that either activate or repress the transcription of a gene of interest. Promoters are regions of a nucleic acid that initiate the transcription of a particular gene. Transcriptional activators typically bind in the vicinity of transcriptional promoters and recruit RNA polymerase to initiate transcription directly. Repressors bind to transcriptional promoters and sterically hinder transcription initiation by RNA polymerase. Other transcriptional regulators can serve as either an activator or a repressor, depending on their binding site and cellular and environmental conditions. Non-limiting examples of classes of transcriptional regulators include, but are not limited to, homeodomain proteins, zinc finger proteins, winged helix proteins (Forkhead proteins), and leucine zipper proteins.

[0079] As used herein, a "repressor protein" or "inducer protein" is a protein that binds to a regulatory sequence element and represses or activates, respectively, the transcription of sequences operably linked to said regulatory sequence element. Preferred repressor proteins and inducer proteins as described herein are sensitive to the presence or absence of at least one input agent or environmental input. Preferred proteins as described herein are modular in form, containing, for example, separable DNA-binding and input-binding or responsive elements or domains.

[0080] As used herein, "carrier" includes any solvents, dispersion media, bases, coatings, diluents, antibacterial and antimycotic agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical actives is well known in the art. Additional active ingredients may also be included in these compositions. The term "pharmaceutically acceptable" refers to molecular entities and compositions that do not result in a toxic, allergic or similar adverse reaction when administered to a host.

[0081] As used herein, an "input agent responsive domain" is a transcription factor domain that binds or otherwise responds to a condition or input agent in such a manner as to cause its associated DNA-binding fusion domain to respond to the presence of said condition or input action. . In one embodiment, the presence of said condition or input action results in a conformational change in the input agent-responsive domain or protein to which it is fused, which modifies the transcription-modulating activity of the transcription factor.

[0082] The term "in vivo" refers to assays or processes that occur in or within an organism, such as a multicellular animal. According to some aspects of the present invention, the method or application may be said to be carried out "in vivo" if a single-celled organism, such as a bacterium, is used. The term "ex vivo" refers to methods and uses that are carried out using a living cell with an intact membrane that is outside the body of a multicellular animal or plant, for example, explants, cultured cells, including primary cells and cell lines, transformed cell lines and extracted tissues or cells, including blood cells, and the like. The term "in vitro" refers to assays and methods that do not require an intact cell membrane, such as cell extracts, and may refer to the introduction of a programmable synthetic biological circuit into a non-cellular system, such as an environment free of cells or cell systems, such as as cell extracts.

[0083] The term "promoter" as used herein refers to any nucleic acid sequence that regulates the expression of another nucleic acid sequence by directing the transcription of said nucleic acid sequence, which may be a heterologous target gene encoding a protein or RNA. Promoters can be constitutive, inducible, repressible, tissue-specific, or any combination of the above. A promoter is a control region of a nucleic acid sequence in which initiation takes place and the level of transcription of the rest of the nucleic acid sequence is determined. The promoter may also contain genetic elements in which binding of regulatory proteins and molecules such as RNA polymerase and other transcription factors can occur. In some embodiments of aspects of this document, a promoter may direct the expression of a transcription factor that regulates the expression of the promoter itself, or a transcription factor of another promoter used in another modular component of the synthetic biological circuits described herein. The promoter sequence contains a transcription initiation site, as well as protein-binding domains responsible for the binding of RNA polymerase. Eukaryotic promoters often, but not always, contain "TATA" boxes and "CAT" boxes. Various promoters, including inducible promoters, can be used to direct the expression of transgenes in the ccDNA vectors described herein.

[0084] The term "enhancer" as used herein refers to a cis-acting regulatory sequence (e.g., 50-1500 base pairs) that binds one or more proteins (e.g., activator proteins or a transcription factor) to increase transcriptional activation of the nucleic acid sequence . Enhancers can be located up to 1,000,000 base pairs upstream of the gene start site or downstream of the gene start site they regulate. An enhancer can be located in the intron region or in the exon region of an unrelated gene.

[0085] A promoter can be said to direct expression or direct transcription of the nucleic acid sequence it regulates. The terms "operably linked", "operably located", "under control", and "under transcriptional control" indicate that the promoter is in the correct functional position and/or orientation relative to the nucleic acid sequence it regulates to control transcription initiation and/or expression of the specified sequence. "Inverted promoter" as used herein refers to a promoter whose nucleic acid sequence is reversed such that the coding strand becomes non-coding, and vice versa. Inverted promoter sequences can be used in various embodiments to regulate the state of a switch. In addition, in various embodiments, a promoter may be used in combination with an enhancer.

[0086] A promoter can be a naturally associated gene or sequence promoter that can be obtained by isolating non-coding 5' sequences located upstream of the coding segment and/or exon of a particular gene or sequence. Such a promoter may be referred to as "endogenous". Similarly, in some embodiments, an enhancer may be a naturally associated enhancer with a nucleic acid sequence located either 3' or 5' from said sequence.

[0087] In some embodiments, a nucleic acid coding segment is placed under the control of a "recombinant promoter" or "heterologous promoter", where both terms refer to a promoter that is not normally associated with the nucleic acid coding sequence to which it is operably linked, under natural conditions. environment. Recombinant or heterologous enhancer refers to an enhancer not normally associated with a specific nucleic acid sequence under natural environmental conditions. Such promoters or enhancers may include promoters or enhancers of other genes; promoters or enhancers isolated from any other prokaryotic, viral or eukaryotic cell; and synthetic promoters or enhancers that are not "naturally occurring" i. containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression introduced using genetic engineering methods known in the art. In addition to producing promoter and enhancer nucleic acid sequences synthetically, promoter sequences can be obtained using recombinant cloning and/or nucleic acid amplification techniques, including PCR, as applied to synthetic biological circuits and modules as described herein (see, for example, US patent No. 4683202, US patent No. 5928906, each of which is incorporated herein by reference). In addition, it is also possible to use control sequences that direct the transcription and/or expression of sequences in non-nuclear organelles such as mitochondria, chloroplasts, and the like.

[0088] As used herein, an "inducible promoter" is a promoter that is characterized by the initiation or enhancement of transcriptional activity in the presence of, exposure to, or contact with an inducer or inducing agent. An "inducer" or "inducing agent" as defined herein can be a normally endogenous or exogenous compound or protein that is or is administered in such a manner as to be active in terms of inducing the transcriptional activity of an inducible promoter. In some embodiments, said inducer or inducing agent, i. the chemical, compound, or protein may itself be derived from the transcription or expression of a nucleic acid sequence (i.e., the inducer may be an inducer protein expressed by another component or module), which may itself be under the control of an inducible promoter. In some embodiments, induction of an inducible promoter occurs in the absence of certain agents, such as a repressor. Examples of inducible promoters include, but are not limited to, tetracycline, metallothione, ecdysone, mammalian viruses (e.g., adenovirus late promoter; and mouse mammary tumor virus long terminal repeat (MMTV-LTR)) and other steroid-responsive promoters, rapamycin-responsive promoters etc.

[0089] The term "subject" as used herein refers to a human or animal being treated, including prophylactic treatment, with an scDNA vector according to the present invention. Typically, said animal is a vertebrate, such as, but not limited to, a primate, rodent, domestic animal, or game animal. Primates include, but are not limited to, chimpanzees, cynomolgus monkeys, coats, and macaques such as the rhesus monkey. Rodents include mice, rats, marmots, ferrets, rabbits and hamsters. Domestic and game animals include, but are not limited to, cows, horses, pigs, deer, bison, buffalo, felines, e.g. domestic cat, canine species, e.g. dog, fox, wolf, bird species, e.g. chicken, emu, ostrich , and fish such as trout, catfish and salmon. In certain embodiments of aspects of the present invention, said subject is a mammal, such as a primate or a human. The subject may be male or female. In addition, the subject may be an infant or child. In some embodiments, said subject may be a neonatal or unborn subject, such as an in utero subject. Preferably, said subject is a mammal. Said mammal may be, but is not limited to, a human, non-human primate, mouse, rat, dog, cat, horse, or cow. Non-human mammals can be successfully used as subjects for animal models of diseases and disorders. In addition, the methods and compositions described herein may be applied to domesticated and/or companion animals. A human subject can be of any age, gender, race, or ethnic group, such as Caucasian (White), Asian, African, Black, African American, African European, Hispanic, Middle Eastern, and the like. In some embodiments, said subject may be a patient or other subject in a clinical setting. In some embodiments, said subject is already undergoing treatment.

[0090] As used herein, the term "antibody" is used in its broadest sense and encompasses various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments. provided that they exhibit the desired antigen-binding activity. "Antibody fragment" refers to a non-intact antibody molecule that contains a portion of an intact antibody that binds the same antigen to which said intact antibody binds. In one embodiment, the antibody or antibody fragment comprises an immunoglobulin chain or fragment thereof and at least one immunoglobulin variable domain sequence. Examples of antibodies or antibody fragments include, but are not limited to, Fv-, scFv-, Fab fragment, Fab'-, F(ab') ₂ , Fab'-SH, single domain antibody (dAb), heavy chain, light chain, heavy and a light chain, a complete antibody (for example, including all of the following: Fc, Fab, heavy chains, light chains, variable regions, etc.), bispecific antibody, diabody, linear antibody, single chain antibody, intrabody, monoclonal antibody, chimeric antibody, multispecific antibody, or multimeric antibody. An antibody or antibody fragment may be of any class, including, but not limited to, IgA, IgD, IgE, IgG, and IgM, and any subclass thereof, including, but not limited to, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. In addition, the antibody may be derived from any mammalian body, such as primates, humans, rats, mice, horses, goats, and the like. In one embodiment, said antibody is a human antibody or a humanized antibody. In some embodiments, said antibody is a modified antibody. In some embodiments, the components of an antibody may be expressed separately such that said antibody is self-assembled after expression of the protein components. In some embodiments, said antibody has a desired function, such as interacting with and inhibiting a desired protein to treat a disease or symptom of a disease. In one embodiment, said antibody or antibody fragment contains a framework or F _c region.

[0091] As used herein, the term "antigen-binding domain" of an antibody molecule refers to the portion of an antibody molecule, such as an immunoglobulin (Ig) molecule, that is involved in antigen binding. In embodiments, the antigen binding site is formed by the amino acid residues of the heavy (H) and light (L) chain variable (V) regions. Three very different segments in the composition of the variable regions of heavy and light chains, called hypervariable regions, are located between more conservative flanking segments, called "framework regions" (FR). FRs are amino acid sequences that are naturally found in immunoglobulins between and adjacent to hypervariable regions. In embodiments, in an antibody molecule, three light chain hypervariable regions and three heavy chain hypervariable regions are arranged relative to each other in three-dimensional space to form an antigen-binding surface that is complementary to the three-dimensional surface of the bound antigen. The three hypervariable regions from each of the heavy and light chains are referred to as "complementarity determining regions" or "CDRs". The framework region and CDR have been defined and described, for example, in Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fife ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917. Each variable chain (eg, variable heavy chain and variable light chain) is typically composed of three CDRs and four FRs, whose amino acid sequences are arranged, in the direction from the amino terminus to the carboxyl terminus, in the following order: FR1, CDR1, FR2, CDR2 , FR3, CDR3 and FR4.

[0092] As used herein, the term "full-length antibody" refers to an immunoglobulin (Ig) molecule (eg, IgG antibodies), for example, naturally occurring, and formed by fragments of immunoglobulin genes during normal recombinatory processes.

[0093] As used herein, the term "functional antibody fragment" refers to a fragment that binds to the same antigen that recognizes an intact (eg, full length) antibody. The terms "antibody fragment" or "functional fragment" also include isolated fragments consisting of variable regions, such as "Fv" fragments consisting of heavy and light chain variable regions, or recombinant single-chain polypeptide molecules in which the heavy and light variable regions chains are connected by a peptide linker ("scFv-proteins"). In some embodiments, the antibody fragment does not contain antibody portions without antigen-binding activity, such as Fc fragments or single amino acid residues.

[0094] As used herein, "an immunoglobulin variable domain sequence" refers to an amino acid sequence that can form an immunoglobulin variable domain structure. For example, said sequence may include all or part of the amino acid sequence of a naturally occurring variable domain. For example, the specified sequence may or may not include one, two or more N- or C-terminal amino acids, or may include other changes that are compatible with the formation of a protein structure.

[0095] As used herein, the term “comprising” or “comprises” is used to refer to compositions, methods, and their respective component(s) that are or are essential to said method or composition, but may include non-specified elements having significant or not.

[0096] As used herein, the term "consisting essentially of" refers to the elements required for a particular implementation. The specified term allows the presence of elements that do not significantly affect the main and new or functional characteristic or characteristics of the specified implementation option.

[0097] The term "consisting of" refers to compositions, methods, and their respective components as described herein, excluding any element not mentioned in the description of the specified embodiment.

[0098] In the present description and the appended claims, the singular forms, including those accompanied by the definition "specified", include the corresponding plural forms, unless otherwise clearly follows from the context. Accordingly, for example, reference to "said method" includes one or more methods and/or steps of the type described herein, and/or methods and/or steps that will be apparent to those skilled in the art upon reading the present description, etc. . Likewise, the term "or" is intended to include "and" unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, suitable methods and materials are described below. The expression "for example" is used herein to describe a non-limiting example.

[0099] With the exception of working examples or otherwise specified, all numbers reflecting the amounts of ingredients or reaction conditions according to the present invention, in all cases should be construed as modified by the term "approximately". The term "approximately" in relation to percentages can mean ±1%. The present invention is further explained in more detail in the following examples, however, the scope of the present invention is not limited thereto.

[00100] It should be understood that the present invention is not limited to the specific methodology, protocols and reagents, and the like described herein, and thus 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.

[00101] Without limitation, the lipid nanoparticle of the present invention includes a lipid formulation that can be used to deliver a non-capsid non-viral DNA vector to a target site of interest (eg, a cell, tissue, organ, etc.). Typically, said lipid nanoparticle contains a non-capsid non-viral DNA vector and an ionizable lipid or salt thereof.

[00102] Accordingly, according to some aspects of the present invention, a lipid nanoparticle comprising scDNA and an ionizable lipid is provided. For example, a lipid nanoparticle formulation is provided that is prepared and loaded with scDNA prepared using the method of Example 1 or otherwise as described herein. This can be done by intensive mixing of ethanol solutions of lipids with an aqueous solution of scDNA at low pH values, which provides protonation of the ionizable lipid and favorable energy parameters for the formation of scDNA bonds with lipid and particle nucleation. These particles can be further stabilized by dilution with water and removal of the organic solvent. These particles can be concentrated to the desired level.

[00103] Typically, lipid particles are prepared with a ratio of total lipids to scDNA (w/w) of about 10:1 to 30:1. In some embodiments, the ratio of lipids to ccDNA (weight ratio; ratio by weight) may range from about 1:1 to about 25:1, from about 10:1 to about 14:1, from about 3:1 to about 15 :1, about 4:1 to about 10:1, about 5:1 to about 9:1; or from about 6:1 to about 9:1. The amounts of lipids and scDNA can be adjusted to provide the desired N/P ratio, for example an N/P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or more. Typically, the total lipid content of a lipid particle formulation may vary from about 5 mg/mL to about 30 mg/mL.

[00104] An ionizable lipid is typically used to condense a loaded nucleic acid, eg, scDNA at low pH, control membrane binding, and fusogenicity. Typically, ionizable lipids are lipids containing at least one amino group that is positively charged or becomes protonated under acidic conditions, such as at a pH value of 6.5 or lower. Ionizable lipids are also referred to herein as cationic lipids.

[00105] Examples of ionizable lipids are described in the PCT and US Patent Publications listed in Table 1, the contents of which are incorporated herein by reference in their entirety.

согласно определению в US2016/0311759, содержание которого включено в настоящий документ полностью посредством ссылки.[00106] In some embodiments, said ionizable lipid is a compound of formula (X),

as defined in US2016/0311759, the contents of which are incorporated herein in their entirety by reference.

, согласно определению в US20150376115 или в US2016/0376224, содержание которых включено в настоящий документ полностью посредством ссылки.[00107] In some embodiments, said ionizable lipid is a compound of formula (I),

, as defined in US20150376115 or US2016/0376224, the contents of which are incorporated herein in their entirety by reference.

, или формулы (II),

, или формулы (III),

, согласно определению в US20160151284, содержание которого включено в настоящий документ полностью посредством ссылки.[00108] In some embodiments, said ionizable lipid is a compound of formula (I),

, or formula (II),

, or formula (III),

, as defined in US20160151284, the contents of which are incorporated herein in their entirety by reference.

, или формулы (IA),

или формулы (II),

или формулы (IIA),

согласно определению в US20170210967, содержание которого включено в настоящий документ полностью посредством ссылки.[00109] In some embodiments, said ionizable lipid is a compound of formula (I),

, or formulas (IA),

or formula (II),

or formulas (IIA),

as defined in US20170210967, the contents of which are incorporated herein in their entirety by reference.

согласно определению в US20150140070, содержание которого включено в настоящий документ полностью посредством ссылки.[00110] In some embodiments, said ionizable lipid is a compound of formula 1-e,

as defined in US20150140070, the contents of which are incorporated herein in their entirety by reference.

, согласно определению в US2013/0178541, содержание которого включено в настоящий документ полностью посредством ссылки.[00111] In some embodiments, said ionizable lipid is a compound of Formula A,

, as defined in US2013/0178541, the contents of which are incorporated herein in their entirety by reference.

согласно определению в US2013/0303587 или в US2013/0123338, содержание которых включено в настоящий документ полностью посредством ссылки.[00112] In some embodiments, said ionizable lipid is a compound of formula (I),

as defined in US2013/0303587 or US2013/0123338, the contents of which are incorporated herein in their entirety by reference.

согласно определению в US2017/0119904, содержание которого включено в настоящий документ полностью посредством ссылки.[00113] In some embodiments, said ionizable lipid is a compound of formula (I),

as defined in US2017/0119904, the contents of which are incorporated herein in their entirety by reference.

, формулы (III),

, формулы (IV),

, или формулы (V),

согласно определению в US2015/0239926, содержание которого включено в настоящий документ полностью посредством ссылки.[00114] In some embodiments, said ionizable lipid is a compound of formula (II),

, formulas (III),

, formulas (IV),

, or formula (V),

as defined in US2015/0239926, the contents of which are incorporated herein in their entirety by reference.

согласно определению в US2015/0141678, содержание которого включено в настоящий документ полностью посредством ссылки.[00115] In some embodiments, said ionizable lipid is a compound of formula (I),

as defined in US2015/0141678, the contents of which are incorporated herein in their entirety by reference.

согласно определению в WO2017/117528, содержание которого включено в настоящий документ полностью посредством ссылки.[00116] In some embodiments, said ionizable lipid is a compound of formula (I) or formula (II), each of which has the structure:

as defined in WO2017/117528, the contents of which are incorporated herein in their entirety by reference.

согласно определению в US2012/0149894, содержание которого включено в настоящий документ полностью посредством ссылки.[00117] In some embodiments, said ionizable lipid is a compound of Formula A,

as defined in US2012/0149894, the contents of which are incorporated herein in their entirety by reference.

согласно определению в US2015/0057373, содержание которого включено в настоящий документ полностью посредством ссылки.[00118] In some embodiments, said ionizable lipid is a compound of formula A,

as defined in US2015/0057373, the contents of which are incorporated herein in their entirety by reference.

согласно определению в WO2013/116126, содержание которого включено в настоящий документ полностью посредством ссылки.[00119] In some embodiments, said ionizable lipid is a compound of formula A,

as defined in WO2013/116126, the contents of which are incorporated herein in their entirety by reference.

согласно определению в US2013/0090372, содержание которого включено в настоящий документ полностью посредством ссылки.[00120] In some embodiments, said ionizable lipid is a compound of Formula A,

as defined in US2013/0090372, the contents of which are incorporated herein in their entirety by reference.

согласно определению в US2013/0274523, содержание которого включено в настоящий документ полностью посредством ссылки.[00121] In some embodiments, said ionizable lipid is a compound of formula A,

as defined in US2013/0274523, the contents of which are incorporated herein in their entirety by reference.

согласно определению в US2013/0274504, содержание которого включено в настоящий документ полностью посредством ссылки.[00122] In some embodiments, said ionizable lipid is a compound of Formula A,

as defined in US2013/0274504, the contents of which are incorporated herein in their entirety by reference.

согласно определению в US2013/0053572, содержание которого включено в настоящий документ полностью посредством ссылки.[00123] In some embodiments, said ionizable lipid is a compound of Formula A,

as defined in US2013/0053572, the contents of which are incorporated herein in their entirety by reference.

согласно определению в WO2013/016058, содержание которого включено в настоящий документ полностью посредством ссылки.[00124] In some embodiments, said ionizable lipid is a compound of Formula A,

as defined in WO2013/016058, the contents of which are incorporated herein in their entirety by reference.

согласно определению в WO2012/162210, содержание которого включено в настоящий документ полностью посредством ссылки.[00125] In some embodiments, said ionizable lipid is a compound of Formula A,

as defined in WO2012/162210, the contents of which are incorporated herein in their entirety by reference.

согласно определению в US2008/042973, содержание которого включено в настоящий документ полностью посредством ссылки.[00126] In some embodiments, said ionizable lipid is a compound of formula (I),

as defined in US2008/042973, the contents of which are incorporated herein in their entirety by reference.

формулы (II),

формулы (III),

или формулы (IV),[00127] In some embodiments, said ionizable lipid is a compound of formula (I),

formulas (II),

formulas (III),

or formula (IV),

согласно определению в US2012/01287670, содержание которого включено в настоящий документ полностью посредством ссылки.

as defined in US2012/01287670, the contents of which are incorporated herein in their entirety by reference.

, согласно определению в US2014/0200257, содержание которого включено в настоящий документ полностью посредством ссылки.[00128] In some embodiments, said ionizable lipid is a compound of formula (I) or formula (II), each of which has the structure:

, as defined in US2014/0200257, the contents of which are incorporated herein in their entirety by reference.

согласно определению в US2015/0203446, содержание которого включено в настоящий документ полностью посредством ссылки.[00129] In some embodiments, said ionizable lipid is a compound of formula (I), formula (II), or formula (III), each of which has the structure:

as defined in US2015/0203446, the contents of which are incorporated herein in their entirety by reference.

или формулы (III),

, согласно определению в US2015/0005363, содержание которого включено в настоящий документ полностью посредством ссылки.[00130] In some embodiments, said ionizable lipid is a compound of formula (I),

or formula (III),

, as defined in US2015/0005363, the contents of which are incorporated herein in their entirety by reference.

формулы (IA),

формулы (IB),

формулы (IC),

формулы (ID,

формулы (II),

формулы (IIA),

, формулы (IIB),

формулы (IIC),

формулы (IID) или формул (III)-(XXIV) согласно определению в US2014/0308304, содержание которого включено в настоящий документ полностью посредством ссылки.[00131] In some embodiments, said ionizable lipid is a compound of formula (I),

formulas (IA),

formulas (IB),

formulas (IC),

formulas (ID,

formulas (II),

formulas (IIA),

, formulas (IIB),

formulas (IIC),

formulas (IID) or formulas (III)-(XXIV) as defined in US2014/0308304, the contents of which are incorporated herein in their entirety by reference.

согласно определению в US2013/0338210, содержание которого включено в настоящий документ полностью посредством ссылки.[00132] In some embodiments, said ionizable lipid is a compound of the formula

as defined in US2013/0338210, the contents of which are incorporated herein in their entirety by reference.

формулы (II),

формулы (III)

или формулы (IV),

согласно определению в WO2009/132131, содержание которого включено в настоящий документ полностью посредством ссылки.[00133] In some embodiments, said ionizable lipid is a compound of formula ((I),

formulas (II),

formulas (III)

or formula (IV),

as defined in WO2009/132131, the contents of which are incorporated herein in their entirety by reference.

, согласно определению в US2012/01011478, содержание которого включено в настоящий документ полностью посредством ссылки.[00134] In some embodiments, said ionizable lipid is a compound of Formula A,

, as defined in US2012/01011478, the contents of which are incorporated herein in their entirety by reference.

или формулы (XXXV),

согласно определению в US2012/0027796, содержание которого включено в настоящий документ полностью посредством ссылки.[00135] In some embodiments, said ionizable lipid is a compound of formula (I),

or formulas (XXXV),

as defined in US2012/0027796, the contents of which are incorporated herein in their entirety by reference.

или формулы (XVII),

согласно определению в US2012/0058144, содержание которого включено в настоящий документ полностью посредством ссылки.[00136] In some embodiments, said ionizable lipid is a compound of formula (XIV),

or formulas (XVII),

as defined in US2012/0058144, the contents of which are incorporated herein in their entirety by reference.

согласно определению в US2013/0323269, содержание которого включено в настоящий документ полностью посредством ссылки.[00137] In some embodiments, said ionizable lipid is a compound of the formula

as defined in US2013/0323269, the contents of which are incorporated herein in their entirety by reference.

, согласно определению в US2011/0117125, содержание которого включено в настоящий документ полностью посредством ссылки.[00138] In some embodiments, said ionizable lipid is a compound of formula (I),

, as defined in US2011/0117125, the contents of which are incorporated herein in their entirety by reference.

формулы (II),

или формулы (III),

согласно определению в US2011/0256175, содержание которого включено в настоящий документ полностью посредством ссылки.[00139] In some embodiments, said ionizable lipid is a compound of formula (I),

formulas (II),

or formula (III),

as defined in US2011/0256175, the contents of which are incorporated herein in their entirety by reference.

формулы (II),

формулы (III),

формулы (IV),

формулы (V),

формулы (VI),

формулы (VII),

формулы (VIII),

формулы (IX),

формулы (X),

формулы (XI),

или формулы (XII),

согласно определению в US2012/0202871, содержание которого включено в настоящий документ полностью посредством ссылки.[00140] In some embodiments, said ionizable lipid is a compound of formula (I),

formulas (II),

formulas (III),

formulas (IV),

formulas (V),

formulas (VI),

formulas (VII),

formula (VIII),

formulas (IX),

formulas (X),

formulas (XI),

or formula (XII),

as defined in US2012/0202871, the contents of which are incorporated herein in their entirety by reference.

формулы (II),

формулы (III),

формулы (IV),

формулы (V),

формулы (VI),

формулы (VII),

формулы (VIII),

формулы (IX),

формулы (X),

, формулы (XI),

формулы (XII),

формулы (XIII),

формулы (XIV),

формулы (XV),

или формулы (XVI),

согласно определению в US2011/0076335, содержание которого включено в настоящий документ полностью посредством ссылки.[00141] In some embodiments, said ionizable lipid is a compound of formula (I),

formulas (II),

formulas (III),

formulas (IV),

formulas (V),

formulas (VI),

formulas (VII),

formula (VIII),

formulas (IX),

formulas (X),

, formulas (XI),

formulas (XII),

formula (XIII),

formulas (XIV),

formulas (XV),

or formulas (XVI),

as defined in US2011/0076335, the contents of which are incorporated herein in their entirety by reference.

