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WO2024249298A2 - Vecteurs d'adn à extrémité fermée (adnce) modifiés, compositions et utilisations associées - Google Patents

Vecteurs d'adn à extrémité fermée (adnce) modifiés, compositions et utilisations associées Download PDF

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Publication number
WO2024249298A2
WO2024249298A2 PCT/US2024/030986 US2024030986W WO2024249298A2 WO 2024249298 A2 WO2024249298 A2 WO 2024249298A2 US 2024030986 W US2024030986 W US 2024030986W WO 2024249298 A2 WO2024249298 A2 WO 2024249298A2
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composition
stem
pharmaceutical composition
itr
sequence
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WO2024249298A3 (fr
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Matthew G. Stanton
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Generation Bio Co
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Generation Bio Co
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • Gene therapy includes the treatment or prevention of medical conditions resulting from defective genes or abnormal regulation or expression, e.g., underexpression or overexpression, that can result in a disorder, disease, malignancy, etc.
  • a disease or disorder caused by a defective gene might be treated, prevented or ameliorated by delivery of a corrective genetic material to a patient resulting in the therapeutic expression of the genetic material within the patient.
  • the basis of gene therapy is to supply a transcription cassette with an active gene product (sometimes referred to as a transgene), e.g., that can result in a positive gain-of-function effect, a negative loss-of-function effect, or another outcome, such as, e.g., an oncolytic effect.
  • Such outcomes can be attributed, e.g., to expression of an activating antibody, therapeutic protein, fusion protein, an inhibitory (neutralizing) antibody.
  • Gene therapy can also be used to treat a disease or malignancy caused by other factors.
  • Human monogenic disorders can be treated by the delivery and expression of a normal gene to the target cells. Delivery and expression of a corrective gene in the patient's target cells can be carried out via numerous methods, including the use of engineered viruses and viral gene delivery vectors.
  • recombinant adeno-associated virus e.g., recombinant retrovirus, recombinant lentivirus, recombinant adenovirus, and the like
  • rAAV recombinant adeno-associated virus
  • rAAV rAAV vectors
  • the second drawback is that as a result of the prevalence of wild-type AAV infection in the population, candidates for rAAV gene therapy have to be screened for the presence of neutralizing antibodies that eliminate the vector from the patient before a therapeutic effect is achieved.
  • a third drawback is 1 ME148589913v.1 related to the capsid immunogenicity that prevents re-administration to patients that were not excluded from an initial treatment.
  • the immune system in the patient can respond to the vector which effectively acts as a “booster” shot to stimulate the immune system generating high titer anti-AAV antibodies that preclude future treatments.
  • Some recent reports indicate concerns with immunogenicity in high dose situations.
  • Another notable drawback is that the onset of AAV-mediated gene expression is relatively slow, given that single-stranded AAV DNA must be converted to double-stranded DNA prior to heterologous gene expression.
  • conventional AAV virions with capsids must be produced in cultured cells, which is inefficient and introduces additional risks, including contamination by infectious agents, including other viruses.
  • Recombinant capsid-free AAV vectors can be obtained as an isolated linear nucleic acid molecule comprising an expressible transgene and promoter regions flanked by two wild-type AAV inverted terminal repeat sequences (ITRs) including the Rep binding and terminal resolution sites.
  • ITRs inverted terminal repeat sequences
  • they avoid many of the problems of AAV-mediated gene therapy in that the transgene capacity is much higher, transgene expression onset is rapid, they still contain the attributes of the AAV-based vectors like the ITR palindromic sequences (e.g., trs, RBE, A, A’, B, B’, C, C’, D and/or D’ regions of AAV ITRs) that are found in the wild-type AAV.
  • ceDNA closed-ended DNA
  • ITR inverted terminal repeat
  • typical AAV ITR structures comprise a palindromic double-stranded T-shaped hairpin structure, in which the double-stranded A-A’ region forms the stem, and the double- stranded B-B’ and C-C’ regions form the cross-arms of the T-shaped structure (see, for example, Ling et al., J. Virology, 89(2):952-961, 2015).
  • the other nucleotides of the typical AAV ITR remain single stranded and are referred to as the single-stranded D(-) sequence (on the 3’ end of the ITR) and the single-stranded D(+) sequence (on the 5’ end of the ITR).
  • the single-stranded regions of the D(+) region and D(-) region undergo second-strand DNA synthesis to turn them into double- stranded D and D’ regions.
  • ITR sequence was required for the replication and packaging of both AAV and ceDNA vectors used in gene therapy applications. More specifically, it was believed that a ceDNA vector minimally required at least one Rep-binding element (RBE) and at least one terminal resolution sequence (trs).
  • RBE Rep-binding element
  • trs terminal resolution sequence
  • the ITR regions D 2 ME148589913v.1 and D’ contain sequences that act as binding sites for host transcription factors, including human transcription factors.
  • Wang et al. in 1996 J. Virol.70:1668-1677, 1996) reported that the D sequence plays a crucial role in high-efficiency rescue, selective replication, and encapsidation of the AAV genome and that a host cell protein, designated the D sequence-binding protein (D-BP), specifically interacts with this sequence.
  • D-BP D sequence-binding protein
  • ceDNA vectors comprising synthetic ITRs comprising no viral sequences showed comparable or even, in some embodiments, enhanced gene expression profiles for their respective transgenes.
  • the ceDNA vectors of the instant disclosure have advantages over ceDNA vectors comprising viral ITRs. Removing such sequences reduced the size of a ceDNA molecule, which may be advantageous for a variety of reasons. For example, it may allow for the inclusion of other desirable sequences or motifs (e.g., functional sequences such as aptamers) without increasing the size of the ceDNA molecule. It may also reduce the cost of manufacturing by reducing the amount of raw materials needed for ceDNA production. It may also reduce the likelihood of provoking an immune response when administered to a subject.
  • the disclosure provides a composition comprising a non-viral, capsid-free DNA vector with covalently-closed ends (ceDNA vector), wherein the ceDNA vector comprises at least one transgene expression cassette flanked by a first stem-loop structure and a second stem-loop structure, each stem-loop structre comprising a partial duplex and at least one loop, and a lipid.
  • the stem-loop structures do not comprise any viral inverted terminal repeat (ITR) sequences.
  • the stem-loop structures do not comprise any AAV ITR sequences.
  • the stem-loop structures do not comprise any wild-type (or naturally occurring) viral inverted terminal repeat (ITR) sequences.
  • the stem-loop structures do not comprise any wild-type (or naturally occurring) AAV inverted terminal repeat (ITR) sequences.
  • ITR AAV inverted terminal repeat
  • 3 ME148589913v.1 disclosed herein is a composition comprising: a non-viral, capsid-free DNA vector with covalently-closed ends (ceDNA vector), wherein the ceDNA vector comprises at least one transgene expression cassette flanked by a first stem-loop structure and a second stem-loop structure, each stem-loop structure comprising a partial duplex and at least one loop, wherein the stem-loop structures do not comprise a viral inverted terminal repeat (ITR) sequence, and a lipid.
  • the composition further comprises a pharmaceutically acceptable excipient or carrier.
  • the ceDNA vector is encapsulated by a lipid nanoparticle (LNP). In one embodiment, the ceDNA vector is associated with an LNP.
  • a pharmaceutical composition comprising: a non-viral, capsid-free DNA vector with covalently-closed ends (ceDNA vector), wherein the ceDNA vector comprises at least one transgene expression cassette flanked by a first stem-loop structure and a second stem-loop structure, each stem-loop structure comprising a partial duplex and at least one loop, wherein the stem-loop structures do not comprise a viral inverted terminal repeat (ITR) sequence, an LNP, and a pharmaceutically acceptable excipient or carrier.
  • ITR viral inverted terminal repeat
  • the ceDNA vector is encapsulated by a lipid nanoparticle (LNP). In one embodiment, the ceDNA vector is associated with an LNP.
  • the stem-loop structures do not comprise a viral ITR sequence comprising a sequence selected from the group consisting of a Rep binding element (RBE), a RBE’, a terminal resolution site (trs), an A ITR region, an A’ ITR region, a B ITR region, a B’ ITR region, a C ITR region, a C’ ITR region, a D ITR region, a D’ ITR region, and a combination thereof.
  • the stem-loop structures do not comprise a viral ITR sequence comprising a Rep binding element (RBE).
  • the stem-loop structures do not comprise a viral ITR sequence comprising an RBE’. In one embodiment, the stem-loop structures do not comprise a viral ITR sequence comprising a trs. In one embodiment, the stem-loop structures do not comprise a viral ITR sequence comprising an A ITR region. In one embodiment, the stem-loop structures do not comprise a viral ITR sequence comprising an A’ ITR region. In one embodiment, the stem-loop structures do not comprise a viral ITR sequence comprising a B ITR region. In one embodiment, the stem-loop structures do not comprise a viral ITR sequence comprising a B’ ITR region.
  • the stem-loop structures do not comprise a viral ITR sequence comprising a C ITR region. In one embodiment, the stem-loop structures do not comprise a viral ITR sequence comprising a C’ ITR region. In one embodiment, the stem-loop structures do not comprise a viral ITR sequence comprising a D ITR region. In one embodiment, the stem-loop structures do not comprise a viral ITR sequence comprising a D’ ITR region.
