Lipid nanoparticle formulations of non-viral, capsid-free DNA vectors

Abstract

To provide lipid nanoparticle formulations of non-viral, capsid-free DNA vectors.SOLUTION: The present description provides a lipid particle comprising an ionizable lipid and a non-viral, capsid-free DNA vector with covalently-closed ends (ceDNA vector), where the ceDNA vector comprises at least one heterologous nucleotide sequence operably positioned between asymmetric inverted terminal repeat sequences (asymmetric ITRs), and at least one of the asymmetric ITRs comprises a functional terminal resolution site and a Rep binding site.SELECTED DRAWING: Figure 9

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Nos. 62 / 556,334, filed September 8, 2017, 62 / 556,333, filed September 8, 2017, 62 / 556,381, filed September 9, 2017, 62 / 675,317, filed May 23, 2018, 62 / 675,322, filed May 23, 2018, 62 / 675,324, filed May 23, 2018, and 62 / 675,327, filed May 23, 2018, the contents of each of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The said ASCII copy, created on September 7, 2018, is named 080170-090660WOPT_SL.txt and is 63,790 bytes in size.

[0003] The present invention is directed to non-viral, capsid-free DNA vector lipid nanoparticles (LNPs) and their use for delivery of exogenous DNA sequences to target cells, tissues, organs, or organisms. [Background technology]

[0004] Recently, non-viral capsid-free DNA vectors with covalently closed ends that contain transgenes adjacent to AAV2 ITR have been reported.However, the targeted delivery of these DNA vectors to cells in vitro and in vivo remains a challenge.Therefore, there remains a need in the art for a formulation that addresses these challenges. Summary of the Invention

[0005] In one embodiment, a novel lipid formulation is provided herein, comprising ionized lipid and capsid-free non-viral vector (ceDNA).The ceDNA vector described herein is a capsid-free linear double-stranded DNA molecule formed from a continuous strand of complementary DNA (linear continuous non-capsidated structure) with covalently closed ends, and comprises 5' inverted terminal repeat (ITR) sequence and 3' ITR sequence that are different or asymmetric with respect to each other.In one embodiment, the non-viral capsid-free DNA vector with covalently closed ends is preferably a linear double-stranded molecule, and can be obtained from a vector polynucleotide that codes for a heterologous nucleic acid that is operably located between two different inverted terminal repeat (ITR) sequences (e.g., AAV ITR), at least one of the ITRs comprises a terminal resolution site and a replication protein binding site (RPS) (sometimes referred to as replication protein binding site), for example, a Rep binding site, and one of the ITRs comprises a deletion, insertion, and / or substitution with respect to the other ITR. That is, one of the ITRs is asymmetric with respect to the other ITRs.In one embodiment, at least one of the ITRs is AAV ITR, for example, wild-type AAV ITR or modified AAV ITR.In one embodiment, at least one of the ITRs is modified ITR with respect to the other ITRs, that is, the ceDNA comprises 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 of the ITRs is not a wild-type ITR.

[0008] In some embodiments, the ceDNA vector, when digested with a restriction enzyme that has a single recognition site on the ceDNA vector and analyzed by both native and denaturing gel electrophoresis, exhibits a characteristic linear, continuous DNA band compared to a linear, discontinuous DNA control.

[0009] In some embodiments, one or more of the asymmetric ITR sequences of ceDNA vector are derived from a virus selected from parvovirus, dependovirus, and adeno-associated virus (AAV).In some embodiments, the asymmetric ITRs are derived from different virus serotypes.For example, one or more of the asymmetric ITRs can be derived 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 ceDNA vector are synthetic.

[0011] In some embodiments, one of the asymmetric ITRs (e.g., one or both) is modified by a 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, the ceDNA vector comprises 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, the ceDNA vector is obtained from a process comprising: (a) incubating a population of insect cells harboring a ceDNA expression construct in the presence of at least one Rep protein, the ceDNA expression construct encoding the ceDNA vector, under conditions effective and for a time sufficient to induce production of the ceDNA vector in the insect cells; and (b) isolating the ceDNA vector from the insect cells. Without being limited thereto, the ceDNA expression construct can be a ceDNA plasmid, a ceDNA bacmid, or a ceDNA baculovirus.

[0014] In general, insect cell insect cell expresses at least one Rep protein.At least one Rep protein can be derived from a virus selected from parvovirus, dependovirus, and adeno-associated virus (AAV).For example, at least one Rep protein can be derived from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

[0015] In some embodiments, the ionizable lipids are lipids described in the publications listed in Table 1.

[0016] In some embodiments of the various aspects disclosed herein, the presence of ceDNA may be confirmed by digestion with a restriction enzyme that has a single recognition site on the ceDNA vector and analyzing the digested DNA material on denaturing and non-denaturing gels to confirm the presence of a characteristic linear, continuous DNA band compared to linear, discontinuous DNA.

[0017] In some embodiments, the DNA vector is derived from a vector polynucleotide, the vector polynucleotide encoding a heterologous nucleic acid operably positioned between two inverted terminal repeats (ITRs), the two ITRs being different from each other (asymmetric), at least one of the ITRs being a functional ITR comprising a functional terminal resolution site and a Rep binding site, one of the ITRs comprising a deletion, insertion, or substitution relative to the functional ITR, and the presence of Rep protein induces replication of the vector polynucleotide.

[0018] In some embodiments, the production of DNA vectors in insect cells. For example, the DNA vectors are obtained from a process that includes: (a) incubating a population of insect cells harboring vector polynucleotides lacking viral capsid coding sequences in the presence of Rep proteins under conditions effective to induce the production of capsid-free, non-viral DNA vectors in the insect cells for a sufficient time, where the insect cells do not contain the production of capsid-free, non-viral DNA in the insect cells in the absence of the vector; and (b) harvesting and isolating the capsid-free, non-viral DNA from the insect cells. In some further embodiments, the presence of the capsid-free, non-viral DNA isolated from the insect cells can be confirmed. For example, the presence of capsid-free, non-viral DNA isolated from insect cells can be confirmed by digesting the DNA isolated from the insect cells with a restriction enzyme that has a single recognition site on the DNA vector and analyzing the digested DNA material on a non-denaturing gel to confirm the presence of a characteristic linear, continuous DNA band compared to linear, discontinuous DNA.

[0019] In some embodiments, the DNA vector is obtained from a vector polynucleotide. For example, the DNA vector is obtained from a vector polynucleotide encoding a heterologous nucleic acid operably located between a first and a second AAV2 inverted terminal repeat DNA polynucleotide sequence (ITR), at least one of which has at least one polynucleotide deletion, insertion or substitution with respect to the corresponding AAV2 wild-type ITR of SEQ ID NO: 1 or SEQ ID NO: 51, and induces the replication of the DNA vector in insect cells in the presence of Rep protein. In some further embodiments, the DNA vector can be obtained from a process comprising: (a) incubating a population of insect cells harboring a vector polynucleotide lacking viral capsid coding sequence in the presence of Rep protein under conditions effective to induce the production of capsid-free non-viral DNA vector in insect cells for a sufficient time, wherein the insect cells do not contain viral capsid coding sequence; and (b) harvesting and isolating the capsid-free non-viral DNA from the insect cells. In some further embodiments, the presence of capsid-free non-viral DNA isolated from insect cells can be confirmed.For example, the presence of capsid-free non-viral DNA isolated from insect cells can be confirmed by digesting the DNA isolated from insect cells with a restriction enzyme that has a single recognition site on DNA vector, and analyzing the digested DNA material on non-denaturing gel to confirm the presence of characteristic linear and continuous DNA bands compared to linear and discontinuous DNA.

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

[0021] In some embodiments, the lipid nanoparticle further comprises a non-cationic lipid, the non-ionic lipid being distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine ... Phosphatidylethanolamine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine (16-O-monomethyl PE, etc.), dimethyl-phosphatidylethanolamine (DMPE ... ethanolamine (e.g. 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), dierucoyl phosphatidylcholine (DEPC), palmitoyloleoylphosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and, for example,The non-cationic lipids are selected from the group consisting of those described in WO2017 / 099823 or US2018 / 0028664.

[0022] In some embodiments, the lipid particle further comprises a complex lipid, the complex lipid being selected from the group consisting of PEG-diacylglycerol (DAG) (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) (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, and those listed in Table 2.

[0023] In some embodiments, the lipid particles further comprise cholesterol or a cholesterol derivative as described in PCT Publication No. WO2009 / 127060 or US Patent Publication No. US2010 / 0130588.

[0024] In some embodiments, the lipid particles include ionized lipids, non-cationic lipids, complex lipids that inhibit particle aggregation, and sterols. The amounts of ionized lipids, non-cationic lipids, complex lipids that inhibit particle aggregation, and sterols can vary independently. In some embodiments, the lipid nanoparticles include ionized lipids in an amount of about 20 mol% to about 90 mol% of the total lipids present in the particles, non-cationic lipids in an amount of about 5 mol% to about 30 mol% of the total lipids present in the particles, complex lipids that inhibit particle aggregation in an amount of about 0.5 mol% to about 20 mol% of the total lipids present in the particles, and sterols in an amount of about 20 mol% to about 50 mol% of the total lipids present in the particles.

[0025] The ratio of total lipids to DNA vectors can vary as desired, for example, the ratio of total lipids to DNA vectors (by mass or weight) can be from about 10:1 to about 30:1.

[0026] Also provided herein is a composition comprising a first lipid nanoparticle and additional compound.The first lipid nanoparticle comprises ionized lipid and a first capsid-free non-viral vector.When the capsid-free non-viral vector is digested with a restriction enzyme that has a single recognition site on DNA vector, it has a characteristic linear and continuous DNA band compared to linear and discontinuous DNA when analyzed on non-denaturing gel.

[0027] In some embodiments, the additional compound is contained in the second lipid nanoparticle.The first and second lipid nanoparticles can be the same or different.In some embodiments, the first and second lipid nanoparticles are different.In some embodiments, the first and second lipid nanoparticles are the same, i.e., the additional compound is contained in the first lipid nanoparticle.

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

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

[0030] These and other aspects of the invention are described in further detail below. [Brief description of the drawings]

[0031] The patent or application file contains at least one color drawing. Copies of this patent or patent application with color drawing(s) will be provided by the Patent Office upon request and payment of the necessary fee.

[0032] [Figure 1A] The exemplary structure of ceDNA vector is shown.In this embodiment, the exemplary ceDNA vector comprises an expression cassette containing CAG promoter, WPRE and BGHpA.The open reading frame (ORF) encoding luciferase transgene is inserted into the cloning site (R3 / R4) between CAG promoter and WPRE.The expression cassette is flanked by two inverted terminal repeats (ITR)-wild type AAV2 ITR at the upstream (5' end) of the expression cassette and modified ITR at the downstream (3' end), and therefore the two ITRs flanking the expression cassette are asymmetric with respect to each other. [Figure 1B] Figure 1 shows an exemplary structure of a ceDNA vector with an expression cassette containing a CAG promoter, a WPRE, and BGHpA. An open reading frame (ORF) encoding a luciferase transgene is inserted into the cloning site between the CAG promoter and the WPRE. The expression cassette is flanked by two inverted terminal repeats (ITRs) - a modified ITR upstream (5' end) and a wild-type ITR downstream (3' end) of the expression cassette. [Figure 1C] 1 shows an exemplary structure of a ceDNA vector with an expression cassette containing an enhancer / promoter, an open reading frame (ORF) for inserting a transgene, a posttranscriptional element (WPRE), and a polyA signal. The open reading frame (ORF) allows the insertion of a transgene into the cloning site between the CAG promoter and the WPRE. The expression cassette is flanked by two inverted terminal repeats (ITRs) that are asymmetric with respect to each other, a modified ITR at the upstream (5' end) and a modified ITR at the downstream (3' end) of the expression cassette, and both the 5'ITR and the 3'ITR are modified ITRs, but with different modifications (i.e., not the same modifications). [Figure 2A]Provided is a T-shaped stem-loop structure of one wild-type ITR of AAV2 with A-A' arms, B-B' arms, C-C' arms, two Rep binding sites (RBE and RBE'), and terminal resolution sites (trs). RBE contains a series of four double tetramers that are believed to interact with either Rep78 or Rep68. In addition, RBE' is also believed to interact with the Rep complex assembled on the wild-type or mutated ITR in the construct. The D and D' regions contain transcription factor binding sites and other conserved structures. [Figure 2B] The proposed Rep catalytic nicking and ligation activities in the wild-type ITR in Figure 2A are shown, including the T-shaped stem-loop structure of the wild-type ITR of AAV2 with the A-A' arm, the B-B' arm, the C-C' arm, two Rep binding sites (RBE and RBE'), and the terminal resolution site (trs), as well as the D and D' regions that contain several transcription factor binding sites, and other conserved structures. [Figure 3A] 1 provides the primary structure (polynucleotide sequence) (left) and secondary structure (right) of the RBE-containing portions of the A-A' arm and the C-C' and B-B' arms of the wild-type left AAV2 ITR (SEQ ID NO:540). [Figure 3B] 1 shows an exemplary mutated ITR (also referred to as modified ITR) sequence of the left ITR. The primary structure (left) and predicted secondary structure (right) of the RBE portion of the A-A', C, and B-B' arms of an exemplary mutant left ITR (ITR-1, left) (SEQ ID NO: 113) are shown. [Figure 3C] The primary structure (left) and secondary structure (right) of the A-A' loop and the RBE-containing portions of the B-B' and C-C' arms of the wild-type right AAV2 ITR (sequence number 541) are shown. [Figure 3D]An exemplary right modified ITR is shown. The primary structure (left) and predicted secondary structure (right) of the A-A' arm and the RBE-containing portion of the B-B' and C arms of an exemplary mutant left ITR (ITR-1, right) (SEQ ID NO: 114) are shown. Any combination of left and right ITRs (e.g., AAV2 ITRs or other viral serotype or synthetic ITRs) can be used, so long as the left ITR is asymmetric or different from the right ITR. Each of the polynucleotide sequences in Figures 3A-3D refers to sequences used in the plasmid or bacmid / baculovirus genome used to produce ceDNA as described herein. The corresponding ceDNA secondary structures deduced from the ceDNA vector configurations and predicted Gibb free energy values ​​in the plasmid or bacmid / baculovirus genome are also included in each of Figures 3A-3D. [Figure 4A]FIG. 4A is a schematic diagram showing an upstream process for making baculovirus-infected insect cells (BIIC), which are useful for the production of ceDNA in the process described in the schematic diagram of FIG. 4B. FIG. 4C shows a biochemical method and process for confirming ceDNA vector production. FIG. 4D and FIG. 4E are schematic diagrams illustrating a process for identifying the presence of ceDNA in DNA harvested from cell pellets obtained during the ceDNA production process of FIG. 4B. FIG. 4E shows DNA with a non-contiguous structure. ceDNA can be cleaved by a restriction endonuclease with a single recognition site on the ceDNA vector, generating two DNA fragments with different sizes (1 kb and 2 kb) in both neutral and denaturing conditions. FIG. 4E also shows ceDNA with a linear and contiguous structure. ceDNA vector can be cleaved by a restriction endonuclease, generating two DNA fragments that migrate as 1 kb and 2 kb in neutral conditions but not in denaturing conditions, producing a single strand in which the strands remain connected and migrate as 2 kb and 4 kb. FIG. 4D shows schematic expected bands of an exemplary ceDNA on the left that is uncut or digested with a restriction endonuclease and then subjected to electrophoresis on either a native or denaturing gel. The leftmost schematic is a native gel and shows multiple bands suggesting that in its duplex and uncut form, the ceDNA exists at least in monomeric and dimeric states and appears as a faster migrating smaller monomer and a slower migrating dimer that is twice the size of the monomer. The second schematic from the left shows that when the ceDNA is cut with a restriction endonuclease, the original band disappears and a faster migrating (e.g., smaller) band appears that corresponds to the expected fragment size remaining after cleavage. Under denaturing conditions, the original duplex DNA is single stranded and migrates as a species twice as large as that observed on a native gel because the complementary strand is covalently attached. Thus, in the second schematic from the right, digested ceDNA shows a similar banding distribution to that observed on the native gel, but the bands migrate as fragments twice the size of their native gel counterparts.The schematic on the far right shows that uncleaved ceDNA under denaturing conditions migrates as a single-stranded open circle, and thus the observed band is twice the size of that observed under native conditions where the circle is not open. In this figure, "kb" is used to indicate the relative size of the nucleotide molecules based on the length of the nucleotide chain (e.g., for single-stranded molecules observed under denaturing conditions) or the number of base pairs (e.g., for double-stranded molecules observed under native conditions), depending on the context. [Figure 4B]FIG. 4A is a schematic diagram showing an upstream process for making baculovirus-infected insect cells (BIIC), which are useful for the production of ceDNA in the process described in the schematic diagram of FIG. 4B. FIG. 4C shows a biochemical method and process for confirming ceDNA vector production. FIG. 4D and FIG. 4E are schematic diagrams illustrating a process for identifying the presence of ceDNA in DNA harvested from cell pellets obtained during the ceDNA production process of FIG. 4B. FIG. 4E shows DNA with a non-contiguous structure. ceDNA can be cleaved by a restriction endonuclease with a single recognition site on the ceDNA vector, generating two DNA fragments with different sizes (1 kb and 2 kb) in both neutral and denaturing conditions. FIG. 4E also shows ceDNA with a linear and contiguous structure. ceDNA vector can be cleaved by a restriction endonuclease, generating two DNA fragments that migrate as 1 kb and 2 kb in neutral conditions but not in denaturing conditions, producing a single strand in which the strands remain connected and migrate as 2 kb and 4 kb. FIG. 4D shows schematic expected bands of an exemplary ceDNA on the left that is uncut or digested with a restriction endonuclease and then subjected to electrophoresis on either a native or denaturing gel. The leftmost schematic is a native gel and shows multiple bands suggesting that in its duplex and uncut form, the ceDNA exists at least in monomeric and dimeric states and appears as a faster migrating smaller monomer and a slower migrating dimer that is twice the size of the monomer. The second schematic from the left shows that when the ceDNA is cut with a restriction endonuclease, the original band disappears and a faster migrating (e.g., smaller) band appears that corresponds to the expected fragment size remaining after cleavage. Under denaturing conditions, the original duplex DNA is single stranded and migrates as a species twice as large as that observed on a native gel because the complementary strand is covalently attached. Thus, in the second schematic from the right, digested ceDNA shows a similar banding distribution to that observed on the native gel, but the bands migrate as fragments twice the size of their native gel counterparts.The schematic on the far right shows that uncleaved ceDNA under denaturing conditions migrates as a single-stranded open circle, and thus the observed band is twice the size of that observed under native conditions where the circle is not open. In this figure, "kb" is used to indicate the relative size of the nucleotide molecules based on the length of the nucleotide chain (e.g., for single-stranded molecules observed under denaturing conditions) or the number of base pairs (e.g., for double-stranded molecules observed under native conditions), depending on the context. [Figure 4C]FIG. 4A is a schematic diagram showing an upstream process for making baculovirus-infected insect cells (BIIC), which are useful for the production of ceDNA in the process described in the schematic diagram of FIG. 4B. FIG. 4C shows a biochemical method and process for confirming ceDNA vector production. FIG. 4D and FIG. 4E are schematic diagrams illustrating a process for identifying the presence of ceDNA in DNA harvested from cell pellets obtained during the ceDNA production process of FIG. 4B. FIG. 4E shows DNA with a non-contiguous structure. ceDNA can be cleaved by a restriction endonuclease with a single recognition site on the ceDNA vector, generating two DNA fragments with different sizes (1 kb and 2 kb) in both neutral and denaturing conditions. FIG. 4E also shows ceDNA with a linear and contiguous structure. ceDNA vector can be cleaved by a restriction endonuclease, generating two DNA fragments that migrate as 1 kb and 2 kb in neutral conditions but not in denaturing conditions, producing a single strand in which the strands remain connected and migrate as 2 kb and 4 kb. FIG. 4D shows schematic expected bands of an exemplary ceDNA on the left that is uncut or digested with a restriction endonuclease and then subjected to electrophoresis on either a native or denaturing gel. The leftmost schematic is a native gel and shows multiple bands suggesting that in its duplex and uncut form, the ceDNA exists at least in monomeric and dimeric states and appears as a faster migrating smaller monomer and a slower migrating dimer that is twice the size of the monomer. The second schematic from the left shows that when the ceDNA is cut with a restriction endonuclease, the original band disappears and a faster migrating (e.g., smaller) band appears that corresponds to the expected fragment size remaining after cleavage. Under denaturing conditions, the original duplex DNA is single stranded and migrates as a species twice as large as that observed on a native gel because the complementary strand is covalently attached. Thus, in the second schematic from the right, digested ceDNA shows a similar banding distribution to that observed on the native gel, but the bands migrate as fragments twice the size of their native gel counterparts.The schematic on the far right shows that uncleaved ceDNA under denaturing conditions migrates as a single-stranded open circle, and thus the observed band is twice the size of that observed under native conditions where the circle is not open. In this figure, "kb" is used to indicate the relative size of the nucleotide molecules based on the length of the nucleotide chain (e.g., for single-stranded molecules observed under denaturing conditions) or the number of base pairs (e.g., for double-stranded molecules observed under native conditions), depending on the context. [Figure 4D]FIG. 4A is a schematic diagram showing an upstream process for making baculovirus-infected insect cells (BIIC), which are useful for the production of ceDNA in the process described in the schematic diagram of FIG. 4B. FIG. 4C shows a biochemical method and process for confirming ceDNA vector production. FIG. 4D and FIG. 4E are schematic diagrams illustrating a process for identifying the presence of ceDNA in DNA harvested from cell pellets obtained during the ceDNA production process of FIG. 4B. FIG. 4E shows DNA with a non-contiguous structure. ceDNA can be cleaved by a restriction endonuclease with a single recognition site on the ceDNA vector, generating two DNA fragments with different sizes (1 kb and 2 kb) in both neutral and denaturing conditions. FIG. 4E also shows ceDNA with a linear and contiguous structure. ceDNA vector can be cleaved by a restriction endonuclease, generating two DNA fragments that migrate as 1 kb and 2 kb in neutral conditions but not in denaturing conditions, producing a single strand in which the strands remain connected and migrate as 2 kb and 4 kb. FIG. 4D shows schematic expected bands of an exemplary ceDNA on the left that is uncut or digested with a restriction endonuclease and then subjected to electrophoresis on either a native or denaturing gel. The leftmost schematic is a native gel and shows multiple bands suggesting that in its duplex and uncut form, the ceDNA exists at least in monomeric and dimeric states and appears as a faster migrating smaller monomer and a slower migrating dimer that is twice the size of the monomer. The second schematic from the left shows that when the ceDNA is cut with a restriction endonuclease, the original band disappears and a faster migrating (e.g., smaller) band appears that corresponds to the expected fragment size remaining after cleavage. Under denaturing conditions, the original duplex DNA is single stranded and migrates as a species twice as large as that observed on a native gel because the complementary strand is covalently attached. Thus, in the second schematic from the right, digested ceDNA shows a similar banding distribution to that observed on the native gel, but the bands migrate as fragments twice the size of their native gel counterparts.The schematic on the far right shows that uncleaved ceDNA under denaturing conditions migrates as a single-stranded open circle, and thus the observed band is twice the size of that observed under native conditions where the circle is not open. In this figure, "kb" is used to indicate the relative size of the nucleotide molecules based on the length of the nucleotide chain (e.g., for single-stranded molecules observed under denaturing conditions) or the number of base pairs (e.g., for double-stranded molecules observed under native conditions), depending on the context. [Figure 4E]FIG. 4A is a schematic diagram showing an upstream process for making baculovirus-infected insect cells (BIIC), which are useful for the production of ceDNA in the process described in the schematic diagram of FIG. 4B. FIG. 4C shows a biochemical method and process for confirming ceDNA vector production. FIG. 4D and FIG. 4E are schematic diagrams illustrating a process for identifying the presence of ceDNA in DNA harvested from cell pellets obtained during the ceDNA production process of FIG. 4B. FIG. 4E shows DNA with a non-contiguous structure. ceDNA can be cleaved by a restriction endonuclease with a single recognition site on the ceDNA vector, generating two DNA fragments with different sizes (1 kb and 2 kb) in both neutral and denaturing conditions. FIG. 4E also shows ceDNA with a linear and contiguous structure. ceDNA vector can be cleaved by a restriction endonuclease, generating two DNA fragments that migrate as 1 kb and 2 kb in neutral conditions but not in denaturing conditions, producing a single strand in which the strands remain connected and migrate as 2 kb and 4 kb. FIG. 4D shows schematic expected bands of an exemplary ceDNA on the left that is uncut or digested with a restriction endonuclease and then subjected to electrophoresis on either a native or denaturing gel. The leftmost schematic is a native gel and shows multiple bands suggesting that in its duplex and uncut form, the ceDNA exists at least in monomeric and dimeric states and appears as a faster migrating smaller monomer and a slower migrating dimer that is twice the size of the monomer. The second schematic from the left shows that when the ceDNA is cut with a restriction endonuclease, the original band disappears and a faster migrating (e.g., smaller) band appears that corresponds to the expected fragment size remaining after cleavage. Under denaturing conditions, the original duplex DNA is single stranded and migrates as a species twice as large as that observed on a native gel because the complementary strand is covalently attached. Thus, in the second schematic from the right, digested ceDNA shows a similar banding distribution to that observed on the native gel, but the bands migrate as fragments twice the size of their native gel counterparts.The schematic on the far right shows that uncleaved ceDNA under denaturing conditions migrates as a single-stranded open circle, and thus the observed band is twice the size of that observed under native conditions where the circle is not open. In this figure, "kb" is used to indicate the relative size of the nucleotide molecules based on the length of the nucleotide chain (e.g., for single-stranded molecules observed under denaturing conditions) or the number of base pairs (e.g., for double-stranded molecules observed under native conditions), depending on the context. [Diagram 5] An illustrative diagram of an example denaturing gel run of ceDNA vectors with (+) or without (-) digestion with endonucleases (EcoRI for ceDNA constructs 1 and 2, BamH1 for ceDNA constructs 3 and 4, SpeI for ceDNA constructs 5 and 6, and XhoI for ceDNA vectors 7 and 8). The sizes of the bands highlighted with asterisks were determined and are shown below the diagram. [Figure 6A] An exemplary Rep-bacmid in pFBDLSR plasmid containing the nucleic acid sequences of Rep proteins Rep52 and Rep78. This exemplary Rep-bacmid contains the polyhedron promoter sequence of the IE1 promoter fragment (SEQ ID NO:66), the Rep78 nucleotide sequence including the Kozak sequence (SEQ ID NO:67), Rep52 (SEQ ID NO:68) and the Rep58 nucleotide sequence beginning with the Kozak sequence (gccgccacc) (SEQ ID NO:69). [Figure 6B] FIG. 1 is a schematic diagram of an exemplary ceDNA-plasmid-1, carrying a wt-L ITR, a CAG promoter, a luciferase transgene, a WPRE and polyadenylation sequence, and a mod-R ITR. [Figure 7A] Shown are results from an in vitro protein expression assay measuring luciferase activity (y-axis, RQ(Luc)) in HEK293 cells 48 hours after transfection with 400 ng (black), 200 ng (grey), or 100 ng (white) of constructs identified on the x-axis (Construct-1, Construct-3, Construct-5, Construct-7) (Table 5 in Example 1). [Figure 7B]Shown is the luciferase activity (y-axis, RQ(Luc)) measured in HEK293 cells 48 hours after transfection with 400 ng (black), 200 ng (gray), or 100 ng (white) of the constructs (construct-2, construct-4, construct-6, construct-8) specified on the x-axis (Table 5). Also provided is the luciferase activity measured in HEK293 cells treated with Fugene ("Fugene") or untreated ("Untreated") without any plasmid. [Figure 8A] Shown is the viability of HEK293 cells (y-axis) 48 hours after transfection with 400 ng (black), 200 ng (grey), or 100 ng (white) of constructs (construct-1, construct-3, construct-5, construct-7) identified on the x-axis. [Figure 8B] Shown is the viability of HEK293 cells (y-axis) 48 hours after transfection with 400 ng (black), 200 ng (grey), or 100 ng (white) of constructs (construct-2, construct-4, construct-6, construct-8) identified on the x-axis. [Figure 9] Figures 9-11 are bar graphs showing the average lipid nanoparticle size and ceDNA encapsulation of several exemplary lipid nanoparticles prepared in buffers containing different salts at a constant N / P ratio (Figure 9) or varying N / P ratios (Figures 10 and 11). [Figure 10] Figures 9-11 are bar graphs showing the average lipid nanoparticle size and ceDNA encapsulation of several exemplary lipid nanoparticles prepared in buffers containing different salts at a constant N / P ratio (Figure 9) or varying N / P ratios (Figures 10 and 11). [Figure 11] Figures 9-11 are bar graphs showing the average lipid nanoparticle size and ceDNA encapsulation of several exemplary lipid nanoparticles prepared in buffers containing different salts at a constant N / P ratio (Figure 9) or varying N / P ratios (Figures 10 and 11). [Figure 12] 1 is a bar graph showing the effect of serum / BSA on encapsulation in exemplary lipid nanoparticles. [Figure 13]1 is a bar graph showing the release of ceDNA from exemplary lipid nanoparticles in the presence of dioleoylphosphatidylserine (DOPS) liposomes. [Figure 14] 1 is a bar graph showing the effect of serum / BSA on encapsulation in exemplary lipid nanoparticles. [Figure 15] 1 is a bar graph showing the release of ceDNA from exemplary lipid nanoparticles in the presence of dioleoylphosphatidylserine (DOPS) liposomes. [Figure 16] 1 shows ApoE binding of several exemplary lipid nanoparticles. [Figure 17] 1 is a bar graph showing HEK293 expression of exemplary ceDNAs. [Figure 18] 1 is a gel electrophoresis photograph showing HEK293 expression of an exemplary ceDNA. [Figure 19] 1 is a bar graph showing HEK293 expression of exemplary ceDNAs. [Figure 20] Some exemplary compounds of formula (I) and formula (II) described in Example 10 are shown. [Figure 21] 1 is a general synthetic scheme for the synthesis of compounds of formula (I) and (II) disclosed in Example 10. [Figure 22] 1 is a general synthetic scheme for the synthesis of compounds of formula (I) and (II) disclosed in Example 10. [Diagram 23] Some exemplary compounds of formula (III) described in Example 11 are shown. [Figure 24] FIG. 1 depicts a general synthetic scheme for the synthesis of compounds of formula (III), formula (IV), and formula (V) as described in Example 11. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] definition Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those skilled in the art to which this disclosure belongs. It is to be understood that the present invention is not limited to the specific methodology, protocols, and reagents, etc. described herein, and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp&Dohme Corp., 2011 (ISBN 978-0-911910-19-3), Robert S.Porter et al.(eds.), Fields Virology, 6th Edition, published by Lippincott Williams&Wilkins, Philadelphia, PA, USA(2013), Knipe, DM and Howley, PM(ed.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908), and Robert A.Meyers(ed.), Molecular Biology and Biotechnology:a Comprehensive Desk Reference,published by VCH Publishers,Inc.,1995(ISBN 1-56081-569-8)、Immunology by Werner Luttmann,published by Elsevier,2006、Janeway‘s Immunobiology,Kenneth Murphy,Allan Mowat,Casey Weaver(eds.),Taylor&Francis Limited,2014(ISBN 0815345305,9780815345305)、Lewin’s Genes XI,published by 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,N.Y.,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), the contents of which are incorporated herein by reference in their entireties.