или формулы (II),

согласно определению в US2006/008378, содержание которого включено в настоящий документ полностью посредством ссылки.[00142] In some embodiments, said ionizable lipid is a compound of formula (I),

or formula (II),

as defined in US2006/008378, the contents of which are incorporated herein in their entirety by reference.

согласно определению в US2013/0123338, содержание которого включено в настоящий документ полностью посредством ссылки.[00143] In some embodiments, said ionizable lipid is a compound of formula (I),

as defined in US2013/0123338, the contents of which are incorporated herein in their entirety by reference.

[00144] In some embodiments, said ionizable lipid is a compound of formula (I), X-A-Y-Z as defined in US2015/0064242, the contents of which are incorporated herein in their entirety by reference.

формулы (XVII),

или формулы (XVIII),

согласно определению в US2013/0022649, содержание которого включено в настоящий документ полностью посредством ссылки.[00145] In some embodiments, said ionizable lipid is a compound of formula (XVLX),

formulas (XVII),

or formula (XVIII),

as defined in US2013/0022649, the contents of which are incorporated herein in their entirety by reference.

, формулы (II),

или формулы (III),

согласно определению в US2013/0116307, содержание которого включено в настоящий документ полностью посредством ссылки.[00146] In some embodiments, said ionizable lipid is a compound of formula (I),

, formulas (II),

or formula (III),

as defined in US2013/0116307, the contents of which are incorporated herein in their entirety by reference.

или формулы (II),

, согласно определению в US2010/0062967, содержание которого включено в настоящий документ полностью посредством ссылки.[00147] In some embodiments, said ionizable lipid is a compound of formula (I),

or formula (II),

, as defined in US2010/0062967, the contents of which are incorporated herein in their entirety by reference.

согласно определению в US2013/0189351, содержание которого включено в настоящий документ полностью посредством ссылки.[00148] In some embodiments, said ionizable lipid is a compound of formula (1)-(X), each of which has the structure:

as defined in US2013/0189351, the contents of which are incorporated herein in their entirety by reference.

согласно определению в US2014/0039032, содержание которого включено в настоящий документ полностью посредством ссылки.[00149] In some embodiments, said ionizable lipid is a compound of formula (I),

as defined in US2014/0039032, the contents of which are incorporated herein in their entirety by reference.

, согласно определению в US2018/0028664, содержание которого включено в настоящий документ полностью посредством ссылки.[00150] In some embodiments, said ionizable lipid is a compound of formula (V),

, as defined in US2018/0028664, the contents of which are incorporated herein in their entirety by reference.

согласно определению в US2016/0317458, содержание которого включено в настоящий документ полностью посредством ссылки.[00151] In some embodiments, said ionizable lipid is a compound of formula (I),

as defined in US2016/0317458, the contents of which are incorporated herein in their entirety by reference.

, согласно определению в US2013/0195920, содержание которого включено в настоящий документ полностью посредством ссылки.[00152] In some embodiments, said ionizable lipid is a compound of formula (I),

, as defined in US2013/0195920, the contents of which are incorporated herein in their entirety by reference.

[00153] In some embodiments, said ionizable lipid is MCZ (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3- DMA or MC3) described in example 9.

[00154] In some embodiments, said ionizable lipid is the ATX-002 lipid described in Example 10.

[00155] In some embodiments, said ionizable lipid is (13Z,16Z)-N,N-dimethyl-3-nonyldocose-13,16-dien-1-amine (Compound 32) described in Example 11.

[00156] In some embodiments, said ionizable lipid is Compound 6 or Compound 22 as described in Example 12.

[00157] Without limitation, the ionizable lipid can be 20-90% (mol.) of the total lipids present in the specified lipid nanoparticle. For example, the molar content of ionizable lipid may be 20-70% (mol.), 30-60% (mol.) or 40-50% (mol.) of the total amount of lipids present in the specified lipid nanoparticle. In some embodiments, the ionizable lipid is from about 50 mole % to about 90 mole % of the total lipids present in said lipid nanoparticle.

[00158] In some aspects, said lipid nanoparticle may further comprise a non-cationic lipid. Nonionic lipids include amphipathic lipids, neutral lipids and anionic lipids. Accordingly, the non-cationic lipid may be a neutral uncharged, zwitterionic or anionic lipid. Non-cationic lipids are generally used to increase fusogenicity.

[00159] Examples of non-cationic lipids include, but are not limited to, distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidyl-ethanolamine (DSPE), monomethylphosphatidylethanolamine ( such as 16-O-monomethyl-PE), dimethylphosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), hydrogenated soybean phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoylphosphatidylcholine (TEMRS), dimyristoylphosphatidylglycerol (DMPG ), дистеароилфосфатидилглицерин (DSPG), диерукоилфосфатидилхолин (ТЭЕРС), пальмитоилолеоилфосфатидилглицерин (POPG), диэлаидоилфосфатидилэтаноламин (DEPE), лецитин, фосфатидилэтаноламин, лизопецитин, лизофосфатидилэтаноламин, фосфатидилсерин, фосфатидилинозитол, сфингомиелин, яичный сфингомиелин (ESM), кефалин, кардиолипин, фосфатидную кислоту, цереброзиды ; dicetyl phosphate, lysophosphatidylchopine, dilinoleoylphosphatidylcholine; or mixtures thereof. It should be understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids may also be used. The acyl groups in said lipids are preferably acyl groups derived from fatty acids with C ₁₀ -C ₂₄ carbon chains, eg lauroyl; myristoyl, papmitoil, stearoyl or oleoyl.

[00160] Other examples of non-cationic lipids suitable for use in lipid nanoparticles include phosphorus-free lipids such as, for example, stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers; triethanolamine lauryl sulfate, alkyl aryl sulfated polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramide, sphingomnelin, and the like.

[00161] In some embodiments, said non-cationic lipid is a phospholipid. In some embodiments, said non-cationic lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some preferred embodiments, said non-cationic lipid is DPSC.

[00162] Examples of non-cationic lipids are described in PCT Publication WO2017/099223 and US Patent Publication US 2018/0028664, the contents of which are incorporated herein in their entirety by reference.

формулы (II)

или формулы (IV),

согласно определению в US2018/0028664, содержание которого включено в настоящий документ полноствю посредством ссылки.[00163] In some examples, said non-cationic lipid is oleic acid or a compound of formula (I),

formulas (II)

or formula (IV),

as defined in US2018/0028664, the contents of which are hereby incorporated by reference in their entirety.

[00164] Said non-cationic lipid may comprise 0-30% (mole) of the total lipids present in said lipid nanoparticle. For example, the content of the specified non-cationic lipid and is 5-20% (mol.) or 10-15% (mol.) of the total amount of lipids present in the specified lipid nanoparticle. In various embodiments, the molar ratio of ionizable lipid to neutral lipid ranges from about 2:1 to about 8:1.

[00165] In some embodiments, said lipid nanoparticles do not contain any phospholipids.

[00166] According to some aspects, the specified lipid nanoparticle may additionally contain a component, such as a sterol, to ensure the integrity of the membrane.

[00167] One example of a sterol that can be used in a lipid nanoparticle is cholesterol and its derivatives. 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, cholestenon, 5α-cholestanone, 5β-cholestanone and cholesteryl decanoate; and their mixtures. In some embodiments, the cholesterol derivative is a polar analogue such as cholesteryl-(4'-hydroxy)-butyl ether.

[00168] Examples of cholesterol derivatives are described in PCT Publication WO2009/127060 and US Patent Publication US2010/0130:533, the contents of which are incorporated herein in their entirety by reference

[00169] A membrane integrity component, such as a sterol, can comprise 0-50% (mole) of the total lipids present in said lipid nanoparticle. In some embodiments, such a component is 20-50% (mol.) 30-40% (mol.) of the total lipid content in the lipid nanoparticle.

[00170] In some aspects, said lipid nanoparticle may further comprise polyethylene glycol (PEG) or a conjugated lipid a molecule. Typically used to inhibit lipid nanoparticle aggregation and/or provide steric stabilization. Examples of conjugated lipids include, but are not limited to, PEG lipid conjugates, lipid polyoxazolines (POZ) conjugates, polyamide lipid conjugates (such as ATTA lipid conjugates), cationic polymer lipid conjugates (CPL); and mixtures of the above. In some embodiments, the lipid conjugated molecule is a PEG-lipid conjugate, such as a (methoxypolyethylene glycol)-conjugated lipid.

[00171] Examples of PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, 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-dialkoxypropylcarbam, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or a mixture thereof Further examples of PEG-lipid conjugates are described in, for example, US5,885,613, US6,287,591, US2003/0077829, US2003/0077829, US2005/0175682, US2008/0020058, US2011/0117125, US2010/0130588 US2017/0119904 and US/099823, the contents of which are incorporated herein in their entirety by reference.

формулы (III-а-I),

формулы (III-а-2),

, формулы (III-b-1),

формулы (III-b-2),

, или формулы (V),

согласно определению в US2018/0028664, содержание которого включено в настоящий документ полностью посредством ссылки.[00172] In some embodiments, the PEG lipid is a compound of formula (III),

formulas (III-a-I),

formulas (III-a-2),

, formulas (III-b-1),

formulas (III-b-2),

, or formula (V),

as defined in US2018/0028664, the contents of which are incorporated herein in their entirety by reference.

согласно определению в US20150376115 или в US2016/0376224, содержание которых включено в настоящий документ полностью по средством ссыпки.[00173] In some embodiments, the PEG lipid has the formula (II),

as defined in US20150376115 or US2016/0376224, the contents of which are incorporated herein in their entirety by reference.

[00174] The PEG-DAA conjugate may be, for example, PEG dilauryloxypropyl, PEG dimyristyloxypropyl, PEG dipalmityloxypropyl or PEG distearyloxypropyl. Said PEG lipid may be any one or more of PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristiglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol ( 1-[8'-(cholest-5-en-3[beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3.4 -Ditetradecoxybenzyl-[omega]-methyl-poly(ethylene glycol) ether) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. According to some examples, said PEG lipid may be selected from the group consisting of PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000],

[00175] Non-PEG conjugated lipids can also be used instead of a PEG lipid. For example, polyoxazoline (POZ) lipid conjugates, polyamide lipid conjugates (such as ATTA lipid conjugates), and cationic polymer lipid (CPL) conjugates may be used instead of or along with PEG lipid.

[00176] Examples of conjugated lipids, ie. PEG lipids, (POZ) lipid conjugates, ATTA-lipid conjugates, and cationic polymer-lipid conjugates are described in the PCT and US patent applications listed in Table 2, the contents of which are incorporated herein in their entirety by reference.

[00177] The PEG or conjugated lipid may comprise 0-20% (mole) of the total lipids present in said lipid nanoparticle. In some embodiments, the content of PEG or conjugated lipid is 0.5-10% or 2-5% (mol.) of the total amount of lipids present in the specified lipid nanoparticle.

[00178] The molar ratios of ionizable lipid, non-cationic lipid, sterol, and PEG/conjugated lipid may vary as needed. For example, said lipid particle may contain 30-70% ionizable lipid based on mole or total weight of the composition, 0-60% cholesterol based on mole or total weight of the composition, 0-30% non-cationic lipid based on mole or total weight of the composition. ; and 1-10% conjugated lipid based on mole or total weight of the composition. Preferably, said composition contains 30-40% ionizable lipid by mole or total weight of the composition, 40-50% cholesterol by mole or total weight of the composition; and 10-20% non-cationic lipid by molar amount or by total weight of the composition. In some other embodiments, said composition contains 50-75% ionizable lipid by mole or total weight of the composition, 20-40% cholesterol by mole or by total weight of the composition, 5-10% non-cationic lipid by mole or by total weight compositions; and 1-10% conjugated lipid based on mole or total weight of the composition. Said composition may contain 60-70% ionizable lipid by mole or total weight of the composition, 25-35% cholesterol by mole or total weight of the composition; and 5-10% non-cationic lipid based on mole or total weight of the composition. Said composition may also contain up to 90% ionizable lipid by molar amount or by total weight of the composition; and 2-15% non-cationic lipid based on mole or total weight of the composition. Said composition may also be a composition based on lipid nanoparticles, for example, containing 8-30% ionizable lipid by mole or total weight of the composition, 5-30% non-cationic lipid by mole or total weight of the composition; and 0-20% cholesterol by molar amount or by total weight of the composition; 4-25% ionizable lipid based on mole or total weight of composition, 4-25% non-cationic lipid based on mole or total weight of composition, 2-25% cholesterol based on mole or total weight of composition, 10-35% lipid conjugate on the molar amount or on the total weight of the composition; and 5% cholesterol by molar amount or by total weight of the composition; or 2-30% ionizable lipid based on mole or total weight of composition, 2-30% non-cationic lipid based on mole or total weight of composition, 1-15% cholesterol based on mole or total weight of composition, 2-35% conjugate lipid on a molar basis or based on the total weight of the composition; and 1-20% cholesterol by molar amount or by total weight of the composition; or even up to 90% ionizable lipid, based on mole or total weight of the composition; and 2-10% non-cationic lipids based on mole or total weight of the composition, or even 100% cationic lipid based on mole or total weight of the composition. In some embodiments, said lipid particle composition comprises an ionizable lipid, a phospholipid, cholesterol, and a pegylated lipid in a molar ratio of 50:10:38.5:1.5. In some other embodiments, said lipid particle composition comprises ionizable lipid, cholesterol, and pegylated lipid in a molar ratio of 60:38.5:1.5.

[00179] In some embodiments, said lipid particle comprises an ionizable lipid, a non-cationic lipid (e.g., a phospholipid), a sterol (e.g., cholesterol), and a pegylated lipid, wherein the lipid molar ratio ranges from 20 to 70 mole percent for the ionizable lipid at the target value 40-60, mole percent non-cationic lipid ranges from 0 to 30 with a target of 0-15, mole percent of sterol ranges from 20 to 70 with a target of 30-50; and the mole percentage of pegylated lipid ranges from 1 to 6 with a target value of 2-5.

[00180] In some embodiments, said lipid particle contains an ionizable lipid/non-cationic lipid/sterol/conjugated lipid in a molar ratio of 50:10:38.5:1.5.

[00181] According to other aspects of the present invention, a lipid nanoparticulate formulation comprising phospholipids, lecithin, phosphatidylcholine, and phosphatidylethanolamine is provided.

[00182] In some embodiments, one or more additional compounds may also be included. These compounds may be administered alone, or these additional compounds may be included in the lipid nanoparticles of the present invention. In other words, these lipid nanoparticles may contain other compounds along with scDNA or at least a second scDNA different from the first. Without limitation, other additional compounds may be selected from the group consisting of small or large organic or inorganic molecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and derivatives, peptidomimetics, nucleic acids, analogs and derivatives of nucleic acids. acids, an extract obtained from biological materials, or any combination thereof.

[00183] In some embodiments, said one or more additional compounds may be a therapeutic agent. Said therapeutic agent may be selected from any class suitable for therapeutic purposes. In other words, said therapeutic agent may be selected from any class suitable for therapeutic purposes. In other words, the specified therapeutic agent can be selected in accordance with the therapeutic purpose and the desired biological effect. For example, if the scDNA in LNP is suitable for cancer treatment, the additional compound may be an anti-cancer agent (e.g., a chemotherapeutic agent, a targeted anti-cancer therapy agent (including, but not limited to, a small molecule, an antibody, or an antibody-drug conjugate). In another example, if an scDNA-containing LNP is suitable for treating an infection, the additional compound may be an antimicrobial agent (e.g., an antibiotic or an antiviral compound).In another example, if an scDNA-containing LNP is suitable for treating an immune disease, or disorders, the additional compound may be a compound that modulates an immune response (e.g., an immunosuppressant, an immunostimulatory compound, or a compound that modulates one or more specific immune pathways).In some embodiments, the compositions and methods of the present invention may use different cocktails of different lipid nanoparticles containing different compounds, such as ccDNAs encoding different proteins, or different compounds, such as therapeutic agents.

[00184] In some embodiments, said additional compound is an immunomodulatory agent. For example, said additional compound is an immunosuppressant. In some embodiments, said additional compound is an immunostimulatory compound.

[00185] Examples of immunomodulators include, but are not limited to, interleukins (e.g., IL-2, IL-7, IL-12), cytokines (e.g., granulocyte colony stimulating factor (G-CSF), interferons), chemokines (e.g., CCL3, CCL26, CXCL7), immunomodulatory imide drugs (IMiDs) (e.g., thalidomide and its analogues (lenalidomide, pomalidomide, and apremilast), other immunomodulators, including, but not limited to: cytosine phosphate guanosine, oligodeoxynucleotides, glucans.

[00186] In some embodiments, said immunomodulator may be an immunosuppressive drug. Examples of immunosuppressive drugs include, but are not limited to, glucocorticoids, cytostatics, antibodies, drugs that act on immunophilins, and other drugs. Glucocorticoids include, but are not limited to, prednisone, dexamethasone, and hydrocortisone. Examples of cytostatics include alkylating agents such as mustargens (eg cyclophosphamide), nitrosoureas and platinum compounds. Cytostatics may also include antimetabolites such as folic acid analogs (eg, methotrexate), purine analogs (eg, azathioprine and mercaptopurine), pyrimidine analogs (eg, fluorouracil), and protein synthesis inhibitors. Other cytostatics include cytotoxic antibiotics such as dactinomycins, anthracyclines, mitomycin C, bleomycin, and mithramycin.

[00187] Immune-suppressing antibodies include, but are not limited to, equine serum-derived atgam and thymoglobulin, antibodies directed to the IL-2 receptor (CD25-) and/or directed to CD3, muromonab-CD3 (MUROMONAB-CD3 ™, Orthoclon-OKT3), basiliximab (Simulect, SIMULECT™), daclizumab (Zenapax, ZENAPAX™), and muromonab.

[00188] Drugs that act on immunophilins include, but are not limited to, cyclosporine, tacrolimus, rapamycin (sirolimus, SIROLIMUS™), and everolimus. Examples of biological agents include abatacept, anakinra, certolizumab, golimumab, ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinumab, and vedolizumab.

[00189] Other drugs suitable for use as immunomodulators or immunosuppressants include, but are not limited to, interferons (e.g., IFN-β), opioids, TNF binding proteins (e.g., TNF-α (tumor necrosis factor alpha) binding protein, infliximab (Remicade, REMICADE™), etanercept (Enbrel, ENBREL™) or adalimumab (HUMIRA™)), curcumin (an ingredient in turmeric) and catechins (found in green tea), mycophenolate, fingolimod, myriocin, antiproliferative agents (eg, myriocin, tacrolimus, mycophenolate mofetil, sodium mycophenolate, azathioprine), mTOR inhibitors (eg, sirolimus and everolimus), calcineurin inhibitors (eg, cyclosporine and tacrolimus), IMDS inhibitors (eg, azathioprine, leflunomide, and mycophenolate), fingolimod, abatacept, anakinru , certolizumab, golimumab, ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinumab, and vedolizumab.

[00190] In some embodiments, immunosuppressive agents suitable for use in compositions and methods as described herein can be selected from one of the following compounds: mycophenolic acid, cyclosporine, azathioprine, tacrolimus, cyclosporin A, FK506, rapamycin, leflunomide, deoxyspergualine, prednisone, azathioprine, mycophenolate mofetil, OKT3, ATAG, or mizoribine.

[00191] In some embodiments, the immunosuppressants are selected from the group consisting of prednisone, methylprednisolone, kenalog, oral medrol, oral medrol tablets, medrol injectable depot injectable, oral prednisolone, medrol injectable solution, oral hydrocortisone administration, Cortef oral, Medrol IV solution, cortisone oral, Celeston injection Celestone Soluspan, oral orapred Orapred ODT, oral Orapred, oral prelon, methylprednisolone acetate injection, prednisone oral Intensol, betamatasone acetate with sodium phosphate for injection, veripred, celeston for oral administration, methylprednisolone sodium succinate for intravenous administration, methylprednisolone sodium succinate for injection, millipred for oral administration, medrol solution (without preservatives) for injection, cortef solution d for injection, aristospan for intra-articular injection, hydrocortisone sodium succinate for injection, prednisolone sodium phosphate for oral administration, methylprednisolone sodium succinate (without preservatives) for intravenous administration, medrol solution (without preservatives) for intravenous administration, triamcinolone hexacetonide for injection , A-Hydrocort Injection, A-Metapred Injection, Millipred DP Oral, Flo-Pred Oral, Aristospan Intralesional Injection, Oral Betamethasone, Methylprednisolone Sodium Succinate (No Preservatives) Injection, Hydrocortisone Sodium succinate (without preservatives) for injection, Cortef solution (without preservatives) for injection, prednisolone acetate for oral administration, dexamethasone in 0.9% NaCl for intravenous administration, Rayos (Rayos), levothyroxine. Of course, immunosuppressants known to those skilled in the art can easily be used instead of those listed, as the list is not to be construed as exhaustive or limiting.

[00192] An immunosuppressant may result in a decrease in the number of immune cells in a subject, for example, a decrease in the number of immune cells that express at least one or more of: CD11b, CD4, CD8, and/or a decrease in the level of pro-inflammatory cytokines selected from , but not limited to, TNFα or MSR-1. In some embodiments, the proinflammatory cytokine is selected from one or a combination of the following: cytokines, lymphokines, monokines, stem cell growth factors, lymphotoxins, hematopoietic factors, colony stimulating factors (CSFs), interferons (IFNs), parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), liver growth factor, prostaglandin, fibroblast growth factor, prolactin, placental lactogen, OB protein, transforming growth factor (TGF), TGF -α, TGF-β, insulin-like growth factor (IGF), erythropoietin, thrombopoietin, tumor necrosis factor (TNF), TNF-α, TNF-β, Müllerian inhibitory substance (MIS), mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor, integrin, interleukin (IL), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF) ), interferon-alpha, interferon-beta, interferon-gamma, factor S1, IL-1, IL-1 cc, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18 IL-21, IL-23 , IL-25, LIF, kit-ligand, FLT-3, angiostatin, thrombospondin and endostatin.

[00193] In some embodiments, the pro-inflammatory cytokine can be selected from any one or combination of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin -8 (IL-8), interferon-γ (IFN-γ), vascular endothelial growth factor (VEGF), leukemia inhibitory factor (LIF), monocytic chemoattractant protein-1 (MCP-1), RANTES, interleukin-10 (IL -10), interleukin-12 (IL-12), matrix metalloproteinase 2 (MMP2), IP-10, macrophage inflammatory protein 1α (MIP1α) and/or macrophage inflammatory protein 1β (MIP1β).

согласно определению в US 20170056448 A1, содержание которого включено в настоящий документ полностью посредством ссылки.[00194] In some embodiments, said immunomodulatory agent is an NLRP3 agonist. Examples of NLRP3 agonists include, but are not limited to, imidazoquinoline; imidazonaphthyridine; pyrazolopyridine; aryl-substituted imidazoquinoline; a compound containing a 1-alkoxy-1H-imidazole ring system; oxazolo[4,5-c]-quinoline-4-amine; thiazolo[4,5-c]-quinoline-4-amine; seleazolo[4,5-c]-quinoline-4-amine; imidazonaphthyridine, imidazoquinolineamine; 1-substituted, 2-substituted 1H-imidazo[4,5-C]quinoline-4-amine; a fused cycloalkylimidazopyridine; 1H-imidazo[4,5-c]quinoline-4-amine; 1-substituted 1H-imidazo-[4,5-c]quinoline-4-amine; imidazo-[4,5-C]quinoline-4-amine; 2-ethyl-1H-imidazo[4,5-c]quinoline-4-amine; olefinic 1H-imidazo[4,5-c]quinoline-4-amine; 6,7-dihydro-8-(imidazol-1-yl)-5-methyl-1-oxo-1H,5H-benzo[ij]quinolizine-2-carboxylic acid; pyridoquinoxaline-6-carboxylic acid; 6,7-dihydro-8-(imidazol-1-yl)-5-methyl-1-oxo-1H,5H-benzo[ij]quinolizine-2-carboxylic acid; substituted naphtho[ij]quinolysine; substituted pyridoquinoxaline-6-carboxylic acid; a 7-hydroxy-benzo[ij]quinolysine-2-carboxylic acid derivative; substituted benzo[ij]quinolysine-2-carboxylic acid; 7-hydroxy-benzo[ij]quinolysine-2-carboxylic acid; substituted pyrido[1,2,3,-de]-1,4-benzoxazine; and tetrahydroquinoline N-methylenemalonate. In some embodiments, said NLRP3 agonist is a compound of the formula:

as defined in US 20170056448 A1, the contents of which are incorporated herein in their entirety by reference.

[00195] In some embodiments, said immunomodulatory agent is a TLR7 and/or TLR8 ligand. In some embodiments, said immunomodulatory agent is imiquimod (1-isobutyl-1H-imidazo[4,5-c]quinoline-4-amine) or resiquimod.

согласно определению в US 9034336 B2, содержание которого включено в настоящий документ полностью посредством ссылки.[00196] In some embodiments, said immunomodulatory agent is an imquidazolquinoline compound of the formula:

as defined in US 9034336 B2, the contents of which are incorporated herein in their entirety by reference.

[00197] In some embodiments, said immunomodulatory agent is an SMAD7 modulator. For example, said SMAD7 modulator may be an SMAD7 antisense oligonucleotide (AON) as defined in WO 2017059225 A1, the contents of which are incorporated herein in their entirety by reference.

[00198] Said immunomodulatory agent may be a TLR modulator. For example, said immunomodulatory agent may be a TLR3, TLR4, TLR7, TLR8, or TLR9 modulator, such as a TLR3, TLR4, TLR7, TLR8, or TLR9 antagonist, or a TLR3, TLR4, TLR7, TLR8, or TLR9 agonist. In some embodiments, said TLR modulator is a TLR3 modulator. In some embodiments, said TLR modulator is a TLR4 modulator. In some embodiments, said TLR modulator is a TLR7 modulator. In some embodiments, said TLR modulator is a TLR8 modulator. In some embodiments, said TLR modulator is a TLR9 modulator. In some embodiments, said TLR modulator is a TLR3 agonist. In some embodiments, said TLR modulator is a TLR4 agonist. In some embodiments, said TLR modulator is a TLR7 agonist. In some embodiments, said TLR modulator is a TLR8 agonist. In some embodiments, said TLR modulator is a TLR9 agonist. In some embodiments, said TLR modulator is a TLR3 antagonist. In some embodiments, said TLR modulator is a TLR4 antagonist. In some embodiments, said TLR modulator is a TLR7 antagonist. In some embodiments, said TLR modulator is a TLR8 antagonist. In some embodiments, said TLR modulator is a TLR9 antagonist. In some embodiments, the TLR modulator described herein may modulate two or more TLRs. In some embodiments, said TLR modulator may activate one or more TLRs and inhibit one or more TLRs. In some embodiments, said TLR modulator is a TLR9 modulator such as kappaproct (KAPPAPROCT ^® ) or monarsen (MONARSEN ^® ). Some examples of TLR modulators are described, for example, in WO 2017059225 A1.