  • the stem-loop structures do not comprise a viral ITR sequence comprising any of a Rep binding element (RBE), a RBE’, a terminal resolution site (trs), an A ITR region, an A’ ITR region, a B ITR region, a B’ ITR region, a C ITR region, a C’ ITR region, a D ITR region, or a D’ ITR region.
  • RBE Rep binding element
  • trs terminal resolution site
  • the ceDNA vector does not comprise a wild-type (or naturally occurring) viral ITR sequence from a virus, e.g., an adeno-associated virus (AAV).
  • AAV adeno-associated virus
  • the ceDNA vector does not comprise a fragment of a wild-type (or naturally occurring) viral ITR sequence from a virus, e.g., an adeno-associated virus (AAV).
  • the at least one transgene expression cassette comprises at least one liver- specific regulatory element operably linked to at least one transgene.
  • the first stem-loop structure is located upstream of the 5’ end of the at least one transgene expression cassette and the second stem-loop structure is located downstream of the 3’ end of the at least one transgene expression cassette.
  • the first stem-loop structure and the second stem-loop structure are symmetric with respect to each other.
  • first stem-loop structure and the second stem-loop structure are asymmetric with respect to each other.
  • first stem-loop structure and the second stem-loop structure each comprise at least one double-stranded stem region and at least one single-stranded loop region.
  • the at least one loop is a single-stranded loop region.
  • the partial duplex comprises the double-stranded stem region.
  • each double-stranded stem region comprises about 1 to about 50 base pairs (bp), or about 1 to about 40 bp, or about 1 to about 30 bp, or about 1 to about 25 bp, or about 1 to about 20 bp, or about 1 to about 15 bp, or about 2 to about 20 bp, or about 2 to about 15 bp, or about 3 to about 20 bp, or about 3 to about 15 bp, or about 4 to about 20 bp, or about 4 to about 15 bp, or about 5 to about 20 bp, or about 5 to about 15 bp, or about 1 bp, or about 2 bp, or about 3 bp, or about 4 bp, or about 5 bp, or about 6 bp, or about 7 bp, or about 8 bp, or about 9 bp, or about 10 bp, or about 11 bp, or about 12 bp, or about 13 bp, or about 14 bp, or about 15
  • each single-stranded loop comprises about 2 to about 100 nucleotides (nt), about 2 to about 90 nt, about 2 to about 80 nt, about 2 to about 70 nt, about 2 to about 60 nt, about 2 to about 50 nt, about 2 to about 40 nt, about 2 to about 30 nt, about 2 to about 20 nt, about 2 to about 15 nt, about 2 to about 12 nt, about 2 to about 10 nt, about 2 to about 8 nt, about 3 to about 20 nt, about 3 to about 15 nt, about 3 to about 12 nt, about 3 to about 10 nt, about 3 to about 8 nt, or about 2 nt, or about 3 nt, or about 4 nt, or about 5 nt, or about 6 nt, or about 7 nt, or about 8 nt, or about 9 nt, or about 10 nt, or about 11 nt, or about 12 nt, or about
  • each stem-loop structure comprises at least one loop structure. In one embodiment, at least one stem-loop structure comprises an aptamer. In one embodiment, each stem- loop structure comprises an aptamer. In one embodiment, each stem-loop structure independently comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to a sequence selected from the group consisting of SEQ ID NO:1 (5’- CTCATCAGAATCACTTTGTGATTCTGA-3’), SEQ ID NO:2 (5’- 5 ME148589913v.1 TCAGAATCACAAAGTGATTCTGATGAG-3’), and SEQ ID NO:4 (5’- CACTCATCAGAATCACTTTGTGATTCTGATG-3’).
  • each stem-loop structure independently comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to a sequence selected from the group consisting of SEQ ID NO:12 (5’- TGAGCCTTGATACCACACTTACCTTTCAAGCTTAATACCTTTAGGAGAGCAATTGCAT-3’) and SEQ ID NO:13 (5’- ATGCAATTGCTCTCCTAAAGGTATTAAGCTTGAAAGGTAAGTGTGGTATCAAGGCTCA- 3’).
  • the ceDNA vector further comprises at least one spacer region, wherein the at least one spacer region comprises an artificial nucleic acid sequence and does not comprise a viral ITR sequence.
  • the at least one spacer region is adjacent to the 5’ end of the at least one transgene expression cassette, or the at least one spacer region is adjacent to the 3’ end of the at least one transgene expression cassette, or both.
  • the at least one spacer region comprises about 1 to 200 bp, or about 5 to 150 bp, or about 10 to 100 bp, or about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 bp.
  • the at least one liver-specific regulatory element is selected from the group consisting of a liver-specific promoter, a liver-specific enhancer, a liver-specific riboswitch, a liver-specific insulator, a liver-specific mir-regulatable element, a liver-specific post-transcriptional regulatory element, a liver-specific transcription factor binding site (or consensus site), and a combination thereof.
  • the at least one liver-specific transcription factor consensus site is FoxA, HNF4, or both.
  • the at least one liver-specific promoter is selected from the group consisting of: a transthyretin (TTR) promoter, minimal TTR promotor (TTRm), an AAT promoter, an albumin (ALB) promotor or minimal promoter, an apolipoprotein A1 (APOA1) promoter or minimal promoter, a complement factor B (CFB) promoter, a ketohexokinase (KHK) promoter, a hemopexin (HPX) promoter or minimal promoter, a nicotinamide N-methyltransferase (NNMT) promoter or minimal promoter, a carboxylesterase 1 (CES1) promoter or minimal promoter, a protein C (PROC) promoter or minimal promoter, an apolipoprotein C3 (APOC3) promoter or minimal promoter, a mannan-binding lectin serine protease 2 (MASP2) promoter or minimal promoter, a hepcidin antimicrobial peptide (TR)
  • the ceDNA vector produces an in vivo expression level of the at least one transgene expression cassette that is equivalent to or higher than the in vivo expression level produced by a reference ceDNA vector comprising a viral ITR sequence.
  • the ceDNA vector produces an in vivo liver expression level of the at least one transgene that is equivalent to or higher than the in vivo liver expression level produced by a reference ceDNA vector comprising a 6 ME148589913v.1 viral ITR sequence.
  • the ceDNA vector elicits an equivalent or lower pro- inflammatory cytokine response than a reference ceDNA vector comprising a viral ITR sequence.
  • the ceDNA vector produces an expression level of the at least one transgene in a cell that is equivalent to or higher than the expression level of the at least one transgene produced by a reference ceDNA vector comprising the same transgene expression cassette and at least one stem-loop structure comprising a viral ITR sequence.
  • the cell is selected from the group consisting of a liver cell, a muscle cell, a T cell, a B cell, a natural killer (NK) cell, and a dendritic cell.
  • the cell is in vitro.
  • the cell is in vivo.
  • the cell is ex vivo.
  • the ceDNA vector is synthetically produced in a cell-free environment.
  • the ceDNA vector is produced using a method comprising: (a) contacting a double-stranded DNA construct having a sense strand and an antisense strand with at least a first restriction endonuclease and at least a second restriction endonuclease, wherein: the double-stranded DNA construct comprises: a transgene expression cassette, a first non-palindromic restriction endonuclease recognition site and a corresponding first cleavage site upstream of the transgene expression cassette, and a second non-palindromic restriction endonuclease recognition site and a corresponding second cleavage site downstream of the transgene expression cassette; and wherein the first restriction endonuclease is capable of cleaving the double-stranded DNA construct at the first cleavage site, and wherein the second restriction endonuclease is capable of cleaving the double- stranded DNA construct at the second cleavage site, and wherein contacting the double-strand
  • the first oligonucleotide and the second oligonucleotide are symmetric with respect to each other. In one embodiment, the first oligonucleotide and the second oligonucleotide are asymmetric with respect to each other. In one embodiment, the first oligonucleotide and the second oligonucleotide each self-anneal to form the one or more stem-loop structures and a single-stranded overhang at the 5’ end of each oligonucleotide or the 3’ end of each oligonucleotide.
  • the single-stranded overhang of the first oligonucleotide and/or the single-stranded overhang of the second oligonucleotide each comprise a 5’ to 3’ nucleic acid sequence selected from the group consisting of CTCT, CTCA, CACT, CTC, and GCT.
  • the first oligonucleotide corresponds to the first stem-loop region in the ceDNA vector and the second oligonucleotide corresponds to the second stem-loop region in the ceDNA vector.
  • At least one of the first and second restriction endonucleases or wherein each of the first and second restriction endonucleases is a Type IIS restriction endonuclease.
  • the first restriction endonuclease and the second restriction endonuclease are the same restriction endonuclease.
  • the first restriction endonuclease and the second restriction endonuclease are different restriction endonucleases.
  • the Type IIS restriction endonuclease is selected from group consisting of AcuI, AlwI, Alw26I, BasI, BbsI, BbvI, BceAI, BcgI, BciVI, BcoDI, BruAI, BmrI, BpiI, BpuEI, BsaI, BsaXI, BseGI, BseRI, BsgI, BsmAI, BsmBI, BsmFI, BsmI, BspCNI, BspMI, BspQI, BsrDI, BsrI, BtgZI, BtsCI, BtsI, MutI, CspCI, EarI, EciI, Eco31I, Esp3I, FauI, FokI, HgaI, HphI, HpyAV, LguI, MboII, MlyI, MmeI, MnlI, Mva1269I, N
  • the Type IIS restriction endonuclease is selected from group consisting of BbsI, BsaI, BsmBI, Esp3I, and SapI, and an isoschizomer thereof. In one embodiment, the Type IIS restriction endonuclease is BsmBI or an isoschizomer thereof; or wherein each of the first and second restriction endonucleases is BsmBI or an isoschizomer thereof.