[0034] As used herein, the term "lipid nanoparticle" refers to a vesicle formed by one or more lipid components. Lipid nanoparticles are typically used as carriers for nucleic acid delivery in the context of formulation development. They act by fusing with cell membranes and rearranging their lipid structure to deliver drugs or active pharmaceutical ingredients (APIs). In general, lipid nanoparticle compositions for such delivery are composed of synthetic ionized or cationic lipids, phospholipids (particularly compounds with phosphatidylcholine groups), cholesterol, and polyethylene glycol (PEG) lipids, although these compositions may also contain other lipids. The overall composition of lipids typically dictates the surface characteristics, and thus the protein (opsonization) content, in biological systems, thus driving the biodistribution and cellular uptake properties.

[0035] As used herein, the term "liposome" refers to lipid molecules assembled in a spherical configuration that encapsulates an internal aqueous volume separated from an aqueous exterior. Liposomes are vesicles with at least one lipid bilayer. Liposomes are typically used as carriers for drug / therapeutic agent delivery in the context of formulation development. They act by fusing with cell membranes and rearranging their lipid structure to deliver drugs or active formulation components. Liposome compositions for such delivery are typically composed of compounds with phospholipids, particularly phosphatidylcholine, although these compositions may also contain other lipids.

[0036] As used herein, the term "ionizable lipid" refers to a lipid having at least one protonatable or proton-releasable group, whereby the lipid is positively charged at a pH below physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably above physiological pH. It will be understood by those skilled in the art that the addition or removal of protons as a function of pH is an equilibrium process, and that references to charged or neutral lipids refer to the nature of the dominant groups, and not all of the lipids need be present in a charged or neutral form. Generally, ionizable lipids have a pK of the protonatable group in the range of about 4 to about 7. a Ionizable lipids are also referred to herein as cationic lipids.

[0037] As used herein, the term "non-cationic lipid" refers to any amphipathic lipid as well as any other neutral or anionic lipid. Thus, non-cationic lipids can be neutral uncharged, zwitterionic, or anionic lipids.

[0038] As used herein, the term "conjugated lipid" refers to a lipid molecule conjugated with a non-lipid molecule, such as PEG, a polyoxazoline, a polyamide, or a polymer (e.g., a cationic polymer).

[0039] As used herein, the term "excipient" refers to a pharmacologically inactive ingredient included in a formulation with an API, e.g., ceDNA and / or lipid nanoparticles to bulk and / or stabilize the formulation when producing a dosage form. General categories of excipients include, for example, bulking agents, fillers, diluents, anti-adhesives, binders, coatings, disintegrants, flavors, colors, lubricants, glidants, adsorbents, preservatives, sweeteners, and products used to promote drug absorption or solubility or for other pharmacodynamic considerations.

[0040] As used herein, "heterologous nucleotide sequence" and "transgene" are used interchangeably and refer to a nucleic acid of interest (other than a nucleic acid encoding a capsid polypeptide) that can be incorporated into and delivered and expressed by the ceDNA vector disclosed herein. Transgenes of interest include, but are not limited to, nucleic acids encoding a polypeptide, preferably a therapeutic agent (e.g., for medical, diagnostic, or veterinary use), or an immunogenic polypeptide (e.g., for a vaccine). In some embodiments, nucleic acids of interest include nucleic acids that are transcribed into therapeutic RNA. Transgenes included for use in the ceDNA vectors of the present invention include, but are not limited to, those that express or code for one or more polypeptides, peptides, ribozymes, aptamers, peptide nucleic acids, siRNAs, RNAi, miRNAs, IncRNAs, antisense oligonucleotides or polynucleotides, antibodies, antigen-binding fragments, or any combination thereof.

[0041] As used herein, the terms "expression cassette" and "transcription cassette" are used interchangeably and refer to a linear stretch of nucleic acid that includes a transgene operably linked to one or more promoters or other regulatory sequences sufficient to direct transcription of the transgene, but does not include capsid-encoding sequences, other vector sequences, or inverted terminal repeat regions. An expression cassette may additionally include one or more cis-acting sequences (e.g., promoters, enhancers, or repressors), one or more introns, and one or more post-transcriptional regulatory elements.

[0042] As used herein, the term "terminal repeat" or "TR" includes any viral or synthetic terminal sequence that contains at least one minimally necessary origin of replication and a region that contains a palindromic hairpin structure. The Rep binding sequence ("RBS") and terminal resolution site ("TRS") together constitute the "minimally necessary origin of replication," and thus a TR contains at least one RBS and at least one TRS. TRs that are reverse complements of each other within a given stretch of a polynucleotide sequence are typically referred to as "inverted terminal repeats" or "ITRs," respectively. In the context of a virus, ITRs mediate replication, viral packaging, integration, and proviral rescue. As unexpectedly found in the present invention herein, TRs that are not reverse complements over their entire length can still perform the traditional functions of ITRs, and thus the term ITR is used herein to refer to TRs in a ceDNA genome or ceDNA vector that can mediate replication of the ceDNA vector. It will be understood by those skilled in the art that there may be more than two ITRs or asymmetric ITR pairs in a composite ceDNA construct. The ITRs may be AAV ITRs or non-AAV ITRs, or AAV ITRs or non-AAV ITRs. ITRs may be derived from the Parvoviridae family, which includes parvoviruses and dependoviruses (e.g., canine parvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus, human parvovirus B-19), or the SV40 hairpin that serves as the origin of SV40 replication may be used as an ITR, which may be further modified by truncation, substitution, deletion, insertion, and / or addition. Parvoviridae viruses consist of two subfamilies, the Parvovirinae subfamily that infect vertebrates, and the Densovirinae subfamily that infects invertebrates. Dependoparvoviruses include the adeno-associated virus (AAV) family of viruses that are capable of replication in vertebrate hosts, including but not limited to humans, primates, bovine, canine, equine, and ovine species.

[0043] As used herein, the term "asymmetric ITR" refers to a pair of ITRs in a single ceDNA genome or ceDNA vector that are not reverse-complementary throughout their entire length. The sequence difference between the two ITRs can be due to nucleotide addition, deletion, truncation, or point mutation. In one embodiment, one ITR of the pair can be a wild-type AAV sequence, and the other can be a non-wild-type or synthetic sequence. In another embodiment, neither ITR of the pair is a wild-type AAV sequence, and the two ITRs differ in sequence from each other. For convenience, the ITR located 5' (upstream of) the expression cassette in the ceDNA vector is referred to as the "5'ITR" or "left ITR", and the ITR located 3' (downstream of) the expression cassette in the ceDNA vector is referred to as the "3'ITR" or "right ITR".

[0044] As used herein, the term "ceDNA genome" refers to an expression cassette that further incorporates at least one inverted terminal repeat region. The ceDNA genome may further comprise one or more spacer regions. In some embodiments, the ceDNA genome is incorporated into a plasmid or virus genome as an intermolecular double-stranded polynucleotide of DNA.

[0045] As used herein, the term "ceDNA spacer region" refers to an intervening sequence that separates functional elements in a ceDNA vector or ceDNA genome. In some embodiments, the ceDNA spacer region holds two functional elements in a desired arrangement for optimal functionality. In some embodiments, the ceDNA spacer region provides or increases the genetic stability of the ceDNA genome, for example, in a plasmid or baculovirus. In some embodiments, the ceDNA spacer region facilitates easy genetic manipulation of the ceDNA genome by providing a convenient location for cloning sites, etc. For example, in certain aspects, an oligonucleotide "polylinker" containing several restriction endonuclease sites, or a non-open reading frame sequence designed to have no known protein (e.g., transcription factor) binding sites, can be positioned in the ceDNA genome to separate cis-acting elements, for example, inserting a 6mer, 12mer, 18mer, 24mer, 48mer, 86mer, 176mer, etc., between the terminal resolution site and the upstream transcriptional regulatory element. Similarly, a spacer can be incorporated between the polyadenylation signal sequence and the 3' terminal resolution site.

[0046] As used herein, "Rep binding site", "Rep binding element", "RBE", and "RBS" are used interchangeably and refer to a binding site of a Rep protein (e.g., AAV Rep78 or AAV Rep68) that, upon binding by the Rep protein, allows the Rep protein to carry out its site-specific endonuclease activity on a sequence incorporating the RBS. The RBS sequence and its reverse complement together form a single RBS. RBS sequences are known in the art and include, for example, the RBS sequence specified in AAV2, 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 531). Any known RBS sequence may be used in embodiments of the invention, including other known AAV RBS sequences and other naturally known or synthetic RBS sequences. Without being bound by theory, it is believed that the nuclease domain of the Rep protein binds to the double-stranded nucleotide sequence GCTC, and thus the two known AAV Rep proteins bind directly to the double-stranded oligonucleotide, 5'-(GCGC)(GCTC)(GCTC)(GCTC)-3' (SEQ ID NO:531), and stably assemble. In addition, the soluble aggregated conformers (i.e., an indefinite number of interrelated Rep proteins) dissociate and bind to oligonucleotides containing Rep binding sites. Each Rep protein interacts with both the nitrogenous bases and the phosphodiester backbone on each strand. The interactions with the nitrogenous bases provide sequence specificity, while the interactions with the phosphodiester backbone are non-sequence specific or low sequence specific, stabilizing the protein-DNA complex.

[0047] As used herein, the terms "terminal resolution site" and "TRS" are used interchangeably herein and refer to the region where Rep forms a tyrosine-phosphodiester bond with 5' thymidine that generates a 3'OH that serves as a substrate for DNA elongation via cellular DNA polymerases, such as DNA pol δ or DNA pol ε. Alternatively, the Rep-thymidine complex can participate in a coordinate ligation reaction. In some embodiments, the TRS minimally includes a non-base-paired thymidine. In some embodiments, the nicking efficiency of the TRS can be controlled at least in part by its distance within the same molecule from the RBS. When the acceptor substrate is a complementary ITR, the resulting product is an intermolecular duplex. TRS sequences are known in the art and include, for example, the hexanucleotide sequence 5'-GGTTGA-3' (SEQ ID NO: 45) identified in AAV2. Any known TRS sequence can be used in embodiments of the invention, including other known AAV TRS sequences, other naturally known 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).

[0048] As used herein, the term "ceDNA-plasmid" refers to a plasmid that contains a ceDNA genome as an intermolecular duplex.

[0049] As used herein, the term "ceDNA-bacmid" refers to an infectious baculovirus genome that contains a ceDNA genome as an intermolecular duplex that can be propagated as a plasmid in E. coli, thereby manipulating it as a shuttle vector for baculovirus.

[0050] As used herein, the term "ceDNA-baculovirus" refers to a baculovirus that contains a ceDNA genome as an intermolecular duplex within the baculovirus genome.

[0051] As used herein, the terms "ceDNA-baculovirus-infected insect cells" and "ceDNA-BIIC" are used interchangeably and refer to invertebrate host cells (including but not limited to insect cells (e.g., Sf9 cells)) infected with a ceDNA-baculovirus.

[0052] As used herein, "closed-end DNA vector," "ceDNA vector," and "ceDNA" are used interchangeably and refer to a non-viral capsid-free DNA vector (i.e., an intermolecular duplex) having at least one covalently closed end. In some embodiments, the ceDNA comprises two covalently closed ends.

[0053] As defined herein, a "reporter" refers to a protein that can be used to provide a detectable readout. Reporters generally produce a measurable signal, such as fluorescence, color, or luminescence. A reporter protein coding sequence encodes a protein whose presence in a cell or organism is easily observed. For example, fluorescent proteins cause cells to fluoresce when excited with a particular wavelength of light, luciferase causes cells to catalyze a reaction that produces light, and enzymes such as β-galactosidase convert a substrate into a colored product. Exemplary reporter polypeptides useful 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.

[0054] As used herein, the term "effector protein" refers to a polypeptide that provides a detectable readout, for example, as a reporter polypeptide, or more appropriately, as a polypeptide that kills a cell, such as a toxin, or an agent that makes the cell susceptible to killing with a selected agent or its deletion. Effector proteins include any protein or peptide that directly targets or damages the DNA and / or RNA of a host cell. For example, effector proteins include, but are not limited to, restriction endonucleases (whether genomic or extrachromosomal) that target host cell DNA sequences, proteases that target polypeptides required for cell survival, DNA gyrase inhibitors, and ribonuclease-type toxins. In some embodiments, the expression of effector proteins controlled by the synthetic biological circuits described herein can participate as a factor in another synthetic biological circuit, thereby expanding the scope and complexity of the reactivity of the biological circuit system.

[0055] Transcriptional regulators refer to transcriptional activators and repressors that activate or suppress the transcription of genes of interest. Promoters are regions of nucleic acids that initiate the transcription of specific genes. Transcriptional activators typically bind near transcriptional promoters and recruit RNA polymerase to directly initiate transcription. Repressors bind transcriptional promoters and sterically hinder the initiation of transcription by RNA polymerase. Other transcriptional regulators can serve as either activators or repressors, depending on where they bind and on cellular and environmental conditions. Non-limiting examples of transcriptional regulator classes include, but are not limited to, homeodomain proteins, zinc finger proteins, winged helix (forkhead) proteins, and leucine-zipper proteins.

[0056] As used herein, "repressor protein" or "inducer protein" refers to a protein that binds to a regulatory sequence element and represses or activates the transcription of the sequence operably linked to the regulatory sequence element, respectively.Preferred repressor and inducer proteins described herein are sensitive to the presence or absence of at least one input agent or environmental input.Preferred proteins described herein are modular in form, for example, comprising separable DNA binding and input agent binding or responsive elements or domains.

[0057] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharma- ceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the composition. The phrase "pharmaceutical acceptable" refers to molecular entities and compositions that do not produce toxic, allergic, or similar untoward reactions when administered to a host.

[0058] As used herein, an "input agent responsive domain" is a domain of a transcription factor that responds to a condition or input agent such that it binds to the condition or input agent or otherwise renders the linked DNA-binding fusion domain responsive to the presence of that condition or input. In one embodiment, the presence of the condition or input results in a conformational change in the input agent responsive domain or the protein to which it is fused, which modifies the transcriptional regulatory activity of the transcription factor.

[0059] The term "in vivo" refers to an assay or process that occurs in or within an organism, such as a multicellular animal. In some of the embodiments described herein, the method or use may be said to occur "in vivo" when a unicellular organism, such as a bacterium, is used. The term "ex vivo" refers to methods and uses carried out using live cells with intact membranes outside the body of a multicellular animal or plant, such as explants, cultured cells (including primary cells and cell lines), transformed cell lines, and extracted tissues or cells (including blood cells), among others. The term "in vitro" refers to assays and methods that do not require the presence of cells with intact membranes, such as cell extracts, and may refer to introducing a programmable synthetic biological circuit into a non-cellular system, such as a cell or cell system-free medium, such as a cell extract.

[0060] As used herein, the term "promoter" refers to any nucleic acid sequence that regulates the expression of another nucleic acid sequence by driving the transcription of the 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 thereof. A promoter is a control region of a nucleic acid sequence where the initiation and rate of transcription of the remainder of the nucleic acid sequence is controlled. Promoters can also contain genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind. In some embodiments of the aspects described herein, a promoter can drive the expression of transcription factors that regulate the expression of itself or of another promoter used in another modular component of the synthetic biological circuit described herein. Within the promoter sequence will be found the transcription initiation site, as well as protein binding domains involved in the binding of RNA polymerase. Eukaryotic promoters often, but not necessarily, contain "TATA" and "CAT" boxes. A variety of promoters, including inducible promoters, can be used to drive the expression of transgenes in the ceDNA vectors disclosed herein.

[0061] As used herein, the term "enhancer" refers to a cis-acting regulatory sequence (e.g., 50-1,500 base pairs) that binds to one or more proteins (e.g., activator proteins or transcription factors) to increase transcriptional activation of a 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 that they regulate. Enhancers can be located within intronic or exon regions of unrelated genes.

[0062] A promoter can be said to drive expression or drive transcription of the nucleic acid sequence it regulates. The phrases "operably linked," "operably positioned," "operably linked," "under control," and "under transcriptional control" indicate that the promoter is in the correct functional location and / or orientation with respect to a nucleic acid sequence and regulates to control transcription initiation and / or expression of that sequence. As used herein, an "inverted promoter" refers to a promoter in which a nucleic acid sequence is in an inverted orientation, whereby what was the coding strand is now the non-coding strand, and vice versa. Inverted promoter sequences are used in various embodiments to regulate the state of a switch. Additionally, in various embodiments, a promoter can be used in conjunction with an enhancer.

[0063] A promoter may be one that is naturally associated with a gene or sequence, which may be obtained by isolating the 5' non-coding sequence located upstream of the coding segment and / or exon of a given gene or sequence. Such a promoter may be referred to as "endogenous". Similarly, in some embodiments, an enhancer may be one that is naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence.

[0064] In some embodiments, the coding nucleic acid segment is placed under the control of a "recombinant promoter" or a "heterologous promoter", both of which refer to a promoter that is not normally associated with an operably linked encoded nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers to an enhancer that is not normally associated with a given nucleic acid sequence in its natural environment. 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.e., may contain different elements of different transcriptional regulatory regions, and / or mutations that alter expression through methods of genetic engineering known in the art. In addition to synthetically producing promoter and enhancer nucleic acid sequences, promoter sequences may be produced using recombinant cloning and / or nucleic acid amplification techniques, including PCR, for the synthetic biological circuits and modules disclosed herein (see, e.g., U.S. Pat. Nos. 4,683,202 and 5,928,906, each of which is incorporated herein by reference). Additionally, it is contemplated that control sequences that direct transcription and / or expression of sequences within non-nuclear organelles, such as mitochondria, chloroplasts, etc., can be used as well.

[0065] As described herein, an "inducible promoter" is one that is characterized by initiating or enhancing transcriptional activity when in the presence of, affected by, or contacted by an inducer or agent. As defined herein, an "inducer" or "inducing agent" can be endogenous or can be a compound or protein, usually exogenous, that is administered in such a way that it is active in inducing transcriptional activity from an inducible promoter. In some embodiments, the inducer or agent, i.e., a chemical, compound, or protein, can itself be the result of transcription or expression of a nucleic acid sequence (i.e., the inducer can be an inducer protein expressed by another component or module) and can itself be under the control of an inducible promoter. In some embodiments, an inducible promoter is induced in the absence of certain agents, such as repressors. Examples of inducible promoters include, but are not limited to, tetracycline, metallothionine, ecdysone, mammalian viruses (e.g., adenovirus late promoter, and mouse mammary tumor virus long terminal repeat (MMTV-LTR)), as well as other steroid-responsive promoters, rapamycin-responsive promoters, and the like.

[0066] As used herein, the term "subject" refers to a human or animal to which treatment, including prophylactic treatment, with the ceDNA vector according to the present invention is provided. Typically, the animal is a vertebrate, such as, but not limited to, a primate, a rodent, a domestic animal, or a game animal. Primates include, but are not limited to, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domestic and game animals include, but are not limited to, cattle, horses, pigs, deer, bison, buffalo, feline species, such as domestic cats, canine species, such as dogs, foxes, wolves, avian species, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon. In certain embodiments of the aspects described herein, the subject is a mammal, such as a primate or a human. The subject can be male or female. Additionally, the subject may be an infant or child. In some embodiments, the subject may be a neonatal or fetal subject, e.g., the subject is present in utero. Preferably, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Non-human mammals may be advantageously used as subjects that represent animal models of diseases and disorders. Additionally, the methods and compositions described herein may be used with domestic animals and / or pets. Human subjects may be of any age, sex, race, or ethnic group, e.g., Caucasian (white), Asian, African, Black, African American, African European, Hispanic, Middle Eastern, etc. In some embodiments, the subject may be a patient or other subject in a clinical setting. In some embodiments, the subject is already undergoing treatment.

[0067] As used herein, the term "antibody" is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. An "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the same antigen that the intact antibody binds. In one embodiment, an antibody or antibody fragment comprises an immunoglobulin chain or a fragment thereof, and at least one immunoglobulin variable domain sequence. Examples of antibodies and antibody fragments include, but are not limited to, Fv, scFv, Fab fragments, Fab', F(ab')2, Fab'-SH, single domain antibodies (dAbs), heavy chains, light chains, heavy and light chains, complete antibodies (e.g., including each of Fc, Fab, heavy chains, sequences, variable regions, etc.), bispecific antibodies, diabodies, linear antibodies, single chain antibodies, intrabodies, monoclonal antibodies, chimeric antibodies, multispecific antibodies, or multimeric antibodies. The antibody or antibody fragment may be of any class, including but not limited to IgA, IgD, IgE, IgG, and IgM, or any subclass, including but not limited to IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Additionally, the antibody may be from any mammal, such as a primate, human, rat, mouse, horse, goat, etc. In one embodiment, the antibody is human or humanized. In some embodiments, the antibody is a modified antibody. In some embodiments, the components of the antibody may be expressed separately such that the antibody itself assembles following expression of the protein components. In some embodiments, the antibody has a desired function, e.g., interaction and inhibition of a desired protein, for purposes of treating a disease or a symptom of a disease. In one embodiment, the antibody or antibody fragment is a fragment of the framework region or F. c Includes the area.

[0068] As used herein, the term "antigen-binding domain" of an antibody molecule refers to the portion of an antibody molecule, e.g., an immunoglobulin (Ig) molecule, that is involved in antigen binding. In embodiments, the antigen-binding site is formed by amino acid residues of the variable regions (V) of the heavy (H) and light (L) chains. Three highly branched stretches in the variable regions of the heavy and light chains, called hypervariable regions, are arranged between more conserved adjacent stretches called "framework regions" (FR). FRs are amino acid sequences naturally found between and adjacent to the hypervariable regions in immunoglobulins. In embodiments, in an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface, which is complementary to the three-dimensional surface of the bound antigen. The three hypervariable regions of each of the heavy and light chains are referred to as "complementarity determining regions" or "CDRs". Framework regions and CDRs are defined and described, for example, in Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment 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 (e.g., variable heavy chain and variable light chain) is typically formed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following amino acid order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0069] As used herein, the term "full length antibody" refers to an immunoglobulin (Ig) molecule (eg, an IgG antibody) that occurs in nature and is formed by normal immunoglobulin gene fragment recombinatorial processes.

[0070] As used herein, the term "functional antibody fragment" refers to a fragment that binds to the same antigen recognized by an intact (e.g., full-length) antibody. The term "antibody fragment" or "functional fragment" also includes isolated fragments consisting of the variable regions, such as an "Fv" fragment consisting of the variable regions of a recombinant single-chain polypeptide molecule in which the heavy and light chains, or the light and heavy chain variable regions, are connected by a peptide linker ("scFv protein"). In some embodiments, an antibody fragment does not include portions of an antibody without antigen-binding activity, such as an Fc fragment or a single amino acid residue.

[0071] As used herein, "immunoglobulin variable domain sequence" refers to an amino acid sequence that can form the structure of an immunoglobulin variable domain. For example, the sequence can include all or part of the amino acid sequence of a naturally occurring variable domain. For example, the sequence may or may not include one, two, or more N-terminal or C-terminal amino acids, or may include other modifications that correspond to the formation of a protein structure.

[0072] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and their respective component(s) that are essential to the method or composition, but are open to the inclusion of unspecified elements, whether essential or not.

[0073] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment.

[0074] The term "consisting of" refers to compositions, methods, and their respective components described herein, excluding any element not recited in the description of the embodiment.

[0075] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to a "method" includes one or more methods, and / or steps of the type described herein and / or that would be apparent to one of ordinary skill in the art upon reading this disclosure. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The abbreviation "eg" is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with "for example."

[0076] Except in the working examples or where otherwise indicated, all numbers expressing amounts of ingredients or reaction conditions used herein should be understood to be modified in all cases by the term "about". When used in relation to percentages, the term "about" can mean ±1%. The present invention is further described in detail by the following examples, but the scope of the present invention should not be limited thereto.

[0077] It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc. described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0078] Without limitation, the lipid nanoparticles of the present invention include lipid formulations that can be used to deliver capsid-free, non-viral DNA vectors to target sites of interest (e.g., cells, tissues, organs, etc.). In general, the lipid nanoparticles comprise a capsid-free, non-viral DNA vector and an ionized lipid or a salt thereof.

[0079] Thus, in some embodiments, the present disclosure provides lipid nanoparticles comprising ceDNA and ionized lipid. For example, lipid nanoparticles made and loaded with ceDNA obtained by the process of Example 1 or other methods disclosed herein. This can be achieved by high-energy mixing of ethanol lipids and aqueous ceDNA at low pH, which protonates ionized lipids and provides favorable energetics for ceDNA / lipid association and nucleation of particles. Particles can be further stabilized by aqueous dilution and removal of organic solvent. Particles can be concentrated to a desired level.

[0080] Generally, lipid particles are prepared with a total lipid to ceDNA (mass or weight) ratio of about 10:1 to 30:1. In some embodiments, the lipid to ceDNA ratio (mass / mass ratio, w / w ratio) can be in the range of about 1:1 to about 25:1, about 10:1 to about 14:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. The amount of lipid and ceDNA can be adjusted to provide a desired N / P ratio, for example, an N / P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or more. Generally, the total lipid content of the lipid particle formulation can be in the range of about 5 mg / mL to about 30 mg / mL.

[0081] Ionized lipids are typically used to condense nucleic acid cargo, such as ceDNA, at low pH and to drive membrane association and fusogenicity.Generally, ionized lipids are lipids that contain at least one amino group that is positively charged or protonated under acidic conditions, such as pH 6.5 or less.Ionized lipids are also referred to herein as cationic lipids.