[00199] In some embodiments, said immunomodulatory agent is a CpG-A or Cpg-B oligonucleotide as described in WO 2017059225 A1.

[00200] Detection in the cytosol of pathogen-derived DNA requires signaling through TANK-binding kinase 1 (TBK1) and its dependent transcription factor, IFN regulatory factor 3 (IRF3). A transmembrane protein called STING (IFN gene stimulator; also known as MITA, ERIS, MPYS, and TMEM173) functions as a signaling receptor for these cyclic purine dinucleotides, causing stimulation of the TBK1-IRF3 signaling axis and a STING-dependent response with type I interferons. Accordingly, in some embodiments, said immunomodulatory agent is a STING modulator. STING directly binds cyclic diguanylate monophosphate, but not other irrelevant nucleotides or nucleic acids. Accordingly, in some embodiments, said STING modulator is a cyclic purine dinucleotide. Examples of cyclic purine dinucleotides and STING modulators are described, for example, in US 9549944 B2, the content of which is incorporated herein in its entirety by reference.

[00201] Additional examples of immunosuppressants include, but are not limited to, abatacept; adalimumab; adenosine receptor agonists; anakinra; aryl hydrocarbon receptor inhibitors; autophagy inhibitors such as 3-methyladenine; calcineurin inhibitors; calcineurin inhibitors such as cyclosporine and tacrolimus; caspase-1 inhibitors; certolizumab; cGAS inhibitors; corticosteroids; corticosteroids such as prednisone, budesonide, prednisolone; cytokine inhibitors; cytokine receptor activators; cytokine receptor inhibitors; etanercept; glucocorticoids; golimumab; G-protein coupled receptor agonists; G-protein coupled receptor antagonists; histone deacetylase inhibitors; histone deacetylase inhibitors such as trichostatin A; IMDH inhibitors such as azathioprine, leflunomide and mycophenolate; infliximab; mitochondrial function inhibitors such as rotenone; ixekizumab; kinase inhibitors; methylprednisolone; monoclonal antibodies such as basiliximab, daclizumab, and muromonab; mTOR inhibitors such as rapamycin or a rapamycin analog, sirolimus and everolimus; natalizumab; NF-κβ inhibitors such as 6Bio, dexamethasone, TCPA-1, IKK VII; OTK3; oxidized ATPs such as P2X receptor blockers; P38 inhibitors; peroxisome proliferator-activated receptor agonists; antagonists of peroxisome proliferator-activated receptors; phosphatase inhibitors; a phosphodiesterase inhibitor such as a phosphodiesterase 4 (PDE4) inhibitor such as rolipram; PI3 KB inhibitors such as TGX-221; prostaglandin E2 (PGE2) agonists such as misoprostol; proteasome inhibitor I (PSI); proteasome inhibitors; retinoids; rituximab; secukinumab; statins; TGF-β receptor agonists; TGF-β signaling agents; tmoglobulin; TLR9 antagonists; tocilizumab; ustekinumab; and vedolizumab. Immunosuppressants also include IDO, vitamin D3, cyclosporins such as cyclosporin A, aryl hydrocarbon receptor inhibitors, resveratrol, azathioprine (Aza), 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), FK506, sangliferin A, salmeterol, mycophenolate mofetil (MMF), aspirin and other COX inhibitors, niflumic acid, estriol and triptolide, interleukins (eg, IL-1, IL-10), cyclosporin A, siRNA-targeting cytokines or cytokine receptors, etc.

[00202] In some embodiments, said immunomodulatory agent is selected from the group consisting of 3-methyladenine, 6-Bio, 6-mercaptopurine (6-MP, 6-thioguanine (6-TG), FK506, sangliferin A, abatacept, adalimumab, anakinra, aryl hydrocarbon receptor inhibitors, aspirin, autophagy inhibitors, azathioprine, basiliximab, budesonide, calcineurin inhibitors, caspase-1 inhibitors, certolizumab, cGAS inhibitors, COX inhibitors, niflumic acid, cyclosporine, cytokine inhibitors, cytokine receptor activators, cytokine receptor inhibitors daclizumab, dexamethasone, estriol, etanercept, everolimus, glucocorticoids, golimumab, G-protein coupled receptor agonists, G-protein coupled receptor antagonists, histone deacetylase inhibitors, IDO, IKK VII, infliximab, interleukin-1, interleukin-10, ixekizumab , kinase inhibitors, leflunomide, methylprednisolone, misoprostol, muromonab, mycophenolate, mycophenolate mofetil (MMF) natalizumab, OTK3, oxidized ATP, P2X receptor blockers, P38 inhibitors, peroxisome proliferator-activated receptor agonists, peroxisome proliferator-activated receptor antagonists, phosphatase inhibitors, PI3 KB inhibitors, prednisolone, prednisone, proteasome inhibitor I (PSI), proteasome inhibitors, rapamycin, and rapamycin analogues, resveratrol, retinoids, rituximab, rolipram, rotenone, salmeterol, secukinumab, sirolimus, statins, tacrolimus, TCPA-1, TGX-221, thymoglobulin, TLR9 antagonists, tocilizumab, trichostatin A, triptolide, ustekinumab, vedolizumab, vitamin D3; and any combination of the above.

[00203] Note that any of the immunomodulatory agents described herein can be used in lipid nanoparticles, scDNA vectors, and/or compositions as described herein. For example, an immunomodulatory agent may be used alone or in combination with one or more (eg, one, two, three, four, five, or more) of the other immunomodulatory agents described herein.

[00204] In some embodiments, said immunomodulatory agent is selected from the group consisting of aryl hydrocarbon receptor inhibitors; caspase-1 inhibitors; cGAS inhibitors; cytokine inhibitors; G-protein coupled receptor agonists; G protein-coupled receptor antagonists; mitochondrial function inhibitors; mTOR inhibitors; NF-κβ inhibitors; agonists of peroxisome proliferator-activated receptors; phosphatase antagonists; a phosphodiesterase inhibitor, TGX-221; prostaglandin E2 (PGE2) agonists; TLR9 antagonists; proteasome inhibitors; TGF-β receptor agonists; and TGF-β signaling agents. Any one or more of the above agents may be used alone or in combination with LNP containing scDNA.

[00205] The present invention also provides a pharmaceutical composition comprising a lipid nanoparticle and a pharmaceutically acceptable carrier or excipient.

[00206] According to some aspects of the present invention, a lipid nanoparticulate formulation further comprising one or more pharmaceutical excipients is provided. In some embodiments, said lipid nanoparticulate formulation further comprises sucrose, Tris, trehalose, and/or glycine.

Some examples of LFO characteristics

[00207] Typically, the lipid nanoparticles of the present invention have an average diameter selected to provide the intended therapeutic effect. Accordingly, in some aspects, said lipid nanoparticle has an average diameter of from about 30 nm to about 150 nm, more often from about 50 nm to about 150 nm, more often from about 60 nm to about 130 nm, more often from about 70 nm to about 110 nm, more often just from about 85 nm to about 105 nm, and preferably about 100 nm. According to some aspects of the present invention, lipid particles with a relative size larger than conventional nanoparticles of about 150-250 nm are provided. The size of lipid nanoparticles can be determined using quasi-elastic light scattering using, for example, the Malvern Zetasizer Nano ZS system (Malvern, UK).

[00208] Depending on the intended use of the lipid particles, the proportions of the components may vary and the delivery efficiency of a particular formulation may be measured using, for example, endosomal release rate (ERP) analysis.

[00209] The scDNA may be complexed with the lipid portion of the particle or encapsulated in the lipid environment of the lipid nanoparticle. In some embodiments, the scDNA can be completely encapsulated in the lipid environment of the lipid nanoparticle, thus protecting it from nuclease degradation, for example, in an aqueous solution. In some embodiments, the scDNA in the lipid nanoparticle is not substantially degraded after exposure of the lipid nanoparticle to nuclease at 37° C. for at least about 20, 30, 45, or 60 minutes. In some embodiments, the scDNA in the lipid nanoparticle is substantially non-degradable after the particle is incubated in serum at 37° C. for at least about 30, 45, or 60 minutes, or 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.

[00210] In some embodiments, said lipid nanoparticles are substantially non-toxic to a subject, eg, a mammal, such as a human.

[00211] In some aspects, said lipid nanoparticulate formulation is a lyophilized powder.

[00212] In some embodiments, the lipid nanoparticles are solid core particles containing at least one lipid bilayer. In other embodiments, said lipid nanoparticles have a structure without bilayers, ie. non-layered (i.e., non-bilayered) morphology. Without limitation, non-bilayer morphology may include, for example, three-dimensional tubes, rods, structures with cubic symmetry, and the like. The non-layered morphology (ie, non-bilayer structure) of lipid particles can be determined using analytical techniques known and used by those skilled in the art. Such techniques include, but are not limited to, cryogenic transmission electron microscopy ("cryo-TEM"), differential scanning calorimetry ("DSC"), X-ray diffraction, and the like. For example, the morphology of lipid nanoparticles (layered or non-layered) can easily be assessed and characterized using, for example, cryo-TEM analysis as described in US 2010/0130588, the contents of which are incorporated herein in their entirety by reference.

[00213] According to some additional embodiments, lipid nanoparticles with a few layered morphology are electron dense.

[00214] According to some aspects of the present invention, a lipid nanoparticle with a single-layer or multi-layer structure is provided. According to some aspects of the present invention, a lipid nanoparticulate formulation is provided that contains multivesicular particles and/or foam-based particles.

[00215] By controlling the composition and concentration of the lipid components, one can control the rate at which the lipid conjugate exits the lipid particle and, in turn, the rate at which said lipid nanoparticle becomes fusogenic. In addition, other variables, including, for example, pH, temperature, or ionic strength, can be used to change and/or control the rate at which the lipid nanoparticle becomes fusogenic. Other methods that can be used to control the rate at which said lipid nanoparticle becomes fusogenic will be apparent to those skilled in the art based on the present disclosure. It is also apparent that by controlling the composition and concentration of the lipid conjugate, the size of the lipid particles can be controlled.

[00216] The pKa value of cationic lipid formulations can be correlated with the effectiveness of LNPs for delivery of nucleic acids (see sources: Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176 (2010), both of which are hereby incorporated by reference in their entirety). The preferred range of pKa values is from ~5 to ~7. The pKa value of a cationic lipid can be determined in lipid nanoparticles using an analysis based on the fluorescence of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS).

[00217] Encapsulation of ccDNA in lipid particles can be determined by size exclusion analysis using a non-membrane permeable fluorescent dye that exhibits enhanced fluorescence when bound to nucleic acid, such as an ^Oligreen® or ^PicoGreen® assay. Typically, encapsulation is determined by adding a dye to a lipid particle formulation, measuring the resulting fluorescence and comparing it to the fluorescence observed with the addition of a small amount of non-ionic detergent. Detergent-mediated disruption of the lipid bilayer releases the encapsulated ccDNA, allowing it to interact with the non-permeable dye. scDNA encapsulation can be calculated as E=(I ₀ -I)/I ₀ , where I and I ₀ refer to the fluorescence intensity before and after detergent addition.

[00218] According to some aspects of the present invention, a liposome formulation is provided that includes one or more polyethylene glycol (PEG) functionalized compounds (so-called "pegylated compounds") that can reduce the immunogenicity/antigenicity of the compound(s), render the compound(s) hydrophilic and hydrophobic (compounds) and/or reducing the therapeutically effective dosing frequency. In other aspects, said liposomal formulation may simply include a polyethylene glycol (PEG) polymer as an additional component. In such aspects, the molecular weight of the PEG or PEG moiety can range from 62 Da to about 5000 Da.

[00219] According to some aspects of the present invention, a liposome formulation is provided that delivers ccDNA with an extended release or controlled release profile over a period of hours to weeks. In some related aspects, said liposomal formulation may comprise aqueous chambers linked by lipid bilayers. In other related aspects, said liposome formulation encapsulates ccDNA with additional components that undergo a physical transition at elevated temperature, releasing said API over a period of hours to weeks.

[00220] In some aspects, said liposomal formulation comprises sphingomyelin and one or more lipids as described herein. In some aspects, said liposomal formulation contains optisomes.

[00221] According to certain aspects of the present invention, a liposomal formulation is provided that includes one or more lipids selected from: N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, (distearoyl- sn-glycero-phosphoethanolamine), MPEG (methoxypolyethylene glycol)-conjugated lipid, HSPC (hydrogenated soy phosphatidylcholine); PEG (polyethylene glycol); DSPE (distearoyl-sn-glycero-phosphoethanolamine); DSPC (distearoylphosphatidylcholine); DOPC (dioleylphosphatidylcholine); DPPG (dipalmitoylphosphatidylglycerol); EPC (egg phosphatidylcholine); DOPS (dioleylphosphatidylserine); POPC (palmitoiloleoylphosphatidylcholine); SM (sphingomyelin); MPEG (methoxy polyethylene glycol); DMPC (dimyristoylphosphatidylcholine); DMPG (dimyristoylphosphatidylglycerol); DSPG (distearoylphosphatidylglycerol); DEPC (dierucoylphosphatidylcholine); DOPE (dioleoli-sn-glycerophosphatidylamine), cholesteryl sulfate (CS), dipalmitoylphosphatidylglycerol (DPPG), DOPC (dioleoli-sn-glycerophosphatidylcholine), or any combination of the above.

[00222] According to some aspects of the present invention, a liposome formulation is provided comprising a phospholipid, cholesterol, and pegylated lipid in a molar ratio of 56:38:5. In some aspects, the total lipid content of said liposomal formulation is 2-16 mg/mL. According to some aspects of the present invention, a liposomal formulation is provided comprising a lipid comprising a phosphatidylcholine functional group, a lipid comprising an ethanolamine functional group, and a pegylated lipid. According to some aspects of the present invention, a liposomal formulation is provided comprising a lipid comprising a phosphatidylcholine functional group, a lipid comprising an ethanolamine functional group, and a pegylated lipid in a molar ratio of 3:0.015:2, respectively. According to some aspects of the present invention, a liposomal formulation is provided comprising a lipid comprising a phosphatidylcholine functional group, cholesterol, and a pegylated lipid. According to some aspects of the present invention, a liposomal formulation is provided comprising a lipid comprising a phosphatidylcholine functional group and cholesterol. In some aspects, said pegylated lipid is PEG-2000-DSPE. In some aspects of the present invention, a liposomal formulation is provided comprising DPPG, soy PC (phosphatidylcholine), an MPEG-DSPE lipid conjugate, and cholesterol.

[00223] According to some aspects of the present invention, a liposomal formulation is provided comprising one or more lipids comprising a phosphatidylcholine functional group and one or more lipids comprising an ethanolamine functional group. According to some aspects of the present invention, a composition is provided comprising one or more of: lipids comprising a phosphatidylcholine functional group, lipids comprising an ethanolamine functional group, and/or sterols, for example, cholesterol. In some aspects, said liposomal formulation contains DOPC/DEPC; and DOPE.

[00224] According to some aspects of the present invention, a liposomal formulation is provided, further comprising one or more pharmaceutical excipients, for example, sucrose and/or glycine.

[00225] According to some aspects of the present invention, a liposomal formulation with a single layer or multilayer structure is provided. According to some aspects of the present invention, a liposome formulation is provided that contains multivesicular particles and/or foam-based particles. In some aspects of the present invention, a liposomal formulation is provided that has a relative particle size larger than conventional nanoparticles, approximately 150-250 nm. In some aspects, said liposomal formulation is a lyophilized powder.

[00226] According to some aspects of the present invention, a liposomal formulation is provided that is prepared and loaded with ccDNA vectors prepared by the method of Example 1 or otherwise described herein by adding a weak base to a mixture containing isolated ccDNA outside the liposome. Said addition increases the pH outside the liposome to about 7.3 and directs said scDNA to the liposome. According to some aspects of the present invention, a liposomal formulation with an acidic pH within the liposome is provided. In such cases, the pH within the liposome may be 4-6.9, more preferably 6.5. According to other aspects of the present invention, a liposomal formulation is provided using intraliposomal drug stabilization technology. In such cases, polymeric or non-polymeric highly charged anions and intraliposomal capturing agents such as sucrose polyphosphate or octasulfate are used.

[00227] According to other aspects of the present invention, a liposomal formulation is provided comprising phospholipids, lecithin, phosphatidylcholine, and phosphatidylethanolamine.

[00228] ZcDNA as described herein can be included in pharmaceutical compositions suitable for administration to a subject for in vivo delivery to cells, tissues or organs of said subject. Typically, said pharmaceutical composition contains ccDNA as described herein and a pharmaceutically acceptable carrier. For example, the ccDNA vectors of the present invention can be incorporated into a pharmaceutical composition suitable for the desired therapeutic route of administration (eg, parenteral administration). Also contemplated are passive tissue transduction by intravenous or intra-arterial infusion under high pressure, as well as intracellular injection, such as intranuclear microinjection or intracytoplasmic injection. Pharmaceutical compositions for therapeutic use may be formulated into a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for a high concentration scDNA vector. Sterile injectable solutions can be prepared by incorporating the ccDNA vector component in the required amount in a suitable buffer, along with one ingredient or combination of ingredients listed above, as needed, followed by sterile filtration.

[00229] The zcDNA vector as described herein can be incorporated into a pharmaceutical composition suitable for topical, systemic, intra-amniotic, intrathecal, intracranial, intra-arterial, intravenous, intralymphatic, intraperitoneal, subcutaneous, tracheal, interstitial (e.g., intramuscular, intracardiac, intrahepatic, intrarenal, intracerebral), intrathecal, intravesical, conjunctival (eg, extraorbital, intraorbital, retroorbital, intraretinal, subretinal, choroidal, subchoroidal, intrastromal, intracameral, and intravitreal), intracochlear, and mucosal (eg, oral, rectal, nasal) administration. Also contemplated are passive tissue transduction by intravenous or intra-arterial infusion under high pressure, as well as intracellular injection, such as intranuclear microinjection or intracytoplasmic injection.

[00230] Pharmaceutically active compositions containing the scDNA vector can be formulated to deliver the transgene in nucleic acid to recipient cells resulting in therapeutic expression of said transgene. Said composition may also include a pharmaceutically acceptable carrier.

[00231] The compositions and vectors of the present invention can be used to deliver a transgene for a variety of purposes. In some embodiments, said transgene encodes a protein or functional RNA for use in scientific research, for example, to create a somatic transgenic animal model carrying said transgene, for example, to study the function of said transgenic product. According to another example, said transgene encodes a protein or functional RNA for use in creating an animal model of a disease. In some embodiments, said transgene encodes one or more peptides, polypeptides, or proteins that are suitable for treating, alleviating, or preventing disease states in a mammalian subject. The specified transgene can be transferred into the patient's body (eg, expressed in the patient) in sufficient quantities to treat diseases associated with reduced expression, lack of expression or gene dysfunction. In some embodiments, said transgene can be transferred to a subject (e.g., expressed in a subject) in sufficient quantity to treat a disease associated with increased expression, gene product activity, or inappropriate upregulation of a gene that said transgene represses or otherwise causes the desired reduction in expression. .

[00232] Pharmaceutical compositions for therapeutic purposes, as a rule, must be sterile and stable under the conditions of receipt and storage. Said composition may be incorporated into a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for a high concentration scDNA vector. Sterile injectable solutions can be prepared by incorporating the ccDNA vector component in the required amount in a suitable buffer, along with one ingredient or combination of ingredients listed above, as needed, followed by sterile filtration.

[00233] The ccDNA vector as described herein may also be introduced into an organism for transduction of cells in vivo.

[00234] Typically, administration is by any of the routes commonly used to bring the molecule into as close contact as possible with blood or tissue cells. Suitable routes for administering such nucleic acids are available and well known to those skilled in the art, and while more than one route may be used to administer a particular composition, a particular route can often provide a faster and more efficient response than another route. Exemplary routes of administration of the ccDNA vector as described herein include oral, rectal, transmucosal, intranasal, inhalation (e.g., e.g., aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intraendothelial, in utero (or in ovo), parenteral (eg, intravenous, subcutaneous, intradermal, intracranial, intramuscular [including injection into skeletal, diaphragmatic, and/or cardiac muscle], intrapleural, intracerebral, and intraarticular), topical (eg, on the skin and mucosal surfaces, including respiratory tract surfaces, and transdermal administration), intralymphatic administration, etc., as well as direct injection into a tissue or organ (for example, the liver, eyes, skeletal muscles, cardiac muscle, diaphragmatic muscle, or head brain).

[00235] The scDNA vector can be introduced at any site in the subject's body, including, without limitation, a site selected from the group consisting of brain, skeletal muscle, smooth muscle, heart, diaphragm, respiratory tract epithelium, liver, kidney , spleen, pancreas, skin and eyes. Can also be the introduction of the specified ccDNA vector in the tumor (for example, in the tumor or near the tumor, or in the lymph node). The most appropriate route in any given case depends on the nature and severity of the condition being treated, alleviated and/or prevented, and on the nature of the particular scDNA vector used. In addition, the use of scDNA allows the introduction of more than one transgene in a single vector or multiple scDNA vectors (eg, an scDNA cocktail).

[00236] Administration of an ccDNA vector as described herein to skeletal muscle includes, but is not limited to, administration to skeletal muscle in the extremities (e.g., upper arm, forearm, thigh, and/or lower leg), back, neck, head (e.g., tongue), chest, abdomen, pelvis/perineum and/or fingers. The ccDNA vector as described herein can be delivered to skeletal muscle by intravenous, intra-arterial, intraperitoneal, limb perfusion (optionally isolated limb, leg, and/or arm perfusion; see e.g., Arruda et al., ( 2005) Blood 105: 3458-3464), and/or direct intramuscular injection. In specific embodiments, the scDNA vector as described herein is administered to a limb (arm and/or leg) of a subject (e.g., a subject with muscular dystrophy such as DMD) by perfusion of the limb, optionally by perfusion of an isolated limb (e.g., by intravenous or intra-articular introductions). In embodiments, the scDNA vector as described herein can be introduced without using "hydrodynamic" techniques.

[00237] Administration of an scDNA vector as described herein to cardiac muscle includes administration to the left atrium, right atrium, left ventricle, right ventricle, and/or septum. The ccDNA vector as described herein can be delivered to the heart muscle by intravenous administration, intra-arterial administration such as intra-aortic administration, direct injection into the heart (e.g., left atrium, right atrium, left ventricle, right ventricle), and/or perfusion coronary artery. The introduction into the diaphragmatic muscle can be carried out by any suitable method, including intravenous administration, intra-arterial administration and/or intraperitoneal administration. Administration to smooth muscle may be by any suitable route, including intravenous administration, intra-arterial administration, and/or intraperitoneal administration. According to one implementation variant, the introduction can be carried out in endothelial cells present in smooth muscles, near and/or on them.

[00238] In some embodiments, the ccDNA vector of the present invention is administered to skeletal muscle, diaphragmatic muscle, and/or cardiac muscle (e.g., to treat, alleviate, and/or prevent muscular dystrophy or heart disease (e.g., PAD or congestive heart disease). insufficiency).

[00239] The ZcDNA vector of the present invention can be introduced into the CNS (eg, the brain or the eye). Said ccDNA vector can be injected into the spinal cord, brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra, pineal gland), cerebellum, telencephalon (striatum, cerebrum occipital, temporal, parietal, and frontal lobes, cerebral cortex, basal ganglia, hippocampus, and amygdala), limbic system, neocortex, striatum, cerebrum, and inferior colliculus. Said scDNA vector may also be introduced into different regions of the eye such as the retina, cornea and/or optic nerve. Said scDNA vector can be delivered to the cerebrospinal fluid (eg, by lumbar puncture). Said ccDNA vector may also be intravascularly introduced into the CNS in situations where the blood-brain barrier has been compromised (eg, in a brain tumor or cerebral infarction).

[00240] In some embodiments, said scDNA vector may be introduced into the desired region or regions of the CNS by any route known in the art, including, but not limited to, intrathecal, intraocular, intracerebral, intraventricular, intravenous (e.g., in the presence of a sugar such as mannitol), intranasal, intra-auricular, intraocular (eg, intravitreal, subretinal, anterior chamber) and periocular (eg, sub-Tenon's space) delivery, as well as intramuscular delivery with retrograde delivery to motor neurons.

[00241] In some embodiments, said scDNA vector is administered as a liquid formulation by direct injection (eg, stereotaxic injection) into the desired area or compartment in the CNS. In other embodiments, said scDNA vector may be administered by topical application to the desired area, or by intranasal administration of an aerosol formulation. Administration to the eye can be accomplished by topical application of liquid drops. In a further alternative, the scDNA vector may be administered as a slow release solid formulation (see, for example, US Pat. No. 7,201,898). In other additional embodiments, said scDNA vector can be used for retrograde transport to treat, alleviate, and/or prevent diseases and disorders affecting motor neurons (e.g., amyotrophic lateral sclerosis (ALS); spinal muscular atrophy (SMA), etc. .). For example, said scDNA vector can be delivered to muscle tissue from which it can migrate to neurons.

Treatment ex vivo

[00242] In some embodiments, cells are removed from the subject, injected with the scDNA vector, and returned to the subject. Methods for extracting cells from a subject's body for ex vivo treatment, followed by introduction back into the subject's body, are known in the art (see, for example, US Pat. No. 5,399,346, the contents of which are fully incorporated herein). Alternatively, the scDNA vector is introduced into cells from another subject, into cultured cells, or into cells from any other suitable source, and said cells are administered to a subject in need thereof.

[00243] Cells transduced with the ccDNA vector are preferably administered to a subject in a "therapeutically effective amount" in combination with a pharmaceutical carrier. Those skilled in the art will appreciate that the therapeutic effects need not be complete or provide a cure, as long as the subject receives some benefit.

[00244] In some embodiments, said scDNA vector may encode a transgene (sometimes referred to as a heterologous nucleotide sequence) that is any polypeptide that is desired to be produced in a cell in vitro, ex vivo, or in vivo. For example, in contrast to the use of said ccDNA vectors in a method of treatment, according to some embodiments, said ccDNA vectors can be introduced into cultured cells and the expressed gene product isolated from them, for example, to produce antigens or vaccines.

[00245] These ccDNA vectors can be used in both veterinary and human medicine. Suitable subjects for the use of ex vivo gene delivery methods as described above include both birds (e.g., chicken, ducks, geese, quails, turkey, and pheasant) and mammals (e.g., humans, cattle, sheep, goats, horses, felines). , canine and lagomorphs), with mammals being preferred. Human subjects are most preferred. Human subjects include neonates, infants, young adults and adults.