  • the lipid or LNP comprises at least one lipid selected from the group consisting of an ionizable lipid, a sterol, non-cationic lipid, and a lipid-anchored polymer. In one embodiment, the LNP comprises a sterol.
  • the sterol is cholesterol or beta-sitosterol.
  • the LNP comprises a non-cationic lipid.
  • the non- cationic lipid is selected from the group consisting of distearoyl-sn-glycero-phosphoethanolamine (DSPE), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexan
  • the non-cationic lipid is selected from the group consisting of dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), and dioleoyl-phosphatidylethanolamine (DOPE).
  • DOPC dioleoylphosphatidylcholine
  • DSPC distearoylphosphatidylcholine
  • DOPE dioleoyl-phosphatidylethanolamine
  • the non-cationic lipid is DSPC.
  • the LNP comprises a lipid-anchored polymer.
  • the lipid-anchored polymer is a PEGylated lipid.
  • the PEGylated lipid is selected from the group consisting of PEG-dilauryloxypropyl; PEG-dimyristyloxypropyl; PEG- dipalmityloxypropyl, PEG-distearyloxypropyl; l-(monomethoxy-polyethyleneglycol)-2,3- dimyristoylglycerol (DMG-PEG); PEG-dilaurylglycerol; PEG-dipalmitoylglycerol; PEG- disterylglycerol; PEG-dilaurylglycamide; PEG-dimyristylglycamide; PEG-dipalmitoylglycamide; PEG-disterylglycamide; (l-[8’-(Cholest-5-en-3[beta]-oxy)carboxamido-3’,6’-dioxaoctanyl] carbamoyl-[omega]
  • the PEGylated lipid is DMG-PEG, DSPE-PEG, or both. In one embodiment, the PEGylated lipid is DMG-PEG2000, DSPE-PEG2000, or both.
  • the LNP comprises an ionizable lipid, a sterol, and a lipid-anchored polymer. In one embodiment, the LNP comprises an ionizable lipid, a sterol, a non-cationic lipid, and a lipid-anchored polymer. In one embodiment, the LNP comprise a tissue-specific targeting ligand. In one embodiment, the tissue-specific targeting ligand is N-acetylgalactosamine (GalNAc) or a GalNAc derivative.
  • GalNAc N-acetylgalactosamine
  • the tissue-specific targeting ligand is covalently linked to the PEGylated lipid to form a PEGylated lipid conjugate.
  • the PEGylated lipid conjugate comprises tetra- or tri-antennary GalNAc covalently linked to the PEGylated lipid.
  • the PEGylated lipid is DSPE-PEG2000
  • the ionizable lipid is present in the LNP at a molar percentage of about 30% to about 70%.
  • the sterol is present in the LNP at a molar percentage of about 20% to about 50%.
  • the non-cationic lipid is present in the LNP at a molar percentage of about 2% to about 20%.
  • the lipid-anchored polymer or PEGylated lipid is present in the LNP at a molar percentage of about 2.1% to about 10%.
  • the PEGylated lipid conjugate is present in the LNP at a molar percentage of about 0.1% to about 10%.
  • the LNP comprises an ionizable lipid, a sterol, a non-cationic lipid, a PEGylated lipid, and a PEGylated lipid conjugate.
  • the LNP has an average diameter ranging from about 40 nm to about 120 nm. In one embodiment, the LNP has an average diameter of less than about 100 nm. In one embodiment, the LNP has an average diameter of about 50 nm to about 80 nm. In one aspect, disclosed herein is an isolated host cell comprising a ceDNA vector of the composition or pharmaceutical compositions disclosed herein.
  • an isolated host cell comprising: a non-viral, capsid-free DNA vector with covalently-closed ends (ceDNA vector), wherein the ceDNA vector comprises at least one transgene expression cassette flanked by a first stem-loop structure and a second stem-loop structure, each stem-loop structure comprising a partial duplex and at least one loop, wherein the stem-loop structures do not comprise any viral ITR sequences.
  • a transgenic animal comprising a ceDNA vector or an isolated host cell.
  • a transgenic animal comprising: a non-viral, capsid-free DNA vector with covalently-closed ends (ceDNA vector), wherein the ceDNA vector comprises at least one transgene expression cassette flanked by a first stem-loop structure and a second stem-loop structure, each stem-loop structure comprising a partial duplex and at least one loop, wherein the stem-loop structures do not comprise any viral ITR sequences.
  • ceDNA vector non-viral, capsid-free DNA vector with covalently-closed ends
  • the ceDNA vector comprises at least one transgene expression cassette flanked by a first stem-loop structure and a second stem-loop structure, each stem-loop structure comprising a partial duplex and at least one loop, wherein the stem-loop structures do not comprise any viral ITR sequences.
  • a method of treating a disorder, disease, or condition in a subject the method comprising administering to the subject a therapeutically effective amount of a composition or pharmaceutical composition disclosed herein.
  • a method of treating a disorder, disease, or condition in a subject comprising administering to the subject a therapeutically effective amount of a non-viral, capsid-free DNA vector with covalently-closed ends (ceDNA vector), wherein the ceDNA vector comprises at least one transgene expression cassette flanked by a first stem-loop structure and a second stem-loop structure, each stem-loop structure comprising a partial duplex and at least one loop, wherein the stem-loop structures do not comprise any viral ITR sequence, wherein the ceDNA vector is encapsulated by an LNP.
  • the disorder, disease, or condition is a genetic disorder, disease, or condition.
  • a method of delivering a therapeutic protein to a subject comprising administering to the subject a therapeutically effective amount of the composition or pharmaceutical composition disclosed herein.
  • a method of delivering a therapeutic protein to a subject comprising administering to the subject a therapeutically effective amount of a non-viral, capsid-free DNA vector with covalently-closed ends (ceDNA vector), wherein the ceDNA vector comprises at least one transgene expression cassette flanked by a first stem-loop structure and a second stem-loop structure, each stem-loop structure comprising a partial duplex and at least one loop, wherein the stem-loop structures do not comprise any viral ITR sequences, and wherein the 10 ME148589913v.1 ceDNA vector is encapsulated by an LNP.
  • ceDNA vector non-viral, capsid-free DNA vector with covalently-closed ends
  • therapeutic protein is selected from the group consisting of an enzyme, a coagulation factor or co-factor, an antibody or an antigen- binding fragment thereof, an antigen, a gene-editing protein, and a cytotoxic protein.
  • a method of producing the ceDNA vector of any one of claims 1 to 72 comprising: (a) contacting a double-stranded DNA construct having a sense strand and an antisense strand with at least a first restriction endonuclease and at least a second restriction endonuclease, wherein: the construct comprises: a transgene expression cassette, a first non-palindromic restriction endonuclease recognition site and a corresponding first cleavage site upstream of the transgene expression cassette, and a second non-palindromic restriction endonuclease recognition site and a corresponding second cleavage site downstream of the transgene expression cassette; and wherein the first restriction endonuclease is
  • the first oligonucleotide and the second oligonucleotide are symmetric with respect to each other. In one embodiment, the first oligonucleotide and the second oligonucleotide are asymmetric with respect to each other. In one embodiment, the first oligonucleotide and the second oligonucleotide each self-anneal to form the one or more stem-loop structures and a single-stranded overhang at the 5’ end of each oligonucleotide or the 3’ end of each oligonucleotide.
  • the single-stranded overhang of the first oligonucleotide and/or the single-stranded overhang of the second oligonucleotide each comprise a 5’ to 3’ nucleic acid sequence selected from the group consisting of CTCT, CTCA, CACT, CTC, and GCT.
  • the first oligonucleotide corresponds to the first stem-loop region in the ceDNA vector and the second oligonucleotide corresponds to the second stem-loop region in the ceDNA vector.
  • at least one of the first and second restriction endonucleases or wherein each of the first and second restriction endonucleases is a Type IIS restriction endonuclease.
  • first restriction endonuclease and the second restriction endonuclease are the same restriction endonuclease. In one embodiment, the first restriction endonuclease and the second restriction endonuclease are different restriction endonucleases.
  • the Type IIS restriction endonuclease is selected from group consisting of AcuI, AlwI, Alw26I, BasI, BbsI, BbvI, BceAI, BcgI, BciVI, BcoDI, BruAI, BmrI, BpiI, BpuEI, BsaI, BsaXI, BseGI, BseRI, BsgI, BsmAI, 11 ME148589913v.1 BsmBI, BsmFI, BsmI, BspCNI, BspMI, BspQI, BsrDI, BsrI, BtgZI, BtsCI, BtsI, MutI, CspCI, EarI, EciI, Eco31I, Esp3I, FauI, FokI, HgaI, HphI, HpyAV, LguI, MboII, MlyI, MmeI, Mnl
  • the Type IIS restriction endonuclease is selected from group consisting of BbsI, BsaI, BsmBI, Esp3I, and SapI, and an isoschizomer thereof. In one embodiment, the Type IIS restriction endonuclease is BsmBI or an isoschizomer thereof; or wherein each of the first and second restriction endonucleases is BsmBI or an isoschizomer thereof.