[0082] Exemplary ionizable lipids are described in the PCT and US patent publications listed in Table 1, the contents of all of which are incorporated herein by reference in their entireties. [Table 1-1] [Table 1-2] [Table 1-3]

[0083] In some embodiments, the ionizable lipid has formula (X): [ka] It is a compound of the formula:

[0084] In some embodiments, the ionizable lipid has formula (I), as defined in US2015 / 0376115 or US2016 / 0376224, the contents of both of which are incorporated herein by reference in their entireties. [ka] It is a compound of the formula:

[0085] In some embodiments, the ionizable lipid has formula (I), as defined in US2016 / 0151284, the contents of which are incorporated herein by reference in their entirety. [ka] Or formula (II) [ka] Or formula (III) [ka] It is a compound of the formula:

[0086] In some embodiments, the ionizable lipid has formula (I), as defined in US2017 / 0210967, the contents of which are incorporated herein by reference in their entirety. [ka] or formula (IA) [ka] Or formula (II) [ka] or formula (IIA) [ka] It is a compound of the formula:

[0087] In some embodiments, the ionizable lipid has formula Ic, as defined in US2015 / 0140070, the contents of which are incorporated herein by reference in their entirety. [ka] It is a compound of the formula:

[0088] In some embodiments, the ionizable lipid has formula A, as defined in US2013 / 0178541, the contents of which are incorporated herein by reference in their entirety. [ka] It is a compound of the formula:

[0089] In some embodiments, the ionizable lipid has formula (I), as defined in US 2013 / 0303587 or US 2013 / 0123338, the contents of both of which are incorporated herein by reference in their entireties. [ka] It is a compound of the formula:

[0090] In some embodiments, the ionizable lipid has formula (I), as defined in US2015 / 0141678, the contents of which are incorporated herein by reference in their entirety. [ka] It is a compound of the formula:

[0091] In some embodiments, the ionizable lipid has formula (II), as defined in US 2015 / 0239926, the contents of which are incorporated herein by reference in their entirety. [ka] , formula (III) [ka] , formula (IV) [ka] or formula (V) [ka] It is a compound of the formula:

[0092] In some embodiments, the ionizable lipid has formula (I), as defined in US2017 / 0119904, the contents of which are incorporated herein by reference in their entirety. [ka] It is a compound of the formula:

[0093] In some embodiments, the ionizable lipid is a compound of formula (I) or formula (II), each having the structure: [ka] has.

[0094] In some embodiments, the ionizable lipid has formula A, as defined in US2012 / 0149894, the contents of which are incorporated herein by reference in their entirety. [ka] It is a compound of the formula:

[0095] In some embodiments, the ionizable lipid has formula A, as defined in US2015 / 0057373, the contents of which are incorporated herein by reference in their entirety. [ka] It is a compound of the formula:

[0096] In some embodiments, the ionizable lipid has formula A, as defined in WO2013 / 116126, the contents of which are incorporated herein by reference in their entirety. [ka] It is a compound of the formula:

[0097] In some embodiments, the ionizable lipid has formula A, as defined in US2013 / 0090372, the contents of which are incorporated herein by reference in their entirety. [ka] It is a compound of the formula:

[0098] In some embodiments, the ionizable lipid has formula A, as defined in US 2013 / 0274523, the contents of which are incorporated herein by reference in their entirety. [ka] It is a compound of the formula:

[0099] In some embodiments, the ionizable lipid has formula A, as defined in US2013 / 0274504, the contents of which are incorporated herein by reference in their entirety. [ka] It is a compound of the formula:

[0100] In some embodiments, the ionizable lipid has formula A, as defined in US2013 / 0053572, the contents of which are incorporated herein by reference in their entirety. [ka] It is a compound of the formula:

[0101] In some embodiments, the ionizable lipid has formula A, as defined in WO2013 / 016058, the contents of which are incorporated herein by reference in their entirety. [ka] It is a compound of the formula:

[0102] In some embodiments, the ionizable lipid has formula A, as defined in WO2012 / 162210, the contents of which are incorporated herein by reference in their entirety. [ka] It is a compound of the formula:

[0103] In some embodiments, the ionizable lipid has formula (I), as defined in US 2008 / 042973, the contents of which are incorporated herein by reference in their entirety. [ka] It is a compound of the formula:

[0104] In some embodiments, the ionizable lipid has formula (I), as defined in US2012 / 01287670, the contents of which are incorporated herein by reference in their entirety. [ka] , formula (II) [ka] , formula (III) [ka] or formula (IV) [ka] It is a compound of the formula:

[0105] In some embodiments, the ionizable lipid is a compound of formula (I) or formula (II), each having the structure: [ka] has.

[0106] In some embodiments, the ionizable lipid is a compound of Formula (I), Formula (II), or Formula (III), each having the structure: [ka] has.

[0107] In some embodiments, the ionizable lipid has formula (I), as defined in US2015 / 0005363, the contents of which are incorporated herein by reference in their entirety. [ka] Or formula (III) [ka] It is a compound of the formula:

[0108] In some embodiments, the ionizable lipid has formula (I), as defined in US2014 / 0308304, the contents of which are incorporated herein by reference in their entirety. [ka] , formula (IA) [ka] , formula (IB) [ka] , formula (IC) [ka] , expression(ID) [ka] , formula (II) [ka] , formula (IIA) [ka] , formula (IIB) [ka] , formula (IIC) [ka] , formula (IID), or a compound of formula (III)-(XXIV).

[0109] In some embodiments, the ionizable lipid has the formula: [ka] It is a compound of the formula:

[0110] In some embodiments, the ionizable lipid has formula (I), as defined in WO2009 / 132131, the contents of which are incorporated herein by reference in their entirety. [ka] , formula (II) [ka] , formula (III) [ka] or formula (IV) [ka] It is a compound of the formula:

[0111] In some embodiments, the ionizable lipid has formula A, as defined in US2012 / 01011478, the contents of which are incorporated herein by reference in their entirety. [ka] It is a compound of the formula:

[0112] In some embodiments, the ionizable lipid has formula (I), as defined in US 2012 / 0027796, the contents of which are incorporated herein by reference in their entirety. [ka] or formula (XXXV) [ka] It is a compound of the formula:

[0113] In some embodiments, the ionizable lipid has formula (XIV), as defined in US2012 / 0058144, the contents of which are incorporated herein by reference in their entirety. [ka] or formula (XVII) [ka] It is a compound of the formula:

[0114] In some embodiments, the ionizable lipid has the formula: [ka] ,or [ka] It is a compound of the formula:

[0115] In some embodiments, the ionizable lipid has formula (I), as defined in US2011 / 0117125, the contents of which are incorporated herein by reference in their entirety. [ka] It is a compound of the formula:

[0116] In some embodiments, the ionizable lipid has formula (I), as defined in US 2011 / 0256175, the contents of which are incorporated herein by reference in their entirety. [ka] , formula (II) [ka] or formula (III)- [ka] It is a compound of the formula:

[0117] In some embodiments, the ionizable lipid has formula (I), as defined in US2012 / 0202871, the contents of which are incorporated herein by reference in their entirety. [ka] , formula (II) [ka] , formula (III) [ka] , formula (IV) [ka] , formula (V) [ka] , formula (VI) [ka] , formula (VII) [ka] , formula (VIII) [ka] , formula (IX) [ka] , formula (X) [ka] , -Formula (XI) [ka] or formula (XII) [ka] It is a compound of the formula:

[0118] In some embodiments, the ionizable lipid has formula (I), as defined in US 2011 / 0076335, the contents of which are incorporated herein by reference in their entirety.

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[0119] In some embodiments, the ionizable lipid has formula (I), as defined in US 2006 / 008378, the contents of which are incorporated herein by reference in their entirety. [ka] Or formula (II) [ka] It is a compound of the formula:

[0120] In some embodiments, the ionizable lipid has formula (I), as defined in US 2013 / 0123338, the contents of which are incorporated herein by reference in their entirety. [ka] It is a compound of the formula:

[0121] In some embodiments, the ionizable lipid is a compound of formula (I), XAYZ, as defined in US2015 / 0064242, the contents of which are incorporated herein by reference in their entirety.

[0122] In some embodiments, the ionizable lipid has formula (XVIX), as defined in US2013 / 0022649, the contents of which are incorporated herein by reference in their entirety. [ka] , formula (XVII) [ka] or formula (XVIII) [ka] It is a compound of the formula:

[0123] In some embodiments, the ionizable lipid has formula (I), as defined in US2013 / 0116307, the contents of which are incorporated herein by reference in their entirety. [ka] , formula (II) [ka] or formula (III) [ka] It is a compound of the formula:

[0124] In some embodiments, the ionizable lipid has formula (I), as defined in US 2010 / 0062967, the contents of which are incorporated herein by reference in their entirety. [ka] Or formula (II) [ka] It is a compound of the formula:

[0125] In some embodiments, the ionizable lipid is a compound of formula (I)-(X), each having the structure as defined in US 2013 / 0189351, the contents of which are incorporated herein by reference in their entirety. [ka] has.

[0126] In some embodiments, the ionizable lipid has formula (I), as defined in US 2014 / 0039032, the contents of which are incorporated herein by reference in their entirety. [ka] It is a compound of the formula:

[0127] In some embodiments, the ionizable lipid has formula (V): [ka] It is a compound of the formula:

[0128] In some embodiments, the ionizable lipid has formula (I), as defined in US2016 / 0317458, the contents of which are incorporated herein by reference in their entirety. [ka] It is a compound of the formula:

[0129] In some embodiments, the ionizable lipid has formula (I), as defined in US2013 / 0195920, the contents of which are incorporated herein by reference in their entirety. [ka] It is a compound of the formula:

[0130] In some embodiments, the ionizable lipid is MC3(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoic acid (DLin-MC3-DMA or MC3), described in Example 9.

[0131] In some embodiments, the ionizable lipid is the lipid ATX-002, described in Example 10.

[0132] In some embodiments, the ionizable lipid is (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine (compound 32), described in Example 11.

[0133] In some embodiments, the ionizable lipid is compound 6 or compound 22 described in Example 12.

[0134] Without limitation, the ionized lipids may comprise 20-90% (mol) of the total lipids present in the lipid nanoparticles. For example, the ionized lipid molar content may be 20-70% (mol), 30-60% (mol), or 40-50% (mol) of the total lipids present in the lipid nanoparticles. In some embodiments, the ionized lipids comprise about 50 mol% to about 90 mol% of the total lipids present in the lipid nanoparticles.

[0135] In some embodiments, lipid nanoparticles can further comprise non-cationic lipid.Non-ionic lipid includes amphipathic lipid, neutral lipid, and anionic lipid.Thus, non-cationic lipid can be neutral uncharged, zwitterionic, or anionic lipid.Non-cationic lipid is typically used to enhance membrane fusogenicity.

[0136] Exemplary non-cationic lipids include distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidyl dioleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (e.g. 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (e.g. 16-O-dimethyl PE), 18 -1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), diercoyl phosphatidylcholine (DEPC), palmitoyl oleoyl phosphatidyl phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof.It will be appreciated that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids may also be used. The acyl groups in these lipids are preferably C. 10 ~C 24 The acyl group is derived from a fatty acid having a carbon chain, for example, lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0137] Other examples of non-cationic lipids suitable for use in lipid nanoparticles include non-phosphorous lipids such as, for example, stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramides, sphingomyelin, and the like.

[0138] In some embodiments, non-cationic lipid is phospholipid.In some embodiments, non-cationic lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM.In some preferred embodiments, non-cationic lipid is DPSC.

[0139] Exemplary non-cationic lipids are described in PCT Publication No. WO2017 / 099823 and U.S. Patent Publication No. US2018 / 0028664, the contents of both of which are incorporated by reference in their entireties.

[0140] In some embodiments, the non-cationic lipid is oleic acid or a cyclic ester of formula (I) as defined in US 2018 / 0028664, the contents of which are incorporated herein by reference in their entirety. [ka] , formula (II) [ka] or formula (IV) [ka] It is a compound of the formula:

[0141] The non-cationic lipid may comprise 0-30% (mol) of the total lipid present in the lipid nanoparticle. For example, the non-cationic lipid content is 5-20% (mol) or 10-15% (mol) of the total lipid present in the lipid nanoparticle. In various embodiments, the molar ratio of ionized lipid to neutral lipid ranges from about 2:1 to about 8:1.

[0142] In some embodiments, the lipid nanoparticles do not contain any phospholipids.

[0143] In some aspects, the lipid nanoparticles may further comprise components such as sterols to provide membrane integrity.

[0144] One exemplary sterol that can be used in lipid nanoparticles 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-cholestan, cholestenone, 5α-cholestanone, 5β-cholestanone, and cholesteryl decanoate, and mixtures thereof.In some embodiments, cholesterol derivative is polar analogs such as cholesteryl-(4'-hydroxy)-butyl ether.

[0145] Exemplary cholesterol derivatives are described in PCT Publication No. WO2009 / 127060 and U.S. Patent Publication No. US2010 / 0130588, the contents of both of which are incorporated herein by reference in their entireties.

[0146] Components that provide membrane integrity, such as sterols, may comprise 0-50% (mol) of the total lipids present in the lipid nanoparticles, in some embodiments, such components comprise 20-50% (mol), 30-40% (mol) of the total lipid content of the lipid nanoparticles.

[0147] In some aspects, lipid nanoparticles may further comprise polyethylene glycol (PEG) or conjugated lipid molecules. These are generally used to inhibit lipid nanoparticle aggregation and / or provide steric stabilization. Exemplary conjugated lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic-polymer lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, such as (methoxypolyethylene glycol)-conjugated lipid.

[0148] Exemplary PEG-lipid conjugates include PEG-diacylglycerol (DAG) (1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG diacylglycerol succinate (PEGS-DAG) (4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbamate, N-(carbonyl-methoxypolyethylene glycol) dialkyloxypropyl ... Examples of PEG-lipid conjugates include, but are not limited to, glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or mixtures thereof. Additional exemplary 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, US2016 / 0376224, US2017 / 0119904, and US / 099823, the contents of all of which are incorporated herein by reference in their entirety.

[0149] In some embodiments, the PEG-lipid has formula (III), as defined in US2018 / 0028664, the contents of which are incorporated herein by reference in their entirety. [ka] , formula (III-aI) [ka] , Formula (III-a-2) [ka] , formula (III-b-1) [ka] , formula (III-b-2) [ka] or formula (V) [ka] It is a compound of the formula:

[0150] In some embodiments, the PEG-lipid has formula (II), as defined in US2015 / 0376115 or US2016 / 0376224, the contents of both of which are incorporated herein by reference in their entireties. [ka] That is why.

[0151] The PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can be PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol (1-[8'-(cholest-5-en-3[β]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl-[ω]-methyl-poly(ethylene glycol). ), PEG-DMB (3,4-ditetradecoxylbenzyl-[ω]-methyl-poly(ethylene glycol) ether), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some examples, the PEG-lipid is one or more of PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], [ka] may be selected from the group consisting of:

[0152] Lipids conjugated with molecules other than PEG can also be used in place of PEG-lipids, for example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic-polymeric lipid (CPL) conjugates can be used in place of or in addition to PEG-lipids.

[0153] Exemplary conjugated lipids, i.e., PEG-lipid, (POZ)-lipid conjugates, ATTA-lipid conjugates, and cationic polymer-lipids, are described in the PCT and U.S. patent applications listed in Table 2, the contents of all of which are incorporated herein by reference in their entireties. [Table 2-1] [Table 2-2]

[0154] The PEG or conjugated lipid may comprise 0-20% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, the PEG or conjugated lipid content is 0.5-10% or 2-5% (mol) of the total lipid present in the lipid nanoparticle.

[0155] The molar ratios of ionizable lipid, non-cationic lipid, sterol, and PEG / complex lipid may vary as needed. For example, the lipid particles may comprise 30-70% ionizable lipid by molar or total weight of the composition, 0-60% cholesterol by molar or total weight of the composition, 0-30% non-cationic lipid by molar or total weight of the composition, and 1-10% complex lipid by molar or total weight of the composition. Preferably, the composition comprises 30-40% ionizable lipid by molar or total weight of the composition, 40-50% cholesterol by molar or total weight of the composition, and 10-20% non-cationic lipid by molar or total weight of the composition. In some other embodiments, the composition comprises 50-75% ionizable lipid by molar or total weight of the composition, 20-40% cholesterol by molar or total weight of the composition, 5-10% non-cationic lipid by molar or total weight of the composition, and 1-10% complex lipid by molar or total weight of the composition. The composition may contain 60-70% ionizable lipid by molar or total weight of the composition, 25-35% cholesterol by molar or total weight of the composition, and 5-10% non-cationic lipid by molar or total weight of the composition. The composition may also contain up to 90% ionizable lipid by molar or total weight of the composition, and 2-15% non-cationic lipid by molar or total weight of the composition.Formulations may also be used that contain, for example, 8-30% ionizable lipid by molar or total weight of the composition, 5-30% noncationic lipid by molar or total weight of the composition, and 0-20% cholesterol by molar or total weight of the composition; 4-25% ionizable lipid by molar or total weight of the composition, 4-25% noncationic lipid by molar or total weight of the composition, 2-25% cholesterol by molar or total weight of the composition, 10-35% complex lipid by molar or total weight of the composition, and 5% cholesterol by molar or total weight of the composition; or or may be lipid nanoparticles comprising 2-30% by molar or total weight of ionized lipid, 2-30% by molar or total weight of non-cationic lipid, 1-15% by molar or total weight of cholesterol, 2-35% by molar or total weight of complex lipid, and 1-20% by molar or total weight of cholesterol; or even up to 90% by molar or total weight of ionized lipid, and 2-10% by molar or total weight of non-cationic lipid; or even 100% by molar or total weight of cationic lipid. In some embodiments, the lipid particle formulation comprises ionized lipid, phospholipid, cholesterol, and PEGylated lipid in a molar ratio of 50:10:38.5:1.5. In some other embodiments, the lipid particle formulation comprises ionized lipid, cholesterol, and PEGylated lipid in a molar ratio of 60:38.5:1.5.

[0156] In some embodiments, the lipid particles comprise an ionizable lipid, a non-cationic lipid (e.g., a phospholipid), a sterol (e.g., cholesterol), and a PEGylated lipid, with the molar ratios of lipids ranging from 20-70 mole percent for the ionizable lipid (target 40-60), the molar percent of the non-cationic lipid ranging from 0-30 (target 0-15), the molar percent of the sterol ranging from 20-70 (target 30-50), and the molar percent of the PEGylated lipid ranging from 1-6 (target 2-5).

[0157] In some embodiments, the lipid particles comprise ionizable lipid / non-cationic lipid / sterol / complex lipid in a molar ratio of 50:10:38.5:1.5.

[0158] In another aspect, the disclosure provides a lipid nanoparticle formulation comprising a phospholipid, a lecithin, a phosphatidylcholine, and a phosphatidylethanolamine.

[0159] In some embodiments, one or more additional compounds may also be included. These compounds may be administered separately, or the additional compounds may be included in the lipid nanoparticles of the present invention. In other words, the lipid nanoparticles may contain other compounds in addition to ceDNA, or at least a second ceDNA that is different from the first one. Without limitation, the 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 their derivatives, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, extracts made from biological materials, or any combination thereof.

[0160] In some embodiments, the one or more additional compounds may be therapeutic agents. The therapeutic agents may be selected from any class suitable for therapeutic purposes. In other words, the therapeutic agents may be selected according to the desired therapeutic purpose and biological action. For example, if the ceDNA in the LNPs is useful for treating cancer, the additional compound may be an anti-cancer agent (e.g., a chemotherapeutic agent, a targeted cancer therapy (including, but not limited to, a small molecule, an antibody, or an antibody-drug conjugate). In another example, if the LNPs containing ceDNA are useful for treating an infectious disease, the additional compound may be an antibacterial agent (e.g., an antibiotic or an antiviral compound). In yet another example, if the LNPs containing ceDNA are useful for treating an immune disease or disorder, 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, different cocktails of different lipid nanoparticles containing different compounds, such as ceDNAs encoding different proteins or different compounds (such as therapeutic agents), may be used in the compositions and methods of the invention.

[0161] In some embodiments, the additional compound is an immunomodulatory agent, e.g., the additional compound is an immunosuppressant, hi some embodiments, the additional compound is immunostimulatory.

[0162] Exemplary immunomodulatory agents 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 analogs (lenalidomide, pomalidomide, and apremilast), other immunomodulatory agents (including, but not limited to, cytosine-guanosine phosphate, oligodeoxynucleotides, glucans).

[0163] In some embodiments, the immunomodulatory agent may be an immunosuppressant. Exemplary immunosuppressants include, but are not limited to, glucocorticoids, cytostatics, antibodies, drugs acting on immunophilins, and other drugs. Glucocorticoids include, but are not limited to, prednisone, dexamethasone, and hydrocortisone. Examples of cytostatic agents include alkylating agents, such as nitrogen mustards (e.g., cyclophosphamide), nitrosoureas, and platinum compounds. Cytostatic agents may also include antimetabolites, such as formic acid analogs (e.g., methotrexate), purine analogs (e.g., azathioprine and mercaptopurine), pyrimidine analogs (e.g., fluorouracil), and protein synthesis inhibitors. Other cytostatic agents include cytotoxic antibiotics, such as dactinomycin, anthracyclines, mitomycin C, bleomycin, and mithramycin.

[0164] Antibodies for immunosuppression include, but are not limited to, Atgam obtained from horse serum, and thymoglobulin, antibodies directed against the IL-2 receptor-(CD25-), and / or CD3-directed antibodies, MUROMONAB-CD3™ (Orthoclone OKT3), basiliximab (SIMULECT™), daclizumab (ZENAPAX™), and muromonab.

[0165] Drugs that act on immunophilins include, but are not limited to, cyclosporine, tacrolimus, rapamycin (SIROLIMUS™), and everolimus. Examples of biologics include abatacept, anakinra, certolizumab, golimumab, ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinumab, and vedolizumab.

[0166] Other drugs useful as immunomodulators or immunosuppressants include interferons (e.g., IFN-β), opioids, TNF binding proteins (e.g., TNF-α (tumor necrosis factor-α) binding proteins, infliximab (REMICADE™), etanercept (ENBREL™), or adalimumab (HUMIRA™), curcumin (a component in turmeric) and catechin (in green tea), mycophenolate, fingolimod, myriocin, antiproliferative agents (e.g., myriocin, tacrolimus, mycophenolate, rifabutin ... These include, but are not limited to, rituximab, secukinumab, tocilizumab, ustekinumab, and vedolizumab, mTOR inhibitors (e.g., sirolimus and everolimus), calcineurin inhibitors (e.g., cyclosporine and tacrolimus), IMDS inhibitors (e.g., azathioprine, leflunomide, and mycophenolate), fingolmod, abatacept, anakinra, certolizumab, golimumab, ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinumab, and vedolizumab.

[0167] In some embodiments, the immunosuppressant useful in the compositions and methods disclosed herein may be selected from one of the following compounds: mycophenolic acid, cyclosporine, azathioprine, tacrolimus, cyclosporine A, FK506, rapamycin, leflunomide, deoxyspergualin, prednisone, azathioprine, mycophenolate mofetil, OKT3, ATAG, or mizoribine.

[0168] In certain embodiments, the immunosuppressant is Prednisone, Methylprednisolone, Kenalog, Medrol (oral), Medrol(Pak) (oral), Depo-Medrol (injection), Prednisolone (oral), Solu-Medrol (injection), Hydrocortisone (oral), Cortef (oral), Solu-Medrol (IV), Cortisone (oral), Celestone Soluspan (injection), Orapred ODT (oral), Orapred (oral), Prelone (oral), Methylprednisolone (injection), Methylprednisolone acetate (injection), Prednisone Intensol (oral), Betamethasone acetate and sodium phosphate (injection), Veripred, Celestone (oral), Methylprednisolone sodium succinate (IV), Methylprednisolone sodium succinate (injection), Millipred (oral), Solu-Medrol (PF) (injection), Solu-Cortef (injection), Aristospan (intra-articular injection), Hydrocortisone sodium succinate (injection), Prednisolone sodium phosphate (oral), Methylprednisolone sodium succinate (PF) (IV), Solu-Medrol (PF) (IV), Triamcinolone hexacetonide (injection), A-Hydrocortisone (injection), A-Methapred (injection), Millipred DP (oral), Flo-Pred (oral), Aristospan (intralesional injection), betamethasone (oral), methylprednisolone sodium succinate (PF) (injection), hydrocortisone sodium succinate (PF) (injection), Solu-Cortef (PF) (injection), prednisolone acetate (oral), dexamethasone (IV) in 0.9% NaCl, Rayos, levothyroxine. Naturally, this list should not be considered exhaustive or limiting, so that immunosuppressants known to those skilled in the art can be easily substituted.

[0169] The immunosuppressant may result in a reduction in immune cells in a subject, for example, a reduction in immune cells expressing at least one or more of CD11b, CD4, CD8, and / or a reduction in pro-inflammatory cytokines selected from, but not limited to, TNFα or MCP-1. In some embodiments, the proinflammatory cytokine is a cytokine, lymphokine, monokine, stem cell growth factor, lymphotoxin, hematopoietic factor, colony stimulating factor (CSF), interferon (IFN), parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), hepatic 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, Mullerian inhibitory substance (MIS), mouse gonadotropin (MG), or a combination thereof. The antibodies are selected from any one or combination of pin-associated peptides, inhibins, activins, vascular endothelial growth factors, integrins, interleukins (ILs), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF), interferon-alpha, interferon-beta, interferon-gamma, S1 factor, IL-1, IL-1cc, 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.

[0170] In some embodiments, the pro-inflammatory cytokine may be selected from any or a combination of interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8), interferon-gamma (IFN-γ), vascular endothelial growth factor (VEGF), leukemia inhibitory factor (LIF), monocyte chemoattractant protein-1 (MCP-1), RANTES, interleukin-10 (IL-10), interleukin-12 (IL-12), matrix metalloproteinase 2 (MMP2), IP-10, macrophage inflammatory protein 1 alpha (MIP1α), and / or macrophage inflammatory protein 1 beta (MIP1β).

[0171] In some embodiments, the immunomodulatory agent is an NLRP3 agonist. Exemplary NLRP3 agonists include imadazoquinolines; imidazonaphthyridines; pyrazolopyridines; aryl-substituted imidazoquinolines; compounds with 1-alkoxy 1H-imidazo ring systems; oxazolo[4,5-c]-quinolin-4-amines; thiazolo[4,5-c]-quinolin-4-amines; selenazolo[4,5-c]-quinolin-4-amines; imidazonaphthyridines, imidazoquinolins, 1H-Imidazo[4,5-C]quinolin-4-amine;1-substituted, 2-substituted 1H-imidazo[4,5-C]quinolin-4-amine;Fused cycloalkylimidazopyridines;1H-Imidazo[4,5-c]quinolin-4-amine;1-substituted 1H-imidazo-[4,5-c]quinolin-4-amine;Imidazo-[4,5-C]quinolin-4-amine;2-Ethyl 1H-imidazo[4,5-xyquinoline-4-amine;Olefin 1H-imidazo[4,5 -c]quinolin-4-amines; 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]quinolizine; substituted pyridoquinoxaline-6-carboxylic acid; 7-hydroxy-benzo[ij]quinolizine-2-carboxylic acid derivatives; substituted benzo[ij]quinolizine-2-carboxylic acid; 7-hydroxy-benzo[ij]quinolizine-2-carboxylic acid; substituted pyrido[1,2,3,-de]-1,4-benzoxazines; and N-methylenemalonates of tetrahydroquinolines. In some embodiments, the NLRP3 agonist is a compound of the formula: [ka] It is a compound of the formula:

[0172] In some embodiments, the immunomodulatory agent is a TLR7 and / or TLR8 ligand. In some embodiments, the immunomodulatory agent is imiquimod (1-isobutyl-1H-imidazo[4,5-c]quinolin-4-amine) or resiquimod.

[0173] In some embodiments, the immunomodulatory agent is of the formula: [ka] It is an imquidazole quinoline compound.

[0174] In some embodiments, the immunomodulatory agent is a SMAD7 modulator. For example, the SMAD7 modulator can be a SMAD7 antisense oligonucleotide (AON), as defined in WO2017 / 059225A1, the contents of which are incorporated herein by reference in their entirety.

[0175] The immunomodulatory agent may be a TLR modulator. For example, the immunomodulatory agent may be a TLR3, TLR4, TLR7, TLR8, or TLR9 modulator, such as a TLR3, TLR4, TLR7, TLR8, or TLR9 agonist, or a TLR3, TLR4, TLR7, TLR8, or TLR9 antagonist. In some embodiments, the TLR modulator is a TLR3 modulator. In some embodiments, the TLR modulator is a TLR4 modulator. In some embodiments, the TLR modulator is a TLR7 modulator. In some embodiments, the TLR modulator is a TLR8 modulator. In some embodiments, the TLR modulator is a TLR9 modulator. In some embodiments, the TLR modulator is a TLR3 agonist. In some embodiments, the TLR modulator is a TLR4 agonist. In some embodiments, the TLR modulator is a TLR7 agonist. In some embodiments, the TLR modulator is a TLR8 agonist. In some embodiments, the TLR modulator is a TLR9 agonist. In some embodiments, the TLR modulator is a TLR3 antagonist. In some embodiments, the TLR modulator is a TLR4 antagonist. In some embodiments, the TLR modulator is a TLR7 antagonist. In some embodiments, the TLR modulator is a TLR8 antagonist. In some embodiments, the TLR modulator is a TLR9 antagonist. In some embodiments, the TLR modulator described herein can modulate two or more TLRs. In some embodiments, the TLR modulator can activate one or more TLRs and inhibit one or more TLRs. In some embodiments, the TLR modulator is a TLR9 modulator, such as KAPPAPROCT® or MONARSEN®. Some exemplary TLR modulators are described, for example, in WO2017 / 059225A1.