[00246] In some embodiments, a transgene in an expression cassette, expression construct, or scDNA vector described herein can be codon-optimized for the host cell. As used herein, the term "codon-optimized" or "codon-optimized" refers to a method of modifying a nucleic acid sequence to improve expression in cells of a vertebrate animal of interest, e.g., a mouse or a human (e.g., humanized), by replacing at least one, more than one or a significant number of codons of natural sequence (eg, prokaryotic sequence) codons, most or most frequently used in the genes of the specified vertebrate. In different species, there is a particular bias with a preference for certain codons for a particular amino acid. Typically, codon optimization does not change the amino acid sequence of the original translated protein. Optimized codons can be determined using, for example, the codon optimization and personalized gene synthesis platform from Aptagen Gene Forge® (Aptagen, Inc.) or other publicly available database.

[00247] In some embodiments, said scDNA vector expresses a transgene in a host cell subject. In some embodiments, said host cell is a human host cell, including, for example, blood cells, stem cells, hematopoietic cells, CD34 ⁺ cells, liver cells, cancer cells, vascular cells, muscle cells, pancreatic cells, nerve retinal cells or cells, epithelial or endothelial cells, dendritic cells, fibroblasts, or any other cell derived from a mammalian body, including, without limitation, hepatocytes (i.e., liver cells), lung cells, heart cells, pancreatic cells, cells Gastrointestinal, diaphragmatic cells, renal (ie, kidney) cells, nerve cells, blood cells, bone marrow cells, or cells from any one or more selected tissues of the subject for whom the gene therapy is intended. In one aspect, the subject host cell is a human host cell.

[00248] One aspect of the technology described herein relates to a method for delivering a transgene into a cell. Typically, in in vitro methods, said scDNA vector can be introduced into a cell using methods as described herein, as well as other methods known in the art. ZcDNA vectors as described herein are preferably introduced into the cell in a biologically effective amount. When an scDNA vector is administered to a cell in vivo (eg, a subject), a biologically effective amount of said scDNA vector is an amount sufficient to allow transduction and expression of said transgene in the target cell.

Dose ranges

[00249] In vivo and/or in vitro assays may optionally be performed to facilitate identification of optimal dosage ranges for use. The precise dosage to be used in the formulation also depends on the route of administration and the severity of the condition, and should be determined based on the judgment of one of ordinary skill in the art and the circumstances of each subject. Effective doses can be extrapolated from dose-response curves obtained in vitro or in test animal models.

[00250] The ZcDNA vector is administered in sufficient amounts to transfect cells of the desired tissue and to ensure sufficient levels of gene transfer and expression without excessive adverse events. Standard and pharmaceutically acceptable routes of administration include, but are not limited to, those described in the Introduction section above, such as direct delivery to a selected organ (e.g. intraportal delivery to the liver), oral, inhalation delivery (including intranasal and intratracheal delivery), intraocular, intravenous, intramuscular, subcutaneous, intradermal, intratumoral delivery; and other routes for parenteral administration. Routes of administration can be combined if desired.

[00251] The dosage amount of scDNA vector required to achieve a particular "therapeutic effect" varies depending on a number of factors, including, but not limited to: the route of administration of the nucleic acid, the level of gene or RNA expression required to achieve a therapeutic effect , the specific disease or disorder being treated, and the stability of said gene or genes, the RNA product or products, or the resulting expressed protein or proteins. A person skilled in the art can easily determine the dosage range of the ccDNA vector for treating a patient with a particular disease or disorder based on the above factors as well as other factors well known in the art.

[00252] The dosage regimen may be adjusted to provide the optimal therapeutic response. For example, the oligonucleotide may be administered multiple times, for example, multiple doses may be administered daily, or the dose may be proportionally reduced to suit the therapeutic need. One of ordinary skill in the art will readily be able to determine suitable doses and administration schedules for the oligonucleotides in question, both for administration to cells and for administration to subjects.

[00253] A "therapeutically effective dose" is a relatively broad range that can be determined in clinical trials and depends on the particular application (neural cells require very small amounts, while systemic injections require significant amounts). For example, for direct in vivo injection into the skeletal or cardiac muscle of a human subject, a therapeutically effective dose would be about 1 μg-100 g of the scDNA vector. Where exosomes or microparticles are used to deliver the ccDNA vector, a therapeutically effective dose can be determined experimentally, but it is expected that between 1 μg and about 100 g of vector will be delivered.

[00254] The formulation of pharmaceutically acceptable excipients and carrier solutions is well known to those skilled in the art, as is the development of suitable dosing and treatment regimens for the use of the particular compositions described herein in various treatment regimens.

[00255] For in vitro transfection, an effective amount of ccDNA vector to deliver into cells (1 x 10 ⁶ cells) is about 0.1-100 μg of ccDNA vector, preferably 1 to 20 μg, more preferably 1 to 15 μg, or 8 to 10 mcg. Larger ccDNA vectors require higher doses. If exosomes or microparticles are used, an effective in vitro dose can be determined experimentally, but should be expected to deliver the same amount of scDNA vector in total.

[00256] Treatment may include the administration of a single dose or multiple doses. In particular embodiments, more than one administration (e.g., two, three, four, or more administrations) at various intervals, such as daily, weekly, monthly, yearly, and the like, may be used to achieve the desired level of gene expression. In some embodiments, more than one dose may be administered to a subject; in fact, multiple doses may be administered as needed, since the ccDNA vector does not elicit an immune response against the capsid in the host due to the absence of a viral capsid. Accordingly, a person skilled in the art can readily determine the appropriate number of doses. The number of doses administered may, for example, be in the order of 1-100, preferably 2-20 doses.

[00257] Without wishing to be bound by any particular theory, the lack of a typical antiviral immune response elicited by administration of an scDNA vector as described herein (i.e., without capsid components) allows said scDNA vector to be repeatedly administered to a host. In some embodiments, the number of administrations of the heterologous nucleic acid to a subject is 2 to 10 times (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times). In some embodiments, the scDNA vector is delivered to a subject more than 10 times.

[00258] In some embodiments, the dose of the scDNA vector is administered to the subject no more than once per calendar day (eg, a 24-hour period). In some embodiments, the dose of the ccDNA vector is administered to the subject no more than once every 2, 3, 4, 5, 6, or 7 calendar days. In some embodiments, the dose of the scDNA vector is administered to the subject no more than once per calendar week (eg, 7 calendar days). In some embodiments, the dose of the scDNA vector is administered to the subject no more than once every two weeks (eg, once every two calendar weeks). In some embodiments, the dose of the scDNA vector is administered to the subject no more than once per calendar month (eg, once every 30 calendar days). In some embodiments, a dose of the ccDNA vector is administered to a subject no more than once every six calendar months. In some embodiments, a dose of the scDNA vector is administered to a subject no more than once per calendar year (eg, 365 days or 366 days in a leap year).

Single dosage forms

[00259] In some embodiments, these pharmaceutical compositions may conveniently be presented in unit dosage form. The unit dosage form is typically adapted to one or more specific routes of administration of the pharmaceutical composition. In some embodiments, said unit dosage form is adapted for administration by inhalation. In some embodiments, said unit dosage form is adapted for administration via a vaporizer. In some embodiments, said unit dosage form is adapted for administration via a nebulizer. In some embodiments, said unit dosage form is adapted for administration with an aerosol generator. In some embodiments, said unit dosage form is adapted for oral administration, for buccal administration, or for sublingual administration. In some embodiments, said unit dosage form is adapted for intravenous, intramuscular, or subcutaneous administration. In some embodiments, said unit dosage form is adapted for intrathecal or intracerebroventricular administration. In some embodiments, said pharmaceutical composition is formulated for topical administration. The amount of active ingredient which can be combined with the carrier material to form a unit dosage form is usually that amount of said compound which provides a therapeutic effect.

Preparation of lipid nanoparticles

[00260] Lipid nanoparticles can spontaneously form by mixing scDNA and a lipid or lipids. Depending on the desired particle size distribution, the final mixture of nanoparticles can be extruded through the membrane (for example, with a cut-off size of 100 nm) using, for example, a Thermobarrel type extruder such as Lipex Extruder (Northern Lipids, Inc.). In some cases, the extrusion step may be skipped. Removal of ethanol and simultaneous replacement of the buffer can be carried out by, for example, dialysis or tangential flow filtration.

[00261] In General, lipid nanoparticles can be obtained by any method known in the art. For example, lipid nanoparticles can be prepared using the methods described in, for example, US 2013/0037977, US 2010/0015218, US 2013/0156845, US 2013/0164400, US 2012/0225129 and US 2010/0130588, the content of each of which is included herein in its entirety by reference. In some embodiments, the lipid nanoparticles can be prepared using a continuous mixing process, a direct dilution process, or an in-line dilution process. Methods and apparatus for producing lipid nanoparticles using direct or in-line dilution processes are described in US 2007/0042031, the contents of which are incorporated herein by reference in their entirety. Methods and apparatus for producing lipid nanoparticles using stepwise dilution processes are described in US 2004/0142025, the contents of which are incorporated herein by reference in their entirety.

[00262] In one non-limiting example, these lipid nanoparticles can be obtained using an impact spray method. Typically, particles are prepared by mixing lipids dissolved in alcohol (eg, ethanol) with ccDNA dissolved in a buffer, such as citrate buffer, sodium acetate buffer, sodium acetate magnesium chloride buffer, malic acid buffer, malic acid buffer and sodium chloride; or buffered with sodium citrate and sodium chloride. The ratio of lipid and scDNA components of the mixture can be approximately 45-55% lipids and approximately 65-45% scDNA.

[00263] The lipid solution may contain an ionizable lipid, a non-cationic lipid (eg, a phospholipid such as DPSC), a PEG or PEG-conjugated molecule (eg, a PEG lipid), and a sterol (eg, cholesterol) for a total lipid concentration of 5-30 mg / ml, more likely - 5-15 mg / ml, most likely - 9-12 mg / ml, in alcohol, for example, in ethanol.

[00264] In a lipid solution, the relative molar lipid content can vary from about 25% to 98% for a cationic lipid, preferably about 35-65%; from about 0% to 15% for non-ionic lipid, preferably about 0-12%; from about 0% to 15% for PEG or PEG-conjugated molecule, preferably about 1-6%; and from about 0% to 75% for the sterol, preferably about 30-50%.

[00265] The ccDNA solution may contain ccDNA in a concentration range of 0.3 to 1.0 mg/ml, preferably 0.3-0.9 mg/ml in a buffered solution, at a pH in the range of 3.5-5.

[00266] To form LNPs, in one non-limiting example, these 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 a shock-spray mixer, with immediate formation LNC. The flow rate during mixing can vary in the range of 10-600 ml/min. The inner diameter (ID) of the tubes can vary in the range from 0.25 to 1.0 mm, and the total flow rate in the range of 10-600 ml/min. The combination of flow rate and ID tubes can provide LNP particle size control in the range of 30 to 200 nm. The solution may then be mixed with the higher pH buffered solution at a mixing ratio ranging from 1:1 to 1:3 by volume, preferably about 1:2 by volume. If necessary, the specified buffered solution may have a temperature in the range from 15-40°C or 30-40°C. Then the stage of anion-exchange filtration of the mixture with LNP can be carried out. Prior to anion exchange, the mixture with LNP can be incubated for a certain period of time, for example, from 30 minutes to 2 hours. The temperature during incubation can be set in the range of 15-40°C or 30-40°C. After incubation, the solution is filtered through a filter, for example, with a pore size of 0.8 μm, while carrying out the step of anion exchange separation. This method can use tubing with an ID in the range of 1 mm to 5 mm and a flow rate of 10 to 2000 ml/min.

[00267] Once LNPs are obtained, they can be concentrated and diafiltered using an ultrafiltration process that removes the alcohol and replaces the buffer with a final buffer solution, such as phosphate buffered saline (PBS) with a pH of approximately 7, for example, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, or about 7.4.

[00268] For the ultrafiltration process, a tangential flow filtration (TFF) format can be used using a membrane with a nominal molecular weight cutoff of 30 to 500 kDa. Membrane format - hollow fiber or flat cassette. In a TFF process with adequate molecular weight cutoff, LNP may be retained in the retentate and the filtrate or permeate contains unnecessary alcohol, citrate buffer, and final buffer. The TFF process is multi-step, with an increase in the initial concentration of scDNA to a concentration of 1-3 mg/ml. After concentration, the LNP solution is diafiltered with 10-20 volumes of the final buffer to remove alcohol and replace the buffer. Then the material can be further concentrated by 1-3 times. A concentrated LNP solution can be sterilized by filtration.

ccDNA

[00269] In various embodiments, said scDNA vectors are capsidless linear duplex DNA molecules formed by a continuous chain of complementary DNA with covalently closed ends (linear, continuous, and capsidless structure) that contain an inverted 5'-terminal repeat (ITR) sequence and sequence of 3'-ITRs that are different from each other, or asymmetric with respect to each other. At least one of the ITRs contains a functional end resolution site and a replication protein (RPS) binding site (sometimes referred to as a replicative protein binding site), for example, a Rep binding site. Typically, the scDNA vector contains at least one modified AAV inverted terminal repeat (ITR) sequence, i.e. deletion, insertion and/or substitution relative to another ITR, and an expressed transgene.

[00270] In one embodiment, at least one of the ITRs is an AAV ITR, eg, a wild-type AAV ITR. In one embodiment, at least one of the ITRs is an ITR modified from the other ITR, ie, the scDNA contains ITRs that are asymmetric with respect to each other. In one embodiment, at least one of the ITRs is a non-functional ITR.

[00271] In some embodiments, said scDNA vector contains: (1) an expression cassette containing a cis regulatory element, a promoter, and at least one transgene; or (2) a promoter operably linked to at least one transgene; and (3) two self-complementary sequences, eg, ITR, flanking said expression cassette, wherein said scDNA vector is not associated with a capsid protein. In some embodiments, said ccDNA vector contains two self-complementary sequences found in the AAV genome, of which at least one contains a functional Rep binding element (RBE) (also sometimes referred to herein as "RBS") and a terminal clearance site (trs ) AAV, or a functional RBE variant, and one or more cis-regulatory elements operably linked to the transgene. In some embodiments, said scDNA vector contains additional components for regulating the expression of the transgene, such as regulatory switches to control and regulate the expression of said transgene, and may include a regulatory switch that is a "safety switch" that provides controlled cell death of the cell containing the scDNA. -vector.

[00272] In some embodiments, the two self-complementary sequences may be ITR sequences from any known parvovirus, eg, dependovirus, such as AAV (eg, AAV1-AAV12). Any AAV serotype may be used, including, but not limited to, a modified AAV2 ITR sequence retaining a Rep binding site (RBS) such as 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 531) and an end clearance site (trs), along with a variable palindromic sequence that provides the formation of a hairpin secondary structure. In some embodiments, the ITR may be synthetic. In one embodiment, the synthetic ITR is based on ITR sequences from more than one AAV serotype. In another embodiment, the synthetic ITR does not include AAV-based sequences. In yet another embodiment, the synthetic ITR retains the ITR structure described above, although it contains only some or no AAV-derived sequence. In some aspects, the synthetic ITR may preferentially interact with a wild-type or serotype-specific Rep, or, in some cases, the synthetic ITR does not recognize a wild-type Rep and only recognizes a mutated Rep. In some embodiments, said ITR is a synthetic ITR sequence that retains a functional Rep binding site (RBS) such as 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 531) and a terminal resolution site (TRS) along with a variable palindromic sequence, providing the formation of a hairpin secondary structure. In some examples, the modified ITR sequence retains the RBS sequence, trs, and the structure and position of the Rep binding element forming the terminal loop portion of the hairpin secondary structure of one of the ITRs from the corresponding wild-type AAV2 ITR sequence. Examples of ITR sequences for use in ccDNA vectors are disclosed in Tables 2-9, 10A and 10B, SEQ ID NOs: 2, 52, 101-449 and 545-547, and in the partial ITR sequences shown in FIG. 26A-26B of PCT Application No. PCT/US18/49996, filed September 7, 2018. In some embodiments, an scDNA vector may contain an ITR with a modification in the ITR corresponding to any of the modifications in the ITR sequences or partial ITR sequences present in any one or more of Tables 2, 3, 4, 5, 6, 7, 8, 9, 10A, and 10B of PCT Application No. PCT/US18/49996, filed September 7, 2018.

[00273] In some embodiments, said closed-ended DNA vector contains a promoter operably linked to the transgene, wherein the scDNA contains no capsid proteins and: (a) is derived from an scDNA plasmid that encodes a mutated AAV2 right-hand ITR with the same number of intramolecular duplex base pairs as in SEQ ID NO: 2, or a mutated AAV2 left ITR with the same number of intramolecular duplex base pairs as in SEQ ID NO: 51 in a hairpin secondary configuration (preferably excluding the deletion of any AAA terminal loop or TTT in the specified configuration with respect to the specified reference sequences); and (b) identified as ccDNA using native and denaturing agarose gel electrophoresis assay to identify ccDNA according to Example 1.

[00274] These zcDNA vectors can be generated using a number of methods known to one of skill in the art after reading this disclosure. ) containing an expression construct polynucleotide template comprising, in that order: a first inverted 5'-terminal repeat (eg, AAV ITR); expression cassette; and 3'-ITR (e.g. AAV ITR), wherein at least one of the 5'- and 3'-ITR is a modified ITR, or if both 5' and 3' ITRs are modified, they contain different modifications and their the sequences are not the same. Without limitation, the expression construct polynucleotide template used to generate scDNA vectors can be a scDNA plasmid, scDNA bacmid, and/or scDNA baculovirus. In some embodiments, the scDNA plasmid contains a cloning restriction site (e.g., SEQ ID NO: 7) operably located between the ITR, where an expression cassette can be inserted that contains, e.g., a promoter operably linked to the transgene, e.g., a reporter gene and/or therapeutic gene).

[00275] For example, non-viral capsidless DNA vectors can be produced in permissive host cells with an expression construct (e.g., plasmid, bacmid, baculovirus; or cell line in an integrated state) containing a heterologous gene located between two different inverted terminal sequences. repetitions (ITR). At least one of the ITRs is modified by deletion, insertion, and/or substitution relative to the wild-type ITR sequence (eg, AAV ITRs); and at least one of the ITRs contains a functional end resolution site (trs) and a Rep binding site. Said scDNA vector is preferably duplex, eg self-complementary, over at least a portion of the molecule, such as an expression cassette. For example, ccDNA is not a double-stranded circular molecule. In some embodiments, said scDNA vector has covalently closed ends and is therefore resistant to exonuclease (eg, exonuclease I or exonuclease III) digestion, for example, for one hour at 37°C.

[00276] In some embodiments, the scDNA vector contains, in the 5' to 3' direction: an adeno-associated virus (AAV) first inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette as described herein), and the second ITR AAV, while the first ITR and the second ITR are asymmetrical with respect to each other, that is, they differ from each other. In an exemplary embodiment, the first ITR may be a wild-type ITR and the second ITR may be a mutated or modified ITR. In some embodiments, the first ITR may be a mutated or modified ITR and the second ITR may be a wild-type ITR. In another embodiment, both the first ITR and the second ITR are modified but have different sequences, or contain different modifications, or are non-identically modified ITRs. In other words, these ITRs are asymmetric, in the sense that any changes in one ITR are not reflected in the other ITR; or, alternatively, where the ITRs differ from each other.

[00277] In some embodiments, the expression cassette is located between two ITRs contained together with one or more of the following, in the following order: promoter operably linked to the transgene, post-transcriptional regulatory element; and a polyadenylation and termination signal. In one embodiment, said promoter is regulable: inducible or repressible. Said promoter may be any sequence that facilitates transcription of the transgene. In one embodiment, said promoter is a CAG promoter (eg SEQ ID NO: 03) or a variant thereof. The specified post-transcriptional regulatory element is a sequence that modulates the expression of a transgene, in a non-limiting example, any sequence that forms a tertiary structure that enhances the expression of the transgene.

[00278] Typically, scDNA vectors do not have packaging restrictions imposed by limited space within the viral capsid. ZcDNA vectors represent a viable alternative for production in eukaryotes instead of production in prokaryotes from plasmid DNA vectors, as opposed to encapsulated AAV genomes. This allows the insertion of control elements, eg, regulatory switches as described herein, large transgenes, multiple transgenes, and the like.

[00279] In some embodiments, said first ITR may be a mutated or modified ITR and the second ITR may be a wild-type ITR. In another embodiment, both the first and second ITRs are modified, but are of different sequences or contain different modifications, or are non-identically modified ITRs. In other words, these ITRs are asymmetric, in the sense that any changes in one ITR are not reflected in the other ITR; or, alternatively, where the ITRs differ from each other. Examples of ITRs in the ccDNA vector and for use in the preparation of the ccDNA plasmid are disclosed in PCT Application PCT/US18/49996, filed September 7, 2018.

[00280] Although the illustrative ITRs in the description and examples herein are the ITRs of AAV2, one skilled in the art will recognize that, as noted above, the ITRs of any known parvovirus, e.g. from the genome of AAV1, AAV2, AAV3, AAV4, AAV5, AAV 5, AAV7, AAV8, AAV9, AAV10, AAV 11, AAV12, AAVrh8, AAVrh10, AAV-DJ and AAV-DJ8 For example, NCBI: NC 002077;NC 001401; NC001729; NC001829; NC006152; NC 006260; NC 006261), chimeric ITR or ITR from any synthetic AAV. In some embodiments, said AAV can infect warm-blooded animals such as avian (AAAV), bovine (BAAV), canine, equine, and ovine adeno-associated viruses. In some embodiments, said ITR is from Parvovirus B19 (GenBank Accession No. NC 000883), Small Mouse Virus (MVM) (GenBank Accession No. NC 001510); geese parvovirus (GenBank Accession No. NC 001701); parvovirus serpent 1 (GenBank Accession No. NC 006148).

[00281] Parvoviruses and other members of the Parvoviridae family are generally described in: Kenneth I. Berns, "Parvoviridae: The Viruses and Their Replication," Chapter 69 in FIELDS VIROLOGY (3d Ed. 1996).

[00282] One of ordinary skill in the art knows that the ITR sequences are similar in structure and contain a double-stranded Holliday structure, which is typically a T-shaped or Y-shaped hairpin structure (see, for example, Fig. 2A and Fig. 3A), with each ITR formed by two palindromic arms or loops (B-B and C-C) embedded in a larger palindromic arm (A-A'), and a single-stranded sequence D (where the order of said palindromic sequences determines the orientation rotation of the ITR ); appropriate modified ITR sequences from any AAV serotype for use in a ccDNA vector or ccDNA plasmid can be readily determined based on the exemplary AAV2 ITR sequences provided by the present invention. See, for example, a description of the structural analysis and comparison of ITR sequences from different AAV serotypes (AAV1-AAV6) in Grimm et al., J. Virology, 2006; 80(1); 426-439; Yan et al., J. Virology, 2005; 364-379; Duan et al., Virology 1999; 261; 8-14. Examples of specific changes and mutations in the ITR are described in detail in PCT Application PCT/US18/49996, filed September 7, 2018. For clarity, in the context of an ITR, "altered" or "mutated" indicates that nucleotides have been inserted, deleted, and/or substituted relative to the wild-type, reference or parent ITR sequence, and can be changed relative to another flanking ITR in an scDNA vector containing two flanking ITRs. The altered or mutated ITR may be an engineered ITR. As used herein, "engineered" refers to an aspect that has been manipulated by human hands. For example, a polypeptide is considered "engineered" if, in at least one aspect, said polypeptide, such as its sequence, has been manipulated by human hands such that it differs from naturally occurring in said aspect.

[00283] Any ITR parvovirus can be used as an ITR or as an ITR base for modification. Preferably, said parvovirus is a dependovirus, more preferably AAV. The choice of serotype may be based on the tissue tropism of said serotype. AAV2 has a tropism for a wide range of tissues, AAV1 preferentially targets neural tissue and skeletal muscle, and AAV5 preferentially targets neural tissue, retinal pigment epithelium, and photoreceptors. AAV6 predominantly targets skeletal muscle and lungs. AAV8 predominantly targets the liver, skeletal muscle, heart, and pancreatic tissue. AAV9 predominantly targets the liver, skeletal tissues, and lung tissues. In one embodiment, the modified ITR is based on the AAV2 ITR. For example, it is selected from the group consisting of: SEQ ID NO: 2 and SEQ ID NO: 52. In one embodiment, each of these aspects of the vector polynucleotide contains an ITR pair selected from the group consisting of: SEQ ID NO: 1 and SEQ ID NO: 52; and SEQ ID NO: 2 and SEQ ID NO: 51. In one embodiment of each of these aspects, the vector polynucleotide or non-viral capsidless DNA vectors with covalently closed ends contain a pair of different ITRs selected from the group consisting of: SEQ ID NO: 101 and SEQ ID NO: 102; SEQ ID NO: 103, and SEQ ID NO: 104, SEQ ID NO: 105, and SEQ ID NO: 106; SEQ ID NO: 107, and SEQ ID NO: 108; SEQ ID NO: 109, and SEQ ID NO: 110; SEQ ID NO: 111 and SEQ ID NO: 112; SEQ ID NO: 113 and SEQ ID NO: 114; and SEQ ID NO: 115 and SEQ ID NO: 116. In some embodiments, the modified ITR is selected from any of SEQ ID NOs: 2, 52, 63, 64, 101,499 and are disclosed in PCT Application No. PCT/US18/ 49996, filed September 7, 2018. In some embodiments, the scDNA vector does not contain a modified ITR selected from any sequence consisting or consisting essentially of: SEQ ID NO: 500-529 as defined herein. In some embodiments, the scDNA vector does not contain an ITR selected from any sequence selected from SEQ ID NOs: 500-529.

[00284] In some embodiments, the scDNA may form an intramolecular duplex secondary structure. Examples of the secondary structure of the first ITR and the asymmetric second ITR are given in the context of wild-type ITRs (see eg FIGS. 2A, 3A and 3C) and modified ITR structures (see eg FIGS. 2B, 3B and 3D). Secondary structures are derived or predicted based on the ITR sequences of the plasmid used to generate the scDNA vector.

[00285] In some embodiments, the left ITR of the scDNA vector is modified or mutated relative to the structure of the AAV wild type (wt) ITR and the right ITR is the wild type ITR. In one embodiment, the right ITR of the scDNA vector is modified to be mutated relative to the structure of the wild type AAV ITR and the left ITR is the wild type AAV ITR. In such embodiments, a modification of an ITR (eg, left or right ITR) can be obtained by deletion, insertion, or substitution of one or more nucleotides in a wild-type ITR derived from the AAV genome.