  • FIG.1A and FIG.1B are schematic diagrams showing, respectively, ceDNAs 384 and ceDNA 558, each representing non-limiting embodiments of non-viral and capsid-free DNA vectors having covalently-closed ends (ceDNA) that contain no viral inverted terminal repeat (ITR) sequences.
  • FIG.2A and FIG.2B are schematic diagrams showing the cell-free synthetic production of, respectively, ceDNA 384 and ceDNA 558, which includes a one-step restriction digestion and ligation reaction.
  • FIG.3A is a graph comparing the in vitro expression of green fluorescence protein (GFP) in human embryonic kidney cells (293T cells) transfected with 30 ng, 90 ng, or 180 ng of reference ceDNA 382 (with 5’ AAV2 ITR and 3’ AAV2 ITR) and ceDNA 558A (with no viral ITR sequences), where the ceDNA vectors each carry a gfp transgene.
  • GFP green fluorescence protein
  • FIG.3B is a graph comparing the in vitro expression of GFP in human fibroblasts (HFF1) transfected with reference ceDNA 382 or ceDNA 558A.
  • FIG.4A and FIG.4B are graphs comparing the in vivo expression of luciferase in C57 mice and Rag2KO mice at Day 7 and Day 14, respectively, after being dosed via intravenous administration with reference ceDNA 382 (with 5’ AAV2 ITR and 3’ AAV2 ITR) and ceDNA 558 (with no viral ITR sequences) that were each formulated as lipid nanoparticles (LNPs).
  • LNPs lipid nanoparticles
  • FIGS.5A-5F and FIGS.5G-5L are images taken using the in vivo imaging system at Day 7 and Day 14 post-dosing, indicating that the luciferase expression was detected in the abdominal area of the mice where the liver is located.
  • FIG.6A and FIG.6B are graphs showing the longitudinal body weight change in C57 mice and Rag2KO mice from Day 0 through Day 28 after being dosed via intravenous administration with reference ceDNA 382 (with 5’ AAV2 ITR and 3’ AAV2 ITR) and ceDNA 558 (with no viral ITR sequences) that were each formulated as lipid nanoparticles (LNPs).
  • LNPs lipid nanoparticles
  • FIGS.7A-7E depict, respectively, blood serum levels of IFN- ⁇ (FIG.7A), IL-18 (FIG.7B), IFN- ⁇ (FIG.7C), TNF- ⁇ (FIG.7D), and IL-6 (FIG.7E) in C57 mice and Rag2KO mice at 6 hours pose-dose following injection of reference ceDNA 382 (with 5’ AAV2 ITR and 3’ AAV2 ITR) and ceDNA 558 (with no viral ITR sequences) that were each formulated as lipid nanoparticles (LNPs).
  • LNPs lipid nanoparticles
  • FIG.8 provides a schematic diagram of the ITR structures of various synthetic ceDNA constructs with one or more complete sections of the ITR regions A and A’, B and B’, C and C’, or D and D’ removed.
  • the structural ITR variant constructs were designated as follows: ceDNA178 (wt- wt); ceDNA4 (wt-wt); ceDNA14 (wt-mu); ceDNA15 (mu-mu); ceDNA16 (blunt-blunt); ceDNA17 (hpin-hpin); ceDNA18 (blunt-wt); ceDNA19 (blunt-mu); ceDNA20 (hpin-wt); ceDNA35 (wt-hpin).
  • FIG.9A and FIG.9B show the results of transfection and expression of ceDNA variants in human leukemia monocytic cells (THP-1) and human fibroblast cell line (HFF1) cells.
  • FIG.10 is a graph that shows expression of ceDNA transgene in 293T cells in suspension.
  • FIG.11 is a graph that shows expression of ceDNA transgene in adherent HFF1 cells.
  • FIG.12 is a graph that shows the results of analysis of different ceDNA constructs shown in FIG.8 by IFN bioassay in THP1-ISG reporter cell. ISG promoter activation was determined by transferring supernatant from 293T cells and supernatant transferred from HFF1 cells.
  • FIG.13A and FIG.13B are graphs that show the results of analysis of ceDNA constructs shown in FIG.8 by Bioassay, and a correlation of transgene expression and IFN response.
  • FIG.14A and FIG.14B are graphs that show the correlation of expression and innate immune response in ceDNA4 (wt-wt) and ceDNA20 (hpin-wt) constructs.
  • FIG.15A is a graph that shows GFP expression of ceDNA20 (hpin-wt) having 3’ left, wt-ITR and ceDNA35 with opposing ITR (wt-hpin) having 5’ right wt-ITR configurations in HFF1 cells.
  • FIG.15B is a graph that shows results obtained in the interferon bioassay in THP1 -ISG reporter cells.
  • FIG.16A and FIG.16B are graphs that show the results of ceDNA expression analysis in retinal pigment epithelial (RPE) for ceDNA4 (wt-wt), ceDNA17 (hpin-hpin), ceDNA20 (hpin-wt) and ceDNA35 (wt-hpin).
  • FIG.16A shows GFP intensity.
  • FIG.16B shows GFP expression over time.
  • FIG.17A and FIG.17B are graphs that show the results of GFP expression analysis in RAW-ISG murine macrophage cell line for ceDNA4 (wt-wt), ceDNA17 (hpin-hpin), ceDNA20 (hpin- 13 ME148589913v.1 wt) and ceDNA35 (wt-hpin).
  • FIG.17A shows GFP intensity.
  • FIG.17B shows GFP expression over time.
  • FIG.18 is a graph that shows GFP expression in HEPaRG cells over time.
  • synthetic ceDNA recapitulated the long-term expression observed with the Sf9-ceDNA (ceDNA178).
  • FIG.19 is a Western blot that shows the results of synthetic ceDNA production in insect cells by transient transfection and the absence of detectable non-ceDNA species.
  • FIG.20 is a Western blot that shows the results of amplification of ceDNA with one ITR (right) in SF9 cells.
  • DETAILED DESCRIPTION One of the biggest hurdles in the development of therapeutics, particularly in rare diseases, is the large number of individual conditions. Around 350 million people on earth are living with rare disorders, defined by the National Institutes of Health as a disorder or condition with fewer than 200,000 people diagnosed. About 80 percent of these rare disorders are genetic in origin, and about 95 percent of them do not have treatment approved by the FDA.
  • the disclosure provides a composition comprising a non-viral, capsid-free DNA vector with covalently-closed ends (ceDNA vector), wherein the ceDNA vector comprises at least one transgene expression cassette flanked by a stem-loop structure comprising a partial duplex and at least one loop, wherein the stem-loop structures do not comprise any viral ITR sequences, and a lipid.
  • ceDNA vector non-viral, capsid-free DNA vector with covalently-closed ends
  • the disclosure provides a method of delivering a therapeutic protein to a subject, the method comprising administering to the subject a therapeutically effective amount of ceDNA vector, wherein the ceDNA vector comprises at least one transgene expression cassette flanked by a stem-loop structure comprising a partial duplex and at least one loop, wherein the stem-loop structures do not comprise any viral ITR sequences.
  • the ceDNA vectors described herein can be used for gene editing.
  • the ceDNA vectors described herein comprise an antibody, or an antigen binding fragment thereof.
  • the ceDNA vectors described herein comprise an aptamer.
  • the ceDNA vectors described herein comprise a regulatory switch, thus allowing for controllable gene expression after delivery. 14 ME148589913v.1 I. Definitions Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this disclosure is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can 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 disclosure, which is defined solely by the claims.
  • AAV adeno-associated virus
  • AAV refers to single-stranded DNA parvoviruses that grow only in cells. Certain functions of AAV are provided only by co-infecting a helper virus. Thirteen serotypes of AAV have been identified. General information and review of AAV can be found, e.g., in Carter, 1989, Handbook of Parvoviruses, Vol.1, p.169-228, and Berns, 1990, Virology, pp.1743-1764, Raven Press, (New York).
  • administering refers to introducing a composition or agent (e.g., nucleic acids, in particular ceDNA) into a subject and includes concurrent and sequential introduction of one or more compositions or agents.
  • administering can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, placebo, and 15 ME148589913v.1 experimental methods. “Administration” also encompasses in vitro and ex vivo treatments.
  • Administration includes self-administration and the administration by another. Administration can be carried out by any suitable route.
  • a suitable route of administration allows the composition or the agent to perform its intended function. For example, if a suitable route is intravenous, the composition is administered by introducing the composition or agent into a vein of the subject.
  • antibody is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
  • An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the same antigen to which the intact antibody binds.
  • the antibody or antibody fragment comprises an immunoglobulin chain or antibody fragment and at least one immunoglobulin variable domain sequence.
  • antibodies or fragments thereof include, but are not limited to, an Fv, an scFv, a Fab fragment, a Fab’, a F(ab’) 2 , a Fab’-SH, a single domain antibody (dAb), a heavy chain, a light chain, a heavy and light chain, a full antibody (e.g., includes each of the Fc, Fab, heavy chains, light chains, variable regions etc.), a bispecific antibody, a diabody, a linear antibody, a single chain antibody, an intrabody, a monoclonal antibody, a chimeric antibody, a multispecific antibody, or a multimeric antibody.