[0176] In some embodiments, the immunomodulatory agent is a CpG-A or Cpg-B oligonucleotide, as described in WO2017 / 059225A1.

[0177] Cytosolic detection of pathogen-derived DNA requires signaling through TANK-binding kinase 1 (TBK1) and its downstream transcription factor IFN regulatory factor 3 (IRF3). A transmembrane protein called STING (also known as stimulator of IFN genes, MITA, ERIS, MPYS, and TMEM173) serves as a signaling receptor for these cyclic purine dinucleotides, leading to stimulation of the TBK1-IRF3 signaling axis and STING-dependent type I interferon responses. Thus, in some embodiments, the immunomodulatory agent is a STING-modulating agent. STING binds regulated to cyclic diguanylate monophosphate but not to other unrelated nucleotides or nucleic acids. Thus, in some embodiments, the STING-modulating agent is a cyclic purine dinucleotide. Exemplary cyclic purine dinucleotides and STING-modulating agents are described, for example, in US9549944B2, the contents of which are incorporated herein by reference in their entirety.

[0178] Additional exemplary immunosuppressants include 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-linked receptor agonists; G -protein-linked receptor antagonists;histone deacetylase inhibitors;histone deacetylase inhibitors, e.g. trichostatin A;IMDH inhibitors, e.g. azathioprine, leflunomide, and mycophenolate;infliximab;inhibitors of mitochondrial function, e.g. rotenone;ixekizumab;kinase inhibitors;methylprednisolone;monoclonal antibodies, e.g. basiliximab, daclizumab, and muromonab;mTOR inhibitors, e.g. rapamycin or rapamycin analogues, sirolimus, and everolimus;natalizumab;NF-κβ inhibitors, e.g. 6Bio, dexamethasone, TCPA-1, IKK VII; OTK3; oxidized ATP, e.g., P2X receptor blockers; P38 inhibitors; peroxisome proliferator-activated receptor agonists; peroxisome proliferator-activated receptor antagonists; phosphatase inhibitors; phosphodiesterase inhibitors, e.g., phosphodiesterase 4 inhibitors (PDE4), e.g., rolipram; PI3 KB inhibitors, e.g., TGX-221; prostaglandin E2 agonists (PGE2), e.g., misoprostol; proteasome inhibitor I (PSI); proteasome inhibitors; retinoids; rituximab; secukinumab; statins; TGF-β receptor agonists; TGF-β signaling agents; thymoglobulins; TLR9 antagonists; tocilizumab; ustekinumab; and vedolizumab.Immunosuppressants can also include IDO, vitamin D3, cyclosporines, such as cyclosporine A, aryl hydrocarbon receptor inhibitors, resveratrol, azathioprine (Aza), 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), FK506, sanglifehrin A, salmeterol, mycophenolate mofetil (MMF), aspirin, and other COX inhibitors, niflumic acid, estriol, and triptolide, interleukins (e.g., IL-1, IL-10), cyclosporine A, siRNA targeting cytokines or cytokine receptors, and the like.

[0179] In some embodiments, the immunomodulatory agent is 3-methyladenine, 6-Bio, 6-mercaptopurine (6-MP, 6-thioguanine (6-TG), FK506, sanglifehrin 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 linked receptor agonists, G-protein linked 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 Selected from the group consisting of KB inhibitors, prednisolone, prednisone, proteasome inhibitor I (PSI), proteasome inhibitors, rapamycin and rapamycin analogs, 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 thereof.

[0180] It should be noted that any one of the immunomodulatory agents described herein can be used with the lipid nanoparticles, ceDNA vectors, and / or compositions disclosed herein. For example, the immunomodulatory agent can be used alone or combined with one or more (e.g., 1, 2, 3, 4, 5, or more) other immunomodulatory agents described herein.

[0181] In some embodiments, the immunomodulatory agent is selected from the group consisting of aryl hydrocarbon receptor inhibitors, caspase-1 inhibitors, cGAS inhibitors, cytokine inhibitors, G-protein-linked receptor agonists, G-protein-linked receptor antagonists, inhibitors of mitochondrial function, mTOR inhibitors, NF-κβ inhibitors, peroxisome proliferator-activated receptor agonists, phosphatase inhibitors, phosphodiesterase inhibitors, TGX-221, prostaglandin E2 agonists (PGE2), TLR9 antagonists, proteasome inhibitors, TGF-β receptor agonists, and TGF-β signaling agents. Any one or more of the above agents can be used alone or in combination with the LNPs containing ceDNA.

[0182] Also provided herein are pharmaceutical compositions comprising the lipid nanoparticles and a pharma- ceutically acceptable carrier or excipient.

[0183] In some aspects, the present disclosure provides lipid nanoparticle formulations further comprising one or more pharmaceutical excipients. In some embodiments, the lipid nanoparticle formulation further comprises sucrose, tris, trehalose, and / or glycine.

[0184] Some exemplary LNP features In general, the lipid nanoparticles of the present invention have an average diameter selected to provide the intended therapeutic effect. Thus, in some embodiments, the lipid nanoparticles have an average diameter of about 30 nm to about 150 nm, more typically about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 85 nm to about 105 nm, and preferably about 100 nm. In some embodiments, the present disclosure provides lipid particles that are larger in relative size to typical nanoparticles, about 150 to 250 nm in size. Lipid nanoparticle particle size can be determined, for example, by quasi-elastic light scattering using a Malvern Zetasizer ZS (Malvern, UK) system.

[0185] Depending on the intended use of the lipid particle, the ratio of the components may vary, and the delivery efficiency of a particular formulation may be measured using, for example, an endosomal release parameter (ERP) assay.

[0186] The ceDNA can be complexed with the lipid portion of the particle or can be encapsulated in the lipid location of the lipid nanoparticle. In some embodiments, the ceDNA can be completely encapsulated in the lipid location of the lipid nanoparticle, thereby protecting it from degradation by nucleases, for example, in aqueous solution. In some embodiments, the ceDNA in the lipid nanoparticle is not substantially degraded after exposure of the lipid nanoparticle to nucleases for at least about 20, 30, 45, or 60 minutes at 37°C. In some embodiments, the ceDNA in the lipid nanoparticle is not substantially degraded after incubation of the particle in serum for at least about 30, 45, or 60 minutes at 37°C, or for at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours.

[0187] In certain embodiments, the lipid nanoparticles are substantially non-toxic to a subject, e.g., a mammal, such as a human.

[0188] In some aspects, the lipid nanoparticle formulation is a lyophilized powder.

[0189] In some embodiments, the lipid nanoparticles are solid core particles with at least one lipid bilayer. In other embodiments, the lipid nanoparticles have a non-bilayer structure, i.e., a non-lamellar (i.e., non-bilayer) morphology. Without limitation, non-bilayer morphology can include, for example, three-dimensional tube, rod, cubic symmetry, etc. The non-lamellar morphology (i.e., 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, cryo-transmission electron microscopy ("Cryo-TEM"), differential scanning calorimetry ("DSC"), X-ray diffraction, etc. For example, the morphology (lamellar vs. non-lamellar) of lipid nanoparticles can be easily evaluated and characterized using Cryo-TEM analysis as described in US2010 / 0130588, the contents of which are incorporated herein by reference in their entirety.

[0190] In some further embodiments, lipid nanoparticles having a non-lamellar morphology are electron dense.

[0191] In some embodiments, the present disclosure provides lipid nanoparticles that are either unilamellar or multilamellar in structure. In some embodiments, the present disclosure provides lipid nanoparticles that include multivesicular particles and / or effervescent particles.

[0192] By controlling the composition and concentration of lipid components, the rate at which lipid complexes exchange out of lipid particles, which in turn controls the rate at which lipid nanoparticles become fusogenic, can be controlled.In addition, other variables, including, for example, pH, temperature, or ionic strength, can be used to change and / or control the rate at which lipid nanoparticles become fusogenic.Other methods that can be used to control the rate at which lipid nanoparticles become fusogenic will be clear to those skilled in the art based on this disclosure.It will also be clear that by controlling the composition and concentration of lipid complexes, lipid particle size can be controlled.

[0193] The pKa of formulated cationic lipids can correlate 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), both of which are incorporated herein by reference in their entireties). A preferred range of pKa is from about 5 to about 7. The pKa of cationic lipids can be determined in lipid nanoparticles using a fluorescence-based assay of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS).

[0194] The encapsulation of ceDNA in lipid particles can be determined by performing a membrane-impermeable fluorescent dye exclusion assay, such as the Oligreen® assay or the PicoGreen® assay, which uses a dye that has enhanced fluorescence when associated with nucleic acid. In general, encapsulation is determined by adding a dye to the lipid particle formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed upon addition of a small amount of non-ionic detergent. Detergent-mediated disruption of the lipid bilayer releases the encapsulated ceDNA, allowing it to interact with the membrane-impermeable dye. The encapsulation of ceDNA can be calculated as E=(I0-I) / I0, where I and I0 refer to the fluorescence intensity before and after the addition of detergent.

[0195] In some embodiments, the present disclosure provides liposomal formulations comprising one or more compounds having polyethylene glycol (PEG) functional groups (so-called "PEGylated compounds") that can reduce immunogenicity / antigenicity, provide hydrophilicity and hydrophobicity to the compound(s), and / or reduce therapeutically effective dosing frequency. In other embodiments, the liposomal formulations may simply include polyethylene glycol (PEG) polymers as additional components. In such embodiments, the molecular weight of the PEG or PEG functional group may be from 62 Da to about 5,000 Da.

[0196] In some embodiments, the present disclosure provides liposomal formulations that deliver ceDNA with extended or controlled release profiles over a period of hours to weeks. In some related embodiments, the liposomal formulations may include aqueous chambers bounded by a lipid bilayer. In other related embodiments, the liposomal formulations encapsulate ceDNA with additional components that undergo a physical transition at elevated temperatures that release the ceDNA over a period of hours to weeks.

[0197] In some embodiments, the liposomal formulation comprises sphingomyelin and one or more lipids disclosed herein. In some embodiments, the liposomal formulation comprises an Optisome.

[0198] In some aspects, the present disclosure provides a method for the preparation of a lipid-rich lipid complex comprising administering to a subject ... The present invention provides a liposome formulation comprising one or more lipids selected from: dioleoylserine; POPC (palmitoyloleoylphosphatidylcholine); SM (sphingomyelin); MPEG (methoxypolyethylene glycol); DMPC (dimyristoylphosphatidylcholine); DMPG (dimyristoylphosphatidylglycerol); DSPG (distearoylphosphatidylglycerol); DEPC (dioleoylphosphatidylcholine); DOPE (dioleoyl-sn-glycero-phosphoethanolamine), cholesteryl sulfate (CS), dipalmitoylphosphatidylglycerol (DPPG), DOPC (dioleoyl-sn-glycero-phosphatidylcholine), or any combination thereof.

[0199] In some embodiments, the present disclosure provides a liposomal formulation comprising a phospholipid, cholesterol, and a PEGylated lipid in a molar ratio of 56:38:5. In some embodiments, the total lipid content of the liposomal formulation is 2-16 mg / mL. In some embodiments, the present disclosure provides a liposomal formulation comprising a lipid containing a phosphatidylcholine functional group, a lipid containing an ethanolamine functional group, and a PEGylated lipid. In some embodiments, the present disclosure provides a liposomal formulation comprising a lipid containing a phosphatidylcholine functional group, a lipid containing an ethanolamine functional group, and a PEGylated lipid in a molar ratio of 3:0.015:2, respectively. In some embodiments, the present disclosure provides a liposomal formulation comprising a phosphatidylcholine functional group, an ethanolamine functional group, and a PEGylated lipid. In some embodiments, the present disclosure provides a liposomal formulation comprising a lipid containing a phosphatidylcholine functional group and cholesterol. In some embodiments, the PEGylated lipid is PEG-2000-DSPE. In some embodiments, the present disclosure provides a liposome formulation comprising DPPG, soy PC, an MPEG-DSPE lipid conjugate, and cholesterol.

[0200] In some embodiments, the present disclosure provides liposomal formulations that include one or more lipids that contain phosphatidylcholine functional groups and one or more lipids that contain ethanolamine functional groups. In some embodiments, the present disclosure provides liposomal formulations that include one or more lipids that contain phosphatidylcholine functional groups, lipids that contain ethanolamine functional groups, and / or sterols, such as cholesterol. In some embodiments, the liposomal formulations include DOPC / DEPC and DOPE.

[0201] In some embodiments, the present disclosure provides liposomal formulations further comprising one or more pharmaceutical excipients, such as sucrose and / or glycine.

[0202] In some embodiments, the present disclosure provides liposomal formulations that are either unilamellar or multilamellar structures. In some embodiments, the present disclosure provides liposomal formulations that include multivesicular particles and / or effervescent particles. In some embodiments, the present disclosure provides liposomal formulations that are larger in size relative to typical nanoparticles, about 150-250 nm in size. In some embodiments, the liposomal formulation is a lyophilized powder.

[0203] In some embodiments, the present disclosure provides liposomal formulations made and loaded with ceDNA vectors obtained by the process of Example 1 or other methods disclosed herein by adding a weak base to a mixture with isolated ceDNA outside the liposomes. This addition increases the pH outside the liposomes to approximately 7.3, driving the ceDNA into the liposomes. In some embodiments, the present disclosure provides liposomal formulations having a pH that is acidic inside the liposomes. In such cases, the inside of the liposomes can be pH 4-6.9, more preferably pH 6.5. In other embodiments, the present disclosure provides liposomal formulations made by using intraliposomal drug stabilization techniques. In such cases, a polymeric or non-polymeric highly charged anion and an intraliposomal entrapment agent, such as polyphosphate or sucrose octasulfate, is utilized.

[0204] In another aspect, the present disclosure provides a liposome formulation comprising a phospholipid, a lecithin, a phosphatidylcholine, and a phosphatidylethanolamine.

[0205] The ceDNA disclosed herein can be incorporated into a pharmaceutical composition suitable for administration to a subject for in vivo delivery to the subject's cells, tissues, or organs. Typically, the pharmaceutical composition comprises the ceDNA disclosed herein and a pharma- ceutically acceptable carrier. For example, the ceDNA vector of the present invention can be incorporated into a pharmaceutical composition suitable for the desired route of therapeutic administration (e.g., parenteral administration). Passive tissue transduction via high pressure intravenous or intra-arterial injection, as well as intracellular injection, e.g., intranuclear microinjection or intracytoplasmic injection, are also contemplated. Pharmaceutical compositions for therapeutic purposes can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high ceDNA vector concentration. Sterile injectable solutions can be prepared by incorporating the required amount of ceDNA vector compounds in a suitable buffer with one or a combination of the above-listed components, and filter sterilization.

[0206] The ceDNA vectors disclosed herein can be incorporated into pharmaceutical compositions suitable for local, systemic, intra-amniotic, intrathecal, intracranial, intra-arterial, intravenous, intralymphatic, intraperitoneal, subcutaneous, bronchial, intratissue (e.g., intramuscular, intracardiac, intrahepatic, intrarenal, intracerebral), intrathecal, intravesical, conjunctival (e.g., extraorbital, intraorbital, retroorbital, intraretinal, subretinal, choroidal, subchoroidal, intrastromal, anterior chamber, and intravitreal), intracochlear, and mucosal (e.g., oral, rectal, nasal) administration. Passive tissue transduction via high pressure intravenous or intra-arterial injection, as well as intracellular injection, e.g., intranuclear microinjection or intracytoplasmic injection, are also contemplated.

[0207] The ceDNA vector-containing pharma- ceutically active composition can be formulated to deliver the transgene in the nucleic acid to the recipient's cells and cause therapeutic expression of the transgene therein. The composition can also include a pharma- ceutically acceptable carrier.

[0208] The compositions and vectors provided herein can be used to deliver transgenes for a variety of purposes. In some embodiments, the transgene encodes a protein or functional RNA intended to be used for research purposes, e.g., to create a somatic transgenic animal model harboring the transgene, e.g., to study the function of the transgene product. In another example, the transgene encodes a protein or functional RNA intended to be used to create an animal model of a disease. In some embodiments, the transgene encodes one or more peptides, polypeptides, or proteins that are useful for treating, ameliorating, or preventing a disease state in a mammalian subject. The transgene can be transferred (e.g., expressed) into a subject in an amount sufficient to treat a disease associated with reduced expression, lack of expression, or dysfunction of the gene in a clinical setting. In some embodiments, the transgene can be transferred (e.g., expressed) into a subject in an amount sufficient to treat a disease associated with increased expression, activity of the gene product, or inappropriate upregulation of a gene (wherein the transgene suppresses or otherwise reduces its expression).

[0209] Pharmaceutical compositions for therapeutic purposes must typically be sterile and stable under the conditions of manufacture and storage.Compositions can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high ceDNA vector concentration.Sterile injectable solutions can be prepared by incorporating the required amount of ceDNA vector compounds in a suitable buffer with one or a combination of the components listed above, and sterilizing by filtration.

[0210] The ceDNA vectors described herein can be administered to an organism for transduction of cells in vivo.

[0211] Generally, administration is by any of the routes normally used to introduce molecules to ultimately contact blood or tissue cells. Suitable methods of administering such nucleic acids are available and well known by those skilled in the art, and while more than one route may be used to administer a particular composition, a particular route may often result in a more immediate and effective response than another route. Exemplary administration forms of the ceDNA vectors disclosed herein include oral, rectal, mucosal, intranasal, inhalation (e.g., via aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intradermal, intrauterine (or intraovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intracranial, intramuscular [including administration to the skeleton, diaphragm, and / or myocardium], intrapleural, intracerebral, and intraarterial), topical (e.g., to skin and mucosal surfaces, including airway surfaces, and transdermal administration), intralymphatic, etc., and direct tissue or organ injection (e.g., to the liver, eye, skeletal muscle, myocardium, diaphragm, muscle, or brain).

[0212] Administration of the ceDNA vector can be to any site of the subject, including, but not limited to, a site selected from the group consisting of the brain, skeletal muscle, smooth muscle, heart, diaphragm, airway epithelium, liver, kidney, spleen, pancreas, skin, and eye. Administration of the ceDNA vector can also be to a tumor (e.g., in or near a tumor or lymph node). The most suitable route in any given case depends on the nature and severity of the condition to be treated, ameliorated, and / or prevented, and the nature of the particular ceDNA vector being used. Additionally, ceDNA allows for the administration of multiple transgenes in a single vector or multiple ceDNA vectors (e.g., ceDNA cocktail).

[0213] Administration of the ceDNA vectors disclosed herein to skeletal muscles includes, but is not limited to, administration to skeletal muscles of the limbs (e.g., upper arms, lower arms, upper legs, and / or lower legs), lower back, neck, head (e.g., tongue), pharynx, abdomen, pelvis / perineum, and / or fingers. The ceDNA vectors disclosed herein can be administered intravenously, intra-arterially, intraperitoneally, via limb perfusion (optionally isolated limb perfusion of the legs and / or arms, e.g., as described by Arruda et al. al., (2005) Blood 105:3458-3464) and / or by direct intramuscular injection into skeletal muscle. In certain embodiments, the ceDNA vectors disclosed herein are administered to a subject (e.g., a subject with a muscular dystrophy such as DMD) by limb perfusion, optionally isolated limb perfusion (e.g., by intravenous or intra-arterial administration). In embodiments, the ceDNA vectors disclosed herein can be administered without the use of "hydrodynamic techniques."

[0214] Administration of the ceDNA vector disclosed herein to the myocardium includes administration to the left atrium, right atrium, left ventricle, right ventricle, and / or septum. The ceDNA vector described herein can be delivered to the myocardium by intravenous administration, intra-arterial administration such as intra-aortic administration, direct cardiac injection (e.g., into the left atrium, right atrium, left ventricle, right ventricle), and / or coronary perfusion. Administration to the diaphragm muscle can be by any suitable method, including intravenous administration, intra-arterial administration, and / or intraperitoneal administration. Administration to the smooth muscle can be by any suitable method, including intravenous administration, intra-arterial administration, and / or intraperitoneal administration. In one embodiment, administration can be to endothelial cells present in, near, and / or on the smooth muscle.

[0215] In some embodiments, a ceDNA vector according to the present invention is administered to skeletal muscle, diaphragm muscle, and / or cardiac muscle (e.g., to treat, ameliorate, and / or prevent muscular dystrophy or cardiac disease (e.g., PAD or congestive heart failure)).

[0216] The ceDNA vector according to the present invention may be administered to the CNS (e.g., brain or eye). The ceDNA vector may be introduced into the spinal cord, brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra, pineal gland), cerebellum, telencephalon (cerebrum including striatum, occipital lobe, temporal lobe, parietal lobe, and frontal lobe, cortex, basal ganglia, hippocampus, and amygdala), limbic system, neocortex, striatum, cerebrum, and inferior colliculus. The ceDNA vector may also be administered to different regions of the eye, such as the retina, cornea, and / or optic nerve. The ceDNA vector may be delivered into the cerebrospinal fluid (e.g., by lumbar puncture). The ceDNA vector may also be administered intravascularly to the CNS in situations where the blood-brain barrier is disrupted (e.g., brain tumor or cerebral infarction).

[0217] The ceDNA vector may be administered to the desired region(s) 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, intraaural, intraocular (e.g., intravitreal, subretinal, anterior chamber), and periocular (e.g., sub-Tenon's area) delivery, as well as intramuscular delivery with retrograde delivery to motor neurons.

[0218] In some embodiments, the ceDNA vector is administered in a liquid formulation by direct injection (e.g., stereotactic injection) into the desired region or compartment in the CNS. In other embodiments, the ceDNA vector can be provided by topical application to the desired region or by intranasal administration of an aerosol formulation. Administration to the eye may be by topical application of liquid drops. As a further alternative, the ceDNA vector can be administered as a solid sustained release formulation (see, e.g., U.S. Pat. No. 7,201,898). In yet further embodiments, the ceDNA vector can be used for retrograde transport to treat, ameliorate, and / or prevent diseases and disorders involving motor neurons (e.g., amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), etc.). For example, the ceDNA vector can be delivered to muscle tissue and translocated from there into neurons.

[0219] Ex vivo treatment In some embodiments, cells are removed from a subject, ceDNA vector is introduced therein, and then the cells are returned to the subject.Methods for removing cells from a subject and then returning them to the subject for ex vivo treatment are known in the art (see, for example, U.S. Patent No. 5,399,346, the disclosure of which is incorporated herein by reference in its entirety).Alternatively, ceDNA vector is introduced into cells from another subject, into cultured cells, or into cells from any other suitable source, and these cells are administered to a subject in need thereof.

[0220] The cells transduced with the ceDNA vector are preferably combined with a pharmaceutical carrier and administered to the subject in a "therapeutically effective amount". Those skilled in the art will understand that the therapeutic effect need not be complete or curative, as long as some benefit is provided to the subject.

[0221] In some embodiments, a ceDNA vector can encode a transgene (sometimes called a heterologous nucleotide sequence), which is any polypeptide that is desirably produced in a cell in vitro, ex vivo, or in vivo. In contrast to the use of ceDNA vectors in, for example, therapeutic methods, in some embodiments, a ceDNA vector can be introduced into a cultured cell and the expressed gene product isolated therefrom, for example, for the production of an antigen or vaccine.

[0222] ceDNA vectors can be used for both veterinary and medical purposes.Suitable subjects for the above ex vivo gene delivery method include birds (e.g. chicken, duck, goose, quail, turkey, and pheasant) and mammals (e.g. human, cow, sheep, goat, horse, cat, dog, and lagomorph), with mammals being preferred.Human subjects are most preferred.Human subjects include neonates, infants, juveniles, and adults.

[0223] In some embodiments, the transgene present in the expression cassette, expression construct, or ceDNA vector described herein may be codon optimized for the host cell. As used herein, the term "optimized codons" or "codon optimization" refers to the process of modifying a nucleic acid sequence by replacing at least one, two or more, or a substantial number of codons of a native sequence (e.g., a prokaryotic sequence) with codons that are more frequently or most frequently used in the genes of a vertebrate of interest, for enhanced expression in the cells of the vertebrate of interest, such as a mouse or human (e.g., humanized). Various species exhibit a particular bias toward certain codons of a particular amino acid. Typically, codon optimization does not change the amino acid sequence of the original translated protein. Optimized codons can be determined, for example, using Aptagen's Gene Forge® codon optimization and custom gene synthesis platform (Aptagen, Inc.) or another publicly available database.

[0224] In some embodiments, the ceDNA vector expresses the transgene in a host cell of interest. In some embodiments, the host cell of interest is a human host cell, such as a blood cell, stem cell, hematopoietic cell, CD34 + The subject host cells may be hepatocytes, cancer cells, vascular cells, muscle cells, pancreatic cells, neural cells, ocular or retinal cells, epithelial or endothelial cells, dendritic cells, fibroblasts, or any other cells of mammalian origin, including, without limitation, hepatic (i.e., liver) cells, lung cells, cardiac cells, pancreatic cells, intestinal cells, diaphragm cells, renal (i.e., kidney) cells, neuronal cells, blood cells, bone marrow cells, or any one or more selected tissues of the subject for which gene therapy is contemplated. In one embodiment, the subject host cells are human host cells.

[0225] One aspect of the technology described herein relates to the method of delivering transgene to cells.Typically, for in vitro method, ceDNA vector can be introduced using the method disclosed herein as well as other methods known in the art.The ceDNA vector disclosed herein is preferably administered to cells in a biologically effective amount.When ceDNA vector is administered to cells in vivo (e.g., to a subject), the biologically effective amount of ceDNA vector is sufficient to cause transduction and expression of transgene in target cells.

[0226] Dose Range In vivo and / or in vitro assays can be optionally used to help identify the optimal dosage range for use.The exact dosage to be used in the formulation also depends on the route of administration and the severity of the condition, and should be determined according to the judgment of the skilled artisan and the circumstances of each subject.Effective dosages can be estimated from dose-response curves derived from in vitro or animal model test systems.

[0227] ceDNA vector is administered in an amount sufficient to transfect cells of desired tissue and to bring about sufficient levels of gene transfer and expression without excessive side effects.Conventional pharmacologic acceptable administration routes include, but are not limited to, those described above in the "administration" section, such as direct delivery to selected organ (e.g., intraportal delivery to liver), oral, inhalation (including intranasal and intratracheal delivery), intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral administration routes.Administration routes can be combined if desired.

[0228] The amount of ceDNA vector dosage required to achieve a particular "therapeutic effect" varies based on several factors, including, but not limited to, the route of nucleic acid administration, the level of gene or RNA expression required to achieve the therapeutic effect, the particular disease or disorder being treated, and the stability of the gene(s), RNA product(s), or resulting expressed protein(s). One of skill in the art can readily determine the ceDNA vector dosage range for treating a subject with a particular disease or disorder based on the aforementioned factors as well as other factors well known in the art.

[0229] Dosage regime can be adjusted to provide optimal therapeutic response.For example, oligonucleotide can be repeatedly administered, for example, several doses can be administered every day, or dose can be proportionally reduced as indicated by the exigencies of therapeutic situation.Those skilled in the art can easily determine the appropriate dose and schedule of administration of subject oligonucleotide, regardless of whether oligonucleotide is administered to cell or to subject.

[0230] A "therapeutically effective amount" falls within a relatively broad range that can be determined through clinical trials and depends on the particular application (neuronal cells require very small amounts, while systemic injection requires large amounts). For example, for direct in vivo injection into skeletal or cardiac muscle of a human subject, a therapeutically effective amount would be approximately about 1 μg to about 100 g of ceDNA vector. When exosomes or microparticles are used to deliver the ceDNA vector, the therapeutically effective amount can be determined empirically, but is expected to deliver 1 μg to about 100 g of vector.

[0231] The formulation of pharma- ceutically acceptable excipients and carrier solutions is well known to those skilled in the art, as is the development of suitable administration and treatment regimens for using the particular compositions described herein in various treatment regimens.

[0232] For in vitro transfection, cells (1 × 10 6An effective amount of ceDNA vector delivered to a cell will be approximately 0.1-100 μg of ceDNA vector, preferably 1-20 μg, more preferably 1-15 μg or 8-10 μg. Larger ceDNA vectors will require larger doses. When exosomes or microparticles are used, the effective in vitro dose is determined empirically, but is generally intended to deliver the same amount of ceDNA vector.