[00286] The ITRs used according to the present invention can be resolvable and non-resolvable, and AAV sequences are preferred for use in ccDNA vectors, with serotypes 1, 2, 3, 4, 5, 6, 7, 8 and 9 being preferred. Resolvable AAV ITRs do not require a wild-type ITR sequence (e.g., endogenous or wild-type AAV ITR sequence can be altered by insertion, deletion, truncation, and/or missense mutations), provided that the terminal repeat mediates the required functions, e.g. , virus replication, virus packaging, virus integration and/or provirus rescue, and the like. Typically, though not necessarily, TRs are from a single AAV serotype, for example, both ITR sequences of a given scDNA vector are from AAV2. Said ITRs may be synthetic sequences that function as AAV inverted terminal repeats. Although not required, the ITRs may be from the same parvovirus, eg both ITR sequences are from AAV2.

[00287] In some embodiments, at least one of the ITRs is a defective ITR for Rep binding and/or Rep nicking. In one embodiment, said defect is at least a 30% reduction relative to wild-type ITR, in other embodiments it is at least a 35%..., 50%..., 65%..., 75%..., 85%... reduction. , 90%…, 95%…, 98%…, or no function at all, or any degree of reduction in between. Host cells do not express viral capsid proteins and the vector polynucleotide template does not contain any viral capsid coding sequences. In one embodiment, the vector polynucleotide template and host cells that do not contain the AAV capsid genes and the resulting protein also do not encode or express the capsid genes of other viruses. In addition, in a specific embodiment, the nucleic acid molecule also does not contain the coding sequences for the Rep AAV protein.

[00288] In some embodiments, the ITR structural element can be any structural element that is involved in the functional interaction of the ITR with a large Rep protein (eg, Rep 78 or Rep 68). In some embodiments, said structural element provides selectivity for the interaction of the ITR with the large Rep protein, i.e. determines, at least in part, which Rep protein functionally interacts with the ITR. In other embodiments, said structural element physically interacts with a large Rep protein upon binding said Rep protein to the ITR. Each building block can be, for example, an ITR secondary structure, an ITR nucleotide sequence, a gap between two or more elements; or a combination of any of the above. In one embodiment, said building blocks are selected from the group consisting of arm A and arm A', arm B and arm B', arm C and arm C, arm D, Rep binding site (RBE) and RBE' (i.e. complementary RBE sequence) and terminal clearance site (trs)

[00289] More specifically, the ability of a structural element to functionally interact with a particular large Rep protein can be changed by modifying said structural element. For example, the nucleotide sequence of a structural element can be modified relative to the wild-type ITR sequence. In one embodiment, a specified structural element (e.g., arm A, arm A', arm B, arm B', arm C, arm C, arm D, RBE, RBE', and trs) of the ITR can be removed and replaced with a wild-type structural element from another parvovirus. For example, the replacement structure can be taken from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, serpentine parvovirus (e.g., king python parvovirus), bovine parvovirus, goat parvovirus , avian parvovirus, canine parvovirus, equine parvovirus, shrimp parvovirus, porcine parvovirus, or insect AAV. For example, the ITR may be an ITR of AAV2 and the arm A or A' or RBE may be replaced by a building block from AAV5. According to another example, the ITR may be an AAV5 ITR and the C or C, RBE and trs arms may be replaced by a building block from AAV2. According to another example, the AAV ITR may be an AAV5 ITR, where the B and B' arms are replaced by the AAV2 ITR arms B and B'.

[00290] In some embodiments, the nucleotide sequence of a building block may be modified (e.g., by modifying 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19 or 20 or more nucleotides, or any number of nucleotides in the available range) to obtain a modified building block.

[00291] According to some implementation options, the structure of the specified structural element can be modified. For example, the height of the stem and/or the number of nucleotides in the loop is changed in the structural element. For example, the stem height may be approximately 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides or more, or any number within the available range. In one embodiment, the stem may be from about 5 nucleotides to about 9 nucleotides in height and is operatively interacting with Rep. In another embodiment, the stem may be approximately 7 nucleotides high and operably interacts with Rep. within the available range.

[00292] In another embodiment, the number of GAGY binding sites or GAGY-related binding sites within an RBE or an extended RBE can be increased or decreased. According to one example, the RBE or extended RBE may contain 1, 2, 3, 4, 5, or 6 or more GAGY binding sites, or any number of binding sites in the available range. Each GAGY binding site can independently be the exact GAGY sequence or a GAGY-like sequence, provided that the sequence is sufficient to bind the Rep proteins.

[00293] In some embodiments, the spacing between two elements (eg, but not limited to RBE and hairpin) can be changed (eg, increased or decreased) to change the functional interaction with the large Rep protein. For example, said span may be approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides, or more than, or contain any number of nucleotides in the available range.

[00294] FIG. 2A and 2B depict one possible mechanism of action for the trs site within the structure of the wild-type ITR portion of the scDNA vector. In some embodiments, said scDNA vector contains one or more functional ITR polynucleotide sequences that contain a Rep binding site (RBS; 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 531) for AAV2) and a terminal resolution site (TRS; 5'-AGTT (SEQ ID NO: 46)). In some embodiments, at least one ITR (wt or modified ITR) is functional. In alternative embodiments, when the scDNA vector contains two modified ITRs that are different or asymmetric from each other, at least one modified ITR is functional and at least one modified ITR is non-functional.

[00295] In some embodiments, the modified ITR (eg, left or right ITR) of the scDNA vector described herein contains modifications in the loop arm, truncated arm, or spacer. Examples of ITR sequences with modifications in the loop arm, truncated arm, or spacer are shown in Tables 2-9, 10A, and 10B of PCT application PCT/US18/49996, filed September 7, 2018.

[00296] These scDNA vectors can be produced from expression constructs that additionally contain a specific combination of cis-regulatory elements. These cis-regulatory elements include, but are not limited to, a promoter, a riboswitch, an insulator, an miRNA-regulated element, a post-transcriptional regulatory element, a tissue-specific and cell-specific promoter, and an enhancer. In some embodiments, said ITR may act as a promoter for the transgene. In some embodiments, said scDNA vector contains additional components to regulate the expression of said transgene, such as regulatory switches as described in PCT Application No. PCT/US18/49996, filed Sept. 7, 2018, to regulate the expression of said transgene, or "emergency "switch" that can ensure the death of a cell containing the scDNA vector.

[00297] Expression cassettes may also include a post-transcriptional element to increase expression of the transgene. In some embodiments, a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) is used to increase transgene expression (eg, SEQ ID NO: 8). Other post-transcriptional processing elements may be used, such as a post-transcriptional element from the herpes simplex virus or hepatitis B virus (HBV) thymidine kinase gene. The transgenes may be fused with secretory sequences, such as the VH-02 and VK-A26 sequences, such as SEQ ID NO: 25 and SEQ ID NO: 26. The expression cassettes may include a polyadenylation sequence known in the art, or a variant thereof, for example, a naturally occurring sequence isolated from bovine growth hormone BGHpA (eg, SEQ ID NO: 74) or SV40pA virus (eg, SEQ ID NO: 10), or a synthetic sequence (eg, SEQ ID NO: 27). Some expression cassettes may also include the SV40 late poly(A) signal (USE) 5' enhancer sequence. In some embodiments, USE may be used in combination with SV40pA or a heterologous poly(A) signal.

[00298] Said expression cassette may contain more than 4,000 nucleotides, 5,000 nucleotides, 10,000 nucleotides, or 20,000 nucleotides, or 30,000 nucleotides, or 40,000 nucleotides, or 50,000 nucleotides, or any number from about 4,000 to 10,000 nucleotides, or 10,000-50,000 nucleotides, or more than 50,000 nucleotides. In some embodiments, said expression cassette may contain a transgene or nucleic acid in the range of 500 to 50,000 nucleotides in length. In some embodiments, said expression cassette may contain a transgene or nucleic acid in the range of 500 to 75,000 nucleotides in length. In some embodiments, said expression cassette may contain a transgene or nucleic acid in the range of 500 to 10,000 nucleotides in length. In some embodiments, said expression cassette may contain a transgene or nucleic acid in the range of 1,000 to 10,000 nucleotides in length. In some embodiments, said expression cassette may contain a transgene or nucleic acid in the range of 500 to 50,000 nucleotides in length. The zcDNA vectors are not size limited as are the capsid-encapsulated AAV vectors and thus allow for the delivery of a large expression cassette for efficient expression of the transgenes. In some embodiments, said scDNA vector is devoid of specific prokaryotic methylation. In some embodiments, the length of the expression cassette in the 5'-3' direction is greater than the known maximum length for the AAV virion capsid sequence. In some embodiments, the length is greater than 4.6 kb, or greater than 5 kb, or greater than 6 kb, or greater than 7 kb.

[00299] FIG. 1A-1C are schematic representations of non-limiting examples of scDNA vectors, or the corresponding sequence of scDNA plasmids. ZcDNA vectors are capsidless and can be generated from a plasmid encoding, in this order: the first ITR, the expressed transgene cassette, and the second ITR, wherein at least one of the first and/or second ITR sequences is mutated relative to the corresponding wild-type AAV2 ITR sequence . Said transgenic cassette to be expressed preferably includes, in that order, one or more of the following: enhancer/promoter, ORF reporter (transgene), post-transcriptional regulatory element (eg, WPRE); and a polyadenylation and termination signal (eg, poly(A) BGH).

[00300] Said expression cassette may contain any transgene of interest. Transgenes of interest include, but are not limited to, nucleic acids encoding polypeptides or non-coding nucleic acids (e.g., RNA interference, miRNAs, etc.), preferably therapeutic (e.g., for medical, diagnostic, or veterinary use) or immunogenic ( for example, for vaccines) polypeptides. In some embodiments, the transgene in the expression cassette encodes one or more of polypeptides, peptides, ribozymes, peptide nucleic acids, siRNA, RNAi, antisense oligonucleotides, antisense polynucleotides, antibodies, antigen-binding fragments; or any combination of the above. In some embodiments, said transgene is a therapeutic gene or marker protein. In some embodiments, said transgene is an agonist or antagonist. In some embodiments, said antagonist is a mimetic or an antibody, or an antibody fragment, or an antigen-binding fragment thereof, such as a neutralizing antibody or antibody fragment, and the like. In some embodiments, said transgene encodes an antibody, including a full length antibody or antibody fragment as defined herein. In some embodiments, said antibody is an immunoglobulin antigen-binding domain or variable domain sequence as defined herein.

[00301] In particular, said transgene may encode one or more therapeutic agents, including, but not limited to, for example, protein ((proteins), polypeptide(s), peptide(s), enzyme(s), antibodies, antigen-binding fragments, as well as variants and/or active fragments thereof, for use in the treatment, prevention and/or alleviation of one or more symptoms of a disease, dysfunction, injury and/or disorder.

[00302] Many structural features distinguish ccDNA vectors from plasmid-based expression vectors. ZcDNA vectors may be characterized by one or more of the following: absence of original (i.e., not inserted) bacterial DNA, absence of a prokaryotic origin of replication, autonomy, i.e. they do not require the presence of any sequences other than two ITRs, including a Rep-binding site and a terminal clearance site (RBS and TRS), and an exogenous sequence between these ITRs, the presence of ITR sequences that form hairpins of eukaryotic origin (i.e. they are produced in eukaryotic cells); and the absence of bacterial-type DNA methylation, or any other methylation perceived as abnormal by the mammalian host. In general, it is preferred that the proposed vectors do not contain any prokaryotic DNA, however, it is possible to insert any prokaryotic DNA as an exogenous sequence, according to a non-limiting example, in the promoter or enhancer region. Another important feature that distinguishes scDNA vectors from plasmid expression vectors is that scDNA vectors are single-stranded linear DNA with closed ends, while plasmids are always double-stranded DNA.

[00303] The ZcDNA vectors preferably have a linear and continuous structure, rather than a discontinuous structure, as judged based on the results of a restriction enzyme digestion assay (FIG. 4D). A linear and continuous structure is considered more stable when attacked by cellular endonucleases, and is also less likely to undergo recombination and cause mutagenesis. Accordingly, an scDNA vector having a linear and continuous structure is a preferred embodiment. A continuous, linear, single-stranded ccDNA vector with intramolecular duplexes may contain covalently linked ends, without sequences encoding AAV capsid proteins. These scDNA vectors are structurally different from plasmids (including the scDNA plasmids described herein), which are circular duplex nucleic acid molecules of bacterial origin. Complementary strands of plasmids can be separated after denaturation to produce two nucleic acid molecules, whereas scDNA vectors, on the contrary, although they contain complementary strands, are one DNA molecule and, accordingly, even after denaturation remain in the form of one molecule. In some embodiments, scDNA vectors as described herein can be generated without methylation of prokaryotic-type DNA bases, unlike plasmids. Accordingly, zcDNA vectors and zcDNA plasmids differ both in structure (in particular, linear or circular), and in the methods that are used to obtain and purify these different objects (see below), as well as in DNA methylation, which occurs in a prokaryotic pattern. in scDNA plasmids and by eukaryotic type in scDNA vector.

[00304] The timing of extraction and harvesting of the DNA vectors described herein from cells can be chosen and optimized to ensure high production of said DNA vectors. For example, the extraction time may be selected based on cell viability, cell morphology, cell growth, and the like. Typically, cells can be harvested long enough after infection with baculovirus to obtain DNA vectors, but before most cells die due to viral toxicity. DNA vectors can be isolated, for example, using plasmid purification kits such as Endo-Free™ kits from Qiagen. Other methods developed for isolating plasmids can also be adapted to DNA vectors. Generally, any method for purifying nucleic acids can be used.

[00305] Promoters: s. Suitable promoters, including those described above, may be derived from viruses and may accordingly be referred to as viral promoters, or they may be derived from any organism, including prokaryotic or eukaryotic organisms. Suitable promoters can be used to drive expression using any RNA polymerase (eg pol I, pol II, pol III). Examples of promoters include, but are not limited to, the SV40 early promoter, the promoter from the mouse mammary tumor virus (LTR) long terminal repeat; adenoviral large late promoter (Ad MLP); herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter such as CMV immediate early promoter region (CMVIE), Rous sarcoma virus (RSV) promoter, human small nuclear RNA U6 promoter (U6, e.g., SEQ ID NO: 18) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep. 1; 31(17)), human H1 promoter (H1) (e.g., SEQ ID NO: 19), CAG promoter, human alpha-1 antitrypsin (HAAT) promoter (e.g., SEQ ID NO: 21), etc. In embodiments, these promoters are changed to contain an intron 3'- end so that they include one or more nuclease cleavage sites. In embodiments, the DNA containing the nuclease cleavage site(s) is foreign to the DNA promoter.

[00306] The promoter may contain one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial and/or temporal characteristics of expression. The promoter may also contain distal enhancer or repressor elements, which may be located up to several thousand base pairs away from the transcriptional start site. The promoter may be derived from sources including viruses, bacteria, fungi, plants, insects and animals. A promoter may regulate the expression of a gene component constitutively or selectively in relation to the cell, tissue, or organ where expression occurs, or in consideration of the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents. Representative examples of promoters include bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lactose operator promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter, or SV40 late promoter and CMV IE promoter as well as the promoters listed below. Such promoters and/or enhancers may be used to express any gene of interest, eg, genome editing molecules, donor sequence, therapeutic proteins, and the like). For example, the vector may contain a promoter that is operably linked to a nucleic acid sequence encoding a therapeutic protein. Said promoter operably linked to the therapeutic protein coding sequence may be the simian virus 40 (SV40) promoter, the mouse mammary tumor virus (MMTV) promoter, the human immunodeficiency virus (HIV) promoter, e.g., the long terminal repeat (LTR) promoter of the virus bovine immunodeficiency (BIV) promoter, Moloney virus promoter, avian leukemia virus (ALV) promoter, cytomegalovirus (CMV) promoter such as the CMV immediate early (IE) promoter, the Epstein-Barr virus (EBV) promoter, or the Rous sarcoma virus promoter ( RSV). Said promoter may also be a human gene promoter, for example, human ubiquitin C (hUbC), human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. Said promoter may also be a tissue-specific promoter such as a liver-specific promoter, eg human alpha-1 antitrypsin (HAAT), natural or synthetic. In one embodiment, delivery to the liver can be achieved using endogenous ApoE-specific targeting of the composition containing the ccDNA vector to hepatocytes via a low density lipoprotein (LDL) receptor present on the surface of the hepatocyte.

[00307] In one embodiment, the promoter used is the natural promoter of the gene encoding the therapeutic protein. Promoters and other regulatory sequences of the relevant genes encoding therapeutic proteins are known and have been characterized. The promoter region used may further include one or more additional (eg, natural) regulatory sequences such as enhancers (eg, SEQ ID NO: 22 and SEQ ID NO: 23).

[00308] Non-limiting examples of suitable promoters for use in accordance with the present invention include the CAG promoter, for example, from (SEQ ID NO: 3), the HAAT promoter (SEQ ID NO: 21), the human EF1-α promoter (SEQ ID NO: 6 ) or a fragment of the EF1a promoter (SEQ ID NO: 15), and the rat EF1-α promoter (SEQ ID NO: 24).

[00309] Polyadenylation sequences: A sequence encoding a polyadenylation sequence can be included in the scDNA vector to stabilize the mRNA expressed from said scDNA vector and to promote nuclear export and translation. In one embodiment, said scDNA vector does not contain a polyadenylation sequence. In other embodiments, said vector includes at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 45, at least 50 or more adenine dinucleotides. In some embodiments, said polyadenylation sequence is about 43 nucleotides, about 40-50 nucleotides, about 40-55 nucleotides, about 45-50 nucleotides, about 35-50 nucleotides, or any number of nucleotides in the ranges indicated.

[00310] In some embodiments, said scDNA can be derived from a vector polynucleotide that encodes a heterologous nucleic acid operably located between two different inverted terminal repeat (ITR) sequences (e.g., AAV ITRs) characterized in that at least one of said The ITR contains an end resolution site and a replicative protein (RPS) binding site, such as the Rep binding site (eg, wt ITR AAV SEQ ID NO: 1 or SEQ ID NO: 2 for AAV2), and one of the ITRs contains a deletion, an insertion, and/ or a substitution with respect to another ITR, such as a functional ITR.

[00311] Host cells do not express viral capsid proteins and the vector polynucleotide template does not contain any viral capsid coding sequences. In one embodiment, the vector polynucleotide template does not only contain the AAV capsid genes but also the capsid genes of other viruses). In addition, in a particular embodiment, said nucleic acid molecule also does not contain the coding sequences for the AAV Rep protein. Accordingly, in a preferred embodiment, the nucleic acid molecule of the present invention contains neither functional AAV cap genes nor functional AAV rep genes.

[00312] According to some embodiments of the present invention, said scDNA vector does not contain modified ITRs containing a nucleotide sequence selected from the group consisting of SEQ ID NOs: 548, 549, 551, 552, 553, 553, 554, 555, 556 and 557.

[00313] In some embodiments, said scDNA vector comprises a regulatory switch as described herein (or in PCT Application No. PCT/US18/49996 filed September 7, 2018) and a modified ITR with a nucleotide sequence selected from the group, consisting of: SEQ ID NOs: 548, 549, 551, 552, 553, 553, 554, 555, 556 and 557.

[00314] Without limitation, the authors of the present invention argue that the above right of refusal applies to at least claims 1-33 in this application and paragraphs [00293] and [00294] in the description.

[00315] Some embodiments of various aspects of the present invention may be defined by any of the following:

1. Liposomal scDNA vector, including a liposome encapsulating the scDNA vector, containing:

an expression cassette containing a cis regulatory element, said cis regulatory element being selected from the group consisting of a post-transcriptional regulatory element and a BGH poly(A) signal; A wild-type ITR at the start (5' end) of the expression cassette, said wild-type ITR comprising a polynucleotide of SEQ ID NO: 51; and a modified ITR at the end (3' end) of the expression cassette, wherein said modified ITR contains a polynucleotide of SEQ ID NO: 2, wherein said vector DNA lacks specific prokaryotic methylation and is not enclosed in an AAV capsid protein.

2. The DNA vector according to claim 1, wherein said DNA vector has a linear and continuous structure.

3. The DNA vector according to any one of paragraphs 1-2, wherein said post-transcriptional regulatory element comprises a WHP post-transcriptional regulatory element (WPRE).

4. A DNA vector according to any one of paragraphs 1-3, wherein said expression cassette further comprises a cloning site.

5. The DNA vector according to any one of items 1-4, wherein said expression cassette contains a promoter selected from the group consisting of a CAG promoter, an AAT promoter, an LP1 promoter, and an EF1a promoter.

6. The DNA vector according to paragraph 1, wherein said expression cassette contains the polynucleotides SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.

7. A DNA vector according to any one of paragraphs 1-6, wherein said expression cassette further comprises a cloning site and an exogenous sequence inserted into the cloning site.

8. DNA vector according to item 7, where the specified exogenous sequence contains at least 2000 nucleotides.

9. DNA vector according to item 7, where the specified exogenous sequence encodes a protein.

10. DNA vector according to item 7, where the specified exogenous sequence encodes a reporter protein.

[00316] Some embodiments of various aspects of the present invention may be defined by any of the following:

1. A lipid particle containing an ionizable lipid and a non-viral non-capsid DNA vector with covalently closed ends (scDNA vector), characterized in that said scDNA vector contains at least one heterologous nucleotide sequence functionally located between the sequences of asymmetric inverted terminal repeats ( asymmetric ITRs), wherein at least one of the asymmetric ITRs contains a functional terminal clearance site and a Rep binding site.

2. The lipid nanoparticle according to claim 1, characterized in that said scDNA vector, after cleavage with a restriction enzyme with a single recognition site on the scDNA vector and analysis using both natural and denaturing gel electrophoresis, demonstrates the characteristic bands of linear and continuous DNA , different from the bands of linear and discontinuous control DNA.

3. Lipid nanoparticle according to claim 1 or 2, characterized in that one or more of the asymmetric ITR sequences are taken from a virus selected from parvovirus, dependdovirus and adeno-associated virus (AAV).

4. Lipid nanoparticle according to claim 3, characterized in that said asymmetric ITRs are taken from different viral serotypes.

5. The lipid nanoparticle according to claim 4, wherein said one or more asymmetric ITRs are from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.

6. Lipid nanoparticle according to any one of claims 1-3, characterized in that one or more of the asymmetric ITR sequences are synthetic.

7. A lipid nanoparticle according to any one of claims 1-6, wherein the one or more ITRs are not wild-type ITRs.

8. Lipid nanoparticle according to any one of claims 17, characterized in that one or both asymmetric ITRs are modified by deletion, insertion and/or substitution in at least one of the ITR regions selected from A, A', B, B' , C, C, D and D'.

9. Lipid nanoparticle according to any one of paragraphs 1-8, characterized in that said scDNA vector contains at least two asymmetric ITRs selected from:

a. SEQ ID NO: 1 and SEQ ID NO: 52; and

b. SEQ ID NO: 2 and SEQ ID NO: 51.

10. Lipid nanoparticle according to any one of paragraphs 1-9, characterized in that said scDNA vector is obtained by a method comprising the following steps:

a. incubation of a population of insect cells bearing an scDNA expression construct in the presence of at least one Rep protein, wherein said scDNA expression construct encodes said scDNA vector, under conditions effective to, and for a time sufficient to induce the production of scDNA- vector in said insect cells; and

b. isolating said scDNA vector from said insect cells.

11. The lipid nanoparticle according to claim 10, characterized in that said scDNA expression construct is selected from scDNA plasmid, scDNA bacmid, and scDNA baculovirus.

12. The lipid nanoparticle according to claim 10 or claim 11, characterized in that said insect cell expresses at least one Rep protein.

13. Lipid nanoparticle according to claim 10, characterized in that at least one Rep protein is taken from a virus selected from parvovirus, dependovirus and adeno-associated virus (AAV)

14. The lipid nanoparticle according to claim 13, characterized in that at least one Rep protein is taken from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.

15. A lipid particle according to any one of paragraphs 1-15, characterized in that said DNA vector is derived from a vector polynucleotide, wherein said vector polynucleotide encodes a heterologous nucleic acid operably located between two inverted terminal repeat (ITR) sequences, wherein said two The ITSs are different from each other (are asymmetric) and at least one of the ITRs is a functional ITR containing a functional terminal clearance site and a Rep binding site, and one of the ITRs contains a deletion, insertion and/or substitution relative to the functional ITR; and the presence of the Rep protein induces replication of the vector polynucleotide and production of a DNA vector in the insect cell, said DNA vector may be a method comprising the following steps:

a. incubation of a population of insect cells carrying said vector polynucleotide, which does not contain viral capsid coding sequences, in the presence of a Rep protein, under conditions effective to, and for a time sufficient to induce production of a capsid-free, non-viral DNA vector in insect cells that differ the fact that the capsidless non-viral DNA produced is not contained in said insect cells in the absence of said vector; and

b. collection and isolation of non-capsid non-viral DNA from insect cells.

16. A lipid particle according to any one of claims 10-15, characterized in that the presence of capsid-free non-viral DNA isolated from insect cells can be confirmed.

17. The lipid particle according to claim 16, characterized in that the presence of capsid-free non-viral DNA isolated from insect cells can be confirmed by cleaving the DNA isolated from insect cells with a restriction enzyme with one recognition site on the specified DNA vector and analyzing the cleaved DNA- material on a non-denaturing gel to confirm the presence of the characteristic bands of linear and continuous DNA, as compared to linear and discontinuous DNA.

18. A lipid particle according to any one of paragraphs 1-17, characterized in that said DNA vector is derived from a vector polynucleotide, wherein said vector polynucleotide encodes a heterologous nucleic acid functionally located between the DNA polynucleotide sequences of the first and second inverted terminal repeats (ITR ) AAV2, wherein at least one of the ITRs contains at least one polynucleotide deletion, insertion and/or substitution relative to the corresponding wild-type AAV2 ITR of SEQ ID NO: 1 or SEQ ID NO: 51 to induce replication of said DNA vector in the cell insect in the presence of the Rep protein, wherein said DNA vector may be a method comprising the following steps:

a. incubation of a population of insect cells carrying said vector polynucleotide, which does not contain viral capsid coding sequences, in the presence of Rep protein, under conditions effective to, and for a time sufficient to induce production of capsid-free non-viral DNA in insect cells, said cells insects do not contain sequences encoding the viral capsid; and

b. collection and isolation of non-capsid non-viral DNA from insect cells.

19. The lipid particle according to claim 18, characterized in that the presence of capsid-free non-viral DNA isolated from insect cells can be confirmed.

20. The lipid particle according to claim 19, characterized in that the presence of capsid-free non-viral DNA isolated from insect cells can be confirmed by cleaving the DNA isolated from insect cells with a restriction enzyme with one recognition site on the specified DNA vector and analyzing the cleaved DNA- material on a non-denaturing gel to confirm the presence of the characteristic bands of linear and continuous DNA, as compared to linear and discontinuous DNA.