  • an antibody or fragment thereof can be of any class, including but not limited to IgA, IgD, IgE, IgG, and IgM, and of any subclass thereof including but not limited to IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
  • an antibody can be derived from any mammal, for example, primates, humans, rats, mice, horses, goats etc.
  • the antibody is human or humanized.
  • the antibody is a modified antibody.
  • the components of an antibody can be expressed separately such that the antibody self-assembles following expression of the protein components.
  • the antibody is “humanized” to reduce immunogenic reactions in a human.
  • the antibody has a desired function, for example, interaction and inhibition of a desired protein for the purpose of treating a disease or a symptom of a disease.
  • the antibody or antibody fragment comprises a framework region or an F c region.
  • the term “antigen-binding domain” of an antibody molecule refers to the part of an antibody molecule, e.g., an immunoglobulin (Ig) molecule, that participates in antigen binding.
  • the antigen binding site is formed by amino acid residues of the variable (V) regions of the heavy (H) and light (L) chains.
  • FRs are amino acid sequences that are naturally found between, and adjacent to, hypervariable regions in immunoglobulins.
  • the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three-dimensional space to form an antigen- binding surface, which is complementary to the three-dimensional surface of a bound antigen.
  • CDRs complementarity- determining regions
  • Each variable chain (e.g., variable heavy chain and variable light chain) is typically made up of three CDRs and four FRs, arranged from amino-terminus to carboxy- terminus in the amino acid order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • anti-therapeutic nucleic acid immune response refers to any undesired immune response against a therapeutic nucleic acid, viral or non-viral in its origin.
  • the undesired immune response is an antigen-specific immune response against the viral transfer vector itself.
  • the immune response is specific to the transfer vector which can be double stranded DNA, single stranded RNA, or double stranded RNA. In other embodiments, the immune response is specific to a sequence of the transfer vector. In other embodiments, the immune response is specific to the CpG content of the transfer vector.
  • the term “aptamer” refers to a nucleic acid molecule that is capable of binding to a particular molecule of interest with high affinity and specificity (Tuerk and Gold, Science 249:505 (1990); Ellington and Szostak, Nature 346:818 (1990)).
  • aptamers may be composed of DNA or RNA, or may comprise nonnatural nucleotides and nucleotide analogs (e.g., locked DNA or peptide nucleic acids [PNAs]) that have high affinity to a protein localized in the nucleus or the membrane thereof.
  • 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 pharmaceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions.
  • ceDNA refers to capsid-free closed-ended linear double stranded (ds) duplex DNA for non-viral gene transfer, synthetic or otherwise. Detailed description of ceDNA is described in International Patent Application No. PCT/US2017/020828, filed March 3, 2017, the 17 ME148589913v.1 entire contents of which are expressly incorporated herein by reference.
  • ceDNA comprising various inverted terminal repeat (ITR) sequences and configurations using cell-based methods
  • ITR inverted terminal repeat
  • synthetic ceDNA vectors comprising various ITR sequences and configurations are described, e.g., in International application PCT/US2019/14122, filed January 18, 2019, the entire content of which is incorporated herein by reference.
  • the terms “ceDNA vector” and “ceDNA” are used interchangeably.
  • the ceDNA is a closed-ended linear duplex (CELiD) CELiD DNA.
  • the ceDNA is a DNA-based minicircle. In one embodiment, the ceDNA is a minimalistic immunological-defined gene expression (MIDGE)-vector. In one embodiment, the ceDNA is a ministering DNA. In one embodiment, the ceDNA is a doggyboneTM DNA. With the exception of the reference ceDNA vectors that are described herein in the Examples and any other non-inventive ceDNA vectors referred herein, the ceDNA vectors of this disclosure contain no viral ITR sequences.
  • MIDGE immunological-defined gene expression
  • reference ceDNA refers to a ceDNA that contains a viral ITR sequence, e.g., a 5’ AAV derived wt-ITR and a 3’ AAV derived wt- ITR, or a partial or a modified AAV-derived ITR sequence.
  • a reference ceDNA may comprise an AAV-derived ITR sequence with a deletion, substitution, or deletion as compared to a wild-type ITR sequence (e.g., a wild-type AAV ITR sequence).
  • a reference ceDNA comprising a partial AAV-derived ITR sequence may comprise, for example, at least one viral ITR sequence, including, but not limited to, a trs, Rep binding element (RBE), A, A’, B, B’, C, C’, D and/or D’ regions of AAV ITRs.
  • a reference ceDNA vector may be prepared using conventional cell-based production methods, such as Sf9 insect cell-based production method, or the reference ceDNA vector may be synthetically produced in a cell-free environment.
  • the terms “closed-ended DNA vector”, “ceDNA vector” and “ceDNA” are used interchangeably and refer to a synthetically made, capsid-free DNA vector, with at least one covalently-closed end (i.e., an intramolecular duplex).
  • the ceDNA comprises two covalently-closed ends.
  • ceDNA (vector) of this invention/disclosure refers to an inventive ceDNA provided herein that contains no viral sequences (e.g., ITR sequences, e.g., does not comprise (is devoid of) any viral ITR sequences, including, but not limited to, trs, RBE, A, A’, B, B’, C, C’, D, and/or D’ regions (e.g., trs, RBE, A and A’, B and B’, C and C’ and/or D and D’ regions of an AAV ITR)).
  • the ceDNA vectors of this disclosure are synthetically produced in a cell-free environment, such as using the procedures as described in Example 1 of this disclosure and also in Examples 2-6 of International Patent Application No. 18 ME148589913v.1 PCT/US2019/014122, filed January 18, 2019, and also in International Patent Application No. PCT/US2022/053868, filed December 22, 2022, each of which is incorporated herein in its entirety by reference.
  • the terms “cell-free,” “cell-free synthesis,” “cell-free production,” “synthetic closed-ended DNA vector production” and “synthetic production” and all other related counterparts are used interchangeably and refer to the production of one or more molecules in a manner that does not involve replication or other multiplication of the molecule by or inside of a cell or using a cellular extract. Synthetic production avoids contamination of the produced molecule with cellular contaminants (e.g., cellular proteins or cellular nucleic acids) and further avoids unwanted cellular- specific modification of the molecule during the production process (e.g., methylation or glycosylation or other post-translational modification).
  • cellular contaminants e.g., cellular proteins or cellular nucleic acids
  • inverted terminal repeat or “ITR” are meant to refer to a nucleic acid sequence located at the 5’ and/or 3’ terminus of an AAV molecule or ceDNA molecule and which comprise at least one stem-loop structure comprising a partial duplex and at least one loop.
  • a wild-type ITR or “WT-ITR” refers to the viral sequence of a naturally occurring ITR sequence in an AAV genome or other dependovirus that retains, e.g., Rep binding activity and Rep nicking ability.
  • the nucleotide sequence of a WT-ITR from any AAV serotype may slightly vary from the canonical naturally occurring sequence due to degeneracy of the genetic code or drift.
  • a wild-type ITR comprises trs, RBE, A, A’, B, B’, C, C’, D, and/or D’ regions (e.g., trs, RBE, A and A’, B and B’, C and C’ and/or D and D’ regions of an AAV ITR).
  • the term “viral inverted terminal repeat,” “viral ITR,” “viral inverted terminal repeat sequence,” “viral ITR sequence,” “virally-derived inverted terminal repeat (ITR)” or “virally- derived ITR sequence” refers to an ITR sequence that is a wild-type ITR sequence, or a fragment thereof, e.g., an AAV wild-type ITR sequence or a fragment thereof.
  • a viral ITR can be, e.g., a naturally occurring ITR sequence or ITR sequence fragment from, e.g., AAV.
  • a viral ITR can be, e.g., a modified ITR sequence or fragment thereof derived from a virus, e.g., wherein the virus is an AAV.
  • a modified ITR sequence derived from a virus can have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a wild-type ITR sequence, e.g., wherein the virus is an AAV.
  • the ITR can be from (i.e., originates from) the family Parvoviridae, which encompasses 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 can be used as an ITR, which can further be modified by truncation, substitution, deletion, insertion and/or addition.
  • Parvoviridae family viruses consist of two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect invertebrates.
  • these ITRs are about 145 nucleotides in length and in nature are inverted with respect to the other.
  • Dependoparvoviruses include the viral family of the adeno-associated viruses (AAV) which are capable of replication in 19 ME148589913v.1 vertebrate hosts including, but not limited to, human, primate, bovine, canine, equine and ovine species.
  • AAV adeno-associated viruses
  • the terminal 125 nucleotides in each ITR form a palindromic double-stranded T-shaped hairpin structure, in which the A-A' palindrome forms the stem, and the two smaller palindromes, B- B' and the C-C', form the cross-arms of the T.
  • a cell-dependent AAV-derived vector typically contains two ITR sequences with trs, RBE, A and A’, B and B’, C and C’ and/or D and/or D’ regions.
  • a viral ITR may comprise one or more of trs, RBE, A, A’, B, B’, C, C’, D, and/or D’ ITR regions (e.g., trs, RBE, A and A’, B and B’, C and C’ and/or D and D’ regions of an AAV ITR).