[0233] Treatment may require administration of a single dose or multiple doses. In certain embodiments, two or more doses (e.g., 2, 3, 4, or more administrations) may be used to achieve the desired gene expression levels over variously spaced time periods, e.g., daily, weekly, monthly, yearly, etc. In some embodiments, two or more doses may be administered to the subject, and indeed multiple doses may be administered as needed, since the ceDNA vector does not elicit an anti-capsid host immune response due to the absence of viral capsid. As such, one of skill in the art can determine the appropriate number of doses. The number of doses administered may be, for example, approximately 1 to 100, preferably 2 to 20 doses.

[0234] Without being bound by any particular theory, the lack of typical anti-viral immune responses elicited by administration of the ceDNA vectors described by the present disclosure (i.e., the absence of capsid components) allows the ceDNA vector to be administered to the host on multiple occasions. In some embodiments, the number of times the heterologous nucleic acid is delivered to the subject is in the range of 2-10 times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 times). In some embodiments, the ceDNA vector is delivered to the subject more than 10 times.

[0235] In some embodiments, the dose of the ceDNA vector is administered to the subject only once per calendar day (e.g., 24 hours). In some embodiments, the dose of the ceDNA vector is administered to the subject only once per 2, 3, 4, 5, 6, or 7 calendar days. In some embodiments, the dose of the ceDNA vector is administered to the subject only once per calendar week (e.g., 7 calendar days). In some embodiments, the dose of the ceDNA vector is administered to the subject only once every 2 weeks (e.g., once every 2 calendar week period). In some embodiments, the dose of the ceDNA vector is administered to the subject only once per calendar month (e.g., once every 30 calendar days). In some embodiments, the dose of the ceDNA vector is administered to the subject only once per 6 calendar months. In some embodiments, the dose of the ceDNA vector is administered to the subject only once per calendar year (e.g., 365 days or 366 days in leap years).

[0236] Unit dosage form In some embodiments, the pharmaceutical composition may be conveniently presented in a unit dosage form. The unit dosage form is typically adapted for one or more routes of administration of the pharmaceutical composition. In some embodiments, the unit dosage form is adapted for administration by inhalation. In some embodiments, the unit dosage form is adapted for administration by a vaporizer. In some embodiments, the unit dosage form is adapted for administration by a nebulizer. In some embodiments, the unit dosage form is adapted for administration by an aerosolizer. In some embodiments, the unit dosage form is adapted for oral administration, buccal administration, or sublingual administration. In some embodiments, the unit dosage form is adapted for intravenous, intramuscular, or subcutaneous administration. In some embodiments, the unit dosage form is adapted for intrathecal or intraventricular administration. In some embodiments, the pharmaceutical composition is formulated for topical administration. The amount of active ingredient that may be combined with a carrier material to produce a single dosage form will generally be the amount of compound that produces a therapeutic effect.

[0237] Preparation of lipid nanoparticles Lipid nanoparticles may be formed simultaneously with mixing of the ceDNA vector and lipid(s). Depending on the desired particle size distribution, the resulting nanoparticle mixture may be extruded through a membrane (e.g., 100 nm cutoff) using, for example, a thermobarrel extruder such as the Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step may be omitted. Ethanol removal and simultaneous buffer exchange may be accomplished, for example, by dialysis or tangential flow filtration.

[0238] In general, lipid nanoparticles can be formed by any method known in the art, including the following. For example, lipid nanoparticles can be prepared by methods described in, for example, US2013 / 0037977, US2010 / 0015218, US2013 / 0156845, US2013 / 0164400, US2012 / 0225129, and US2010 / 0130588 (the contents of each of which are incorporated herein by reference in their entirety). In some embodiments, lipid nanoparticles can be prepared using a continuous mixing method, a direct dilution process, or an in-line dilution process. Processes and apparatus for preparing lipid nanoparticles using direct dilution and in-line dilution processes are described in US2007 / 0042031 (the contents of which are incorporated herein by reference in their entirety). A process and apparatus for preparing lipid nanoparticles using a serial dilution process is described in US2004 / 0142025, the contents of which are incorporated herein by reference in their entirety.

[0239] In one non-limiting example, lipid nanoparticles can be prepared by an impinging jet process. Generally, the particles are formed by mixing lipids dissolved in alcohol (e.g., ethanol) with ceDNA dissolved in a buffer, such as citrate buffer, sodium acetate buffer, sodium acetate and magnesium chloride buffer, malic acid buffer, malic acid and sodium chloride buffer, or sodium citrate and sodium chloride buffer. The mixture ratio of lipid to ceDNA can be about 45-55% lipid and about 65-45% ceDNA.

[0240] The lipid solution may contain ionizable lipids, non-cationic lipids (e.g., phospholipids such as DPSC), PEG or PEG-conjugated molecules (e.g., PEG-lipids), and sterols (e.g., cholesterol) at a total lipid concentration of 5-30 mg / mL in alcohol, e.g., ethanol, more preferably 5-15 mg / mL, and most preferably 9-12 mg / mL.

[0241] The molar ratio of lipids in the lipid solution may range from about 25-98% for cationic lipids, preferably about 35-65%; about 0-15% for nonionic lipids, preferably about 0-12%; about 0-15% for PEG or PEG-conjugated molecules, preferably about 1-6%; and about 0-75% for sterols, preferably about 30-50%.

[0242] The ceDNA solution may contain ceDNA in a concentration range of 0.3-1.0 mg / mL, preferably 0.3-0.9 mg / mL, in a buffer solution having a pH in the range of 3.5-5.

[0243] To form the LNPs, in one exemplary but non-limiting embodiment, the two liquids are heated to a temperature in the range of about 15-40°C, preferably about 30-40°C, and then mixed, for example, in an impinging jet mixer, to instantly form the LNPs. The mixing flow rate can be in the range of 10-600 mL / min. The tubing ID can have a range of 0.25-1.0 mm and a total flow rate of 10-600 mL / min. The combination of flow rate and tubing ID can have the effect of controlling the particle size of the LNPs between 30-200 nm. The solution can then be mixed with a buffer at a higher pH in a mixing ratio ranging from 1:1 to 1:3 vol:vol, preferably about 1:2 vol:vol. If necessary, this buffer can be at a temperature in the range of 15-40°C or 30-40°C. The mixed LNPs can then undergo an anion exchange filtration step. Prior to the anion effect, the mixed LNPs can be incubated for a period of, for example, 30 minutes to 2 hours. The temperature during incubation can range from 15-40 °C or 30-40 °C. After incubation, the solution is filtered through a filter, such as a 0.8 µm filter, and includes an anion exchange separation step. This process can use tubing IDs ranging from 1 mm ID to 5 mm ID and flow rates from 10 to 2000 mL / min.

[0244] After formation, the LNPs can be concentrated and diafiltered via an ultrafiltration process in which the alcohol is removed and the buffer is exchanged for a final buffer, for example, phosphate buffered saline (PBS) at about pH 7, for example, about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

[0245] The ultrafiltration process can use a tangential flow filtration format (TFF), using a membrane nominal molecular weight cutoff range of 30-500 KD. The membrane format is a hollow fiber or flat sheet cassette. A TFF process using the appropriate molecular weight cutoff can retain the LNPs in the retentate, while the filtrate or permeate contains alcohol, citrate buffer, and final solution buffer waste. The TFF process is a multi-step process, with an initial concentration for ceDNA concentration of 1-3 mg / mL. Following concentration, the LNP solution is diafiltered against 10-20 volumes of final buffer to remove alcohol and perform a buffer exchange. The material can then be further concentrated 1-3 times. The concentrated LNP solution can be sterile filtered.

[0246] ceDNA In various embodiments, a ceDNA vector is a non-encapsidated linear double-stranded DNA molecule formed from a continuous strand of complementary DNA (linear continuous non-encapsidated structure) with covalently closed ends, and includes 5' inverted terminal repeat (ITR) and 3' ITR sequences that are different or asymmetric with respect to each other. At least one of the ITRs includes a functional terminal resolution site and a replication protein binding site (RPS) (sometimes referred to as a replication protein binding site), e.g., a Rep binding site. In general, a ceDNA vector includes at least one modified AAV inverted terminal repeat (ITR), i.e., a deletion, insertion, and / or substitution with respect to the other ITR and the expressible transgene.

[0247] In one embodiment, at least one of the ITRs is AAV ITR, for example, wild-type AAV ITR.In one embodiment, at least one of the ITRs is a modified ITR with respect to other ITRs, i.e., the ceDNA comprises ITRs that are asymmetric with respect to each other.In one embodiment, at least one of the ITRs is non-functional ITR.

[0248] In some embodiments, the ceDNA vector comprises (1) an expression cassette comprising 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, e.g., ITRs, flanking the expression cassette, wherein the ceDNA vector is not associated with a capsid protein. In some embodiments, the ceDNA vector comprises two self-complementary sequences found in the AAV genome, at least one of which comprises an operational Rep-binding element (RBE) and a terminal resolution site (trs) of AAV or a functional variant of the RBE, and a cis-regulatory element operably linked to one or more transgenes. In some embodiments, the ceDNA vector may comprise additional components for regulating the expression of the transgene, e.g., a regulatory switch for controlling and regulating the expression of the transgene, and a regulatory switch that is a kill switch that allows for controlled cell death of cells containing the ceDNA vector.

[0249] In some embodiments, the two self-complementary sequences can be ITR sequences from any known parvovirus, such as a depend virus, such as AAV (e.g., AAV1-AAV12). Any AAV serotype can be used, including, but not limited to, modified AAV2 ITR sequences that retain a Rep binding site (RBS) and terminal resolution site (trs), such as 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 531), in addition to variable palindromic sequences that allow for hairpin secondary structure formation. In some embodiments, the ITRs can be synthetic. In one embodiment, the synthetic ITRs are based on ITR sequences from two or more AAV serotypes. In another embodiment, the synthetic ITRs do not contain AAV-based sequences. In yet another embodiment, the synthetic ITRs preserve the ITR structure described above, but have little or no AAV source sequences. In some aspects, the synthetic ITRs can preferentially interact with wild-type Rep or Rep of a particular serotype, or in some cases, are not recognized by wild-type Rep, but only by mutant Rep. In some embodiments, the ITR is a synthetic ITR sequence that retains a functional Rep binding site (RBS) and terminal resolution site (TRS), such as 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 531), in addition to a variable palindrome sequence that allows for hairpin secondary structure formation. In some examples, the modified ITR sequence retains the sequence of the RBS, trs, and the structure and position of the Rep binding element that forms one terminal loop portion of the ITR hairpin secondary structure from the corresponding sequence of the wild-type AAV2 ITR. Exemplary ITR sequences for use in ceDNA vectors are disclosed in Tables 2-9, 10A and 10B, with SEQ ID NOs: 2, 52, 101-449, and 545-547, as well as partial ITR sequences shown in Figures 26A-26B of PCT Application No. PCT / US18 / 49996, filed September 7, 2018.In some embodiments, the ceDNA vector may include an ITR having an ITR modification corresponding to any of the ITR sequence or ITR subsequence modifications set forth 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.

[0250] In some embodiments, the closed-end DNA vector comprises a promoter operably linked to a transgene, and the ceDNA is produced from a ceDNA-plasmid that lacks capsid protein and encodes (a) a mutated right AAV2 ITR having the same number of intramolecularly duplexed base pairs as SEQ ID NO:2 or a mutated left AAV2 ITR having the same number of intramolecularly duplexed base pairs as SEQ ID NO:51 in its hairpin secondary configuration (preferably excluding the deletion of any AAA or TTT terminal loops in this configuration compared to these reference sequences), and (b) is identified as ceDNA using the assay for identification of ceDNA by agarose gel electrophoresis under native and denaturing conditions in Example 1.

[0251] The ceDNA vector can be obtained by several means known to the skilled artisan upon reading this disclosure. For example, the capsid-free non-viral DNA vector can be obtained from a plasmid (herein referred to as "ceDNA-plasmid") that contains a polynucleotide expression construct template that contains a first 5' inverted terminal repeat (e.g., AAV ITR), an expression cassette, and a 3' ITR (e.g., AAV ITR) in that order, where at least one of the 5' and 3' ITRs is a modified ITR, or when both the 5' and 3' ITRs are modified, they have different modifications from each other and are not the same sequence. Without limitation, the polynucleotide expression construct template used to generate the ceDNA vector can be a ceDNA-plasmid, a ceDNA-bacmid, and / or a ceDNA-baculovirus. In some embodiments, the ceDNA-plasmid contains a restriction cloning site (e.g., SEQ ID NO: 7) operably located between the ITRs, and can insert, for example, an expression cassette that contains a promoter operably linked to a transgene, for example, a reporter gene and / or a therapeutic gene.

[0252] For example, non-viral capsid-free DNA vectors can be produced in permissive host cells from expression constructs (e.g., plasmids, baculoviruses, or integrative cell lines) that contain a heterologous gene located between two different inverted terminal repeat (ITR) sequences. At least one of the ITRs is modified by deletion, insertion, and / or substitution compared to the wild-type ITR sequence (e.g., AAV ITR), and at least one of the ITRs contains a functional terminal resolution site (trs) and a Rep binding site. The ceDNA vector is preferably double-stranded, e.g., self-complementary, over at least a portion of the molecule, such as an expression cassette. For example, the ceDNA is not a double-stranded circular molecule. In some embodiments, the ceDNA vector has covalently closed ends and is therefore resistant to exonuclease digestion (e.g., exonuclease I or exonuclease III), e.g., at 37°C for 1 hour or more.

[0253] In some embodiments, the ceDNA vector comprises, in the 5' to 3' direction, a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette as described herein), and a second AAV ITR, where the first ITR and the second ITR are asymmetric with respect to each other, i.e., they are different from each other. In an exemplary embodiment, the first ITR can be a wild-type ITR, and the second ITR can be a mutant or modified ITR. In some embodiments, the first ITR can be a mutant or modified ITR, and the second ITR is a wild-type ITR. In another embodiment, the first ITR and the second ITR are both modified, but are of different sequences, or have different modifications, or are not identical modified ITRs. In other words, the ITRs are asymmetric, where any change in one ITR is not reflected in the other ITR, or the ITRs are different with respect to each other.

[0254] In some embodiments, the expression cassette is located between two ITRs and includes one or more of a promoter, a post-transcriptional regulatory element, and a polyadenylation and termination signal, in that order, operably linked to a transgene. In one embodiment, the promoter is regulatable-inducible or repressible. The promoter can be any sequence that promotes the transcription of the transgene. In one embodiment, the promoter is a CAG promoter (e.g., SEQ ID NO: 03) or a variant thereof. The post-transcriptional regulatory element is a sequence that regulates the expression of the transgene, including, but not limited to, any sequence that creates a tertiary structure that enhances the expression of the transgene.

[0255] Generally, ceDNA vector does not have the packaging constraint imposed by the limited space in viral capsid.In contrast to the enclosed AAV genome, ceDNA vector represents a versatile eukaryotically produced alternative to the prokaryotically produced plasmid DNA vector, in contrast to the enclosed AAV genome.This allows the insertion of control elements, such as the regulatory switch disclosed herein, large transgenes, multiple transgenes, etc.

[0256] In some embodiments, the first ITR can be a mutant or modified ITR, and the second ITR is a wild type ITR. In another embodiment, the first ITR and the second ITR are both modified, but are different sequences, or have different modifications, or are not the same modified ITR. In other words, the ITRs are asymmetric, where any change in one ITR is not reflected in the other ITR, or the ITRs are different with respect to each other. Exemplary ITRs in ceDNA vectors for use in generating ceDNA-plasmids are disclosed in PCT Application No. PCT / US18 / 49996, filed September 7, 2018.

[0257] ITRs are illustrated in the specification, and an example herein is the AAV2 ITR, but one of skill in the art will recognize that ITRs from any known parvovirus, e.g., dependovirus, e.g., AAV (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV5, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8 genomes, see, e.g., NCBI: NC002077; NC001401; NC001729; NC001829; NC006152; NC006260; NC006261), chimeric ITRs, or ITRs from any synthetic AAV, as described above. In some embodiments, AAV can infect warm-blooded animals, such as avian (AAAV), bovine (BAAV), canine, equine, and ovine adeno-associated viruses. In some embodiments, the ITRs are derived from B19 parvovirus (GenBank Accession No. NC000883), minute virus of mice (MVM) (GenBank Accession No. NC001510), goose parvovirus (GenBank Accession No. NC001701), snake parvovirus 1 (GenBank Accession No. NC006148).

[0258] 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).

[0259] The skilled artisan will understand that ITR sequences have the general structure of a double-stranded Holliday junction, typically a T-shaped or Y-shaped hairpin structure (e.g., Figures 2A and 3A), with each ITR formed by two palindromic arms or loops (B-B' and C-C') embedded in a larger palindromic arm (A-A'), as well as a single-stranded D sequence (the order of these palindromic sequences defines the flip or flop orientation of the ITR). Based on the exemplary AAV2 ITR sequences provided herein, the skilled artisan can easily determine the corresponding modified ITR sequences from any AAV serotype for use in ceDNA vectors or ceDNA-plasmids. See, for example, structural analysis and sequence comparison of ITRs from different AAV serotypes (AAV1-AAV6). 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. Exemplary specific modifications and mutations in ITRs are detailed in PCT Application No. PCT / US18 / 49996, filed September 7, 2018. For clarity, in the context of ITRs, "modified" or "mutated" indicates that nucleotides have been inserted, deleted, and / or substituted relative to the wild-type sequence, reference sequence, or original ITR sequence, and may be modified relative to other adjacent ITRs in a ceDNA vector having two adjacent ITRs. A modified or mutated ITR may be an engineered ITR. As used herein, "engineered" refers to an aspect that has been manipulated by the hand of man. For example, a polypeptide is considered to be "engineered" when at least one aspect of the polypeptide, e.g., its sequence, has been manipulated by the hand of man and differs from the aspect it occurs in nature.

[0260] Any parvovirus ITR can be used for modification as an ITR or as a base ITR. Preferably, the parvovirus is a dependovirus. More preferably, it is an AAV. The serotype selected can be based on the tissue tropism of the serotype. AAV2 has a broad tissue tropism, AAV1 preferentially targets neurons and skeletal muscle, and AAV5 targets neurons, retinal pigment epithelium, and photoreceptors. AAV6 preferentially targets skeletal muscle and lung. AAV8 preferentially targets liver, skeletal muscle, heart, and pancreatic tissue. AAV9 preferentially targets liver, skeletal, and lung tissue. 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 of each of these aspects, the vector polynucleotide comprises a pair of ITRs 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 capsid-free DNA vector with covalently closed ends comprises 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 ITRs are 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 ceDNA vector does not have a modified ITR selected from any sequence consisting of or consisting essentially of SEQ ID NOs:500-529 provided herein. In some embodiments, the ceDNA vector has no ITRs selected from any sequence selected from SEQ ID NOs: 500-529.

[0261] In some embodiments, ceDNA can form intramolecular double-stranded secondary structure.The secondary structure of the first ITR and asymmetric second ITR is illustrated in the context of wild-type ITR (see, for example, Figures 2A, 3A, and 3C) and modified ITR structure (see, for example, Figures 2B, 3B, and 3D).The secondary structure is estimated or predicted based on the ITR sequence of the plasmid used to produce ceDNA vector.

[0262] In some embodiments, the left ITR of the ceDNA vector is modified or mutated with respect to the wild-type (wt) AAV ITR structure, and the right ITR is the wild-type ITR.In one embodiment, the right ITR of the ceDNA vector is modified with respect to the wild-type AAV ITR structure, and the left ITR is the wild-type AAV ITR.In such an embodiment, the modification of the ITR (left or right ITR) can be generated by the deletion, insertion, or substitution of one or more nucleotides from the wild-type ITR derived from the AAV genome.

[0263] The ITRs used herein can be degradable and non-degradable and are selected for use in ceDNA vectors, preferably AAV sequences, with serotypes 1, 2, 3, 4, 5, 6, 7, 8, and 9 being preferred. Degradable AAV ITRs do not require wild-type ITR sequences (e.g., endogenous or wild-type AAV ITR sequences can be altered by insertion, deletion, truncation, and / or missense mutations) as long as the terminal repeats mediate the desired function, such as replication, viral packaging, integration, and / or proviral rescue. Typically, but not necessarily, the TRs are from the same AAV serotype, e.g., both ITR sequences of the ceDNA vector are from AAV2. The ITRs may be synthetic sequences that function as AAV inverted terminal repeats. Although not necessarily, the ITRs can be from the same parvovirus, e.g., both ITR sequences are from AAV2.

[0264] In some embodiments, at least one of the ITRs is defective with respect to Rep binding and / or Rep nicking. In one embodiment, the defect is at least 30% relative to the wild-type reduced ITR, and in other embodiments, at least 35%..., 50%..., 65%..., 75%..., 85%..., 90%..., 95%..., 98%..., or completely lacking in function or any point in between. The host cell does not express viral capsid proteins, and the polynucleotide vector template lacks any viral capsid coding sequence. In one embodiment, the polynucleotide vector template and host cell lacking AAV capsid genes, and the resulting proteins also do not encode or express capsid genes of other viruses. Additionally, in certain embodiments, the nucleic acid molecule also lacks AAV Rep protein coding sequence.

[0265] In some embodiments, the structural element of the ITR can be any structural element involved in the functional interaction of the ITR with a Rep protein (e.g., Rep 78 or Rep 68). In certain embodiments, the structural element provides selectivity for the interaction of the ITR with a large Rep protein, i.e., determines, at least in part, which Rep protein functionally interacts with the ITR. In other embodiments, the structural element physically interacts with the large Rep protein when the Rep protein is bound to the ITR. Each structural element can be, for example, the secondary structure of the ITR, the nucleotide sequence of the ITR, the space between two or more elements, or any combination of the above. In one embodiment, the structural element is selected from the group consisting of A and A' arms, B and B' arms, C and C' arms, D arms, Rep binding sites (RBE) and RBE', and terminal resolution sites (trs).

[0266] More specifically, the ability of a structural element to functionally interact with a particular large Rep protein can be altered by modifying the structural element. For example, the nucleotide sequence of the structural element can be modified compared to the wild-type sequence of the ITR. In one embodiment, the structural elements of the ITR (e.g., A arm, A' arm, B arm, B' arm, C arm, C' arm, D arm, RBE, RBE', and trs) can be removed and replaced with wild-type structural elements from a different parvovirus. For example, the replacement structure can be from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, snake parvovirus (e.g., royal python parvovirus), bovine parvovirus, caprine parvovirus, avian parvovirus, canine parvovirus, equine parvovirus, shrimp parvovirus, porcine parvovirus, or insect AAV. For example, the ITRs can be AAV2 ITRs and the A or A' arms or RBE can be replaced with structural elements from AAV5. In another embodiment, the ITRs can be AAV5 ITRs and the C or C' arms, RBE, and trs can be replaced with structural elements from AAV2. In another embodiment, the AAV ITRs can be AAV5 ITRs and the B and B' arms are replaced with the AAV2 ITR B and B' arms.

[0267] In some embodiments, the nucleotide sequence of a structural element can be modified (e.g., 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 range therein) to produce a modified structural element.

[0268] In some embodiments, the structure of the structural element may be modified. For example, the structural element may be modified in the height of the stem and / or the number of nucleotides in the loop. For example, the height of the stem may be about 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides or more, or any range therein. In some embodiments, the stem height may be about 5 nucleotides to about 9 nucleotides and functionally interact with Rep. In another embodiment, the stem height may be about 7 nucleotides and functionally interact with Rep. In another example, the loop may be 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides or more, or any range therein.

[0269] In some embodiments, the number of GAGY binding sites or GAGY-related binding sites in an RBE or extended RBE can be increased or decreased. In one example, an RBE or extended RBE can contain 1, 2, 3, 4, 5, 6 or more GAGY binding sites, or any range therein. Each GAGY binding site can independently be the exact GAGY sequence or a sequence similar to GAGY, so long as the sequence is sufficient to bind to the Rep protein.

[0270] In some embodiments, the spacing between two elements (such as, but not limited to, an RBE and a hairpin) can be altered (e.g., increased or decreased) to alter functional interactions with a large Rep protein. For example, the spacing can be about 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, or any range therein.

[0271] 2A and 2B show one possible mechanism for the manipulation of trs sites within the wild-type ITR structure portion of a ceDNA vector. In some embodiments, the ceDNA vector comprises one or more functional ITR polynucleotide sequences, including a Rep-binding site (RBS, 5'-GCGCGCTCGCTCGCTC-3' for AAV2, SEQ ID NO: 531) and a terminal resolution site (TRS, 5'-AGTT, SEQ ID NO: 46). In some embodiments, at least one ITR (wild-type or modified ITR) is functional. In alternative embodiments, where the ceDNA vector comprises 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.

[0272] In some embodiments, a modified ITR (e.g., left or right ITR) of a ceDNA vector described herein has a modification in the loop arm, cleavage arm, or spacer. Exemplary sequences of ITRs with modifications in the loop arm, cleavage arm, or spacer are listed in Tables 2-9, 10A, and 10B of PCT Application No. PCT / US18 / 49996, filed September 7, 2018.

[0273] ceDNA vectors can be produced from expression constructs that further comprise a specific combination of cis-regulatory elements. Cis-regulatory elements include, but are not limited to, promoters, riboswitches, insulators, mir regulatory elements, post-transcriptional regulatory elements, tissue and cell type specific promoters, and enhancers. In some embodiments, ITRs can act as promoters of transgenes. In some embodiments, ceDNA vectors comprise additional components for regulating the expression of transgenes, such as the regulatory switches described in PCT Application No. PCT / US18 / 49996, filed September 7, 2018, to regulate the expression of transgenes, or kill switches that can kill cells that contain ceDNA vectors.

[0274] The expression cassette may also include post-transcriptional elements to increase expression of the transgene. In some embodiments, the Woodchuck Hepatitis Virus (WHP) post-transcriptional regulatory element (WPRE) (e.g., SEQ ID NO: 8) is used to increase expression of the transgene. Other post-transcriptional processing elements, such as post-transcriptional elements from the thymidine kinase gene of Herpes Simplex Virus or Hepatitis B Virus (HBV), can be used. Secretory sequences can be linked to the transgene, such as VH-02 and VK-A26 sequences, such as SEQ ID NO: 25 and SEQ ID NO: 26. The expression cassette may include polyadenylation sequences known in the art or variations thereof, such as naturally occurring sequences isolated from bovine BGHpA (e.g., SEQ ID NO: 74) or viral SV40pA (e.g., SEQ ID NO: 10), or synthetic sequences (e.g., SEQ ID NO: 27). Some expression cassettes may also include an SV40 late polyA signal upstream enhancer (USE) sequence. USE can be used in combination with SV40pA or a heterologous polyA signal.

[0275] An expression cassette may comprise more than 4000 nucleotides, 5000 nucleotides, 10,000 nucleotides, or 20,000 nucleotides, or 30,000 nucleotides, or 40,000 nucleotides, or 50,000 nucleotides, or any range between about 4000-10,000 nucleotides, or 10,000-50,000 nucleotides, or more than 50,000 nucleotides. In some embodiments, an expression cassette may comprise a transgene or nucleic acid in the range of 500-50,000 nucleotides in length. In some embodiments, an expression cassette may comprise a transgene or nucleic acid in the range of 500-75,000 nucleotides in length. In some embodiments, an expression cassette may comprise a transgene or nucleic acid in the range of 500-10,000 nucleotides in length. In some embodiments, an expression cassette may comprise a transgene or nucleic acid in the range of 1000-10,000 nucleotides in length. In some embodiments, the expression cassette may contain a transgene or nucleic acid ranging from 500 to 5,000 nucleotides in length. ceDNA vectors do not have the size limitations of encapsidated AAV vectors, thus allowing delivery of large size expression cassettes resulting in efficient expression of the transgene. In some embodiments, the ceDNA vector lacks prokaryotic cell-specific methylation. In some embodiments, the expression cassette length in the 5' to 3' orientation exceeds the maximum length known to be encapsidated in AAV virions. 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.

[0276] 1A-1C show schematic diagrams of non-limiting exemplary ceDNA vectors, or corresponding sequences of ceDNA plasmids. The ceDNA vector is obtained from a plasmid that is capsid-free and encodes a first ITR, an expressible transgene cassette, and a second ITR in this order, and at least one of the first and / or second ITR sequences is mutated with respect to the corresponding wild-type AAV2 ITR sequence. The expressible transgene cassette preferably includes one or more of an enhancer / promoter, an ORF reporter (transgene), a post-transcriptional regulatory element (e.g., WPRE), and a polyadenylation and termination signal (e.g., BGH polyA) in this order.