21. A lipid particle according to any one of paragraphs 1-20, characterized in that said lipid particle further comprises one or more of a non-cationic lipid; a PEG-conjugated lipid; and sterol.

22. A lipid particle according to any one of paragraphs 1-21, characterized in that said ionizable lipid is the lipid described in Table 1.

23. A lipid particle according to any one of paragraphs 1-22, characterized in that said lipid particle further comprises a non-cationic lipid, wherein said non-ionic lipid is selected from the group consisting of distearoyl-vp-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleylphosphatidylcholine (DOPC ), dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal ), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine (such as 16-O-monomethyl PE), dimethylphosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1-trans PE, 1- stearoyl-2-oleoylphosphatidylethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg th phosphatidylcholine (EPC), dioleylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierukoylphosphatidylcholine (DEPC), palmitoyloleoylphosphatidylglycerol (POPG), dielaidoylphosphatidylethanolamine (DEPE), phosphatidylethanolamine, lecithin, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, lysophosphatidylcholine, and dilinoleoylphosphatidylcholine.

24. A lipid particle according to any one of paragraphs 1-23, characterized in that said lipid particle further comprises a conjugated lipid, wherein said conjugated lipid is selected from the group consisting of PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethylene glycol )-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, 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-DMH)), PEG-dialkoxypropylcarbam and N-(sodium salt carbonylmethoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine.

25. Lipid particle according to any one of paragraphs 1 to 24, characterized in that said lipid particle additionally contains cholesterol or a cholesterol derivative.

26. Lipid particle according to any one of paragraphs 1-25, characterized in that said lipid particle contains:

(i) an ionizable lipid;

(ii) a non-cationic lipid;

(iii) a conjugated lipid that inhibits particle aggregation; and

(iv) sterol.

27. Lipid particle according to any one of paragraphs 1-26, characterized in that said lipid particle contains:

(a) ionizable lipid in an amount of from about 20 mol. % to about 90 mol. % of the total lipids present in said particle;

(b) non-cationic lipid in an amount of from about 5 mol. % to about 30 mol. % of the total lipids present in said particle;

(c) a conjugated lipid that inhibits particle aggregation, in an amount of from about 0.5 mol. % to about 20 mol. % of the total lipids present in said particle; and

(d) sterol in an amount of from about 20 mol. % to about 50 mol. % of total lipids present in said particle.

28. A lipid particle according to any one of paragraphs 1-27, characterized in that the ratio of total lipids to vector DNA (w/w) is from about 10:1 to about 30:1.

29. A composition comprising a first lipid nanoparticle and an additional compound, wherein said first lipid nanoparticle comprises a first non-capsid non-viral vector and is a lipid nanoparticle according to any one of paragraphs 1-28.

30. The composition according to claim 29, characterized in that said additional compound is included in the second lipid nanoparticle and in that said first and second lipid nanoparticles are different.

31. The composition according to claim 28 or 29, characterized in that said additional compound is included in the first lipid nanoparticle.

32. A composition according to any one of paragraphs 28-30, characterized in that said additional compound is a therapeutic agent.

33. The composition of claim 28, wherein said additional compound is a second non-viral non-viral vector, said first and second non-viral non-viral vectors are different.

[00317] The examples below are intended to be illustrative and not restrictive.

EXAMPLES

Example 1: Method Examples for Obtaining scDNA Vectors

Construction of scDNA Plasmids

[00318] Obtaining scDNA vectors using a polynucleotide template construct is described. For example, the polynucleotide template construct used to generate the scDNA vectors of the present invention may be a scDNA plasmid, scDNA bacmid, and/or scDNA baculovirus. Without being limited by theory, in a permissive host cell in the presence of, for example, Rep, a polynucleotide template construct with two ITRs and an expression construct, with at least one of the ITRs modified, replicates to produce scDNA vectors. Obtaining the scDNA vector involves two steps: first, excision (“rescue”) of the template from the matrix backbone (e.g., zcDNA plasmids, scDNA bacmids, scDNA baculovirus genome, etc.) by means of Rep proteins, and second, Rep-mediated replication of the excised scDNA vector.

[00319] An example of a method for obtaining scDNA vectors is provided by the preparation of an scDNA plasmid as described herein. 1A and 1B, the polynucleotide template construct of each of the zcDNA plasmids includes both a left ITR and a right mutated ITR, between which are: (i) an enhancer/promoter; (ii) a cloning site for the transgene; (iii) a post-transcriptional response element (eg, woodchuck hepatitis virus post-transcriptional regulatory element (WPRE)); and (iv) a polyadenylation signal (e.g., from the bovine growth hormone (BGHpA) gene. Unique restriction endonuclease recognition sites (R1-R6) (shown in FIGS. 1A and 1B) were also introduced between all components to facilitate the introduction of new genetic components into specific sites in the construct The enzyme sites R3 (PmeI) GTTTAAAC (SEQ ID NO: 7) and R4 (PacI) TTAATTAA (SEQ ID NO: 542) are inserted into the cloning site to introduce the open reading frame of the transgene These sequences were cloned into plasmid pFastBac HT B obtained from ThermoFisher Scientific.

[00320] Briefly, a series of ccDNA vectors were generated from the ccDNA plasmid constructs shown in Table 3 using the method shown in FIG. 4A-4C. Table 5 lists the corresponding polynucleotide sequence numbers for each component, including sequences with protein replication site (RPS) activity (eg, Rep binding site) at either end of the promoter operably linked to the transgene. The numbers in Table 3 refer to SEQ ID NOs herein corresponding to the sequences of each component.

[00321] In some embodiments, a construct for producing ccDNA vectors contains a promoter that is a regulatory switch, such as an inducible promoter. Other constructs containing the MND or HLCR promoter functionally linked to the luciferase transgene were also used to obtain ccDNA vectors.

Obtaining scDNA-bacmid:

[00322] As shown in FIG. 4A, DH10Bac competent cells (MAX EFFICIENCY® DH10Bac™ Competent Cells, Thermo Fisher) were transformed with either test or control plasmids following the protocol according to the manufacturer's instructions. Recombination was induced between the plasmid and the baculovirus shuttle vector in DH10Bac cells to produce recombinant scDNA bacmids. Recombinant bacmids were selected by a positive selection screen based on a blue-white screen in E. coli (marker <E>80dlacZΔM15 provides α-complementation of the β-galactosidase gene from the bacmid vector) on a bacterial agar plate containing X-gal and IPTG with antibiotics for selection of transformants, and maintenance of bacmids and transposase plasmids. White colonies resulting from a transposition that destroys the β-galactoside indicator gene were collected and cultured in 10 ml of medium.

[00323] Recombinant scDNA bacmids were isolated from E. coli and transfected into Sf9 or Sf21 insect cells using FugeneHD to generate infectious baculovirus. Adhesive insect cells Sf9 or Sf21 were cultured in 50 ml of medium in T25 flasks at 25°C. After 4 days, the culture medium (containing the P0 virus) was separated from the cells, filtered through a filter with a pore size of 0.45 μm, separating infectious baculovirus particles from cells or cell debris.

[00324] Optionally, first generation baculovirus (P0) was amplified

by infecting intact insect cells with Sf9 or Sf21 in medium volumes from 50 to 500 ml. The cells were maintained in suspension cultures in an incubator with an orbital shaker at 130 rpm and 25°C, monitoring the cell diameter and viability until the cells reached a diameter of 18–19 nm (from the diameter of intact cells 14–15 nm) and a density of ~4. 0E+6 cells/ml. 3-8 days after infection, P1 baculovirus particles in the medium were collected after centrifugation to remove cells and debris, followed by filtration through a filter with a pore size of 0.45 μm.

[00325] The scDNA baculovirus containing test constructs was harvested and the infectivity, or titer, of the baculovirus was determined. In particular, 4×20 ml of cultures of Sf9 cells with a density of 2.5E+6 cells/ml were treated with baculovirus P1 in the following dilutions: 1/1000, 1/10,000, 1/50,000, 1/100,000, and incubated at 25- 27°C. Infectivity was determined daily for 4-5 days in terms of cell diameter increase and cell cycle arrest, as well as changes in cell viability.

[00326] As shown in FIG. 4A, the "Rep plasmid" according to FIG. 6A was produced in the pFASTBAC™-Dual expression vector (ThermoFisher) containing both Rep78 (SEQ ID NO: 13) or Rep68 (SEQ ID NO: 12) and Rep52 (SEQ ID NO: 14).

[00327] Competent DH10Bac cells (MAX EFFICIENCY® DH10Bac™ Competent Cells (Thermo Fisher)) were transformed with the indicated Rep plasmid following the manufacturer's protocol. recombinant bacmids were selected using positive selection, which included a blue-white screen for E. coli (marker <E>80dlacZΔM15 provides α-complementation of the β-galactosidase gene from the bacmid vector) on a bacterial agar plate containing X-gal and IPTG.Isolated white colonies were recovered and inoculated into 10 ml of selective medium (kanamycin, gentamicin, tetracycline in LB broth).Recombinant bacmids (Rep-bacmids) were isolated from E. coli and these Rep-bacmids were transfected into Sf9 or Sf21 insect cells to obtain infectious baculovirus.

[00328] Sf9 or Sf21 insect cells were cultured in 50 ml of medium for 4 days, and infectious recombinant baculovirus ("Rep-baculovirus") was isolated from the culture. Optionally, the first generation of Rep-baculovirus (P0) was amplified by infecting intact insect cells with Sf9 or Sf21 and cultured in 50-500 ml of medium. 3-8 days after infection, P1 baculovirus particles in the medium were collected by separating the cells by centrifugation or filtration, or using another fractionation method. Collected Rep-baculovirus and determined the infectious activity of the baculovirus. Specifically, 4×20 ml of Sf9 cell cultures at a density of 2.5×10 ⁶ cells/ml were treated with baculovirus P1 in the following dilutions: 1/1000, 1/10000, 1/50000, 1/100000, and incubated. Infectivity was determined daily for 4-5 days in terms of cell diameter increase and cell cycle arrest, as well as changes in cell viability.

Obtaining and Characterizing the scDNA Vector

[00329] As shown in FIG. 4B, then Sf9 insect cell culture medium containing either (1) a sample containing scDNA bacmid or scDNA baculovirus, or (2) Rep baculovirus, as described above, was added to fresh Sf9 cell culture (2.5E+6 cells /ml, 20 ml) at a ratio of 1:1000 and 1:10000, respectively. The cells were then cultured at 130 rpm at 25°C. 4-5 days after coinfection, the diameter and viability of the cells are detected. When the cell diameter reached 18–20 nm with a viability of ~70–80%, cell cultures were centrifuged, the medium was removed, and cell pellets were collected. The cell pellets were first resuspended in an appropriate volume of aqueous medium, either water or buffer. The ccDNA vector was isolated and purified from cells using the purification protocol from Qiagen MIDI PLUS™ (Qiagen, treatment with 0.2 mg cell pellet weight per column).

[00330] Yields for scDNA vectors produced and purified from Sf9 insect cells were initially determined based on UV absorbance at 260 nm. Yields for various scDNA vectors based on UV absorption are shown in Table 4 below.

[00331] Evaluation of ccDNA vectors can be performed by identification using agarose gel electrophoresis under native or denaturing conditions, as illustrated in FIG. 4D, where the scDNA vectors produce several bands on native gels, eg see FIG. 4D. Each band may correspond to vectors in a different conformation, such as monomeric, dimeric, and the like. The presence of a single band under denaturing conditions and double bands (corresponding to monomeric and dimeric forms) under non-denaturing conditions is indicative of the presence of the scDNA vector.

[00332] The structures of the isolated scDNA vectors were further analyzed by digesting DNA derived from coinfected Sf9 cells (as described herein) with restriction endonucleases selected based on a) the presence of only one cut site in the scDNA vectors, and b) obtaining the final fragments , large enough to be clearly visible when fractionated on a 0.8% denaturing agarose gel (>800 bp). As shown in FIG. 4E, the linear scatter DNA vectors and the scDNA linear and contiguous vector can be distinguished by the size of their reaction products—for example, the scatter DNA vector is expected to produce 1 kb fragments. and 2 kb, while the non-capsid-embedded contiguous vector is expected to produce fragments of 2 kb. and 4 kb.

[00333] Accordingly, in order to qualitatively demonstrate that the isolated zcDNA vectors have covalently closed ends, as required by definition, these samples were digested with a restriction endonuclease identified in the context of a specific vector DNA sequence as having a single restriction site, preferably producing two products splits of unequal size (for example, 1000 bp and 2000 bp). After cleavage and electrophoresis on a denaturing gel (separating two complementary strands of DNA), linear covalently open DNA will be separated into 1000 bp fragments. and 2000 bp, while covalently closed DNA (i.e., the ZcDNA vector) will be divided into fragments twice as large (2000 bp and 4000 bp), since the two DNA strands are connected, and in under these circumstances, are unfolded and are twice as long (although they are single-stranded). In addition, when monomeric, dimeric and n-meric forms of DNA vectors are cleaved, they will all be divided into fragments of the same size due to the binding of the ends of multimeric DNA vectors (see Fig. 4D)

[00334] FIG. Figure 5 shows an example image of a denaturing gel with the following scDNA vectors: construct-1, construct-2, construct-3, construct-4, construct-5, construct-6, construct-7, and construct-8 (all described in Table 12 above), with cleavage (+) or without cleavage (-) by endonuclease. Each scDNA vector from constructs 18 produced two bands (*) after reaction with endonuclease. The sizes of their fragments in two bands, determined by the size marker, are shown at the bottom of the image. Band sizes confirm that each of the scDNA vectors produced from plasmids containing construct 18 has a contiguous structure.

[00335] As used herein, "assay for identifying DNA vectors by agarose gel electrophoresis under native and denaturing gel conditions" refers to an assay for assessing the presence of closed ends of ccDNA by performing restriction endonuclease digestion followed by electrophoretic evaluation of cleavage products. One example of such an assay is provided below, although one of ordinary skill in the art will appreciate that many variations of this example known in the art are possible. The restriction endonuclease is chosen to be an enzyme that cuts the scDNA vector of interest at one site, resulting in products approximately 1/3× and 2/3× the length of the DNA vector. This ensures band separation on both native and denaturing gels. Before denaturing, it is important to remove the buffer from the sample. Qiagen PCR purification kit or desalting "spin columns" such as columns from GE HEALTHCARE ILUSTRA™ MICROSPIN™ G-25 are some of the options known in the art for endonuclease digestion. Said assay includes, for example, i) digestion of the DNA with an appropriate restriction endonuclease(s), 2) use of, for example, a Qiagen PCR purification kit, eluting with distilled water, iii) addition of 10× denaturing solution (10×=0.5 M NaOH, 10 mM EDTA), addition of 10X unbuffered dye, and analysis along with DNA ladders prepared by adding 10X denaturing solution in 4×, on a 0.8-1.0% gel previously incubated with 1 mM EDTA and 200 mM NaOH to ensure homogeneity of NaOH concentration in the gel and gel chamber, and running the gel in the presence of 1x denaturing solution (50 mM NaOH, 1 mM EDTA). A person skilled in the art will understand what voltage to use for electrophoresis, depending on the size and the required time to obtain results. After electrophoresis, the gels are dried and neutralized in 1× TBE or TAE, transferred to distilled water or 1× TBE/TAE with 1× SYBR Gold. The bands can then be visualized, for example, using a stain from Thermo Fisher, SYBR® Gold Nucleic Acid Gel Stain (10000X concentrate in DMSO) and epifluorescent light (blue) or UV (312 nm).

[00336] Evaluation of the purity of the resulting scDNA vector can be carried out using any method known in the art. According to one non-limiting example of the method, the contribution of the ccDNA plasmid to the total UV uptake of the sample can be calculated by comparing the fluorescence intensity of the ccDNA vector and the standard. For example, if, based on UV absorbance, 4 μg of the ccDNA vector was loaded onto the gel and the fluorescence intensity of the ccDNA vector is equivalent to a 2 kb band. with a known mass of 1 µg, then there is 1 µg of the scDNA vector, and the scDNA vector makes up 25% of the total UV absorbing material. Then plot the dependence of the intensity of the band on the gel from the calculated injected amount to which the band corresponds. be 25% of the total amount administered, which in this case will be 0.25 μg for 1.0 μg administered. Using a dilution of the scDNA vector-carrying plasmid to build a standard curve, a regression line equation is obtained, which is then used to calculate the amount of the specified scDNA vector in the corresponding band, which can then be used to determine the percentage of the total amount of scDNA vector injected, or percent purity.

Example 2 ccDNA Vectors Express the Luciferase Transgene in Vitro

[00337] Constructs were generated by introducing an open reading frame encoding a luciferase reporter gene into the cloning site of the scDNA plasmid constructs: construct-1, construct-3, construct-5, and construct-7. The 3cDNA plasmids (see Table 3 above) comprising the luciferase coding sequence are referred to as the 1-Luc plasmid construct, the 3-Luc plasmid construct, the 5-Luc plasmid construct, and the 7-Luc plasmid construct, respectively.

[00338] HEK293 cells were cultured and transfected with 100 ng, 200 ng or 400 ng of plasmid constructs 1, 3, 5 and 7 using FUGENE® (Promega Corp.) as the transfection agent. The expression of luciferase with each of the plasmids was determined based on the activity of luciferase in each cell culture; the results are shown in FIG. 7A. Luciferase activity was not detected in untreated control cells ("untreated") or cells treated with Fugene alone ("Fugene"), confirming that luciferase activity is the result of gene expression from plasmids. As shown in FIG. 7A and 7BB, sustained expression of luciferase from constructs 1 and 7 was detected. When luciferase was expressed from construct-7, a dose-dependent increase in luciferase activity was detected.

[00339] Growth and viability of cells transfected with each of the plasmids were also determined and are shown in FIG. 8A and 8B. Growth and viability of transfected cells did not differ significantly between groups of cells treated with different constructs.

[00340] Accordingly, luciferase activity measured in each group and normalized for cell growth and viability did not differ from unnormalized luciferase activity. The zcDNA plasmid with the 1-Luc construct showed the most consistent expression of luciferase, with or without normalization.

[00341] Accordingly, the data presented in FIG. 7A, 7B, 8A and 8B demonstrate that construct 1 containing in the 5' to 3' direction WT-ITR (SEQ ID NO: 51), CAG promoter (SEQ ID NO: 3), R3/R4 cloning site (SEQ ID NO: 7), WPRE (SEQ ID NO: 8), BGHpA (SEQ ID NO: 9), and modified ITR (SEQ ID NO: 2) efficiently produce a ccDNA vector that can express a transgene protein included in composition of said scDNA vector.

Example 3: Lipid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA)

[00342] Lipid particles containing scDNA can be prepared or formulated in combination with the lipid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino )-butanoate (DLin-MC3-DMA), also referred to as "MC3" herein; having the structure:

[00343] The DLin-MC3-DMA lipid is described in: Jayaraman et al., Angew. Chem. hit. Ed Engl. (2012), 51(34): 8529-8533, the contents of which are hereby incorporated by reference in their entirety. It is synthesized as follows:

[00344] Synthesis of octadec-9,12-dienyl ester of methanesulfonic acid: To a solution of alcohol 1 (26.6 g, 100 mmol) in dichloromethane (100 ml) is added triethylamine (13.13 g, 130 mmol) and the solution is cooled in an ice bath. To this cold solution was added dropwise a solution of mesyl chloride (12.6 g, 110 mmol) in dichloromethane (60 ml); after the addition was complete, the reaction mixture was allowed to warm to ambient temperature and stirred overnight. TLC of the reaction mixture shows completion of the reaction. The reaction mixture was diluted with dichloromethane (200 ml), washed with water (200 ml), saturated NaHCO ₃ (200 ml), brine (100 ml) and dried (NaSO ₄ ). The organic layer was concentrated to give the crude product, which was purified by column chromatography (silica gel) using 0-10% Et ₂ O in hexanes. The pure product fractions are combined and concentrated to give the pure product of methanesulfonic acid octadeca-9,12-dienyl ester as a colorless oil. ¹ H NMR (CDCl ₃ , 400 MHz) δ 5.42-5.21 (m, 4H), 4.20 (t, 2H), 3.06 (s, 3H), 2.79 (t, 2H) , 2.19-2.00 (m, 4H), 1.90-1.70 (m, 2H), 1.06-1.18 (m, 18H), 0.88 (t, 3H). ¹³ C NMR (CDCl ₃ ) δ 130.76, 130.54, 128.6, 128.4, 70.67, 37.9, 32.05, 30.12, 29.87, 29.85, 29, 68, 29.65, 29.53, 27.72, 27.71, 26.15, 25.94, 23.09, 14.60. MS. Molecular weight calculated for C ₁₉ H ₃₆ O ₃ S: 344.53.

[00345] Synthesis of 18-bromo-octadeca-6,9-diene: Mesylate (octadeca-9,12-dienyl ester of methanesulfonic acid, 13.44 g, 39 mmol) is dissolved in anhydrous ether (500 ml) and added to it complex MgBr.Et ₂ O (30.7 g, 118 mmol) in an argon atmosphere; the mixture is condensed in an argon atmosphere for 26 hours, after which TLC shows the completion of the reaction. The reaction mixture was diluted with ether (200 ml), ice water (200 ml) was added to the mixture and the layers were separated. The organic layer was washed with 1% aqueous K ₂ CO ₃ (100 ml), brine (100 ml) and dried (anhydrous Na ₂ SO ₄ ). Concentration of the organic layer gave a crude product which was further purified by column chromatography (silica gel) using 0-1% Et ₂ O in hexanes to isolate the product, 18-bromo-octadeca-6,9-diene, as a colorless oil. ¹ H NMR (CDCl ₃ , 400 MHz) δ 5.41-5.29 (m, 4H), 4.20 (d, 2H), 3.40 (t, J=7 Hz, 2H), 2.77 (t, J=6.6 Hz, 2H), 2.09-2.02 (m, 4H), 1.88-1.00 (m, 2H), 1.46-1.27 (m, 18H ), 0.88 (t, J=3.9 Hz, 3H). ¹³ C NMR (CDCl ₃ ) δ 130.41, 130.25, 128.26, 128.12, 34.17, 33.05, 31.75, 29.82, 29.57, 29.54, 29, 39, 28.95, 28.38, 27.42, 27.40, 25.84, 22.79, 14.28.

[00346] Synthesis of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (DLin-MeOH): Add freshly activated Mg(2 .4 g, 100 mmol) and the flask is equipped with a magnetic stirrer, addition funnel and reflux condenser. This system is degassed, purged with argon, and 10 ml of anhydrous ether is added to the flask using a syringe. 18-bromo-octadeca-6,9-diene (26.5 g, 80.47 mmol) was dissolved in anhydrous ether (50 mL) and added to an addition funnel. Approximately 5 ml of this ethereal solution is added to the Mg shavings with vigorous stirring. An exothermic reaction is noted (5 mg of iodine is added to confirm/accelerate the formation of the Grignard reagent and an immediate discoloration is observed, confirming the formation of the Grignard reagent) and the ether begins to condense. The remainder of the bromide solution is added dropwise while continuing to carefully condense the reaction mixture while cooling the flask in water. After completion of the addition, the reaction mixture was kept at 35°C for 1 hour, after which it was cooled in an ice bath. Ethyl formate (2.68 g, 36.2 mmol) was dissolved in anhydrous ether (40 ml), transferred to an addition funnel and added dropwise to the reaction mixture, with stirring. An exothermic reaction is observed and the reaction mixture begins to condense. After the start of the reaction, the remainder of the ethereal formate solution is quickly added in a stream, and the reaction mixture is stirred for an additional 1 hour at ambient temperature. The reaction was quenched by adding 10 ml of acetone dropwise, followed by the addition of ice water (60 ml). The reaction mixture is treated with an aqueous solution of H ₂ SO ₄ (10% by volume, 300 ml) until the solution is homogeneous and the layers are separated. The aqueous phase is extracted with ether (2×100 ml). The combined ether layers are dried (Na ₂ SO ₄ ) and concentrated to give the crude product, which is treated with 1 g of sodium in methanol (200 ml) at room temperature overnight. After completion of the reaction, most of the solvent is evaporated. The final mixture is poured into 150 ml of 5% hydrochloric acid solution. The aqueous phase is extracted with ether (2×150 ml). The combined ether extract was washed with water (2×100 ml), brine (100 ml) and dried over anhydrous sodium sulfate. Evaporation of the solvent gave a crude product which was purified by column chromatography (silica gel, 0-10% ether in hexanes) and pure product fractions were evaporated to give the product DLin-MeOH as a colorless oil. ¹ H NMR (400 MHz, CDCl ₃ ) δ 5.47-5.24 (m, 8H), 3.56 (dd, J=6.8, 4.2, 1H), 2.85-2.66 (m, 4H), 2.12-1.91 (m, 9H), 1.50-1.17 (m, 46H), 0.98-0.76 (m, 6H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 130.41, 130.37, 128.18, 128.15, 77.54, 77.22, 76.91, 72.25, 37.73, 31.75 , 29.94, 29.89, 29.83, 29.73, 29.58, 29.53, 27.46, 27.43, 25.89, 25.86, 22.80, 14.30.

[00347] Synthesis of 6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (MC3): DLin-MeOH (144 g, 272 mmol) is dissolved in 1 L of dichloromethane and dimethylaminobutyric acid hydrochloride salt 7 (55 g, 328 mmol) was added thereto, followed by diisopropylethylamine (70 ml) and DMAP (4 g). After stirring for 5 minutes at ambient temperature, EDCI (80 g, 417 mmol) is added and the reaction mixture is stirred at room temperature overnight, after which TLC analysis (silica gel, 5% MeOH in CH ₂ Cl ₂ ) shows complete disappearance original alcohol. The reaction mixture was diluted with CH ₂ Cl ₂ (500 ml) and washed with saturated NaHCO ₃ (400 ml), water (400 ml) and brine (500 ml). The combined organic layers are dried over anhydrous Na ₂ SO ₄ and the solvents are removed in vacuo. The crude product thus obtained is purified by flash column chromatography [2.5 kg silica gel using the eluents listed below: i) the column is packed with 6 L of 0.1% Net ₃ in DCM; after loading ii) 4 L 0.1% Net ₃ in DCM; iii) 16 L 2% MeOH - 98% 0.1% Net ₃ in DCM; iv) 4 L 2.5% MeOH - 97.5% 0.1% Net ₃ in DCM; v) 12 L 3% MeOH - 97% 0.1% Net ₃ in DCM] to isolate the pure product MC3 as a colorless oil. ¹ H NMR (400 MHz, CDCl ₃ ): δ 5.46-5.23 (m, 8H), 4.93-4.77 (m, 1H), 2.83-2.66 (m, 4H) , 2.37-2.22 (m, 4H), 2.20 (s, 6H), 2.10-1.96 (m, 9H), 1.85-1.69 (m, 2H), 1 .49 (d, J=5.4, 4H), 1.39-1.15 (m, 39H), 0.95-0.75 (m, 6H). ¹³ C NMR (101 MHz, CDCl ₃ ): δ 173.56, 130.38, 130.33, 128.17, 128.14, 77.54, 77.22, 76.90, 74.44, 59, 17, 45.64, 34.36, 32.69, 31.73, 29.87, 29.76, 29.74, 29.70, 29.56, 29.50, 27.44, 27.41, 25.84, 25.55, 23.38, 22.78, 14.27. EI-MS (+ve): MW calc. for C ₄₃ H ₇₉ NO ₂ (M+H) ⁺ ; 642.6.