  • spacer or “spacer region” refer to an intervening sequence that separates functional elements in the ceDNA vector.
  • ceDNA spacer regions keep two functional elements at a desired distance for optimal functionality.
  • ceDNA spacer regions provide or add to the genetic stability of the ceDNA within e.g., a plasmid or baculovirus.
  • ceDNA spacer regions facilitate ready genetic manipulation of the ceDNA molecule by providing a convenient location for cloning sites and the like.
  • 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 molecule to separate the cis – acting factors, e.g., inserting a 6mer, 12mer, 18mer, 24mer, 48mer, 86mer, 176mer, etc. between the terminal resolution site and the upstream transcriptional regulatory element.
  • the spacer may be incorporated between the polyadenylation signal sequence and the 3’-terminal resolution site.
  • the 5’ or 3’ end of the spacer region of a ceDNA vector contains partial sequences of one or more recognition sites for a restriction endonuclease, such as a Type IIS restriction endonuclease.
  • a restriction endonuclease such as a Type IIS restriction endonuclease.
  • exogenous refers to a substance present in a cell other than its native source.
  • the term “exogenous” when used herein can refer to a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found and one wishes to introduce the nucleic acid or polypeptide into such a cell or organism.
  • exogenous can refer to a nucleic acid or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is found in relatively low amounts and one wishes to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to create ectopic expression or levels.
  • endogenous refers to a substance that is native to the biological system or cell.
  • effector protein refers to a polypeptide that provides a detectable read-out, either as, for example, a reporter polypeptide, or more appropriately, as a polypeptide that kills a cell, e.g., a toxin, or an agent that renders a cell susceptible to killing with a chosen agent or lack thereof. Effector proteins include any protein or peptide that directly targets or damages the host 20 ME148589913v.1 cell’s DNA and/or RNA.
  • effector proteins can include, but are not limited to, a restriction endonuclease that targets a host cell DNA sequence (whether genomic or on an extrachromosomal element), a protease that degrades a polypeptide target necessary for cell survival, a DNA gyrase inhibitor, and a ribonuclease-type toxin.
  • a restriction endonuclease that targets a host cell DNA sequence (whether genomic or on an extrachromosomal element)
  • protease that degrades a polypeptide target necessary for cell survival
  • a DNA gyrase inhibitor a DNA gyrase inhibitor
  • ribonuclease-type toxin ribonuclease-type toxin.
  • the expression of an effector protein controlled by a synthetic biological circuit as described herein can participate as a factor in another synthetic biological circuit to thereby expand the range and complexity of a biological circuit system’s responsiveness.
  • transgene expression cassette As used herein, the terms “transgene expression cassette,” “expression cassette,” “transcription cassette” and “gene expression unit” are used interchangeably and refer to a linear stretch of nucleic acids that includes a transgene that is operably linked to one or more regulatory elements such as promoters or other regulatory sequences sufficient to direct transcription of the transgene, but which does not comprise capsid-encoding sequences, other vector sequences or inverted terminal repeat regions.
  • An expression cassette may additionally comprise one or more regulatory genetic elements including cis-acting sequences (e.g., promoters, enhancers, or repressors), one or more introns, one or more polyadenylation signals and one or more post-transcriptional regulatory elements such as a WHP post-transcriptional regulatory element (WPRE).
  • cis-acting sequences e.g., promoters, enhancers, or repressors
  • introns e.g., one or more introns
  • polyadenylation signals e.g., a post-transcriptional regulatory element (WPRE).
  • WHP post-transcriptional regulatory element WHP post-transcriptional regulatory element
  • flanking refers to terminal repeats at each end of the linear single strand synthetic AAV vector.
  • full length antibody refers to an immunoglobulin (Ig) molecule (e.g., an IgG antibody), for example, that is naturally occurring, and formed by normal immunoglobulin gene fragment recombinatorial processes.
  • functional antibody fragment refers to a fragment that binds to the same antigen as that recognized by the intact (e.g., full-length) antibody.
  • antibody fragment or “functional fragment” also include isolated fragments consisting of the variable regions, such as the “Fv” fragments consisting of the variable regions of the heavy and light chains or recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker (“scFv proteins”).
  • an antibody fragment does not include portions of antibodies without antigen binding activity, such as Fc fragments or single amino acid residues.
  • gene is used broadly to refer to any segment of nucleic acid associated with expression of a given RNA or protein, in vitro or in vivo.
  • genes include regions encoding expressed RNAs (which typically include polypeptide coding sequences) and, often, the 21 ME148589913v.1 regulatory sequences required for their expression.
  • Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have specifically desired parameters.
  • the phrase “genetic disease” or “genetic disorder” refers to a disease, partially or completely, directly or indirectly, caused by one or more abnormalities in the genome, including and especially a condition that is present from birth.
  • the abnormality may be a mutation, an insertion or a deletion in a gene.
  • the abnormality may affect the coding sequence of the gene or its regulatory sequence.
  • heterologous refers to a nucleotide or polypeptide sequence that is not found in the native nucleic acid or protein, respectively.
  • 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 is incorporated into and may be delivered and expressed by a ceDNA vector as disclosed herein.
  • a heterologous nucleic acid sequence may be linked to a naturally occurring nucleic acid sequence (or a variant thereof) (e.g., by genetic engineering) to generate a chimeric nucleotide sequence encoding a chimeric polypeptide.
  • a heterologous nucleic acid sequence may be linked to a variant polypeptide (e.g., by genetic engineering) to generate a nucleotide sequence encoding a fusion variant polypeptide.
  • Transgenes of interest include, but are not limited to, nucleic acids encoding polypeptides, preferably therapeutic (e.g., for medical, diagnostic, or veterinary uses) or immunogenic polypeptides (e.g., for vaccines).
  • nucleic acids of interest include nucleic acids that are transcribed into therapeutic RNA.
  • Transgenes included for use in the ceDNA vectors of the disclosure include, but are not limited to, those that express or encode one or more polypeptides, peptides, ribozymes, aptamers, peptide nucleic acids, siRNAs, RNAis, miRNAs, lncRNAs, antisense oligo- or polynucleotides, antibodies, antigen binding fragments, or any combination thereof.
  • sequence identity refers to the relatedness between two nucleic acid sequences, that is to the percentage of nucleotide or amino acid residues that are identical to the nucleotide or amino acid residues in another nucleotide or amino acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleotide sequence homology can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ClustalW2 or Megalign (DNASTAR) software.
  • a nucleic acid sequence (e.g., DNA sequence), for example of a homology arm of a repair template, is considered “homologous” when the sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to the 22 ME148589913v.1 corresponding native or unedited nucleic acid sequence (e.g., genomic sequence) of the host cell.
  • a nucleic acid sequence e.g., DNA sequence
  • a homology arm of a repair template is considered “homologous” when the sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%
  • the terms “homology” or “homologous” may be used interchangeably with the terms “identity” or “identical”, respectively.
  • the term “host cell” refers to any cell type that is susceptible to transformation, transfection, transduction, and the like with nucleic acid therapeutics of the present disclosure.
  • a host cell can be an isolated primary cell, pluripotent stem cells, CD34 + cells, induced pluripotent stem cells, or any of a number of immortalized cell lines (e.g., HepG2 cells).
  • a host cell can be an in situ or in vivo cell in a tissue, organ or organism.
  • a host cell can be a target cell of, for example, a mammalian subject (e.g., human patient in need of gene therapy).
  • an “immunoglobulin variable domain sequence” refers to an amino acid sequence which can form the structure of an immunoglobulin variable domain.
  • the sequence may include all or part of the amino acid sequence of a naturally-occurring variable domain.
  • the sequence may or may not include one, two, or more N- or C-terminal amino acids, or may include other alterations that are compatible with formation of the protein structure.
  • an “inducible promoter” is one that is characterized by initiating or enhancing transcriptional activity when in the presence of, influenced by, or contacted by an inducer or inducing agent.
  • An “inducer” or “inducing agent,” as defined herein, can be endogenous, or a normally exogenous compound or protein that is administered in such a way as to be active in inducing transcriptional activity from the inducible promoter.
  • the inducer or inducing agent i.e., a chemical, a compound or a protein
  • the inducer or inducing agent can itself be the result of transcription or expression of a nucleic acid sequence (i.e., an inducer can be an inducer protein expressed by another component or module), which itself can be under the control or an inducible promoter.
  • an inducible promoter is induced in the absence of certain agents, such as a repressor.
  • inducible promoters include, but are not limited to, tetracycline, metallothionine, ecdysone, mammalian viruses (e.g., the adenovirus late promoter; and the mouse mammary tumor virus long terminal repeat (MMTV-LTR)) and other steroid-responsive promoters, rapamycin responsive promoters and the like.
  • an “input agent responsive domain” is a domain of a transcription factor that binds to or otherwise responds to a condition or input agent in a manner that renders a linked DNA binding fusion domain responsive to the presence of that condition or input.
  • the presence of the condition or input results in a conformational change in the input agent responsive domain, or in a protein to which it is fused, that modifies the transcription-modulating activity of the transcription factor.
  • the term “in vivo” refers to assays or processes that occur in or within an organism, such as a multicellular animal. In some of the aspects described herein, a method or use can be said to occur 23 ME148589913v.1 “in vivo” when a unicellular organism, such as a bacterium, is used.