[0277] The expression cassette may include any transgene of interest. Transgenes of interest include, but are not limited to, nucleic acids encoding polypeptides or non-coding nucleic acids (e.g., RNAi, miRs, etc.), preferably therapeutic agents (e.g., for medical, diagnostic, or veterinary applications), or immunogenic polypeptides (e.g., for vaccines). In certain embodiments, the transgene in the expression cassette encodes one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNAs, RNAis, antisense oligonucleotides, antisense polynucleotides, antibodies, antigen-binding fragments, or any combination thereof. In some embodiments, the transgene is a therapeutic gene or a marker protein. In some embodiments, the transgene is an agonist or antagonist. In some embodiments, the antagonist is an antibody mimic, or an antibody fragment, or an antigen-binding fragment thereof, such as a neutralizing antibody or antibody fragment. In some embodiments, the transgene encodes an antibody, including a full-length antibody or an antibody fragment, as defined herein. In some embodiments, the antibody is an antigen-binding domain or an immunoglobulin variable domain sequence, as defined herein.

[0278] In particular, the transgene can encode one or more therapeutic agent(s) including, but not limited to, protein(s), polypeptide(s), peptide(s), enzyme(s), antibodies, antigen-binding fragments, and variants and / or active fragments thereof, for use in the treatment, prevention, and / or amelioration of one or more symptoms of a disease, dysfunction, injury, and / or disorder, for example.

[0279] There are many structural features of ceDNA vectors that are different from plasmid-based expression vectors. ceDNA vectors may have one or more of the following features: lack of original (i.e., uninserted) bacterial DNA, lack of a prokaryotic replication origin, self-contained (i.e., no sequences other than the two ITRs containing Rep binding and terminal resolution sites (RBS and TRS) or exogenous sequences between the ITRs), presence of hairpin-forming ITR sequences of eukaryotic origin (i.e., they are produced in eukaryotic cells), and absence of bacterial-type DNA methylation, or indeed any other methylation that would be considered abnormal by a mammalian host. In general, it is preferred that the vector does not contain any prokaryotic DNA, although it is contemplated that some prokaryotic DNA may be inserted as exogenous sequences, as a non-limiting example in a promoter or enhancer region. Another important feature that distinguishes ceDNA vectors from plasmid expression vectors is that ceDNA vectors are single-stranded linear DNA with closed ends, whereas plasmids are always double-stranded DNA.

[0280] The ceDNA vector preferably has a linear, continuous structure, as determined by a restriction enzyme digestion assay, rather than a discontinuous structure (FIG. 4D). A linear, continuous structure is believed to be more stable against attack by cellular endonucleases, while at the same time less likely to recombine and cause mutagenesis. Thus, a linear, continuous structure is a preferred embodiment. A continuous, linear, single-stranded intramolecular duplex ceDNA vector may be covalently linked at the termini without sequences encoding AAV capsid proteins. These ceDNA vectors are structurally different from plasmids (including the ceDNA plasmids described herein), which are circular, double-stranded nucleic acid molecules of bacterial origin. Complementary strands of a plasmid can be separated following denaturation to produce two nucleic acid molecules, whereas, conversely, a ceDNA vector has complementary strands, but is a single DNA molecule, and thus remains a single molecule even when denatured. In some embodiments, ceDNA vectors, unlike plasmids, can be produced without prokaryotic cell-type DNA base methylation. Thus, ceDNA vectors and ceDNA-plasmids differ both in terms of structure (specifically, linear vs. circular) and the methods used to produce and purify these different objects, as well as in terms of their DNA methylation, which in the case of ceDNA-plasmids is of prokaryotic cell type and in the case of ceDNA vectors is of eukaryotic cell type.

[0281] The time for harvesting the DNA vector described herein from cells can be selected and optimized to achieve high yield production of DNA vector. For example, harvest time can be selected with respect to viability, cell morphology, cell growth, etc. Usually, cells can be harvested after baculovirus infection for sufficient time to produce DNA-vector, but before the majority of cells start to die due to viral toxicity. DNA-vector can be isolated using a plasmid purification kit, such as, for example, Qiagen Endo-Free™ Plasmid Kit. Other methods developed for plasmid isolation can also be adapted for DNA-vector. In general, any nucleic acid purification method known in the art can be employed.

[0282] Promoters: Suitable promoters, including those mentioned above, may be derived from viruses and therefore may be referred to as viral promoters, or they may be derived from any organism, including prokaryotes or eukaryotes. Suitable promoters can be used to drive expression by any RNA polymerase (e.g., pol I, pol II, pol III). Exemplary promoters include the SV40 early promoter, mouse mammary tumor virus long terminal sequence (LTR) promoter, adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter, such as the CMV immediate early promoter region (CMVIE), Rous sarcoma virus (RSV) promoter, human U6 micronucleus 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-antitypsin (HAAT) promoter (e.g., SEQ ID NO: 21), and the like. In embodiments, these promoters are modified at their downstream intron-containing ends to contain one or more nuclease cleavage sites. In embodiments, the DNA containing the nuclease cleavage site(s) is foreign to the promoter DNA.

[0283] A promoter may contain one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. A promoter may also contain distal enhancer or repressor elements, which may be located as far away as several thousand base pairs from the start site of transcription. Promoters may be derived from sources including viral, bacterial, myocardial, plant, insect, and animal. A promoter may constitutively or differentially regulate the expression of a gene component with respect to the cell, tissue, or organ in which expression occurs, or with respect to the developmental stage in which expression occurs, or in response to an external stimulus, such as a physiological stress, a pathogen, a metal ion, or an inducer. Representative examples of promoters include bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac 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 can be used for the expression of any gene of interest (e.g., gene editing molecules, donor sequences, therapeutic proteins, etc.). For example, the vector can include a promoter operably linked to a nucleic acid sequence encoding a therapeutic protein. The promoter operably linked to a sequence encoding a therapeutic protein can be a promoter from Simian Virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter, such as a bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter, such as a CMV immediate early promoter, an Epstein-Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter.The promoter can also be a promoter from a human gene, such as human ubiquitin C (hUbC), human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionine. The promoter can also be a tissue-specific promoter, such as a liver-specific promoter, such as human alpha 1 antitypsin (HAAT) (natural or synthetic). In one embodiment, delivery to the liver can be achieved using endogenous ApoE-specific targeting of the composition comprising the ceDNA vector to hepatocytes via the low-density lipoprotein (LDL) receptor present on the surface of hepatocytes.

[0284] In one embodiment, the promoter used is the native promoter of the gene that codes for therapeutic protein.The promoter and other regulatory sequences of each gene that codes for therapeutic protein are known and characterized.The promoter region used may further comprise one or more additional regulatory sequences (e.g., native), such as enhancers (e.g., SEQ ID NO:22 and SEQ ID NO:23).

[0285] Non-limiting examples of suitable promoters for use in accordance with the present invention include, for example, the CAG promoter (SEQ ID NO: 3), the HAAT promoter (SEQ ID NO: 21), the human EF1-α promoter (SEQ ID NO: 6), or fragments of the EF1a promoter (SEQ ID NO: 15) and the rat EF1-α promoter (SEQ ID NO: 24).

[0286] Polyadenylation sequence: A sequence encoding a polyadenylation sequence may be included in a ceDNA vector to stabilize the mRNA expressed from the ceDNA vector and to aid in nuclear transport and translation. In one embodiment, the ceDNA vector does not contain a polyadenylation sequence. In other embodiments, the vector contains 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, the polyadenylation sequence contains about 43 nucleotides, about 40-50 nucleotides, about 40-55 nucleotides, about 45-50 nucleotides, about 35-50 nucleotides, or any range therebetween.

[0287] In some embodiments, the ceDNA may be obtained from a vector polynucleotide encoding a heterologous nucleic acid operably positioned between two different inverted terminal repeats (ITRs) (e.g., AAV ITRs), where at least one of the ITRs contains a terminal resolution site and a replication protein binding site (RPS), e.g., a Rep binding site (e.g., wt AAV ITR SEQ ID NO:1 or SEQ ID NO:2 of AAV2), and one of the ITRs contains a deletion, insertion, and / or substitution with respect to the other ITR, e.g., the functional ITR.

[0288] The host cell does not express viral capsid protein, and the polynucleotide vector template lacks any viral capsid coding sequence.In one embodiment, the polynucleotide vector template lacks not only AAV capsid gene, but also other viral capsid genes.In addition, in certain embodiments, the nucleic acid molecule also lacks AAV Rep protein coding sequence.Therefore, in a preferred embodiment, the nucleic acid molecule of the present invention lacks both functional AAV cap gene and AAV rep gene.

[0289] In certain embodiments of the present invention, the ceDNA vector does not have a modified ITR comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 548, 549, 551, 552, 553, 553, 554, 555, 556, and 557.

[0290] In some embodiments, the ceDNA vector comprises a regulatory switch as disclosed herein (or PCT Application No. PCT / US18 / 49996, filed September 7, 2018) and a modified ITR having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 548, 549, 551, 552, 553, 553, 554, 555, 556, and 557.

[0291] Without limitation, the above disclaimer applies to at least claims 1-33 of this application, and paragraphs set forth at

[0293] and

[0294] .

[0292] Some embodiments of the various aspects disclosed herein can be defined by any one of the following paragraphs. 1. A liposomal ceDNA vector comprising a liposome encapsulating a ceDNA vector, the ceDNA vector being: An expression cassette comprising a cis-regulatory element, the cis-regulatory element being selected from the group consisting of a post-transcriptional regulatory element and a BGH polyA signal; A wild-type ITR upstream (5' end) of the expression cassette, the wild-type ITR comprising the polynucleotide of SEQ ID NO: 51; a modified ITR downstream (3' end) of the expression cassette, the modified ITR comprising the polynucleotide of SEQ ID NO:2; A liposomal ceDNA vector, wherein the DNA vector lacks prokaryotic cell-specific methylation and is not encapsidated in AAV capsid proteins. 2. A DNA vector according to paragraph 1, wherein the DNA vector has a linear, contiguous structure. 3. The DNA vector of paragraph 1 or 2, wherein the post-transcriptional regulatory element comprises a WHP post-transcriptional regulatory element (WPRE). 4. The DNA vector of any of paragraphs 1 to 3, wherein the expression cassette further comprises a cloning site. 5. The DNA vector according to any of paragraphs 1 to 4, wherein the expression cassette comprises a promoter selected from the group consisting of the CAG promoter, the AAT promoter, the LP1 promoter, and the EF1a promoter. 6. The DNA vector described in paragraph 1, wherein the expression cassette comprises the polynucleotides of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9. 7. The DNA vector of any of paragraphs 1 to 6, wherein the expression cassette further comprises a cloning site and an exogenous sequence inserted into the cloning site. 8. A DNA vector according to paragraph 7, wherein the exogenous sequence comprises at least 2000 nucleotides. 9. A DNA vector according to paragraph 7, wherein the exogenous sequence encodes a protein. 10. A DNA vector according to paragraph 7, wherein the exogenous sequence encodes a reporter protein.

[0293] Some embodiments of the various aspects disclosed herein can be defined by any one of the following paragraphs. 1. A lipid particle comprising an ionizable lipid and a non-viral capsid-free DNA vector (ceDNA vector) having covalently closed ends, wherein the ceDNA vector comprises at least one heterologous nucleotide sequence operably positioned between asymmetric inverted terminal repeats (asymmetric ITRs), at least one of the asymmetric ITRs comprising a functional terminal resolution site and a Rep binding site. 2. The lipid nanoparticle described in paragraph 1, wherein the ceDNA vector, when digested with a restriction enzyme having a single recognition site on the ceDNA vector and analyzed by both native and denaturing gel electrophoresis, exhibits a characteristic linear and continuous DNA band compared to a linear and discontinuous DNA control. 3. The lipid nanoparticle of paragraph 1 or 2, wherein one or more of the asymmetric ITR sequences are derived from a virus selected from a parvovirus, a dependovirus, and an adeno-associated virus (AAV). 4. The lipid nanoparticle of paragraph 3, wherein the asymmetric ITRs are derived from different viral serotypes. 5. The lipid nanoparticle of paragraph 4, wherein one or more asymmetric ITRs are derived from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. 6. The lipid nanoparticle of any one of paragraphs 1 to 3, wherein one or more of the asymmetric ITR sequences is synthetic. 7. The lipid nanoparticle of any one of paragraphs 1 to 6, wherein one or more of the ITRs is not a wild-type ITR. 8. The lipid nanoparticle of any one of paragraphs 1 to 7, wherein one or more of the asymmetric ITRs are modified by a 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. ceDNA vectors, a. SEQ ID NO:1 and SEQ ID NO:52, and b. The lipid nanoparticle of any one of paragraphs 1 to 8, comprising at least two asymmetric ITRs selected from SEQ ID NO:2 and SEQ ID NO:51. 10. ceDNA vectors, a. incubating a population of insect cells harboring a ceDNA expression construct in the presence of at least one Rep protein, the ceDNA expression construct encoding a ceDNA vector, under conditions effective, and for a time sufficient, to induce production of the ceDNA vector in the insect cells; b. The lipid nanoparticles according to any one of paragraphs 1 to 9, obtained from a process comprising the steps of: isolating the ceDNA vector from an insect cell. 11. The lipid nanoparticle of paragraph 10, wherein the ceDNA expression construct is selected from a ceDNA plasmid, a ceDNA bacmid, and a ceDNA baculovirus. 12. The lipid nanoparticle of paragraph 10 or paragraph 11, wherein the insect cells express at least one Rep protein. 13. The lipid nanoparticle of paragraph 10, wherein at least one Rep protein is derived from a virus selected from a parvovirus, a dependovirus, and an adeno-associated virus (AAV). 14. The lipid nanoparticle of paragraph 13, wherein at least one Rep protein is derived from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. 15. A DNA vector is obtained from a vector polynucleotide, the vector polynucleotide encodes a heterologous nucleic acid operably positioned between two inverted terminal repeats (ITRs), the two ITRs are different from each other (asymmetric), at least one of the ITRs is a functional ITR containing a functional terminal resolution site and a Rep binding site, one of the ITRs contains a deletion, insertion, and / or substitution relative to the functional ITR, the presence of Rep protein induces replication of the vector polynucleotide in an insect cell and production of a DNA vector, and the DNA vector is a. incubating a population of insect cells harboring a vector polynucleotide lacking a viral capsid coding sequence in the presence of Rep protein under conditions effective, and for a time sufficient, to induce production of capsid-free, non-viral DNA vector in the insect cells, wherein the insect cells, in the absence of the vector, do not contain production of capsid-free, non-viral DNA in the insect cells; b. harvesting and isolating capsid-free, non-viral DNA from insect cells. 16. A lipid particle according to any one of paragraphs 10 to 15, in which the presence of capsid-free non-viral DNA isolated from insect cells can be confirmed. 17. The lipid particle described in paragraph 16, wherein the presence of capsid-free, non-viral DNA isolated from the insect cells can be confirmed by digesting the DNA isolated from the insect cells with a restriction enzyme that has a single recognition site on the DNA vector and analyzing the digested DNA material on a non-denaturing gel to confirm the presence of a characteristic linear, continuous DNA band compared to linear, discontinuous DNA. 18. A DNA vector is obtained from a vector polynucleotide, the vector polynucleotide encoding a heterologous nucleic acid operably positioned between a first and a second AAV2 inverted terminal repeat DNA polynucleotide sequence (ITR), at least one of the ITRs having at least one polynucleotide deletion, insertion, and / or substitution relative to the corresponding AAV2 wild-type ITR of SEQ ID NO:1 or SEQ ID NO:51, and directs replication of the DNA vector in an insect cell in the presence of Rep protein, the DNA vector being a. incubating a population of insect cells harboring a vector polynucleotide lacking a viral capsid coding sequence in the presence of Rep protein under conditions effective, and for a time sufficient, to induce production of a capsid-free, non-viral DNA vector in the insect cells, wherein the insect cells do not contain viral capsid coding sequences; b. harvesting and isolating capsid-free, non-viral DNA from insect cells. 19. The lipid particle according to paragraph 18, wherein the presence of capsid-free non-viral DNA isolated from insect cells can be confirmed. 20. The lipid particle described in paragraph 19, wherein the presence of capsid-free, non-viral DNA isolated from the insect cells can be confirmed by digesting the DNA isolated from the insect cells with a restriction enzyme that has a single recognition site on the DNA vector and analyzing the digested DNA material on a non-denaturing gel to confirm the presence of a characteristic linear, continuous DNA band compared to linear, discontinuous DNA. 21. The lipid particle of any one of paragraphs 1-20, wherein the lipid particle further comprises one or more of a non-cationic lipid, a PEG-conjugated lipid, and a sterol. 22. The lipid particle of any one of paragraphs 1 to 21, wherein the ionizable lipid is a lipid listed in Table 1. 23.The lipid particles further comprise a non-cationic lipid, the non-ionic lipid being selected from the group consisting of distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylcholine (DPPC), and the like. Leoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (e.g. 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (e.g. 16-O-dimethyl PE) , 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), diercoyl phosphatidylcholine (DEPC), palmitoyl oleoyl phosphatidylglycerol (DSPG), diethyl ... 23. The lipid particle of any one of paragraphs 1 to 22, wherein the lipid particle is selected from the group consisting of oleyl phosphatidylethanolamine (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, lysophosphatidylcholine, and dilinoleoylphosphatidylcholine. 24. The lipid particle according to any one of paragraphs 1 to 23, further comprising a complex lipid, the complex lipid being selected from the group consisting of PEG-diacylglycerol (DAG) (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) (4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, and N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt. 25. The lipid particle according to any one of paragraphs 1 to 24, wherein the lipid particle further comprises cholesterol or a cholesterol derivative. 26. A lipid particle is (i) an ionizable lipid; (ii) a non-cationic lipid; and (iii) a conjugated lipid that inhibits particle aggregation; (iv) a sterol. 27. A lipid particle is (a) an ionized lipid in an amount of about 20 mol% to about 90 mol% of the total lipid present in the particle; (b) a non-cationic lipid in an amount of about 5 mol % to about 30 mol % of the total lipid present in the particle; and (c) a complex lipid that inhibits particle aggregation in an amount of about 0.5 mol % to about 20 mol % of the total lipid present in the particle; (d) a sterol in an amount of about 20 mol % to about 50 mol % of the total lipid present in the particle. 28. The lipid particle of any one of paragraphs 1 to 27, wherein the ratio (by mass or weight) of total lipid to DNA vector is from about 10:1 to about 30:1. 29. A composition comprising a first lipid nanoparticle and an additional compound, wherein the first lipid nanoparticle comprises a first capsid-free, non-viral vector and is a lipid nanoparticle described in any one of paragraphs 1 to 28. 30. The composition of paragraph 29, wherein the additional compound is incorporated into a second lipid nanoparticle, and the first and second lipid nanoparticles are different. 31. The composition of paragraph 28 or 29, wherein the additional compound is incorporated into the first lipid nanoparticle. 32. The composition of any one of paragraphs 28 to 30, wherein the additional compound is a therapeutic agent. 33. The composition of paragraph 28, wherein the additional compound is a second capsid-free non-viral vector, and the first and second capsid-free non-viral vectors are different.

[0294] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0295] Example 1: Exemplary methods for producing ceDNA vectors Construction of ceDNA plasmids The production of ceDNA vector using polynucleotide construct template is described. For example, the polynucleotide construct template used to generate the ceDNA vector of the present invention can be ceDNA-plasmid, ceDNA-bacmid, and / or ceDNA-baculovirus. Without being limited by theory, in permissive host cell, for example, in the presence of Rep, the polynucleotide construct template with two ITRs (at least one of the ITRs is modified) and an expression construct is compounded to produce ceDNA vector. ceDNA vector production goes through two steps: first, excision (rescue) of the template from the template backbone (for example, ceDNA-plasmid, ceDNA-bacmid, ceDNA-baculovirus genome, etc.), and second, Rep-mediated replication of the excised ceDNA vector.

[0296] An exemplary method for producing ceDNA vectors is derived from the ceDNA-plasmid described herein. Referring to Figures 1A and 1B, each polynucleotide construct template of the ceDNA-plasmid includes both the left ITR and the right mutant ITR, and has the following between the ITR sequences: (i) enhancer / promoter, (ii) transgene cloning site, (iii) post-transcriptional response element (e.g., Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE)), and (iv) polyadenylation signal (e.g., from the bovine growth hormone gene (BGHpA). Unique restriction endonuclease recognition sites (R1-R6) (shown in Figures 1A and 1B) were also introduced between each component to facilitate the introduction of new gene components at specific sites in the construct. R3 (PmeI) GTTTAAAC (SEQ ID NO: 7) and R4 (PacI) TTAATTAA (SEQ ID NO: 542) enzyme sites are designed into the cloning sites to introduce the open reading frame of the transgene. These sequences were cloned into pFastBac HT B plasmid obtained from Thermo Fisher Scientific.

[0297] In summary, a series of ceDNA vectors were obtained from the ceDNA-plasmid constructs shown in Table 3 using the process shown in Figures 4A-4C. Table 5 shows the number of the corresponding polynucleotide sequence of each component, which contains a sequence active as a replication protein site (RPS) on each end of the promoter operably linked to the transgene. The numbers in Table 3 refer to the SEQ ID NOs in this document that correspond to the sequence of each component. [Table 3]

[0298] In some embodiments, the construct for making the ceDNA vector includes a promoter that is a regulatory switch, e.g., an inducible promoter. Other constructs were used to make ceDNA vectors that include the MND or HLCR promoter operably linked to the luciferase transgene.

[0299] Production of ceDNA-bacmid: Referring to FIG. 4A, DH10Bac competent cells (MAX EFFICIENCY® DH10Bac™ competent cells, Thermo Fisher) were transformed with either the test or control plasmids according to the protocol provided by the manufacturer. Recombination between the plasmids and the baculovirus shuttle vector in DH10Bac cells was induced to generate recombinant ceDNA-bacmids. Recombinant bacmids were selected by screening for positive selection based on blue-white screening in E. coli on bacterial agar plates containing X-gal and IPTG (Φ80dlacZΔM15 marker provides α-complementation of the β-galactosidase gene from the bacmid vector) with antibiotics to select for bacmid and transposase transformants and maintenance. White colonies caused by transposition disrupting the β-galactosidase indicator gene were picked and cultured in 10 mL of medium.

[0300] Recombinant ceDNA-bacmids were isolated from E. coli and transfected into Sf9 or Sf21 insect cells using FugeneHD to produce infectious baculovirus. Adherent Sf9 or Sf21 insect cells were cultured in 50 mL of medium in a T25 flask at 25°C. After 4 days, the culture medium (containing P0 virus) was removed from the cells and filtered through a 0.45 μm filter to separate infectious baculovirus particles from cells or cell debris.

[0301] Optionally, first generation baculovirus (P0) was amplified in 50-500 mL of medium by infecting naive Sf9 or Sf21 insect cells. Cells in suspension culture in an orbital shaker incubator were maintained at 130 rpm and 25 °C, and cell diameter and viability were monitored until the cells reached a diameter of 18-19 nm (from a naive diameter of 14-15 nm), as well as a density of approximately 4.0E+6 cells / mL. Between 3 and 8 days post-infection, P1 baculovirus particles in the medium were collected after centrifugation to remove cells and debris, followed by filtration through a 0.45 μm filter.

[0302] ceDNA-baculovirus containing the test constructs was harvested and the infectious activity or titer of the baculovirus was determined. Specifically, 4 x 20 mL Sf9 cell cultures at 2.5E+6 cells / mL were treated with the following dilutions of P1 baculovirus: 1 / 1000, 1 / 10,000, 1 / 50,000, 1 / 100,000 and incubated at 25-27°C. Infectivity was determined daily over a period of 4-5 days by changes in the rate of cell diameter increase and cell cycle arrest, as well as cell viability.

[0303] Referring to Figure 4A, "Rep-plasmids" according to Figure 6A were generated in pFASTBAC™-dual expression vectors (ThermorFisher) containing both Rep78 (SEQ ID NO: 13) or Rep68 (SEQ ID NO: 12) and Rep52 (SEQ ID NO: 14).

[0304] Rep-plasmids were transformed into DH10Bac competent cells (MAX EFFICIENCY®) or DH10Bac™ competent cells (Thermo The bacmid was transformed into 100-kDa plasmid (Fisher). Recombination between the Rep-plasmid and the baculovirus shuttle vector in DH10Bac cells was induced to generate a recombinant bacmid ("Rep-bacmid"). The recombinant bacmid was selected by positive selection involving blue-white screening in E. coli (Φ80dlacZΔM15 marker provides α-complementation of the β-galactosidase gene from the bacmid vector) on bacterial agar plates containing X-gal and IPTG. Isolated white colonies were picked and plated in 10 mL of selection medium (kanamycin, gentamycin, tetracycline in LB broth). The recombinant bacmid (Rep-bacmid) was isolated from E. coli, and the Rep-bacmid was transfected into Sf9 or Sf21 insect cells to produce infectious baculovirus.

[0305] 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 Rep-baculovirus (P0) was amplified by infecting naive Sf9 or Sf21 insect cells and cultured in 50-500 mL of medium. Between 3 and 8 days after infection, the P1 baculovirus particles in the medium were collected either by separating the cells by centrifugation or by filtration or another fractionation process. The Rep-baculovirus was collected and the infectious activity of the baculovirus was determined. Specifically, 2.5 × 10 6 4 × 20 mL Sf9 cell cultures at 100 cells / mL were treated and incubated with P1 baculovirus at the following dilutions: 1 / 1000, 1 / 10,000, 1 / 50,000, 1 / 100,000. Infectivity was determined daily over 4-5 days by the rate of cell diameter increase and cell cycle arrest, as well as changes in cell viability.

[0306] ceDNA vector generation and characterization Referring to Figure 4B, next, Sf insect cell culture medium containing either (1) the sample containing ceDNA-bacmid or ceDNA-baculovirus, and (2) the Rep-baculovirus described above was added to a fresh culture of Sf9 cells (2.5E+6 cells / mL, 20 mL) at a ratio of 1:1000 and 1:10,000, respectively. Then, the cells were cultured at 25°C and 130 rpm. Four to five days after coinfection, cell diameter and viability were detected. When the cell diameter reached 18-20 nm and the viability was about 70-80%, the cell culture was centrifuged, the medium was removed, and the cell pellet was collected. First, the cell pellet was resuspended in an appropriate amount of aqueous medium (either water or buffer). The ceDNA vector was isolated and purified from the cells using the Qiagen MIDI PLUS™ purification protocol (Qiagen, 0.2 mg processed cell pellet mass per column).

[0307] The yields of ceDNA vectors produced and purified from Sf9 insect cells were first determined based on UV absorbance at 260 nm. The yields of various ceDNA vectors determined based on UV absorbance are provided in Table 4. [Table 4]

[0308] The ceDNA vector can be evaluated by identifying it by agarose gel electrophoresis under native or denaturing conditions as shown in Figure 4D, where the ceDNA vector purifies multiple bands based on the native gel. See Figure 4D. Each band may represent a vector with a different structure, e.g., monomer, dimer, etc. The presence of a single band under denaturing conditions and a double band (corresponding to the monomer and dimer forms) under non-denaturing conditions is characteristic of the presence of the ceDNA vector.

[0309] The structure of the isolated ceDNA vectors was further analyzed by digesting DNA obtained from coinfected Sf9 cells (described herein) with restriction endonucleases for a) the presence of only a single cleavage site within the ceDNA vector, and b) the resulting fragments large enough to be clearly visible when fractionated on a 0.8% denaturing agarose gel (>800bp). As shown in Figure 4E, linear DNA vectors with discontinuous structures and ceDNA vectors with linear and continuous structures can be distinguished by the size of their reaction products, e.g., DNA vectors with discontinuous structures are expected to produce 1kb and 2kb fragments, while non-encapsidated vectors with continuous structures are expected to produce 2kb and 4kb fragments.

[0310] Therefore, to qualitatively demonstrate that the isolated ceDNA vector is covalently closed, as required by definition, the sample is digested with a restriction endonuclease identified in the context of the specific DNA vector sequence as having a single restriction site, preferably resulting in two cleavage products of unequal size (e.g., 1000bp and 2000bp). Following digestion and electrophoresis on a denaturing gel (separating the two complementary DNA strands), the linear non-covalently closed DNA will dissolve at 1000bp and 2000bp in size, whereas the covalently closed DNA (i.e., the ceDNA vector) will dissolve at twice the size (2000bp and 4000bp) when the two DNA strands are ligated and unfolded to twice the length (but still single stranded). Furthermore, digestion of the monomeric, dimeric, and n-meric forms of the DNA vector will all dissolve as fragments of the same size due to the end-to-end ligation of the multimeric DNA vector (see FIG. 4D).

[0311] FIG. 5 provides an exemplary diagram of a denaturing gel with ceDNA vectors: Construct-1, Construct-2, Construct-3, Construct-4, Construct-5, Construct-6, Construct-7, and Construct-8 (all described in Table 3 above) with (+) or without (-) digestion with an endonuclease. Each ceDNA vector from Construct-1 to Construct-8 produced two bands (*) after the endonuclease reaction. The sizes of those two bands, determined based on the size markers, are provided at the bottom of the figure. The band sizes confirm that each of the ceDNA vectors produced from the plasmids containing Construct-1 to Construct-8 have a continuous structure.