Example 4 Lipid ATX-002

[00348] Lipid particles containing ccDNA can be prepared or formulated in combination with an ATX-002 lipid having the structure:

[00349] The ATX-002 lipid is described in WO 2015/074085, the contents of which are hereby incorporated by reference in their entirety. It is synthesized as follows:

[00350] Synthesis of methyl 8-bromooctanoate: Under N ₂ atmosphere, 8-bromooctanoic acid (60 g, 1 eq.) is dissolved in 400 ml of dry methanol. 10 drops of concentrated H ₂ SO ₄ are added dropwise and the reaction mixture is stirred at reflux for three hours.

[00351] The reaction is monitored by thin layer chromatography (TLC) to completion. The solvent is completely removed under vacuum. The reaction mixture is diluted with ethyl acetate and washed with water. The aqueous layer was re-extracted with ethyl acetate. The entire organic layer was washed with saturated NaHCO ₃ solution. The organic layer is washed again with water and finally washed with brine. The product is dried over anhydrous Na ₂ SO ₄ and concentrated.

[00352] Synthesis of dimethyl 8,8'-(benzanediyl)dioctanoate: take dry K ₂ CO ₃ (104.7 g, 6 eq.) and add to dry dimethylformamide under N ₂ atmosphere. Benzylamine (13.54 g, 1 eq.) in dimethylformamide is added slowly. Then add methyl-8-bromooctanoate (60 g, 2 eq.), dissolved in dimethylformamide, at room temperature. The reaction mixture is heated to 80°C and support the reaction for 36 hours with stirring.

[00353] The reaction is monitored by thin layer chromatography to completion. The reaction product is cooled to room temperature and water is added. The compound is extracted with ethyl acetate. The aqueous layer was re-extracted with ethyl acetate. The entire organic layer was washed with water and finally with brine. The product is dried over anhydrous Na ₂ SO ₄ and concentrated.

[00354] The reaction product is purified by silica gel column chromatography in 3% methanol in chloroform. Using a TLC system with 10% methanol in chloroform, the product migrates with a retention factor of Rf: 0.8 when visualized by carbonization in ninhydrin. This compound is a light brown liquid. The structure is confirmed by ¹ H-NMR.

[00355] Synthesis of dimethyl 8,8'-azandiyldioctanoate: Dimethyl 8,8'-(benzanediyl)dioctanoate (3.5 g, 1 eq.) was transferred to a glass hydrogenation vessel and 90 ml of ethanol was added followed by 10% Pd/C (700 mg). The reaction mixture was shaken in a Parr mixer at 50 psi atmospheric pressure. inch H ₂ for two hours at room temperature. The reaction product is filtered through celite and washed with hot ethyl acetate. The filtrate is concentrated under vacuum.

[00356] Synthesis of dimethyl 8,8'-((tert-butoxycarbonyl)azandiyl)dioctanoate:

Dimethyl 8,8'-azandiyldioctanoate (32 g, 1 eq.) was taken up in dry DCM (700 ml) and dry Et ₃ N (9 g; 4 eq.) into the reaction mass and cooled to 0°C. Boc anhydride (31.3 g, 1.5 eq.) diluted in DCM was added dropwise to the above reaction. After completion of the addition, the reaction mixture was stirred at room temperature for three hours.

[00357] Quench the reaction with water and separate the layer in DCM. The aqueous phase is re-extracted with DCM and the combined DCM layers are washed with brine and dried over Na ₂ SO ₄ . After concentration, the crude compound is collected. The crude reaction product was purified by column chromatography using 012% ethyl acetate in hexane. The only product migrates on TLC in 20% ethyl acetate in hexane with an Rf of 0.5 upon carbonization with ninhydrin.

[00358] Synthesis of 8,8'-((tert-butoxycarbonyl)azandiyl)dioctanoic acid:

Dimethyl 8,8'-((tert-butoxycarbonyl)azandiyl)dioctanoate (21 g, 1 eq) was taken up in dry THF (200 ml). A 6N aqueous sodium hydroxide solution (175 ml) was added at room temperature. The reaction is maintained with stirring overnight at room temperature.

[00359] The reaction mass is evaporated under vacuum at 25°C to remove THF. The reaction product is acidified with 5N HCl. Ethyl acetate is added to the aqueous layer. The separated organic layer was washed with water and the aqueous layer was re-extracted with ethyl acetate. The combined organic layers are washed with brine and dried over anhydrous Na ₂ SO ₄ . The concentration of this solution gives the crude product.

[00360] Synthesis of di((Z)-non-2-en-1-yl)8,8'((tert-butoxycarbonyl)azandiyl): 8,8'-((tert-butoxycarbonyl)azandiyl)dioctanoic acid (18 g, 1 eq.) was dissolved in dry DCM (150 ml). To this solution is added HATU (26.15 g, 2.1 eq.). D-isopropylethylamine (14.81 g, 3.5 eq.) was slowly added to the reaction mixture at room temperature. The internal temperature rises to 40°C and a pale yellow solution forms. DMAP (400 mg, 0.1 eq.) was added to the reaction mixture, followed by a solution of cis-2-nonen-1-ol (9.31 g, 2 eq.) in dry DCM. The reaction mixture changes color to brown. The reaction mixture is stirred for five hours at room temperature.

[00361] The reaction is checked by thin layer chromatography to completion. Water is added to the reaction product, and the reaction product is extracted with DCM. The DCM layer is washed with water and then with brine. The organic layer was dried over anhydrous Na ₂ SO ₄ and concentrated to give the crude compound.

[00362] Synthesis of ATC-002: Di((Z)-non-2-en-1-yl)8,8'((tert-butoxycarbonyl)azandiyl)dioctanoate (13.85 mmol, 9 grams) dissolved in dry DCM (150 ml). TFA is added at 0°C to initiate the reaction. The reaction temperature is allowed to rise slowly to room temperature over 30 minutes with stirring. Thin layer chromatography shows that the reaction is complete. The reaction product is concentrated under vacuum at 40°C, the crude residue is diluted with DCM and washed with 10% NaHCO ₃ solution. The aqueous layer is re-extracted with DCM, the combined organic layers are washed with brine, dried over Na ₂ SO ₄ and concentrated. The collected crude product was dissolved in dry DCM (85 ml) under nitrogen gas. Add triphosgene, the reaction mixture is cooled to 0°and add Et ₃ N dropwise. The reaction mixture is stirred overnight at room temperature. Thin layer chromatography shows that the reaction is complete. The DCM solvent is removed from the reaction mass by distillation under N ₂ . The reaction product was cooled to 0° C., diluted with DCM (50 ml) and 2-(dimethylamino)ethanediol-HCl (0.063 mol, 8.3 g) was added followed by Et ₃ N (dry). The reaction mixture was then stirred overnight at room temperature. Thin layer chromatography shows that the reaction is complete. The reaction product was diluted with 0.3 M HCl solution (75 ml) and the organic layer was separated. The aqueous layer was re-extracted with DCM and the combined organic layers were washed with 10% aqueous K ₂ CO ₃ (75 ml) and dried over anhydrous Na ₂ SO ₄ . The concentration of the solvent gives the crude product. The crude compound was purified on a silica gel column (100-200 mesh) using 3% MeOH/DCM.

Example 5: (13Z,16Z)-N,N-dimethyl-3-nonyldocose-13,16-diene-1-amine (compound 32)

Lipid particles containing scDNA can be prepared or formulated in combination with (13Z,16Z)-N,N-dimethyl-3-nonyldocose-13,16-dien-1-amine (compound 32) having the structure:

[00364] Compound 32 is described in WO 2012/040184, the contents of which are incorporated herein in their entirety by reference. It is synthesized as follows:

[00365] Synthesis of α,β-unsaturated amide (vii):

[00366] Peterson's silylamide reagent (3.1 g, 16.7 mmol) was dissolved in THF (35 ml) and cooled to -63°C. To this solution is added nBuLi (16.7 mmol, 6.7 ml of a 2.5M solution). The reaction is heated to ambient temperature for 30 minutes. Ketone (iii) (5.0 g, 11.9 mmol) is dissolved in THF (25 ml) in a second flask. The ketone solution is transferred to Peterson's reagent for 30 minutes while maintaining the temperature between -60°C and -40°C. The reaction is heated to -40°C for 1 hour, then heated to 0°C for 30 minutes. The reaction is quenched with sodium bicarbonate, diluted with additional water and partitioned between water/hexanes. The organic layers are washed with brine, dried over sodium sulfate, filtered and evaporated in vacuo. Purification using flash chromatography (0-40% MTBE/hexanes) gives α,β-unsaturated amide (vii). ¹ H NMR (400 MHz, CDCl ₃ ) δ 5.75 (s, 1H), 5.36 (m, 4H), 3.01 (s, 3H), 2.99 (s, 3H), 2.78 (t, 2H), 2.28 (t, 2H), 2.05 (m, 6H), 1.35 (m, 34H), 0.89 (m, 6H).

[00367] α,β-unsaturated amide (vii) (1 g, 2.1 mmol) and LS-Selectride (4.1 mmol, 4.1 ml of 1M solution) are combined in a closed tube and heated to 60°C for 24 hours. The reaction is cooled to ambient temperature and divided between solutions of ammonium chloride and heptane. The organic layers are dried over sodium sulfate, filtered and evaporated in vacuo to give the amide (viii). This intermediate product is directly transferred to the next reaction without purification.

[00368] Alternatively, the reduction of the α,β-unsaturated amide conjugate (vii) involves the use of reduction with copper hydride.

[00369] [In a 5 L round bottom flask, the copper catalyst (9.77 g, 17.13 mmol) is dissolved in toluene (1713 mL) under a nitrogen atmosphere. To this solution was added PMHS from Aldrich (304 mL, 1371 mmol) in one portion. The reaction is maintained for 5 minutes. α,β-unsaturated amide (vii) (167.16 g, 343 mmol) is added to the solutions. Then, tert-amyl alcohol (113 ml, 1028 mmol) was added to this mixture over 3 hours using a syringe pump. After completion of the addition to the reaction solution, add ~1700 ml of 20% NH ₄ OH in small portions. Attention: at the beginning of quenching, intensive bubbling and foaming occurs, careful observation and slow, small addition of ammonium hydroxide is necessary. The reaction mixture is partitioned between water and hexanes. The organic layers are filtered through celite and evaporated in vacuo. The resulting elastic solid material is pulverized using a mechanical agitator in hexanes to give small solids which are then filtered and washed with hexanes. The organic layers are then evaporated in vacuo and purified using flash chromatography (silica gel, 0-15% ethyl acetate/hexanes) to give the desired amide (viii). LC/MS (M+H)=490.7.

[00370] Synthesis of compound 32: To a solution of amide (viii) (2.85 g, 5.8 mmol) was added lithium aluminum hydride (8.7 mmol, 8.7 ml of a 1M solution). The reaction mixture is stirred at ambient temperature for 10 minutes, after which the reaction is quenched by slowly adding sodium sulfate decahydrate solution. The solids are filtered and washed with THF, the filtrate is evaporated in vacuo. The crude mixture was purified using reverse phase preparative chromatography (column C8) to give (13Z,16Z)-N,N-dimethyl-3-nonyldocose-13,16-dien-1-amine (compound 32) as an oil. HRMS (M+H) calculation. 476.5190. ¹ H NMR (400 MHz, CDCl ₃ ) δ 5.37 (m, 4H), 2.78 (t, 2H), 2.42 (m, 8H), 2.05 (q, 4H), 1.28 (m, 41H), 0.89 (m, 6H).

Example 6 Compounds 6 and 22

[00371] Lipid particles containing ccDNA can be prepared or formulated in combination with compounds 6 or 22 having the structures:

[00372] Compounds 6 and 22 are described in WO 2015/199952, the contents of which are incorporated herein in their entirety by reference. Compounds 6 and 22 were synthesized as follows:

[00373] Synthesis of compound 6: A solution of nonane-1,9-diol (12.6 g) in methylene chloride (80 ml) was treated with 2-hexyldecanoic acid (10.0 g), DCC (8.7 g), and DMAP ( 5.7 g). The solution is stirred for two hours. The reaction mixture is filtered and the solvent is removed. The residue is dissolved in warm hexane (250 ml) and allowed to crystallize. The solution is filtered and the solvent is removed. The residue is taken up in methylene chloride and washed with diluted hydrochloric acid. The organic layer is dried over anhydrous magnesium sulfate, filtered and the solvent is removed. The residue was passed through a silica gel (75 g) column using 0-12% ethyl acetate/hexane as eluent to give 9-(2'-hexyldecanoyloxy)nonan-1-ol as an oil.

[00374] The product was dissolved in methylene chloride (60 ml) and treated with pyridinium chlorochromate (6.4 g) for four hours. Add diethyl ether (200 ml) and filter the supernatant through a pad of silica gel. Remove the solvent from the filtrate and pass the resulting oil through a silica gel (75 g) column in an ethyl acetate/hexane (0-12%) gradient to give 9-(2'-ethylhexanoyloxy)nonanal as an oil.

[00375] A solution of the crude product (6.1 g), acetic acid (0.34 g) and 2-N,N-dimethylaminoethylamine (0.46 g) in methylene chloride (20 ml) was treated with sodium triacetoxyborohydride (2.9 g) for two hours. This solution is diluted with methylene chloride, washed with aqueous sodium hydroxide and then with water. The organic phase is dried over anhydrous magnesium sulfate, filtered and the solvent is removed. The residue was passed through a silica gel column (75 g) in a methanol/methylene chloride (0-8%) gradient and then through a second column (20 g) in a methylene chloride/acetic acid/methanol gradient. The purified fractions were dissolved in methylene chloride, washed with diluted aqueous sodium hydroxide, dried over anhydrous magnesium sulfate, filtered, and the solvent removed to give the desired product as a colorless oil.

[00376] Synthesis of compound 22: In step 1 to a solution of 6-bromohexanoic acid (20 mmol, 3.901 g), 2-hexyl-1-decanol (1.8 eq., 36 mmol, 8.72 g) and 4-dimethylaminopyridine (DMAP 0.5 eq., 10 mmol, 1.22 g) DCC (1.1 eq., 22 mmol, 4.54 g) was added in DCM (80 ml). The final mixture is stirred at room temperature for 16 hours. The precipitate is removed by filtration. The filtrate is concentrated. The residue was purified by silica gel column chromatography eluting with a gradient of ethyl acetate in hexanes (0-2%). The result is the desired product in the form of a colorless oil.

[00377] In step 2, a mixture of bromide from step 1 (1.34 eq., 7.88 g, 18.8 mmol), N,N-diisopropylethylamine (1.96 eq., 27.48 mmol, 4.78 ml ) and N,N-dimethylethylenediamine (1 eq, 14.02 mmol, 1.236 g, 1.531 ml) in acetonitrile (70 ml) in a 250 ml flask equipped with a condenser, heated at 79°C (oil bath) for 16 hours. The reaction mixture is cooled to room temperature and concentrated. The residue is taken up in a mixture of ethyl acetate and hexanes (1:9) and water. The phases are separated, washed with water (100 ml) and brine. Then dried over sodium sulfate and concentrated (8.7 g oil). The crude oil (8.7 g) was purified by silica gel column chromatography (0-3% MeOH in chloroform). Fractions containing the desired product are combined and concentrated. The residue is dissolved in 1 ml of hexane and filtered through a plug of silica gel (34 mm, washed with 8 ml of hexane). The filtrate is dried under a stream of Ar and dried in vacuo overnight. The desired product is obtained as a colorless oil. ¹ H NMR (400 MHz, CDCl ₃ ) δ 3.96 (d, 5.8 Hz, 4H), 2.55-2.50 (m, 2H), 2.43-2.39 (m, 4H) , 3.37-3.32 (m, 2H), 2.30 (t, 7.5 Hz, 4H), 2.23 (s, 6H), 1.63 (quintet-like, 7.6 Hz, 6H) , 1.48-1.40 (m, 4H), 1.34-1.20 (52H), 0.88 (t-like, 6.8 Hz, 12H).

Example 7 Preparation of Lipid Formulations

[00378] Lipid nanoparticles (LNPs) can be prepared with a total lipid to scDNA weight ratio of approximately 10:1-30:1. Briefly, an ionizable lipid (e.g., MC3, ATX-002, compound 6, compound 22, a compound of formula (A') or (A''), or a compound of formula (B'), (B') or (B'') ), a non-cationic lipid (such as distearoylphosphatidylcholine (DSPC)), a membrane integrity component (such as a sterol, such as cholesterol), and a conjugated lipid molecule (such as a PEG lipid, such as 1-(monomethoxypolyethylene glycol)-2,3- dimyristoylglycerol, PEG 2000 average molecular weight ("PEG-DMG")) is solubilized in an alcohol (eg ethanol) in a molar ratio of 50:10:38.5:1.5. scDNA is diluted to the required concentration in a buffer solution. For example, ccDNA can be diluted to a concentration of 0.1 mg/ml - 0.25 mg/ml in a buffer solution containing sodium acetate, sodium acetate with magnesium chloride, citrate, malic acid, or malic acid with sodium chloride. According to one example, ccDNA is diluted to 0.2 mg/ml in 10-50 mM citrate buffer, pH 4. An alcoholic solution of lipids is mixed with an aqueous solution of ccDNA using, for example, syringe pumps or an impact-spray mixer, in a ratio of approximately 1:5 -1:3 (by volume) at a total flow rate above 10 ml/min. In one example, an alcoholic lipid solution is mixed with an aqueous solution of scDNA in a ratio of approximately 1:3 (by volume) at a flow rate of 12 ml/min. The alcohol is removed and the buffer is replaced with PBS by dialysis. Alternatively, the buffer can be exchanged for PBS using centrifuge tubes. The removal of the alcohol and the simultaneous replacement of the buffer can be carried out, for example, by dialysis or tangential flow filtration. The obtained lipid nanoparticles are filtered through a sterile filter with a pore size of 0.2 μm until further use.

Example 8 Lipid Particulate Composition Analysis

[00379] The size of lipid nanoparticles can be determined using quasi-elastic light scattering in the Malvem Zetasizer Nano ZS system (Malvern, UK); their diameter is about 55-95 nm or about 70-90 nm diameter.

[00380] The pKa value of a cationic lipid formulation can be correlated with the effectiveness of LNP for delivery of nucleic acids (see sources: Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al. Nature Biotechnology 28, 172-176 (2010, both of which are incorporated by reference in their entirety). The preferred range of pKa values is from ~5 to ~7. The pKa value for each cationic lipid is determined in lipid nanoparticles using an analysis based on the fluorescence of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). Lipid nanoparticles containing cationic lipid/DSPC/cholesterol/PEG lipid (50/10/38.5/1.5 mol %) in PBS at a total lipid concentration of 0.4 mM can be prepared using an in-line process according to described in this document and other sources. TNS can be obtained as a 100 μM stock solution in distilled water. Vesicles can be diluted to 24 µM lipids in 2 ml buffered solutions containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, 130 mM NaCl, where the pH varies from 2.5 to 11. An aliquot of TNS solution can be added to obtain a final concentration of 1 μM, and after vortexing, the fluorescence intensity is measured at room temperature on an SLM Aminco Series 2 fluorescent spectrophotometer at an excitation wavelength and an emission wavelength of 321 nm and 445 nm. Fluorescence data can be processed by best fit using the sigmoid function; pKa is measured as pH, giving half-maximal fluorescence intensity values.

[00381] Encapsulation of ccDNA into lipid particles can be determined by assaying with Oligreen®. Oligreen® is an ultrasensitive fluorescent nucleic acid stain for the quantification of oligonucleotides and single-stranded DNA or RNA in solution (available from Invitrogen Corporation; Carlsbad, Calif.). Alternatively, PicoGreen® may be used. Briefly, encapsulation can be determined by performing a size exclusion assay with a non-membrane permeable fluorescent dye using a dye that exhibits increased fluorescence when bound to a nucleic acid. Encapsulation is determined by adding said dye to the lipid particle formulation, measuring the resulting fluorescence and comparing it with the fluorescence observed after adding a small amount of non-ionic detergent. Detergent-mediated disruption of the lipid bilayer releases the encapsulated ccDNA, allowing it to interact with the non-permeable dye. Encapsulation of scDNA can be calculated as E=(I ₀ -I)/I ₀ where I and I ₀ refer to the fluorescence intensity before and after detergent addition.

[00382] Relative activity can be determined by measuring luciferase expression in the liver 4 hours after administration by tail vein injection. The activity is compared at a dose of 0.3 and 1.0 mg ccDNA/kg and is expressed as ng luciferase/g liver when measured 4 hours after administration.

Example 9 Evaluation of scDNA-LNP Examples

[00383] Received lipid nanoparticles containing examples of scDNA, with various N/P ratios (for example, 3, 4, 5 and 6) using a solution of lipids containing MC3, DSPC, cholesterol and DMG-PEG2000 (molar ratio 50:10: 38.5:1.5). Aqueous solutions of the ccDNA vector were prepared in buffered solutions containing salts such as sodium acetate, sodium acetate with magnesium chloride, citrate, malic acid, or malic acid with sodium chloride. The lipid solution and the ccDNA solution were mixed using a proprietary nano-assembler procedure at a total flow rate of 12 ml/min at a lipid to ccDNA ratio of 1:3 (v/v).

Characterization

[00384] The size of the lipid nanoparticles and the encapsulation of the ccDNA in the lipid nanoparticles were determined. Particle size was determined using dynamic light scattering (ZEN3600, Malvern Instruments). Encapsulation efficiency was calculated by determining the content of unencapsulated scDNA by measuring the fluorescence after adding PicoGreen (Thermo Scientific) to the LNP suspension (C _free ) and comparing this value with the total scDNA content obtained after LNP lysis using 1% Triton X-100 (C _total ) where % encapsulation = (C _total -C _free )/C _total × 100. The results are shown in FIG. 9-11.

Endosomal release assay

[00385] The effect of serum and BSA on the encapsulation and release of ccDNA from LNP was determined. Endosome-mimicking anionic liposomes were prepared by mixing DOPS:DOPC:DOPE (molar ratio 1:1:2) in chloroform, followed by evaporation of the solvent under vacuum. The dried lipid film was resuspended in DPBS with a brief sonication followed by filtration through a 0.45 µm syringe filter to obtain an anionic liposome.

[00386] LNPs containing scDNA and BSA or serum were mixed in equal volumes. The mixture was incubated at 37°C for 20 minutes. Then, an anionic liposome with the required molar ratio of anionic/cationic lipid in DPBS at pH 7.5 or 6.0 was added to the mixture of LNP with BSA or serum. The final combination was then incubated at 37°C for another 15 minutes. As a control, an equal volume of DPBS instead of an anionic liposome was added to a mixture of LNP with BSA or serum. The amount of free scDNA at different treatments at pH7.5 or pH6.0 was calculated by determining the content of unencapsulated scDNA, measuring the fluorescence after adding PicoGreen (Thermo Scientific) to the LNP suspension (C _free ) and comparing this value with the total content of scDNA obtained after LNP lysis using 1% Triton X-100 (C _total ), where % Free ccDNA = C _free /C _total × 100. % ccDNA released after incubation with anionic liposomes was calculated using the equation below:

[00387] The results are shown in FIG. 12-15 and summarized in Table 5.

ApoE binding

[00388] The binding of lipid nanoparticles to ApoE was determined. LNP (10 μg/ml) were incubated at 37°C for 20 minutes with an equal volume of recombinant ApoE3 (500 μg/ml) in 1x DPBS. After incubation, LNP samples were diluted 10-fold using 1x DPBS and analyzed using heparin-sepharose chromatography on AKTA pure 150 (GE Healthcare) under the conditions below:

Chromatography conditions HiTrap Column HiTrap Heparin Sepharose HP 1 ml Balance Buffer 1×DPBS Wash Buffer 1×DPBS Elution buffer 1 M NaCl in 10 mM sodium phosphate buffer, pH 7.0 Flow rate 1 ml/min Volume of administration 500 µl detection 260 nm

[00389] The results are shown in FIG. 16.

Expression of HEK293

[00390] Expression of ccDNA encapsulated in lipid nanoparticles was analyzed as follows.

[00391] HEK293 cells were maintained at 37° C. with 5% CO ₂ in DMEM + GlutaMAX™ culture medium (Thermo Scientific) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were seeded in 96-well plates at a density of 30,000 cells/well the day before transfection. Lipofectamine™ 3000 Transfection Reagent (Thermo Scientific) was used to transfect control ccDNA at 100 ng/well according to the manufacturer's protocol. Control ccDNA was diluted in Opti-MEM™ (Thermo Scientific) and reagent P3000™ was added. Lipofectamine™ 3000 was then diluted to a final concentration of 3% in Opti-MEM™. Diluted Lipofectamine™ 3000 was added to the diluted ccDNA at a ratio of 1:1 and incubated for 15 minutes at room temperature. The required amount of scDNA lipid complex or LNP was then added directly to each well containing cells. Cells were incubated at 37°C with 5% CO ₂ for 72 hours. Expression levels of secreted factor IX in HEK293-conditioned medium were determined using the VisuLize FIX Antigen ELISA Kit (Affinity Biologies) according to the manufacturer's instructions. The results are shown in FIG. 17-19.

Example 10 Compounds for Formulation A

[00392] Lipid nanoparticles (LNPs) containing scDNA can be prepared or formulated in combination with one or more compounds of formula (I) or (II), preferably forming a liposome or lipid nanoparticle suitable for storage and therapeutic delivery of scDNA- vector.