  • ex vivo refers to methods and uses that are performed using a living cell with an intact membrane that is outside of the body of a multicellular animal or plant, e.g., explants, cultured cells, including primary cells and cell lines, transformed cell lines, and extracted tissue or cells, including blood cells, among others.
  • in vitro refers to assays and methods that do not require the presence of a cell with an intact membrane, such as cellular extracts, and can refer to the introducing of a programmable synthetic biological circuit in a non-cellular system, such as a medium not comprising cells or cellular systems, such as cellular extracts.
  • the term “local delivery” refers to delivery of an active agent such as ceDNA or LNP encapsulating a ceDNA molecule directly to a target site within an organism.
  • an agent can be locally delivered by direct injection into a disease site such as a tumor or other target site such as a site of inflammation or a target organ such as the liver, heart, pancreas, kidney, skin, and the like.
  • neDNA or “nicked ceDNA” refers to a closed-ended DNA having a nick or a gap of 1-100 bases in a stem region or spacer region 5’ upstream of an open reading frame (e.g., a promoter and transgene to be expressed).
  • nucleic acid refers to a polymer containing at least two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in either single- or double-stranded form and includes DNA, RNA, and hybrids thereof.
  • DNA may be in the form of, e.g., antisense molecules, plasmid DNA, DNA-DNA duplexes, pre-condensed DNA, PCR products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups.
  • DNA may be in the form of minicircle, plasmid, bacmid, minigene, ministring DNA (linear covalently closed DNA vector), closed-ended linear duplex DNA (CELiD or ceDNA), doggyboneTM DNA, dumbbell shaped DNA, minimalistic immunological-defined gene expression (MIDGE)-vector, viral vector or nonviral vectors.
  • RNA may be in the form of small interfering RNA (siRNA), Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, gRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof.
  • Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid.
  • analogs and/or modified residues include, without limitation, phosphorothioates, phosphorodiamidate morpholino oligomer (morpholino), phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2’-O-methyl ribonucleotides, locked nucleic acid (LNATM), and peptide nucleic acids (PNAs).
  • nucleic acids Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively 24 ME148589913v.1 modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • nucleotides contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups.
  • nucleic acid therapeutic As used herein, the phrases “nucleic acid therapeutic”, “therapeutic nucleic acid” and “TNA” are used interchangeably and refer to any modality of therapeutic using nucleic acids as an active component of therapeutic agent to treat a disease or disorder. As used herein, these phrases refer to RNA-based therapeutics and DNA-based therapeutics.
  • Non-limiting examples of RNA-based therapeutics include mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNAs (RNAi), Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA).
  • Non-limiting examples of DNA-based therapeutics include minicircle DNA, minigene, viral DNA (e.g., Lentiviral or AAV genome) or non-viral synthetic DNA vectors, closed-ended linear duplex DNA (ceDNA / CELiD), plasmids, bacmids, doggyboneTM DNA vectors, minimalistic immunological-defined gene expression (MIDGE)-vector, nonviral ministring DNA vector (linear-covalently closed DNA vector), or dumbbell-shaped DNA minimal vector (“dumbbell DNA”).
  • liver-specific regulatory element refers to a genetic element, including but not limited to a promoter, an enhancer, a riboswitch, an insulator, a mir- regulatable element, a post-transcriptional regulatory element, a transcription factor binding site (or consensus site), that confers or enhances liver-specific expression (e.g., in vivo expression) of a (trans)gene, i.e.
  • the at least one liver-specific regulatory element is selected from the group consisting of a liver-specific promoter, a liver-specific enhancer, a liver-specific riboswitch, a liver-specific insulator, a liver- specific mir-regulatable element, a liver-specific post-transcriptional regulatory element, a liver-specific transcription factor binding site (or consensus site), and a combination thereof.
  • liver-specific regulatory elements are as described in International Patent Application Publication No.
  • WO2023/044059 (including any one of SEQ ID NOs: 1-80, 138, 139 and 155-1689 as disclosed therein), which is incorporated herein by reference in its entirety.
  • the term “promoter,” as used herein, refers to any nucleic acid sequence that regulates the expression of another nucleic acid sequence by driving transcription of the nucleic acid sequence, which can be a heterologous target gene encoding a protein or an RNA. Promoters can be constitutive, inducible, repressible, tissue-specific, or any combination thereof.
  • a promoter is a control region of a nucleic acid sequence at which initiation and rate of transcription of the remainder of a nucleic acid sequence are controlled.
  • a promoter can also contain genetic elements at which regulatory proteins and molecules can bind, such as RNA polymerase and other transcription factors.
  • a promoter can drive the expression of a transcription factor that regulates the expression of the promoter itself, or that of another promoter used in another modular component of the synthetic biological circuits described herein.
  • ME148589913v.1 sequence will be found within the promoter 25 ME148589913v.1 sequence will be found a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain “TATA” boxes and “CAT” boxes.
  • Various promoters, including inducible promoters may be used to drive the expression of transgenes in the ceDNA vectors disclosed herein.
  • liver-specific promoter encompasses any promoter that confers or enhances liver- specific expression (e.g., in vivo expression) of a (trans)gene, i.e., expression (e.g., in vivo expression) of the (trans)gene in the liver.
  • liver-specific promoters are provided on the Liver-specific Gene Promoter Database (LSPD, rulai.cshl.edu/LSPD/), and include, for example, the transthyretin (TTR) promoter or TTR-minimal promoter (TTRm), the alpha 1-antitrypsin (AAT) promoter, the albumin (ALB) promotor or minimal promoter, the apolipoprotein A1 (APOA1) promoter or minimal promoter, the complement factor B (CFB) promoter, the ketohexokinase (KHK) promoter, the hemopexin (HPX) promoter or minimal promoter, the nicotinamide N- methyltransferase (NNMT)promoter or minimal promoter, the (liver) carboxylesterase 1 (CES1) promoter or minimal promoter, the protein C (PROC) promoter or minimal promoter, the apolipoprotein C3 (APOC3) promoter or minimal promoter, the mannan
  • TTR
  • an enhancer refers a cis-acting regulatory sequence (e.g., about 50- 1000 base pairs) that bind one or more proteins (e.g., activator proteins or transcription factors) to increase transcriptional activation of a nucleic acid sequence.
  • Endogenous enhancers may be positioned up to 1,000,000 base pairs upstream of the gene start site or downstream of the gene start site that they regulate.
  • An enhancer can be positioned within an intronic region, or in the exonic region of an unrelated gene.
  • an enhancer may be located anywhere within the expression cassette (e.g., upstream or downstream, with or without spacers, etc.).
  • a promoter can be said to drive expression or drive transcription of the nucleic acid sequence that it regulates.
  • “operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression.
  • the phrases “operably linked,” “operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” indicate that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence it regulates to control transcriptional initiation and/or expression of that sequence.
  • inverted promoter refers to a promoter in which the nucleic acid sequence is in the reverse orientation, such that what was the coding strand is now the non-coding strand, and vice versa. Inverted promoter sequences can be used in various embodiments to regulate the state of a switch. In addition, in various embodiments, a promoter can be used in conjunction with an enhancer. 26 ME148589913v.1 A promoter can be one naturally associated with a gene or sequence, as can be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and/or exon of a given gene or sequence.
  • an enhancer can be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence.
  • a coding nucleic acid segment is positioned under the control of a “recombinant promoter” or “heterologous promoter,” both of which refer to a promoter that is not normally associated with the encoded nucleic acid sequence it is operably linked to in its natural environment.
  • a recombinant or heterologous enhancer refers to an enhancer not normally associated with a given nucleic acid sequence in its natural environment.
  • promoters or enhancers can 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., comprise different elements of different transcriptional regulatory regions, and/or mutations that alter expression through methods of genetic engineering that are known in the art.
  • promoter sequences can be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR, in connection with the synthetic biological circuits and modules disclosed herein (see, e.g., U.S. Pat.
  • Rep binding site e.g., AAV Rep 78 or AAV Rep 68
  • RBE a binding site for Rep protein
  • RBS an RBS sequence and its inverse complement together form a single RBS.
  • RBS sequences are known in the art, and include, for example, 5’- GCGCGCTCGCTCGCTC-3′, an RBS sequence identified in AAV2. Without being bound by theory it is thought that he nuclease domain of a Rep protein binds to the duplex nucleotide sequence GCTC, and thus the two known AAV Rep proteins bind directly to and stably assemble on the duplex oligonucleotide, 5’-(GCGC)(GCTC)(GCTC)(GCTC)-3’.
  • the ceDNA vectors of the instant disclosure are devoid of any viral ITR sequences, and because RBE sequences are located within viral ITRs (e.g., AAV ITRs), the ceDNA vectors of the instant disclosure should not comprise any RBE sequences.
  • reference ceDNA vectors e.g., reference ceDNA vectors used as controls for comparison purposes
  • reporter refers to a nucleic acid or protein sequence that can be used to provide a detectable read-out. Reporters generally produce a measurable signal such as fluorescence, color, or luminescence. Reporter protein coding sequences encode proteins whose 27 ME148589913v.1 presence in the cell or organism is readily observed.