[0312] As used herein, the phrase "assay for identification of DNA vectors by agarose gel electrophoresis under native and denaturing conditions" refers to an assay for evaluating the closed end of ceDNA by performing restriction endonuclease digestion followed by electrophoretic evaluation of the digestion products. One such exemplary assay is shown below, but one skilled in the art will understand that many variations of this example known in the art are possible. A restriction endonuclease is selected to be a single-cutting enzyme for the ceDNA vector of interest that will generate products of approximately 1 / 3x and 2 / 3x the DNA vector length. This will dissolve the bands in both native and denaturing gels. It is important to remove the buffer from the sample before denaturation. Qiagen PCR clean-up kits or desalting "spin columns", such as GE HEALTHCARE ILUSTRA™ MICROSPIN™ G-25 columns, are some options known in the art for endonuclease digestion. The assay may, for example, include i) digesting the DNA with the appropriate restriction endonuclease(s), 2) applying, for example, to a Qiagen PCR cleanup kit and eluting with distilled water, iii) adding 10x denaturing solution (10x = 0.5M NaOH, 10mM EDTA), adding 10x dye, not buffering, and running together with a DNA ladder prepared by adding 10x denaturing solution to 4x on a 0.8-1.0% gel previously incubated with 1mM EDTA and 200mM NaOH to ensure that the NaOH concentration is uniform in the gel and gel box and allowing the gel to run in the presence of 1x denaturing solution (50mM NaOH, 1mM EDTA). One of skill in the art will know what voltage to use to run electrophoresis based on size and desired timing results. After electrophoresis, the gel is drained, neutralized in 1x TBE or TAE, and transferred to distilled water or 1x TBE / TAE with 1x SYBR Gold.Bands can then be visualized using, for example, Thermo Fisher's SYBR® Gold nucleic acid gel stain (10,000X concentrate in DMSO) and epifluorescent light (blue) or UV (312 nm).

[0313] The purity of the generated ceDNA vector can be evaluated using any method known in the art. As one exemplary non-limiting method, the contribution of ceDNA-plasmid to the overall UV absorbance of a sample can be estimated by comparing the fluorescence intensity of ceDNA vector with a standard. For example, based on UV absorbance, if 4 μg of ceDNA vector is loaded on a gel and the ceDNA vector fluorescence intensity corresponds to a 2 kb band known to be 1 μg, then there is 1 μg of ceDNA vector, and the ceDNA vector is 25% of the total UV-absorbing material. The band intensity on the gel is then plotted against the calculated input that the band represents. For example, if the total ceDNA vector is 8 kb and the excised comparative band is 2 kb, the band intensity is plotted as 25% of the total input, in this case 0.25 μg for 1.0 μg input. When a standard curve is plotted using ceDNA vector plasmid titration, the regression line equation can be used to calculate the amount of ceDNA vector band, which can then be used to determine the percent of total input represented by ceDNA vector, or the percent purity.

[0314] Example 2: ceDNA vectors express luciferase transgene in vitro The constructs were generated by introducing an open reading frame encoding a luciferase reporter gene into the cloning sites of the ceDNA-plasmid constructs: construct-1, construct-3, construct-5, and construct-7. The ceDNA-plasmids containing the luciferase coding sequence (see above in Table 3) are referred to as plasmid construct-1-Luc, plasmid construct-3-Luc, plasmid construct-5-Luc, and plasmid construct-7-Luc, respectively.

[0315] HEK293 cells were cultured and transfected with 100ng, 200ng, or 400ng of plasmid constructs 1, 3, 5, and 7 using FUGENE® (Promega Corp.) as a transfection agent. Expression of luciferase from each of the plasmids was determined based on luciferase activity in each cell culture, and the results are provided in FIG. 7A. No luciferase activity was detected from untreated control cells ("Untreated") or cells treated with Fugene alone ("Fugene"), confirming that the luciferase activity resulted from gene expression from the plasmids. As shown in FIG. 7A and 7BB, robust expression of luciferase was detected from constructs 1 and 7. Expression from construct-7 expressed luciferase, and a dose-dependent increase in luciferase activity was detected.

[0316] The growth and viability of cells transfected with each of the plasmids was also determined and is presented in Figures 8A and 8B. Cell growth and viability of the transfected cells did not differ significantly between the different groups of cells treated with the different constructs.

[0317] Thus, the luciferase activity measured in each group and normalized based on cell proliferation and viability did not differ from the luciferase activity without normalization. The ceDNA-plasmid carrying construct 1-Luc showed the most robust expression of luciferase with or without normalization.

[0318] Thus, the data presented in Figures 7A, 7B, 8A, and 8B demonstrate that construct 1, comprising the 5' to 3'-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), is effective in producing a ceDNA vector capable of expressing a transgene protein within the ceDNA vector.

[0319] Example 3: Lipid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA) The lipid particles containing ceDNA were prepared using the lipid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)-butanoate (DLin-MC3-DMA) (also referred to herein as "MC3" and having the following structure): [ka] It may be prepared or formulated in combination with.

[0320] The lipid DLin-MC3-DMA is described in Jayaraman et al., Angew. Chem. Int. Ed Engl. (2012), 51(34):8529-8533, the contents of which are incorporated herein by reference in their entirety. It is synthesized as follows.

[0321] Synthesis of methanesulfonic acid octadeca-9,12-dienyl ester To a solution of alcohol 1 (26.6 g, 100 mmol) in dichloromethane (100 mL), triethylamine (13.13 g, 130 mmol) is added and the solution is cooled in an ice bath. To this cold solution, a solution of mesyl chloride (12.6 g, 110 mmol) in dichloromethane (60 mL) is added dropwise and after the addition is complete, the reaction mixture is allowed to warm to ambient temperature and stirred overnight. TLC of the reaction mixture indicates completion of the reaction. The reaction mixture is diluted with dichloromethane (200 mL), washed with water (200 mL), saturated NaHCO3 (200 mL), brine (100 mL), and dried (NaSO4). The organic layer is concentrated to 0-10% in hexanes. The crude product was purified by column chromatography (silica gel) using Et 2 O. The pure product fractions were combined and concentrated to give the pure methanesulfonic acid octadeca-9,12-dienyl ester as a colorless oil. 1H NMR(CDCl3,400MHz)δ5.42-5.21(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(CDCl3)δ130.76,130.54,128.6,128.4,70.67,37.9,32.05,30.12,29. 87,29.85,29.68,29.65,29.53,27.72,27.71,26.15,25.94,23.09,14.60. M.S.C. 19 H 36 Calculated molecular weight of O3S: 344.53.

[0322] Synthesis of 18-bromo-octadeca-6,9-diene: The mesylate (methanesulfonic acid octadeca-9,12-dienyl ester, 13.44 g, 39 mmol) is dissolved in anhydrous ether (500 mL) to which MgBr.Et2O complex (30.7 g, 118 mmol) is added under argon, and the mixture is refluxed under argon for 26 h, after which TLC indicates completion of the reaction. The reaction mixture is diluted with ether (200 mL), ice-cold water (200 mL) is added to the mixture, and the layers are separated. The organic layer is washed with 1% aqueous K2CO3 (100 mL), brine (100 mL), and dried (anhydrous Na2SO4). Concentration of the organic layer provides the crude product, which is further purified by column chromatography (silica gel) using 0-1% EtO in hexanes to isolate the product, 18-bromo-octadeca-6,9-diene, as a colorless oil. 1 H NMR(CDCl3,400MHz)δ5.41-5.29(m,4H),4.20(d,2H),3.40(t,J=7Hz,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=3.9Hz,3H). 13C NMR(CDCl3)δ130.41,130.25,128.26,128.12,34.17,33.05,31.75,29.82,29.57,29.54,29.39,28.95,28.38,27.42,27.40,25.84,22.79,14.28。

[0323] Synthesis of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (DLin-MeOH): Add freshly activated Mg turnings (2.4 g, 100 mmol) to a flame-dried 500 mL RB flask equipped with a magnetic stir bar, addition funnel, and reflux condenser. The setup is degassed and filled with argon, and 10 mL of anhydrous ether is added to the flask via syringe. 18-Bromo-octadeca-6,9-diene (26.5 g, 80.47 mmol) is dissolved in anhydrous ether (50 mL) and added to the addition funnel. Approximately 5 mL of this ether solution is added to the Mg turnings with vigorous stirring. An exothermic reaction is observed (to confirm / accelerate Grignard reagent formation, 5 mg of iodine is added and immediate decolorization is observed, confirming the formation of the Grignard reagent) and the ether begins to reflux. The remainder of the bromide solution is added dropwise while keeping the reaction under gentle reflux by cooling the flask in water. After the addition is complete, the reaction mixture is kept at 35° C. for 1 hour and then cooled in an ice bath. Ethyl formate (2.68 g, 36.2 mmol) is dissolved in anhydrous ether (40 mL) and 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 reflux. After the reaction begins, the remainder of the ethereal solution of formic acid is added quickly as a stream and the reaction mixture is stirred at ambient temperature for an additional hour. The reaction is quenched by the dropwise addition of 10 mL of acetone followed by ice-cold water (60 mL). The reaction mixture is treated with aqueous H2SO4 (10% by volume, 300 mL) until the solution becomes homogenous and the layers are separated. The aqueous phase is extracted with ether (2 x 100 mL). The combined ether layers are dried (Na2SO4) and concentrated to give the crude product, which is treated with 1 g of sodium in methanol (200 mL) at room temperature overnight. Upon completion of the reaction, most of the solvent is evaporated. The resulting mixture is poured into 150 mL of 5% hydrochloric acid solution. The aqueous phase is extracted with ether (2 x 150 mL). The combined ether extracts are washed with water (2 x 100 mL), brine (100 mL) and dried over anhydrous sodium sulfate.Evaporation of the solvent gives the crude product, which is purified by column (silica gel, 0-10% ether in hexanes) chromatography and the pure product fractions are evaporated to yield the product DLin-MeOH as a colorless oil. 1 H NMR (400MHz, CDCl3) δ5.47-5.24(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(101MHz,CDCl3)δ130.41,130.37,128.18,128.15,77.54,77.22,76.91,72.25,37.73,3 1.75,29.94,29.89,29.83,29.73,29.58,29.53,27.46,27.43,25.89,25.86,22.80,14.30.

[0324] 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 to which is added the hydrochloride salt of dimethylaminobutyric acid 7 (55 g, 328 mmol), followed by diisopropylethylamine (70 mL) and DMAP (4 g). After stirring for 5 min at ambient temperature, EDCI (80 g, 417 mmol) is added and the reaction mixture is stirred at room temperature overnight, after which TLC (silica gel, 5% MeOH in CHCl) analysis shows complete disappearance of the starting alcohol. The reaction mixture is diluted with CHCl (500 mL) and washed with saturated NaHCO (400 mL), water (400 mL), and brine (500 mL). The combined organic layer is dried over anhydrous Na2SO4 and the solvent is removed in vacuum. The crude product thus obtained is purified by flash column chromatography [2.5Kg silica gel, column packed with the following eluents: i) 6L 0.1% NEt3 in DCM; after loading, ii) 4L 0.1% NEt3 in DCM; iii) 16L 2% MeOH-98% 0.1% NEt3 in DCM; iv) 4L 2.5% MeOH-97.5% 0.1% NEt3 in DCM; v) 12L 3% MeOH-97% 0.1% NEt3 in DCM] to isolate the pure product MC3 as a colorless oil. 1 H NMR (400MHz, CDCl3): δ5.46-5.23(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). 13C NMR (101MHz, CDCl3): δ173.56,130.38,130.33,128.17,128.14,77.54,77.22,76.90,74.44,59.17,45.64,34. 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):C 43 H 79 Calculated MW of NO2 (M+H) + :642.6.

[0325] Example 4: Lipid ATX-002 Lipid particles containing ceDNA can be prepared or formulated in combination with the lipid ATX-002, which has the following structure: [ka]

[0326] The lipid ATX-002 is described in WO2015 / 074085, the contents of which are incorporated herein by reference in their entirety, and is synthesized as follows.

[0327] Synthesis of methyl 8-bromooctanoate: Under N2 atmosphere, 8-bromooctanoic acid (60 gm, 1 eq.) was dissolved in 400 mL of dry methanol. 10 drops of concentrated H2SO4 were added dropwise and the reaction mixture was stirred under reflux for 3 hours.

[0328] The reaction is monitored by thin layer chromatography (TLC) until completion. The solvent is completely removed under vacuum. The reaction mixture is diluted with ethyl acetate and washed with water. The aqueous layer is re-extracted with ethyl acetate. The total organic layer is washed with saturated NaHCO3 solution. The organic layer is washed again with water and finally with brine. The product is dried over anhydrous Na2SO4 and concentrated.

[0329] Synthesis of Dimethyl 8,8'-(Benzanediyl)dioctanoate: Take dry K2CO3 (104.7 gm, 6 eq) and add it to dry dimethylformamide under N2. Add benzylamine (13.54 gm, 1 eq) in dimethylformamide slowly. Then add methyl 8-bromooctanoate (60 gm, 2 eq) dissolved in dimethylformamide at room temperature. Heat the reaction mixture to 80°C and maintain the reaction with stirring for 36 hours.

[0330] The reaction is monitored by thin layer chromatography until completion. The reaction product is cooled to room temperature and water is added. The compound is extracted with ethyl acetate. The aqueous layer is re-extracted with ethyl acetate. The combined organic layers are washed with water and finally with a brine solution. The product is dried over anhydrous Na2SO4 and concentrated.

[0331] The reaction product is purified by silica gel column chromatography in 3% methanol in chloroform. Using a TLC system of 10% methanol in chloroform, the product migrates at Rf: 0.8 and is visualized by charring in ninhydrin. The compound is a light brown liquid. The structure is: 1 Confirmed by H-NMR.

[0332] Synthesis of dimethyl 8,8'-azanediyldioctanoate: Dimethyl 8,8'-(benzandiyl)dioctanoate (3.5 gm, 1 eq.) is transferred to a hydrogenation glass vessel and 90 mL of ethanol is added followed by 10% Pd / C (700 mg). The reaction mixture is stirred in a Parr stirrer under 50 psi H2 atmospheric pressure for 2 hours at room temperature. The reaction product is filtered through Celite and washed with hot ethyl acetate. The filtrate is concentrated under vacuum.

[0333] Synthesis of Dimethyl 8,8'-((tertbutoxycarbonyl)azanediyl)dioctanoate: Dimethyl 8,8'-azanediyl-dioctanoate (32 gm, 1 eq) is transferred to reaction mass in dry DCM (700 mL) and dry Et3N (9 gm, 4 eq) and cooled to 0°C. Boc anhydride (31.3 gm, 1.5 eq) diluted in DCM is added dropwise to the above reaction. After the addition is complete, the reaction mixture is stirred at room temperature for 3 hours.

[0334] The reaction is quenched with water and the DCM layer is separated. The aqueous phase is re-extracted with DCM and the combined DCM layers are washed with brine solution and dried over Na2SO4. After concentration, the crude compound is collected. The crude reaction product is purified by column chromatography using 0-12% ethyl acetate in hexanes. A single product is migrated by thin layer chromatography in 20% ethyl acetate in hexanes with an Rf of 0.5 and charred with ninhydrin.

[0335] Synthesis of 8,8'-((tertbutoxycarbonyl)azanediyl)dioctanoic acid: Dimethyl 8,8'-((tertbutoxy-carbonyl)azanediyl)dioctanoate (21 gm, 1 eq.) is transferred into dry THF (200 mL). 6N aqueous sodium hydroxide solution (175 mL) is added at room temperature. Keep the reaction under stirring at room temperature overnight.

[0336] 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 is washed with water and the aqueous layer is re-extracted with ethyl acetate. The combined organic layers are washed with brine solution and dried over anhydrous Na2SO4. Concentration of the solution gives the crude product.

[0337] Synthesis of di((Z)-non-2-en-1-yl)8,8'((tertbutoxycarbonyl)azanediyl): Dissolve 8,8'-((tertbutoxycarbonyl)azanediyl)dioctanoic acid (18 gm, 1 eq) in dry DCM (150 mL). Add HATU (26.15 gm, 2.1 eq) to this solution. Add D-isopropylethylamine (14.81 gm, 3.5 eq) slowly to the reaction mixture at room temperature. The internal temperature rises to 40° C. and a pale yellow solution forms. Add DMAP (400 mg, 0.1 eq) followed by a solution of cis-2-nonen-1-ol (9.31 gm, 2 eq) in dry DCM to the reaction mixture. The reaction turns brown. Stir the reaction at room temperature for 5 hours.

[0338] The reaction is checked by thin layer chromatography until completion. Water is added to the reaction product, which is extracted with DCM. The DCM layer is washed with water, followed by brine solution. The organic layer is dried over anhydrous Na2SO4 and concentrated to give the crude compound.

[0339] Synthesis of ATX-002: Di((Z)-non-2-en-1-yl) 8,8'((tertbutoxycarbonyl)azanediyl) dioctanoate (13.85mmol, 9 grams) is dissolved in dry DCM (150mL). TFA is added at 0°C to initiate the reaction. The reaction temperature is allowed to slowly warm to room temperature over 30 minutes with stirring. Thin layer chromatography shows 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% NaHCO3 solution. The aqueous layer is re-extracted with DCM, the combined organic layers are washed with brine solution, dried over Na2SO4 and concentrated. The collected crude product is dissolved in dry DCM (85mL) under nitrogen gas. Triphosgene is added, the reaction mixture is cooled to 0°C and Et3N is added dropwise. The reaction mixture is allowed to stir at room temperature overnight. Thin layer chromatography shows the reaction is complete. The DCM solvent is removed from the reaction mass by distillation under N2. The reaction product is cooled to 0°C, diluted with DCM (50 mL) and 2-(dimethylamino)ethanethiol HCl (0.063 mol, 8.3 g) is added followed by Et3N (dry). The reaction mixture is then stirred at room temperature overnight. Thin layer chromatography indicates that the reaction is complete. The reaction product is diluted with 0.3 M HCl solution (75 mL) and the organic layer is separated. The aqueous layer is re-extracted with DCM and the combined organic layers are washed with 10% K2CO3 aqueous solution (75 mL) and dried over anhydrous Na2SO4. Concentration of the solvent gives the crude product. The crude compound is purified by silica gel column (100-200 mesh) using 3% MeOH / DCM. Example 5: (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine (compound 32)

[0340] Lipid particles containing ceDNA can be prepared or formulated in combination with (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine (compound 32), having the following structure: [ka]

[0341] Compound 32 is described in WO2012 / 040184, the contents of which are incorporated herein by reference in their entirety, and is synthesized as follows:

[0342] Synthesis of α,β-unsaturated amide (vii): [ka]

[0343] Silylamide Peterson reagent (3.1 g, 16.7 mmol) is dissolved in THF (35 mL) and cooled to -63°C. To this solution is added nBuLi (16.7 mmol, 6.7 mL of a 2.5 M solution). The reaction is allowed to warm to ambient temperature for 30 min. Ketone (iii) (5.0 g, 11.9 mmol) is dissolved in THF (25 mL) in a second flask. The ketone solution is transferred to the Peterson reagent over 30 min while maintaining the temperature between -60°C and -40°C. The reaction is allowed to warm to -40°C for 1 h and then to 0°C for 30 min. The reaction is quenched with sodium bicarbonate, diluted with additional water, and partitioned between water and hexanes. The organics are washed with brine, dried over sodium sulfate, filtered, and evaporated in vacuo. Purification by flash column chromatography (0-40% MTBE / hexanes) affords the α,β-unsaturated amide (vii). 1 H NMR (400MHz, CDCl3) δ5.75(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). [ka]

[0344] The α,β-unsaturated amide (vii) (1 g, 2.1 mmol) and LS-Selectride (4.1 mmol, 4.1 mL of a 1 M solution) are combined in a sealed tube and heated to 60° C. for 24 hours. The reaction is cooled to ambient temperature and partitioned between ammonium chloride solution and heptane. The organics are dried over sodium sulfate, filtered, and evaporated in vacuo to give the amide (viii). This intermediate is carried directly into the next reaction crude product.

[0345] An alternative conjugate reduction of the α,β-unsaturated amide (vii) involves the use of copper hydride reduction. [ka]

[0346] In a 5 L RB, dissolve copper catalyst (9.77 g, 17.13 mmol) in toluene (1713 mL) under nitrogen. To this, add PMHS from Aldrich (304 mL, 1371 mmol) in a single portion. Allow the reaction to stagnate for 5 min. To this solution, add α,β-unsaturated amide (vii) (167.16 g, 343 mmol). To this mixture is then added t-amyl alcohol (113 mL, 1028 mmol) via syringe pump over 3 h. After the addition is complete, add approximately 1700 mL of 20% NH4OH to the solution in small portions to the reaction. CAUTION: There will be vigorous boiling and foaming at the beginning of the quench, which must be carefully monitored, and ammonium hydroxide is added slowly in small portions. Partition the reaction between water and hexane. Filter the organics through Celite and evaporate in vacuo. The resulting rubbery solid material is ground in hexane using a mechanical stirrer to produce small particles which are then filtered and washed with hexane. The organics are then evaporated in vacuo and purified by flash column chromatography (silica, 0-15% ethyl acetate / hexane) to give the desired amide (viii). LC / MS (M+H)=490.7.

[0347] Synthesis of compound 32: To a solution of amide (viii) (2.85 g, 5.8 mmol) is added lithium aluminum hydride (8.7 mmol, 8.7 mL of a 1 M solution). The reaction is stirred at ambient temperature for 10 min and then quenched by slow addition of sodium sulfate decahydrate solution. The solid is filtered, washed with THF, and the filtrate is evaporated in vacuo. The crude mixture is purified by reverse phase preparative chromatography (C8 column) to give (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine (compound 32) as an oil. HRMS (M+H) calculated 476.5190. 1 H NMR (400MHz, CDCl3) δ5.37 (m, 4H), 2.78 (t, 2H), 2.42 (m, 8H), 2.05 (q, 4H), 1.28 (m, 41H), 0.89 (m, 6H).

[0348] Example 6: Compounds 6 and 22 Lipid particles containing ceDNA can be prepared or formulated in combination with compounds 6 or 22, having the following structures: [ka]

[0349] Compounds 6 and 22 are described in WO2015 / 199952, the contents of which are incorporated herein by reference in their entirety. Compounds 6 and 22 were synthesized as follows.

[0350] Synthesis of compound 6: Treat a solution of nonane-1,9-diol (12.6 g) in methylene chloride (80 mL) with 2-hexyldecanoic acid (10.0 g), DCC (8.7 g), and DMAP (5.7 g). Stir the solution for 2 h. Filter the reaction mixture and remove the solvent. Dissolve the residue in warm hexane (250 mL) and allow to crystallize. Filter the solution and remove the solvent. Dissolve the residue in methylene chloride and wash with dilute hydrochloric acid. Dry the organic fraction over anhydrous magnesium sulfate, filter, and remove the solvent. Load the residue onto a silica gel column (75 g) using 0-12% ethyl acetate / hexane as the eluent to obtain 9-(2'-hexyldecanoyloxy)nonan-1-ol as an oil.

[0351] The product is dissolved in methylene chloride (60 mL) and treated with pyridinium chromium chloride (6.4 g) for 4 h. Diethyl ether (200 mL) is added and the supernatant is filtered through a bed of silica gel. The solvent is removed from the filtrate and the resulting oil is transferred to a silica gel (75 g) column using an ethyl acetate / hexane (0-12%) gradient to give 9-(2'-ethylhexanoyloxy)nonanal as an oil.

[0352] 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) is treated with sodium triacetoxyborohydride (2.9 g) for 2 h. The solution is diluted with methylene chloride and washed with aqueous sodium hydroxide followed by water. The organic phase is dried over anhydrous magnesium sulfate, filtered, and the solvent removed. The residue is transferred to a silica gel (75 g) column using a methanol / methylene chloride (0-8%) gradient followed by a second column (20 g) using a methylene chloride / acetic acid / methanol gradient. The purified fraction is dissolved in methylene chloride, washed with dilute aqueous sodium hydroxide, dried over anhydrous magnesium sulfate, filtered, and the solvent removed to give the desired product as a colorless oil.

[0353] Synthesis of compound 22: In step 1, to 6-bromohexanoic acid (20 mmol, 3.901 g), 2-hexyl-1-decanol (1.8 equiv, 36 mmol, 8.72 g), and 4-dimethylaminopyridine (DMAP 0.5 equiv, 10 mmol, 1.22 g) in DCM (80 mL), add DCC (1.1 equiv, 22 mmol, 4.54 g). Stir the resulting mixture at room temperature for 16 h. Discard the precipitate by filtration. Concentrate the filtrate. Purify the residue by column chromatography on silica gel eluted with gradient mixtures of ethyl acetate in hexanes (0-2%). This gives the desired product as a colorless oil.

[0354] In step 2, a mixture of the bromide from step 1 (1.34 equiv, 7.88 g, 18.8 mmol), N,N-diisopropylethylamine (1.96 equiv, 27.48 mmol, 4.78 mL), and N,N-dimethylethylenediamine (1 equiv, 14.02 mmol, 1.236 g, 1.531 mL) in acetonitrile (70 mL) in a 250 mL flask equipped with a condenser is heated at 79 °C (oil bath) for 16 h. The reaction mixture is cooled to room temperature and concentrated. The residue is taken up in a mixture of ethyl acetate and hexane (1:9), and water. The phases are separated and washed with water (100 mL) and brine. It is then dried over sodium sulfate and concentrated (8.7 g of oil). The crude product (8.7 g of oil) is purified by column chromatography on silica gel (0-3% MeOH in chloroform). Combine and concentrate the fractions containing the desired product. Dissolve the residue in 1 mL of hexane and filter through a layer of silica gel (3-4 mm, washed with 8 mL of hexane). Blow the filtrate dry by spraying with a stream of Ar and thoroughly dry in vacuum overnight. Obtain the desired product as a colorless oil. 1H NMR(400MHz,CDCl3)δ3.96(d,5.8Hz,4H),2.55-2.50(m,2H),2.43-2.39(m,4H),3.37-3.32(m,2H),2.30(t, 7.5Hz, 4H), 2.23 (s, 6H), 1.63 (quintet-like, 7.6Hz, 6H), 1.48-1.40 (m, 4H), 1.34-1.20 (52H), 0.88 (t-like, 6.8Hz, 12H).

[0355] Example 7: Preparation of lipid formulations Lipid nanoparticles (LNPs) can be prepared at total lipid to ceDNA weight ratios of approximately 10:1 to 30:1. Briefly, ionizable lipids (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")), non-cationic lipids (e.g., distearoylphosphatidylcholine (DSPC)), components for providing membrane fusogenicity (such as sterols, e.g., cholesterol), and conjugated lipid molecules (such as PEG-lipids, e.g., 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol, with an average PEG molecular weight of 2000 ("PEG-DMG")) are solubilized in alcohol (e.g., ethanol) at a molar ratio of 50:10:38.5:1.5. The ceDNA is diluted to the desired concentration in a buffer solution. For example, ceDNA can be diluted to a concentration of 0.1 mg / mL to 0.25 mg / mL in a buffer solution containing sodium acetate, sodium acetate and magnesium chloride, citrate, malic acid, or malic acid and sodium chloride. In one example, ceDNA is diluted to 0.2 mg / mL in 10-50 mM citrate buffer (pH 4). The alcoholic lipid solution is mixed, for example, using a syringe pump or an impinging jet mixer, at a ratio of about 1:5 to 1:3 (vol / vol) at a total flow rate of more than 10 mL / min. In one example, the alcoholic lipid solution is mixed at a ratio of about 1:3 (vol / vol) 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 replaced with PBS using a centrifuge tube. Alcohol removal and simultaneous buffer exchange can be achieved, for example, by dialysis or tangential flow filtration. The resulting lipid nanoparticles are filtered through a 0.2 μm pore sterile filter before further use.

[0356] Example 8: Analysis of lipid particle formulations Lipid nanoparticle particle size can be determined by quasi-elastic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK) and is approximately 55-95 nm in diameter or approximately 70-90 nm in diameter.

[0357] The pKa of the formulated cationic lipids can correlate with the efficacy of the LNPs for delivery of nucleic acids (see Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176 (2010), both of which are incorporated herein by reference in their entireties). The preferred range of pKa is about 5 to about 7. The pKa of each cationic lipid is determined in lipid nanoparticles using a fluorescence-based assay of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). Lipid nanoparticles consisting of cationic lipid / DSPC / cholesterol / PEG-lipid (50 / 10 / 38.5 / 1.5 mol%) in PBS at a concentration of 0.4 mM total lipid can be prepared using the in-line process described herein and elsewhere. TNS can be prepared as a 100 μM stock solution in distilled water. Vesicles can be diluted to 24 μM lipid in 2 mL of buffer solution containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, 130 mM NaCl, with a pH ranging from 2.5 to 11. An aliquot of TNS solution can be added to a final concentration of 1 μM, followed by vortex mixing. Emission intensity is measured at room temperature in an SLM Aminco Series 2 emission spectrophotometer using an excitation wavelength of 321 nm and an emission wavelength of 445 nm. A sigmoidal best-fit analysis can be applied to the fluorescence data, and the pKa is determined as the pH that produces half-optimal fluorescence intensity.