[00393] Compounds of formula (I) suitable for formulating lipid particles have the structure:

where

each of R ¹ , R ² , R ⁴ and R ⁵ is independently selected from the group consisting of hydrogen, C ₁ -C ₂₀ alkyl substituted with C ₁ -C ₂₀ alkyl, C ₂ -C ₂₀ alkenyl substituted with C ₂ -C ₂₀ alkenyl, C ₂ -C ₂₀ alkynyl and cholesteryl;

R ³ is selected from the group consisting of C ₁ -C ₂₀ alkyl, substituted C ₁ -C ₂₀ alkyl, C ₂ -C ₂₀ alkenyl, substituted C ₂ -C ₂₀ alkenyl, and C ₂ -C ₂₀ alkynyl;

L is selected from the group consisting of S, O, C ₁ -C ₂₀ alkenyl substituted with C ₁ -C ₂₀ alkenyl;

, или

;X ¹ is absent, represents

, or

;

, или

; иX ² is absent, represents

, or

; and

each R6 is independently selected from the group consisting of C ₁ -C ₂₀ alkyl, substituted C ₁ -C ₂₀ alkyl, C ₂ -C ₂₀ alkenyl, substituted C ₂ -C ₂₀ alkenyl, and C ₂ -C ₂₀ alkynyl;

or their salts.

[00394] A compound of formula (I) suitable for preparing a lipid particle formulation has the structure:

where

each of R ¹ , R ² and R ⁴ is independently selected from the group consisting of hydrogen, C ₁ -C ₂₀ alkyl, substituted C ₁ -C ₂₀ alkyl, C ₂ -C ₂₀ alkenyl, substituted C ₂ -C ₂₀ alkenyl, C ₂ -C ₂₀ alkynyl and cholesteryl;

R ³ is selected from the group consisting of C ₁ -C ₂₀ alkyl, substituted C ₁ -C ₂₀ alkyl, C ₂ -C ₂₀ alkenyl, substituted C ₂ -C ₂₀ alkenyl, and C ₂ -C ₂₀ alkynyl;

R ⁵ is absent or selected from the group consisting of C ₁ -C ₂₀ alkyl, substituted C ₁ -C ₂₀ alkyl, C ₂ -C ₂₀ alkenyl, substituted C ₂ -C ₂₀ alkenyl and C ₂ -C ₂₀ alkynyl;

L is selected from the group consisting of S, O, C ₁ -C ₂₀ alkenyl substituted with C ₁ -C ₂₀ alkenyl;

или

;X ¹ is absent, represents

or

;

или

;X ² is absent, represents

or

;

each R6 is independently selected from the group consisting of hydrogen, C ₁ -C ₂₀ alkyl, substituted C ₁ -C ₂₀ alkyl, C ₂ -C ₂₀ alkenyl, substituted C ₂ -C ₂₀ alkenyl, and C ₂ -C ₂₀ alkynyl; and

представляет собой ковалентную связь с R⁵, если присутствует R⁵, или

отсутствует, если отсутствует R⁵;

is a covalent bond with R ⁵ if R ⁵ is present, or

absent if R ⁵ is absent;

or their salts.

[00395] In some examples of compounds of formula (I) or formula (II), R ¹ is branched C ₁₂ -C ₂₀ alkyl; R ² is linear C ₅ -C ₁₀ alkyl or branched C ₁₂ -C ₂₀ alkyl; each of R ⁴ and R ³ independently represents a linear C ₁ -C ₅ alkyl; each R6 is independently linear or branched C ₁ -C ₅ alkyl; and L is linear C ₁ -C ₃ alkyl.

[00396] Examples of compounds of formula (I) or (II) may be one or more compounds selected from those shown in FIG. twenty.

[00397] The general synthesis of compounds of formula (I) and (II) is shown in FIG. 21 and 22, where each R value is independently selected.

[00398] In general, the starting compounds described above and other compounds can be purchased from commercial sources or obtained in accordance with methods known to a person skilled in the art. One skilled in the art will be able to design even highly substituted scaffolds for the compounds described herein using standard synthetic techniques and reference to reference literature and databases on chemical compounds and chemical reactions as known to one skilled in the art. Suitable reference literature and studies detailing the synthesis of reactants suitable for the preparation of compounds as described herein or citing references to articles describing the preparation of compounds as described herein include, for example, "Synthetic Organic Chemistry", John Wiley and Sons, Inc. New York; S.R. Sandier et al, "Organic Functional Group Preparations," rd. Ed., Academic Press, New York, 1983; H.O. House, "Modem Synthetic Reactions," 2nd Ed., W.A. Benjamin Inc. Menio Park, Calif., 1972; T.L. Glichrist, "Heterocyclic Chemistry," 2nd Ed. John Wiley and Sons, New York, 1992; J. March, "Advanced Organic Chemistry: reactions, Mechanisms and Structure," 5th Ed., Wiley hiterscience, New York, 2001; Specific and similar reactants can also be identified in the Chemical Abstracts Index of Known Chemicals compiled by the American Chemical Society Chemical Abstracts Service, which is available from most public and university libraries and Internet databases (more information can be obtained from the American Chemical Society). , Washington, DC). Chemicals known but not commercially available in catalogs can be manufactured by custom synthesis companies, with many standard chemical companies providing custom synthesis services in addition.

Example 11 Compounds for Formulation B

[00399] Other lipid particles containing ccDNA can be prepared or formulated in combination with one or more compounds of formula (III), (IV) or (V) described in this example, preferably forming a liposome or lipid nanoparticle, suitable for storage and therapeutic delivery of the scDNA vector.

[00400] Compounds of formula (III) suitable for use in the preparation of a lipid particle formulation have the structure:

where

представляет собой циклическое кольцо, выбранное из группы, состоящей из С₃-С₁₀ циклоалкила, С₃-С₁₀ циклоалкенила, С₃-С₁₀ гетероциклоалкила, С₃-С₁₀ гетероциклоалкенила, С₆-С₁₀ арила и С₅-С₁₀ гетероарила;

is a cyclic ring selected from the group consisting of C ₃ -C ₁₀ cycloalkyl, C ₃ -C ₁₀ cycloalkenyl, C ₃ -C ₁₀ heterocycloalkyl, C ₃ -C ₁₀ heterocycloalkenyl, C ₆ -C ₁₀ aryl, and C ₅ -C ₁₀ heteroaryl;

each of R ¹ , R ² , R ³ and R ⁴ is independently selected from the group consisting of hydrogen, C ₁ -C ₂₀ alkyl substituted with C ₁ -C ₂₀ alkyl, C ₂ -C ₂₀ alkenyl substituted with C ₂ -C ₂₀ alkenyl, C ₂ -C ₂₀ alkynyl and cholesteryl; and X ¹ is absent, is C ₁ -C ₂₀ alkyl substituted with C ₁ -C ₂₀ alkyl,

или

;

or

;

X ² is absent, is C ₁ -C ₂₀ alkyl substituted by C ₁ -C ₂₀ alkyl,

, или

;

, or

;

or their salt.

[00401] In some examples of compounds of formula (III), at least one of R ¹ , R ² , R ³ and R ⁴ is:

[00402] The compound of formula (III) may be a compound selected from the compounds shown in FIG. 23.

[00403] Compounds of formula (IV) suitable for use in the preparation of a lipid particle formulation have the structure:

where:

n is 0-150; and each of R ¹ , R ² and R ³ is independently selected from the group consisting of hydrogen, C ₁ -C ₂₀ alkyl, substituted C ₁ -C ₂₀ alkyl, C ₂ -C ₂₀ alkenyl, substituted C ₂ -C ₂₀ alkenyl, C ₂ -C ₂₀ alkynyl and cholesteryl;

or their salts.

[00404] Compounds of formula (V) suitable for use in the preparation of a lipid particle formulation have the structure:

where each of R ¹ , R ² , R ³ , R ⁴ and R ⁵ is independently selected from the group consisting of: hydrogen, C ₁ -C ₂₀ alkyl, substituted C ₁ -C ₂₀ alkyl, C ₂ -C ₂₀ alkenyl, substituted C ₂ -C ₂₀ alkenyl, C ₂ -C ₂₀ alkynyl and cholesteryl;

X ¹ is absent, is C ₁ -C ₂₀ alkyl, substituted with C ₁ -C ₂₀ alkyl, or

X ² is absent, is C ₁ -C ₂₀ alkyl substituted by C ₁ -C ₂₀ alkyl,

или

;

or

;

X ³ is absent, is C ₁ -C ₂₀ alkyl substituted with C ₁ -C ₂₀ alkyl,

или

;

or

;

or their salt.

[00405] Examples of compounds of formula (V) include, but are not limited to, the following:

where n and m are independently 0-20; and

each of R ¹ and R ² is independently selected from the group consisting of: hydrogen, C ₁ -C ₂₀ alkyl, substituted C ₁ -C ₂₀ alkyl, C ₂ -C ₂₀ alkenyl, substituted C ₂ -C ₂₀ alkenyl, C ₂ - C ₂₀ alkynyl and cholesteryl;

or their salt.

[00406] The general synthesis of compounds of formula (III), formula (IV) and (V) is shown in FIG. 24, where each R value is independently selected.

[00407] Those skilled in the art will appreciate that in the process described herein, the functional groups of intermediates may need to be protected with suitable protecting groups. Such functional groups include hydroxyl groups, amino groups, mercapto groups and carboxylic acid. Suitable protecting groups for hydroxyl groups include trialkylsilyl or diarylalkylsilyl (eg, t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino and guanidino groups include t-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto groups include -C(O)-R'' (where R'' is alkyl, aryl or arylalkyl), p-methoxybenzyl, trityl, and the like. Suitable protecting groups for a carboxylic acid include alkyl, aryl or arylalkyl esters. Protecting groups may be added or removed in accordance with standard techniques known to those skilled in the art and as described herein. The use of protecting groups is described in detail in Green, T.W. and P.G.M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley (incorporated herein by reference in its entirety). As will be understood by one of skill in the art, the protecting group may also be a polymeric resin such as Wang resin, Rink resin or 2-chlorotrityl chloride resin. In general, starting components can be obtained from sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, Fluorochem USA, etc., or synthesized according to sources known to those skilled in the art. techniques (see, for example, source: Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5e ed. ition (Wiley, December 2000), incorporated herein in its entirety by reference).

Example 12 Preparation of Lipid Formulations

[00408] Liposomal nanoparticles (LNPs) can be prepared. The cationic lipid, DSPC, cholesterol and PEG lipid can be solubilized in ethanol in a molar ratio of 50:10:38.5:1.5. Lipid nanoparticles (LNPs) can be prepared with a total lipid to scDNA weight ratio of approximately 10:1-30:1. Briefly, ccDNA is diluted to 0.2 mg/mL in 10-50 mM citrate buffer, pH 4. Syringe pumps can be used to mix an ethanolic lipid solution with an aqueous solution of ccDNA in a ratio of approximately 1:5-1:3 (v/v) at a total flow rate above 15 ml/min. The ethanol can then be removed and the external buffer replaced with PBS by dialysis. Finally, the lipid nanoparticles can be filtered through a sterile filter with a pore size of 0.2 µm. The diameter of lipid nanoparticles is approximately 55-95 nm, in some cases approximately 70-90 nm, which can be determined using quasi-elastic light scattering using the Malvem Zetasizer Nano ZS system (Malvern, UK).

[00409] The pKa value of cationic lipid formulations can be correlated with the effectiveness of LNPs for delivery of nucleic acids (see Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al. Nature Biotechnology 28, 172-176 (2010), incorporated by reference in their entirety). The preferred range of pKa values is from ~5 to ~7. The pKa value of each cationic lipid in the lipid nanoparticles is determined using an analysis based on the fluorescence of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). Lipid nanoparticles containing cationic lipid/DSPC/cholesterol/PEG lipid (50/10/38.5/1.5 mol %) in PBS at a total lipid concentration of 0.4 mM can be prepared using an in-line process as described in this document and elsewhere. TNS can be obtained as a 100 μM stock solution in distilled water. Vesicles can be diluted to a lipid concentration of 24 µM in 2 ml buffered solutions containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, 130 mM NaCl, where the pH varies from 2.5 to 11. An aliquot of TNS solution can be added to obtain a final concentration of 1 μm, and after mixing on a vortex, measure the fluorescence intensity at room temperature on an SLM Aminco Series 2 luminescent spectrophotometer at an excitation wavelength and an emission wavelength of 321 nm and 445 nm. Fluorescence data can be processed by best fit using the sigmoid function; pKa is measured as pH, giving half-maximal fluorescence intensity values.

[00410] The composition with lipid nanoparticles can also be obtained using the following molar ratio: 50% cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG-lipid ("PEG-DMG", t e.e. (1-(monomethoxypolyethylene glycol)-2,3-dimyristoylglycerol, at average molecular weight PEG 2000) Relative potency can be determined by measuring luciferase expression in the liver 4 hours after administration by tail vein injection as described herein The activity is compared at 0.3 and 1.0 mg ccDNA/kg and is expressed as ng luciferase/g liver as measured 4 hours after administration.

[00411] Compositions with therapeutic compounds containing ccDNA constructs can be prepared using the following molar ratio: 50% cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipid (“PEG-DMA ", 2-[2-(w-Methoxy(polyethylene glycol-2000)ethoxy]-N,N-ditradecylacetamide). Relative potency can be determined by measuring luciferase expression in the liver 4 hours after administration by tail vein injection as described above This activity can be measured at a dose of 0.3 and 1.0 mg mRNA or DNA/kg and is expressed as ng luciferase/g liver when measured 4 hours after administration, as described above.

Example 13 In Vivo Expression of Luciferase Transgene Protein from ccDNA Vectors

[00412] In vivo expression of the transgene protein from ccDNA vectors derived from constructs 1-8 described above was evaluated in mice. The scDNA vector derived from the scDNA plasmid construct 1 (as described in Table 5 in Example 1) was tested and demonstrated sustained and sustained transgenic expression of luciferase in a mouse model following hydrodynamic injection of a composition containing the scDNA vector contained in lipid nanoparticles. , into the tail vein. Luciferase transgene expression was measured by IVIS imaging following intravenous administration to CD-1® IGS mice (Charles River Laboratories; wild-type mice).

[00413] The study evaluates the biodistribution of a hydrodynamically administered luciferase-expressing non-viral IVIS gene therapy vector following intravenous administration to CD-1® IGS mice. The basis is a sterile FSB. In this study, two groups of five (5) CD-1 mice were injected with either PBS or 0.35 mg/kg of scDNA encoding luciferase by hydrodynamic injection of 1.2 ml into the tail vein. Luciferase expression was assessed by IVIS imaging at days 3, 4, 7, 14, 21, 28, 31, 35 and 42. Briefly, mice are intraperitoneally injected with 150 mg/kg of luciferin substrate and then whole body luminescence is assessed using IVIS imaging.

[00414] In vivo expression of luciferase: 5-7 week old male CD-1 IGS mice (Charles River Laboratories) are administered 0.35 mg/kg 0.35 mg/kg scDNA vector construct 14 Luc (Group 1) in volume 1 .2 ml by intravenous hydrodynamic administration on day 0. Some animals in each group are re-injected with an identical dose of the 1-4 Luc ccDNA vector construct (Group 1) on day 28.

[00415] Expression of luciferase from the ccDNA vector is assessed by in vivo chemiluminescence using IVIS® instrumentation after intraperitoneal (ip) injection of 150 mg/kg of luciferase substrate. IVIS imaging is performed on day 3, day 4, day 7, day 14, day 21, day 28, day 31, day 35 and day 42, and harvested organs are imaged ex vivo after sacrifice at day 42.

[00416] Throughout the study, animals are weighed daily and monitored for general health and well-being. At sacrifice, the blood of each animal is collected by terminal cardiac puncture, divided into two parts and processed to obtain 1) plasma and 2) serum; the plasma is flash frozen and the serum is used for a liver enzyme panel and then flash frozen. In addition, the liver, spleen, kidney and inguinal lymph nodes (LN) are harvested and visualized ex vivo using IVIS.

[00417] Hepatic luciferase expression is assessed using MAXDISCOVERY® luciferase ELISA ELISA (BIOO Scientific/PerkmElmer), qPCR for luciferase in liver samples, histopathological analysis of liver samples, and/or a panel of liver enzymes in serum (VetScanVS2; Abaxis Preventative Care Profile Plus).

Sources

[00418] All sources listed and disclosed in the present description and examples, including patents, patent applications, international patent applications and publications, are incorporated herein in their entirety by reference.

Claims (53)

1. A lipid nanoparticle for delivery of a capsid-free non-viral DNA vector to a target site, containing an ionizable lipid and a non-viral capsid-free DNA vector with covalently closed ends (zcDNA vector), characterized in that said scDNA vector contains at least one heterologous nucleotide sequence , functionally located between asymmetric inverted terminal repeats (asymmetric ITRs), where the ITRs are asymmetric with respect to each other and where one or more of the asymmetric ITRs are modified by a deletion, insertion and/or replacement in at least one of the regions selected from A, A', B, B', C, C', D and D', and moreover, at least one of the asymmetric ITR contains a functional terminal resolution site and a Rep binding site. 2. The lipid nanoparticle of claim 1, wherein said scDNA vector, when cleaved with a restriction enzyme with a single recognition site on the scDNA vector and analyzed using both native and denaturing gel electrophoresis, exhibits the characteristic bands of linear and continuous DNA compared with linear and discontinuous DNA controls. 3. A lipid nanoparticle according to claim 1 or 2, wherein one or more of the asymmetric ITRs are from a virus selected from parvovirus, dependdovirus, and adeno-associated virus (AAV). 4. A lipid nanoparticle according to claim 3, wherein said asymmetric ITRs are from different viral serotypes. 5. The lipid nanoparticle of claim 4, wherein said one or more of the asymmetric ITRs are from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. 6. Lipid nanoparticle according to any one of paragraphs. 1-3, where one or more of the asymmetric ITRs are synthetic. 7. Lipid nanoparticle according to any one of paragraphs. 1-6, where one or more ITRs are not wild-type ITRs. 8. Lipid nanoparticle according to any one of paragraphs. 1-7, wherein one or more ITRs are wild-type ITRs. 9. Lipid nanoparticle according to any one of paragraphs. 1-7, where both asymmetric ITRs are modified by deletion, insertion and/or substitution in at least one of the regions selected from A, A', B, B', C, C', D and D'. 10. Lipid nanoparticle according to any one of paragraphs. 1-9, where said scDNA vector contains at least two asymmetric ITRs selected from: a) SEQ ID NO: 1 and SEQ ID NO: 52 and b) SEQ ID NO: 2 and SEQ ID NO: 51. 11. Lipid nanoparticle according to any one of paragraphs. 1-10, where the indicated scDNA vector is obtained by a method including the following steps: a) incubating a population of insect cells carrying the scDNA expression construct in the presence of at least one Rep protein, said scDNA expression construct encoding the scDNA vector, under conditions effective to, and for a time sufficient to induce scDNA production a vector in said insect cells; and b) isolating said scDNA vector from said insect cells. 12. A lipid nanoparticle according to claim 11, wherein said scDNA expression construct is selected from scDNA plasmid, scDNA bacmid, and scDNA baculovirus. 13. The lipid nanoparticle according to claim 11 or 12, wherein said insect cells express at least one Rep protein. 14. The lipid nanoparticle of claim 11 wherein at least one Rep protein is from a virus selected from parvovirus, dependdovirus, and adeno-associated virus (AAV). 15. The lipid nanoparticle of claim 14 wherein at least one Rep protein is from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12. 16. Lipid nanoparticle according to any one of paragraphs. 1-15, wherein said DNA vector is derived from a vector polynucleotide, wherein said vector polynucleotide encodes a heterologous nucleic acid operably located between two inverted terminal repeats (ITRs), where the ITRs are asymmetric with respect to each other and at least one of the ITRs is a functional ITR comprising a functional terminal clearance site and a Rep binding site, and wherein one of the asymmetric ITRs is modified by a deletion, insertion and/or substitution in at least one of the regions selected from A, A', B, B', C, C ', D and D' relative to the specified functional ITR; wherein the presence of the Rep protein induces replication of the vector polynucleotide and production of a vector DNA in the insect cell, said vector DNA can be obtained by a method comprising the following steps: a) incubating a population of insect cells carrying said vector polynucleotide, which does not contain viral capsid coding sequences, in the presence of a Rep protein, under conditions effective to, and for a time sufficient to induce production of a capsid-free, non-viral DNA vector in the cells insects, characterized in that the capsid-free non-viral DNA produced is not contained in said insect cells in the absence of said vector; and b) collection and isolation of non-capsid non-viral DNA from insect cells. 17. Lipid nanoparticle according to any one of paragraphs. 11-16, where the presence of non-capsid non-viral DNA isolated from insect cells can be confirmed. 18. The lipid nanoparticle according to claim 17, wherein the presence of capsid-free non-viral DNA isolated from insect cells can be confirmed by cleaving the DNA isolated from insect cells with a restriction enzyme with one recognition site on said DNA vector and analyzing the cleaved DNA material for non-denaturing gel to confirm the presence of characteristic bands of linear and continuous DNA compared to linear and discontinuous DNA. 19. Lipid nanoparticle according to any one of paragraphs. 1-18, wherein said DNA vector is derived from a vector polynucleotide, wherein said vector polynucleotide encodes a heterologous nucleic acid operably located between the first and second inverted terminal repeats (ITRs) of AAV2, wherein the ITRs are asymmetric with respect to each other and wherein one or more of asymmetric ITRs are modified by deletion, insertion and/or substitution in at least one of the regions selected from A, A', B, B', C, C', D and D' relative to the corresponding wild-type AAV2 ITR SEQ ID NO: 1 or SEQ ID NO: 51 to induce replication of said DNA vector in an insect cell in the presence of a Rep protein, wherein said vector DNA can be obtained by a method comprising the following steps: a) incubating a population of insect cells carrying said vector polynucleotide, which does not contain viral capsid coding sequences, in the presence of a Rep protein, under conditions effective to, and for a time sufficient to induce the production of capsid-free, non-viral DNA in the insect cells, wherein said insect cells do not contain viral capsid coding sequences; and b) collection and isolation of non-capsid non-viral DNA from insect cells. 20. The lipid nanoparticle of claim 19, wherein the presence of capsid-free, non-viral DNA isolated from insect cells can be confirmed. 21. The lipid nanoparticle according to claim 20, wherein the presence of capsid-free non-viral DNA isolated from insect cells can be confirmed by cleaving the DNA isolated from insect cells with a restriction enzyme with one recognition site on said DNA vector and analyzing the cleaved DNA material for non-denaturing gel to confirm the presence of characteristic bands of linear and continuous DNA compared to linear and discontinuous DNA. 22. Lipid nanoparticle according to any one of paragraphs. 1-21, wherein said lipid nanoparticle further comprises one or more of a non-cationic lipid. 23. Lipid nanoparticle according to any one of paragraphs. 1-22 wherein the non-cationic lipid is selected from the group consisting of distearoyl-sn-glycerophosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleylphosphatidylethanolamine (DOPE) , palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethylethanolphosphatidylethanolamine, dimethylphosphatidylethanolamine, 18-1-trans-PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) ), distearoylphosphatidylglycerol (DSPG), dierucoilphospha тидилхолина (DEPC), пальмитоилолеоилфосфатидилглицерина (POPG), диэлаидоилфосфатидилэтаноламина (DEPE), лецитина, фосфатидилэтаноламина, лизолецитина, лизофосфатидилэтаноламина, фосфатидилсерина, фосфатидилинозитола, сфингомиелина, яичного сфингомиелина (ESM), кефалина, кардиолипина, фосфатидной кислоты, цереброзидов, дицетилфосфата, лизофосфатидилхолина и дилинолеоилфосфатидилхолина. 24. Lipid nanoparticle according to any one of paragraphs. 1-23, where the specified lipid nanoparticle additionally contains a PEG-conjugated lipid, while the specified PEG-conjugated lipid is selected from the group consisting of PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide ( Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG-diacylglycerol succinate (PEGS-DAG), PEG-dialkoxypropylcarbam and N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt. 25. Lipid nanoparticle according to any one of paragraphs. 1-24, where said lipid nanoparticle additionally contains cholesterol or a cholesterol derivative. 26. Lipid nanoparticle according to any one of paragraphs. 1-25, where said lipid nanoparticle contains: (i) an ionizable lipid; (ii) a non-cationic lipid; (iii) a conjugated lipid that inhibits particle aggregation; and (iv) sterol. 27. Lipid nanoparticle according to any one of paragraphs. 1-26, where said lipid nanoparticle contains: (a) an ionizable lipid in an amount of from about 20 mol.% to about 90 mol.% of the total amount of lipids present in the specified particle; (b) a non-cationic lipid in an amount of from about 5 mol.% to about 30 mol.% of the total amount of lipids present in the specified particle; (c) a conjugated lipid that inhibits particle aggregation, in an amount of from about 0.5 mol.% to about 20 mol.% of the total amount of lipids present in the specified particle; and (d) sterol in an amount of from about 20 mol.% to about 50 mol.% of the total amount of lipids present in the specified particle. 28. A lipid nanoparticle according to claim 27, wherein the ionizable lipid is contained in an amount of from about 20 mol.% to about 70 mol.% of the total lipids present in the specified particle, optionally where the ionizable lipid is contained in an amount of from about 30 mol.% up to about 60 mol.% of the total amount of lipids present in the specified particle. 29. A lipid nanoparticle according to claim 27, wherein the ionizable lipid is contained in an amount of from about 30 mol.% to about 60 mol.% of the total amount of lipids present in the specified particle. 30. A lipid nanoparticle according to claim 27, wherein the ionizable lipid is present in an amount of from about 40 mol% to about 50 mol% of the total amount of lipids present in said particle. 31. Lipid nanoparticle according to any one of paragraphs. 1-27, where the ratio of total lipids to vector DNA (w/w) is from about 10:1 to about 30:1. 32. A lipid nanoparticle according to claim 27, wherein the conjugated lipid is present in an amount of from about 2 mol% to about 5 mol% of the total amount of lipids present in said particle. 33. A lipid nanoparticle according to claim 32, wherein the conjugated lipid is a PEG-lipid conjugate. 34. A lipid nanoparticle according to claim 27, wherein the sterol is contained in an amount of from about 30 mol.% to about 40 mol.% of the total amount of lipids present in the specified particle. 35. Pharmaceutical composition for delivering a heterologous nucleic acid, containing a first lipid nanoparticle and an additional compound, where the additional compound is a therapeutic agent, and wherein said first lipid nanoparticle contains the first non-capsid non-viral vector and is a lipid nanoparticle according to any one of paragraphs. 1-34, where said lipid nanoparticle contains a heterologous nucleic acid and an excipient. 36. The composition of claim 35, wherein said additional compound is included in a second lipid nanoparticle and said first and second lipid nanoparticles are different. 37. Pharmaceutical composition for delivering a heterologous nucleic acid, containing the first lipid nanoparticle and an additional compound, where the additional compound is included in the first lipid nanoparticle and where the additional compound is a therapeutic agent, and wherein the specified first lipid nanoparticle contains the first non-capsid non-viral vector and is lipid nanoparticle according to any one of paragraphs. 1-34, where said lipid nanoparticle contains a heterologous nucleic acid and an excipient.

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