[0358] Encapsulation of ceDNA in lipid particles can be determined by Oligreen® assay. Oligreen® is an ultrasensitive fluorescent nucleic acid stain for quantifying oligonucleotides and single-stranded DNA or RNA in solution (available from Invitrogen Corporation, Carlsbad, Calif.). Alternatively, PicoGreen® can be used. Briefly, encapsulation can be determined by performing a membrane-impermeable fluorescent dye exclusion assay, which uses a dye that has enhanced fluorescence when associated with nucleic acid. Encapsulation is determined by adding the dye to the lipid particle formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed upon addition of a small amount of non-ionic detergent. Detergent-mediated disruption of the lipid bilayer releases the encapsulated ceDNA, allowing it to interact with the membrane-impermeable dye. Encapsulation of ceDNA can be calculated as E=(I0-I) / I0, where / and I0 refer to the fluorescence intensity before and after addition of detergent.

[0359] Relative activity can be determined by measuring luciferase expression in the liver 4 hours after dosing via tail vein injection. Activity is compared at doses of 0.3 and 1.0 mg ceDNA / kg and is expressed as ng luciferase / g liver measured 4 hours after dosing.

[0360] Example 9: Evaluation of exemplary ceDNA-LNPs Exemplary ceDNA-containing lipid nanoparticles were prepared at various N / P ratios (e.g., 3, 4, 5, and 6) using lipid solutions containing MC3, DSPC, cholesterol, and DMG-PEG2000 (molar ratios of 50:10:38.5:1.5). Aqueous solutions of ceDNA vectors in buffer solutions containing salts such as sodium acetate, sodium acetate and magnesium chloride, citrate, malic acid, or malic acid and sodium chloride were prepared. Lipid and ceDNA solutions were mixed using an in-house procedure on a NanoAssembler at a lipid to eDNA ratio of 1:3 (v / v) and a total flow rate of 12 mL / min.

[0361] Characterization The lipid nanoparticle size and encapsulation of ceDNA in the lipid nanoparticles were determined. Particle size was determined by dynamic light scattering (ZEN3600, Malvern Instruments). Encapsulation efficiency was measured by the fluorescence (C 遊離 ) was measured and this value was expressed as the total ceDNA content (C 全 ) to determine the non-encapsulated ceDNA content. % Encapsulated = (C 全 -C 遊離 ) / C 全 × 100. The results are shown in Figures 9 to 11.

[0362] Endosomal escape assay The effects of serum and BSA on ceDNA encapsulation and release from LNPs were determined. Endosome-mimetic anionic liposomes were prepared by mixing DOPS:DOPC:DOPE (1:1:2 molar ratio) in chloroform, followed by solvent evaporation in vacuum. The dried lipid film was resuspended in DPBS with brief sonication, followed by filtration through a 0.45 μm syringe filler to form anionic liposomes.

[0363] Equal volumes of LNPs or serum containing ceDNA and BSA were mixed together. The mixtures were incubated at 37°C for 20 minutes. Anionic liposomes were then added to the LNP-BSA or serum mixtures at the desired anionic / cationic lipid molar ratio in DPBS at either pH 7.5 or 6.0. The resulting complexes were then incubated for an additional 15 minutes at 37°C. As a control, an equal volume of DPBS was added to the LNP-BSA or serum mixtures instead of the anionic liposomes. Released ceDNA by different treatments at pH 7.5 or pH 6.0 was measured by the fluorescence (C 遊離 ) was measured, and this value was converted to 1% Triton X-100 (C 全The % free = C 遊離 / C 全 × 100. The % ceDNA released after incubation with anionic liposomes was calculated based on the following equation: %Released ceDNA = %Free ceDNA アニオン性リポソームと混合 -% Free ceDNA DPBSと混合

[0364] The results are shown in Figures 12-15 and summarized in Table 5. [Table 5]

[0365] ApoE binding The binding of lipid nanoparticles to ApoE was determined. LNPs (10 μg / mL) were incubated with an equal amount of recombinant ApoE3 (500 μg / mL) in 1×DPBS for 20 min at 37° C. After incubation, LNP samples were diluted 10-fold using 1×DPBS and analyzed by heparin sepharose chromatography on an AKTA Pure 150 (GE Healthcare) according to the conditions described below. [Table 6]

[0366] The results are shown in Figure 16.

[0367] HEK293 expression The expression of ceDNA encapsulated in lipid nanoparticles was assayed as follows.

[0368] HEK293 cells were maintained at 5% CO2, 37°C 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 (Thermo Scientific) transfection reagent was used to transfect 100ng / well of control ceDNA according to the manufacturer's protocol. Control ceDNA was diluted in Opti-MEM™ (Thermo Scientific) and P3000™ reagent was added. Lipofectamine™ 3000 was then diluted to a final concentration of 3% in Opti-MEM™. The diluted Lipofectamine™ 3000 was added to the diluted ceDNA in a 1:1 ratio and incubated at room temperature for 15 minutes. The desired amount of ceDNA-lipid complexes or LNPs were then added directly to each well containing cells. The cells were incubated at 37°C, 5% CO2 for 72 hours. The expression levels of secreted factor IX in HEK293 conditioned medium were determined by VisuLize FIX antigen ELISA kit (Affinity Biologics) according to the manufacturer's instructions. The results are shown in Figures 17-19.

[0369] Example 10: Compounds of Formulation A Lipid nanoparticles (LNPs) containing ceDNA can be prepared or formulated in combination with one or more compounds of formula (I) or (II), preferably to form liposomes or lipid nanoparticles suitable for storage and therapeutic delivery of ceDNA vectors.

[0370] Compounds of formula (I) useful for preparing lipid particle formulations, having the structure: [ka] During the ceremony, R 1 , R 2 , R 4 , and R5 Each of the is independently hydrogen, C1-C 20 Alkyl, substituted C1-C 20 Alkyl, C2-C 20 Alkenyl, substituted C2-C 20 Alkenyl, C2-C 20 alkynyl, and cholesteryl; R 3 is C1~C 20 Alkyl, substituted C1-C 20 Alkyl, C2-C 20 Alkenyl, substituted C2-C 20 Alkenyl, and C2-C 20 alkynyl, L is S, O, C1~C 20 Alkenyl, substituted C1-C 20 alkenyl, X 1 is non-existent, [ka] and X 2 is non-existent, [ka] and Each R 6 are independent, C1~C 20 Alkyl, substituted C1-C 20 Alkyl, C2-C 20 Alkenyl, substituted C2-C 20 Alkenyl, and C2-C 20 A compound selected from the group consisting of alkynyl, Or its salt.

[0371] Compounds of formula (I) useful for preparing lipid particle formulations, having the structure: [ka] During the ceremony, R1 , R 2 , and R 4 Each of the is independently hydrogen, C1-C 20 Alkyl, substituted C1-C 20 Alkyl, C2-C 20 Alkenyl, substituted C2-C 20 Alkenyl, C2-C 20 alkynyl, and cholesteryl; R 3 is C1~C 20 Alkyl, substituted C1-C 20 Alkyl, C2-C 20 Alkenyl, substituted C2-C 20 Alkenyl, and C2-C 20 alkynyl, R 5 is non-existent or C1~C 20 Alkyl, substituted C1-C 20 Alkyl, C2-C 20 Alkenyl, substituted C2-C 20 Alkenyl, and C2-C 20 alkynyl, L is S, O, C1~C 20 Alkenyl, substituted C1-C 20 alkenyl, X 1 is non-existent, [ka] and X 2 is non-existent, [ka] and Each R 6 are independently hydrogen, C1 to C 20 Alkyl, substituted C1-C 20 Alkyl, C2-C 20 Alkenyl, substituted C2-C 20 Alkenyl, and C2-C 20alkynyl, R 5 When exists, [ka] is R 5 or R 5 When is non-existent, [ka] is non-existent, compound, Or its salt.

[0372] In some exemplary compounds of formula (I) or formula (II), R 1 is branch C 12 ~C 20 is alkyl, R 2 is a straight chain C5~C 10 Alkyl or branched C 12 ~C 20 is alkyl, R 4 and R 3 are each independently a linear C1-C5 alkyl group, and each R 6 is independently a straight chain or branched C1 to C5 alkyl, and L is a straight chain C1 to C3 alkyl.

[0373] Exemplary compounds of formula (I) or (II) can be one or more compounds selected from the compounds depicted in FIG.

[0374] The general synthesis of compounds of formula (I) and (II) is shown in Figures 21 and 22, where each R value is independently selected.

[0375] In general, the starting compounds and other compounds disclosed above can be purchased from commercial sources or prepared according to methods familiar to those skilled in the art. A skilled person would be able to use conventional synthesis methods and through reference books and databases of chemical compounds and reactions known to those skilled in the art to construct the most substituted scaffolds in the compounds described herein. Suitable reference books and articles that detail the synthesis of reactants useful in the preparation of the compounds disclosed herein or provide references to articles that describe the preparation of the compounds disclosed herein include, for example, "Synthetic Organic Chemistry", John Wiley and Sons, Inc. New York; S.R. Sandler et al., "Organic Functional Group Preparations," rd. Ed., Academic Press, 1999; Press, New York, 1983; HOHouse, "Modem Synthetic Reactions," 2nd Ed., WA Benjamin, Inc. Menlo Park, Calif., 1972; TLGlichrist, "Heterocyclic Chemistry," 2nd Ed. John Wiley and Sons, New York, 1992; J. March, "Advanced Organic Chemistry: reactions, mechanisms and structure," 5th Ed., Wiley Interscience, New York, 2001. Specific similar reactants can also be identified through the index of known chemicals prepared by the Chemical Abstract Service of the American Chemical Society, available in most public and university libraries, as well as through online databases (for further details, the American Chemical Society (Washington, DC) can be contacted). Chemicals that are known but not commercially available in catalogs may be prepared by custom chemical synthesis companies, and many of the standard chemical supply companies also offer custom synthesis services.

[0376] Example 11: Compounds of Formulation B Other lipid particles containing ceDNA can be prepared or formulated in combination with one or more compounds of formula (III), (IV), or (V) described in this Example, preferably to form liposomes or lipid nanoparticles suitable for storage and therapeutic delivery of ceDNA vectors.

[0377] Compounds of formula (III) useful for preparing lipid particle formulations, having the following structure: [ka] During the ceremony, [ka] is C3~C 10 Cycloalkyl, C3-C 10 Cycloalkyl, C3-C 10 Heterocycloalkyl, C3 to C 10 Heterocycloalkenyl, C6-C 10 Aryl, and C5-C 10 represents a cyclic ring selected from heteroaryl, R 1 , R 2 , R 3 , and R 4 Each of the is independently hydrogen, C1-C 20 Alkyl, substituted C1-C 20 Alkyl, C2-C 20 Alkenyl, substituted C2-C 20 Alkenyl, C2-C 20 alkynyl, and cholesteryl; X 1 is non-existent, C1~C 20 Alkyl, substituted C1-C 20 Alkyl, [ka] and X 2 is non-existent, C1~C 20 Alkyl, substituted C1-C 20 Alkyl, [ka] A compound, Or its salt.

[0378] In some exemplary compounds of formula (III), R 1 , R 2 , R 3 , and R 4 At least one of the [ka] It is.

[0379] The compound of formula (III) may be a compound selected from the compounds shown in FIG.

[0380] Compounds of formula (IV) useful for preparing lipid particle formulations, having the following structure: [ka] During the ceremony, n is 0 to 150; R 1 , R 2 , and R 3 Each of the is independently hydrogen, C1-C 20 Alkyl, substituted C1-C 20 Alkyl, C2-C 20 Alkenyl, substituted C2-C 20 Alkenyl, C2-C 20 A compound selected from the group consisting of alkynyl, and cholesteryl, Or its salt.

[0381] Compounds of formula (V) useful for preparing lipid particle formulations, having the following structure: [ka] During the ceremony, R 1 , R 2 , R 3 , R 4 , and R 5 Each of the is independently hydrogen, C1-C 20 Alkyl, substituted C1-C 20 Alkyl, C2-C 20 Alkenyl, substituted C2-C 20 Alkenyl, C2-C 20 alkynyl, and cholesteryl; X 1 is non-existent, C1~C 20 Alkyl, substituted C1-C 20Alkyl, or [ka] and X 2 is non-existent, C1~C 20 Alkyl, substituted C1-C 20 Alkyl, [ka] and X 3 is non-existent, C1~C 20 Alkyl, substituted C1-C 20 Alkyl, [ka] A compound, Or its salt.

[0382] Exemplary compounds of formula (V) include, but are not limited to, [ka] In the formula, n and m each independently represent 0 to 20. Each R 1 and R 2 are independently hydrogen, C1 to C 20 Alkyl, substituted C1-C 20 Alkyl, C2-C 20 Alkenyl, substituted C2-C 20 Alkenyl, C2-C 20 A compound selected from the group consisting of alkynyl, and cholesteryl, Or its salt.

[0383] A general synthesis of compounds of formula (III), formula (IV), and formula (V) is shown in FIG. 24, where each R value is independently selected.

[0384] It will be appreciated by those skilled in the art that in the processes described herein, functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxy, amino, mercapto, and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl, or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino, and guanidino include t-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto include -C(O)-R" (wherein R" is alkyl, aryl, or arylalkyl), p-methoxybenzyl, trityl, and the like. Suitable protecting groups for carboxylic acid include alkyl, aryl, or arylalkyl esters. Protecting groups can be added or removed according to standard techniques known to those skilled in the art and described herein. The use of protecting groups is described in detail in Green, TW and PGM Hutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley (incorporated herein in its entirety by reference). As will be appreciated by those skilled in the art, the protecting group can also be a polymeric resin, such as Wang resin, Rink resin, or 2-chlorotrityl-chloride resin. In general, the starting components can be obtained from sources such as, for example, Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, or can be synthesized according to sources known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley, December 2000), which is incorporated herein in its entirety by reference).

[0385] Example 12: Preparation of lipid formulations Liposomal nanoparticles (LNPs) can be prepared. Cationic lipid, DSPC, cholesterol, and PEG-lipid can be solubilized in ethanol at a molar ratio of 50:10:38.5:1.5. Lipid nanoparticles (LNPs) can be prepared at a total lipid to ceDNA weight ratio of approximately 10:1 to 30:1. Briefly, ceDNA is diluted to 0.2 mg / mL in 10-50 mM citrate buffer (pH 4). A syringe pump can be used to mix the ethanol lipid solution with the aqueous ceDNA solution at a ratio of approximately 1:5 to 1:3 (vol / vol) at a total flow rate of more than 15 mL / min. The ethanol can then be removed and the external buffer is replaced with PBS by dialysis. Finally, the lipid nanoparticles can be filtered through a 0.2 μm pore sterile filter. The lipid nanoparticles have a particle size of approximately 55-95 nm in diameter, and in some cases approximately 70-90 nm in diameter, as can be determined by quasi-elastic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK).

[0386] The pKa of the formulated cationic lipids can correlate with the efficacy of the LNPs for delivery of nucleic acids (see Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176 (2010), both of which are incorporated herein by reference in their entireties). The preferred range of pKa is about 5 to about 7. The pKa of each cationic lipid is determined in lipid nanoparticles using a fluorescence-based assay of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). Lipid nanoparticles consisting of cationic lipid / DSPC / cholesterol / PEG-lipid (50 / 10 / 38.5 / 1.5 mol%) in PBS at a concentration of 0.4 mM total lipid can be prepared using the in-line process described herein and elsewhere. TNS can be prepared as a 100 μM stock solution in distilled water. Vesicles can be diluted to 24 μM lipid in 2 mL of buffer solution containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, 130 mM NaCl, with a pH ranging from 2.5 to 11. An aliquot of TNS solution can be added to a final concentration of 1 μM, followed by vortex mixing. Emission intensity is measured at room temperature in an SLM Aminco Series 2 emission spectrophotometer using an excitation wavelength of 321 nm and an emission wavelength of 445 nm. A sigmoidal best-fit analysis can be applied to the fluorescence data, and the pKa is determined as the pH that produces half-optimal fluorescence intensity.

[0387] Lipid nanoparticles can also be formulated using the following molar ratio: 50% cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipid ("PEG-DMG", i.e., (1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol, average PEG molecular weight 2000). Relative activity can be determined by measuring luciferase expression in the liver 4 hours after administration via tail vein injection as described herein. Activity is compared at doses of 0.3 and 1.0 mg ceDNA / kg and is expressed as ng luciferase per gram of liver measured 4 hours after administration.

[0388] Therapeutic ceDNA construct compounds may be formulated 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-ditetradecylacetamide). Relative activity may be determined by measuring luciferase expression in the liver 4 hours after administration via tail vein injection, as described above. Activity is compared at doses of 0.3 and 1.0 mg mRNA or DNA / kg and is expressed as ng luciferase per g liver measured 4 hours after administration, as described above.

[0389] Example 13: In vivo protein expression of luciferase transgene from ceDNA vector In vivo protein expression of transgenes from ceDNA vectors produced from constructs 1-8 above is evaluated in mice. ceDNA vectors obtained from ceDNA-plasmid construct 1 (described in Table 5 of Example 1) were tested and demonstrated sustained and resistant luciferase transgene expression in a mouse model following hydrodynamic injection of a composition comprising the ceDNA vector contained in lipid nanoparticles into the tail vein. Luciferase transgene expression was measured in CD-1® IGS mice (Charles River The results were measured by IVIS imaging after intravenous administration to mice (Davidson Laboratories, WT mice).

[0390] This study evaluates the biodistribution of hydrodynamic luciferase-expressing non-viral gene therapy vectors by IVIS after intravenous administration in CD-1® IGS mice. The vehicle is sterile PBS. In this study, two groups of five CD-1 mice were administered either PBS or 0.35 mg / kg luciferase-encoding ceDNA via 1.2 mL hydrodynamic injection into the tail vein. Luciferase expression was assessed by IVIS imaging on days 3, 4, 7, 14, 21, 28, 31, 35, and 42. Briefly, mice were intraperitoneally injected with 150 mg / kg luciferin substrate, and then whole-body luminescence was assessed via IVIS imaging.

[0391] In vivo luciferase expression: Five- to seven-week-old male CD-1 IGS mice (Charles River Laboratories) are administered 0.35 mg / kg of ceDNA vector construct 1-4 Luc (group 1) in a 1.2 mL volume via intravenous hydrodynamic administration on day 0. Some animals from each group are re-administered with the same dose of ceDNA vector construct 1-4 Luc (group 1) on day 28.

[0392] Luciferase expression from the ceDNA vector is assessed by in vivo chemiluminescence using an IVIS® instrument following intraperitoneal (ip) injection of 150 mg / kg of luciferase substrate. IVIS imaging is performed on days 3, 4, 7, 14, 21, 28, 31, 35, and 42, and harvested organs are imaged ex vivo after sacrifice on day 42.

[0393] During the course of the study, animals are weighed and monitored daily for general health and well-being. After sacrifice, blood is collected from each animal by terminal cardiac puncture and divided into two portions and processed into 1) plasma and 2) serum, which is flash frozen and used for liver enzyme panels and then flash frozen. Additionally, liver, spleen, kidneys, and inguinal lymph nodes (LN) are collected and imaged ex vivo by IVIS.

[0394] Luciferase expression was measured using the MAXDISCOVERY® Luciferase ELISA assay (BIOO Scientific / PerkinElmer), qPCR for luciferase in liver samples, histopathology of liver samples, and / or a serum liver enzyme panel (VetScanVS2, Abaxis Preventative Care). The liver is assessed by the IL-1 Profile Plus.

[0395] References All references cited and disclosed in the specification and examples, including patents, patent applications, international patent applications and publications, are hereby incorporated by reference in their entirety. The present invention provides, for example, the following items. (Item 1) A lipid particle comprising an ionized lipid and a non-viral capsid-free DNA vector (ceDNA vector) having a covalently closed end, the ceDNA vector comprising at least one heterologous nucleotide sequence operably positioned between asymmetric inverted terminal repeats (asymmetric ITRs), at least one of the asymmetric ITRs comprising a functional terminal resolution site and a Rep binding site. (Item 2) The lipid nanoparticle described in item 1, wherein the ceDNA vector, when digested with a restriction enzyme having a single recognition site on the ceDNA vector and analyzed by both native and denaturing gel electrophoresis, exhibits a characteristic linear and continuous DNA band compared to a linear and discontinuous DNA control. (Item 3) The lipid nanoparticle of item 1 or 2, wherein one or more of the asymmetric ITR sequences are derived from a virus selected from a parvovirus, a dependovirus, and an adeno-associated virus (AAV). (Item 4) The lipid nanoparticle described in item 3, wherein the asymmetric ITRs are derived from different viral serotypes. (Item 5) The lipid nanoparticle of item 4, wherein the one or more asymmetric ITRs are derived from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. (Item 6) The lipid nanoparticle according to any one of items 1 to 3, wherein one or more of the asymmetric ITR sequences are synthetic. (Item 7) The lipid nanoparticle according to any one of items 1 to 6, wherein one or more of the ITRs are not wild-type ITRs. (Item 8) The lipid nanoparticle according to any one of items 1 to 7, wherein one or more of the 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'. (Item 9) The ceDNA vector, c. SEQ ID NO:1 and SEQ ID NO:52, and d. The lipid nanoparticle of any one of items 1 to 8, comprising at least two asymmetric ITRs selected from SEQ ID NO: 2 and SEQ ID NO: 51. (Item 10) The ceDNA vector, a. incubating a population of insect cells harboring a ceDNA expression construct in the presence of at least one Rep protein, the ceDNA expression construct encoding the ceDNA vector, under conditions effective, and for a time sufficient, to induce production of the ceDNA vector in the insect cells; b. Isolating the ceDNA vector from the insect cell. The lipid nanoparticle of any one of items 1 to 9, obtained from a process comprising the steps of: (Item 11) The lipid nanoparticle of item 10, wherein the ceDNA expression construct is selected from a ceDNA plasmid, a ceDNA bacmid, and a ceDNA baculovirus. (Item 12) The lipid nanoparticle of claim 10 or 11, wherein the insect cells express at least one Rep protein. (Item 13) The lipid nanoparticle described in item 10, wherein at least one Rep protein is derived from a virus selected from a parvovirus, a dependovirus, and an adeno-associated virus (AAV). (Item 14) 14. The lipid nanoparticle of item 13, wherein at least one Rep protein is derived from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. (Item 15) The DNA vector is derived from a vector polynucleotide, the vector polynucleotide encoding a heterologous nucleic acid operably positioned between two inverted terminal repeats (ITRs), the two ITRs being different from each other (asymmetric), at least one of the ITRs being a functional ITR comprising a functional terminal resolution site and a Rep binding site, one of the ITRs comprising a deletion, insertion and / or substitution relative to the functional ITR, the presence of a Rep protein induces replication of the vector polynucleotide in an insect cell and production of the DNA vector, and the DNA vector is a. incubating a population of insect cells harboring the vector polynucleotide lacking viral capsid coding sequences in the presence of Rep protein under conditions effective, and for a time sufficient, to induce production of the capsid-free, non-viral DNA vector in the insect cells, wherein the insect cells, in the absence of the vector, do not contain production of capsid-free, non-viral DNA in the insect cells; b. harvesting and isolating the capsid-free, non-viral DNA from the insect cells. The lipid particle according to any one of claims 1 to 15, (Item 16) 16. The lipid particle according to any one of items 10 to 15, wherein the presence of the capsid-free non-viral DNA isolated from the insect cells can be confirmed. (Item 17) 17. The lipid particle according to item 16, wherein the presence of the capsid-free, non-viral DNA isolated from the insect cells can be confirmed by digesting the DNA isolated from the insect cells with a restriction enzyme having a single recognition site on the DNA vector and analyzing the digested DNA material on a non-denaturing gel to confirm the presence of a characteristic linear, continuous DNA band compared to linear, discontinuous DNA. (Item 18) The DNA vector is derived from a vector polynucleotide, the vector polynucleotide encoding a heterologous nucleic acid operably positioned between first and second AAV2 inverted terminal repeat DNA polynucleotide sequences (ITRs), at least one of the ITRs having at least one polynucleotide deletion, insertion, and / or substitution relative to a corresponding AAV2 wild-type ITR of SEQ ID NO:1 or SEQ ID NO:51, and directs replication of the DNA vector in an insect cell in the presence of Rep protein, the DNA vector comprising: a. incubating a population of insect cells harboring said vector polynucleotide lacking viral capsid coding sequences in the presence of Rep protein under conditions effective, and for a time sufficient, to induce production of said capsid-free, non-viral DNA vector in said insect cells, wherein said insect cells do not contain viral capsid coding sequences; b. harvesting and isolating the capsid-free, non-viral DNA from the insect cells. The lipid particle according to any one of claims 1 to 17, (Item 19) 19. The lipid particle according to item 18, wherein the presence of the capsid-free non-viral DNA isolated from the insect cells can be confirmed. (Item 20) 20. The lipid particle according to item 19, wherein the presence of the capsid-free, non-viral DNA isolated from the insect cells can be confirmed by digesting the DNA isolated from the insect cells with a restriction enzyme having a single recognition site on the DNA vector and analyzing the digested DNA material on a non-denaturing gel to confirm the presence of a characteristic linear, continuous DNA band compared to linear, discontinuous DNA. (Item 21) 21. The lipid particle according to any one of items 1 to 20, wherein the lipid particle further comprises one or more of a non-cationic lipid, a PEG-conjugated lipid, and a sterol. (Item 22) 22. The lipid particle according to any one of items 1 to 21, wherein the ionizable lipid is a lipid listed in Table 1. (Item 23) The lipid particles further comprise a non-cationic lipid, the non-ionic lipid being selected from the group consisting of distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), and the like. ), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, 18-trans-PE, 1 -Stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), diercoyl phosphatidylcholine (DEPC), palmitoyl oleoyl phosphatidylglycerol (P 23. The lipid particle according to any one of items 1 to 22, wherein the lipid particle is selected from the group consisting of dilinoleoyl-phosphatidylethanolamine (DPPE), dielaidoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, lysophosphatidylcholine, and dilinoleoylphosphatidylcholine. (Item 24) The lipid particle according to any one of items 1 to 23, further comprising a complex lipid, the complex lipid being 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. (Item 25) 25. The lipid particle according to any one of items 1 to 24, wherein the lipid particle further comprises cholesterol or a cholesterol derivative. (Item 26) The lipid particles are (v) an ionizable lipid; and (vi) a non-cationic lipid; and (vii) a conjugated lipid that inhibits particle aggregation; (viii) A sterol. (Item 27) The lipid particle according to any one of Items 1 to 25, The lipid particles are (e) an ionized lipid in an amount of about 20 mol% to about 90 mol% of the total lipid present in the particle; (f) a non-cationic lipid in an amount of about 5 mol% to about 30 mol% of the total lipid present in the particle; (g) a complex lipid that inhibits particle aggregation in an amount of about 0.5 mol % to about 20 mol % of the total lipid present in the particle; (h) a sterol in an amount of about 20 mol % to about 50 mol % of the total lipid present in the particle. The lipid particle according to any one of items 1 to 26, (Item 28) 28. The lipid particle according to any one of items 1 to 27, wherein the ratio (by mass or weight) of total lipid to DNA vector is from about 10:1 to about 30:1. (Item 29) A composition comprising a first lipid nanoparticle and an additional compound, wherein the first lipid nanoparticle comprises a first capsid-free non-viral vector, and is the lipid nanoparticle according to any one of items 1 to 28. (Item 30) 30. The composition of claim 29, wherein the additional compound is incorporated into a second lipid nanoparticle, and the first and second lipid nanoparticles are different. (Item 31) 30. The composition of claim 28 or 29, wherein the additional compound is incorporated into the first lipid nanoparticle. (Item 32) 31. The composition according to any one of items 28 to 30, wherein the additional compound is a therapeutic agent. (Item 33) 29. The composition of claim 28, wherein the additional compound is a second capsid-free, non-viral vector, and the first and second capsid-free, non-viral vectors are different.

Claims

[Claim 1] A lipid particle comprising an ionized lipid and a non-viral capsid-free DNA vector (ceDNA vector) having covalently closed ends, wherein the ceDNA vector comprises at least one heterologous nucleotide sequence operably positioned between asymmetric inverted terminal repeats (asymmetric ITRs), at least one of the asymmetric ITRs comprising a functional terminal resolution site and a Rep binding site.

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