  • reporter polypeptides useful for experimental or diagnostic purposes include, but are not limited to, ⁇ -lactamase, ⁇ -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.
  • a “repressor protein” or “inducer protein” is a protein that binds to a regulatory sequence element and represses or activates, respectively, the transcription of sequences operatively linked to the regulatory sequence element.
  • Preferred repressor and inducer proteins as described herein are sensitive to the presence or absence of at least one input agent or environmental input.
  • Preferred proteins as described herein are modular in form, comprising, for example, separable DNA-binding and input agent-binding or responsive elements or domains.
  • the terms “sense” and “antisense” are meant to refer to the orientation of the structural element on the polynucleotide. The sense and antisense versions of an element are the reverse complement of each other.
  • subject refers to a human or animal, to whom treatment, including prophylactic treatment, with the ceDNA vector according to the present disclosure, is provided.
  • animal is a vertebrate such as, but not limited to, a primate, rodent, domestic animal or game animal.
  • Primates include, but are not limited to, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus.
  • Rodents include, but are not limited to, mice, rats, woodchucks, ferrets, rabbits and hamsters.
  • domestic and game animals include, but are not limited to, cows, horses, pigs, goats, deer, bison, buffalo, feline species (e.g., domestic cat), canine species (e.g., dog, fox, wolf), avian species (e.g., chicken, emu, ostrich), fish (e.g., zebrafish, trout, catfish and salmon), or amphibians (e.g., frogs such as Xenopus laevis).
  • the subject is a mammal, e.g., a non-human primate or a human.
  • a subject can be male or female. Additionally, a subject can be an infant or a child.
  • the subject can be a neonate or an unborn subject, e.g., the subject is in utero. Mammals other than humans can be advantageously used as subjects that represent animal models of diseases and disorders.
  • the methods and compositions described herein can be used for domesticated animals and/or pets.
  • a human subject can be of any age, gender, race or ethnic group, e.g., Caucasian (white), Asian, African, black, African American, African European, Hispanic, Mideastern, etc.
  • the subject can be a patient or other subject in a clinical setting. In some embodiments, the subject is already undergoing treatment.
  • the term “suppress,” “decrease,” “interfere,” “inhibit” and/or “reduce” generally refers to the act of reducing, either directly or indirectly, a concentration, 28 ME148589913v.1 level, function, activity, or behavior relative to the natural, expected, or average, or relative to a control condition.
  • asymmetric ITRs also referred to as “asymmetric ITR pairs” refers to a pair of ITRs within a single ceDNA or reference ceDNA vector that are not inverse complements across their full length.
  • an asymmetric ITR pair does not have a symmetric three-dimensional spatial organization to their cognate ITR such that their 3D structures are different shapes in geometrical space.
  • the term “symmetric ITRs” refers to a pair of ITRs within a single ceDNA molecule or reference ceDNA vector that are inverse complements across their full length.
  • a symmetric ITR pair has a symmetric three-dimensional spatial organization with respect to each other such that their 3D structures are the same shapes in geometrical space.
  • an ITR located 5’ to (upstream of) an expression cassette in a ceDNA vector is referred to as a “5’ ITR” or a “left ITR”
  • an ITR located 3’ to (downstream of) an expression cassette in a ceDNA vector is referred to as a “3’ ITR” or a “right ITR”.
  • the term “symmetric” when referring to the 5’ and 3’ stem-loop regions of the ceDNA vectors of the present disclosure refers to a pair of stem-loop regions that comprise only artificial sequences, and do not comprise viral ITR sequences, and that are inverse complements of each other across their full length.
  • ceDNA 384 whereby the 5’ and 3’ stem-loop regions of the vector are inverse complements of each other (e.g., SEQ ID NO: 1 and SEQ ID NO: 2).
  • asymmetric when referring to the 5’ and 3’ stem-loop regions of the ceDNA vectors of the present disclosure, refers to a pair of stem-loop regions that comprise only artificial sequences, and do not comprise viral ITR sequences, that are not inverse complements of each other across their full length. See e.g., ceDNA 558 whereby the 5’ and 3’ stem-loop regions of the vector are not inverse complements of each other (e.g., SEQ ID NO: 1 and SEQ ID NO: 4).
  • Asymmetric stem-loop regions may be of the same length or different lengths.
  • the terms “synthetic vector”, “synthetic ceDNA vector” and “synthetic production of ceDNA vector” are meant to refer to a ceDNA vector, such as the ceDNA vectors of the present disclosure, and synthetic production methods thereof in an entirely cell-free environment.
  • the term “terminal repeat” or “TR” includes any viral terminal repeat or synthetic sequence that comprises at least one minimal required origin of replication and a region comprising a palindrome hairpin structure.
  • a Rep-binding sequence (“RBS”) (also referred to as RBE (Rep-binding element)) and a terminal resolution site (“TRS”) together constitute a “minimal required origin of replication” and thus the TR comprises at least one RBS and at least one TRS.
  • TRs that are the inverse complement of one another within a given stretch of polynucleotide sequence are typically each referred to as an “inverted terminal repeat” or “ITR”.
  • an ITR may generally include ITRs that are viral (e.g., wild-type or modified AAV ITRs) and ITRs that comprise no viral 29 ME148589913v.1 ITR sequences.
  • the ceDNA vectors of the instant disclosure comprise no viral ITR sequences and are preferably synthetic.
  • an ITR of the disclosure may be a minimal ITR comprising only a number of nucleotides needed to close an end of a ceDNA vector.
  • an ITR may comprise a stem-loop structure comprising a stem of as few as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 base pairs, and a loop comprising as few as 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.
  • an ITR may comprise at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or more total nucleotides.
  • the stem region of an ITR may comprise at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 or more base pairs.
  • the ITR may comprise more than one stem-loop region (e.g., a “doggy-bone”, “dumbbell”, “hammerhead”, or “cruciform” structure).
  • a loop region of an ITR may comprise at least 5, 10, 15, 20, 25 or more nucleotides.
  • an ITR may comprise an aptamer.
  • terminal resolution site refers to a region at which Rep forms a tyrosine-phosphodiester bond with the 5’ thymidine generating a 3’ OH that serves as a substrate for DNA extension via a cellular DNA polymerase, e.g., DNA pol delta or DNA pol epsilon.
  • a TRS minimally encompasses a non-base-paired thymidine.
  • TRS sequences are known in the art, and include, for example, 5’-GGTTGA-3’, the hexanucleotide sequence identified in AAV2.
  • TRS sequences include, for example, AGTT, GGTTGG, AGTTGG, AGTTGA, and other motifs such as RRTTRR.
  • the ceDNA vectors of the instant disclosure are devoid of any viral ITR sequences, and because TRS sequences are located within viral ITRs (e.g., AAV ITRs), the ceDNA vectors of the instant disclosure should not comprise any TRS sequences.
  • reference ceDNA vectors e.g., reference ceDNA vectors used as controls for comparison purposes
  • transcriptional regulator refers to transcriptional activators and repressors that either activate or repress transcription of a gene of interest.
  • Promoters are regions of nucleic acid that initiate transcription of a particular gene.
  • Transcriptional activators typically bind nearby to transcriptional promoters and recruit RNA polymerase to directly initiate transcription.
  • Repressors bind to transcriptional promoters and sterically hinder transcriptional initiation by RNA polymerase.
  • Other transcriptional regulators may serve as either an activator or a repressor depending on where they bind and cellular and environmental conditions.
  • transcriptional regulator classes include, but are not limited to, homeodomain proteins, zinc-finger proteins, winged-helix (forkhead) proteins, and leucine-zipper proteins.
  • the terms “treat,” “treating,” and/or “treatment” include abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical symptoms of a condition, or substantially preventing the appearance of clinical symptoms of a condition, obtaining beneficial or desired clinical results.
  • Treating further refers to accomplishing one 30 ME148589913v.1 or more of the following: (a) reducing the severity of the disorder; (b) limiting development of symptoms characteristic of the disorder(s) being treated; (c) limiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting recurrence of symptoms in patients that were previously asymptomatic for the disorder(s).
  • Beneficial or desired clinical results include, but are not limited to, preventing the disease, disorder or condition from occurring in a subject that may be predisposed to the disease, disorder or condition but does not yet experience or exhibit symptoms of the disease (prophylactic treatment), alleviation of symptoms of the disease, disorder or condition, diminishment of extent of the disease, disorder or condition, stabilization (i.e., not worsening) of the disease, disorder or condition, preventing spread of the disease, disorder or condition, delaying or slowing of the disease, disorder or condition progression, amelioration or palliation of the disease, disorder or condition, and combinations thereof, as well as prolonging survival as compared to expected survival if not receiving treatment.
  • proliferative treatment preventing the disease, disorder or condition from occurring in a subject that may be predisposed to the disease, disorder or condition but does not yet experience or exhibit symptoms of the disease (prophylactic treatment), alleviation of symptoms of the disease, disorder or condition, diminishment of extent of the disease, disorder or condition, stabilization (i.e., not worsening) of
  • the terms “therapeutic amount”, “therapeutically effective amount”, an “amount effective”, or “pharmaceutically effective amount” of an active agent are used interchangeably to refer to an amount that is sufficient to provide the intended benefit of treatment.
  • dosage levels are based on a variety of factors, including the type of injury, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular active agent employed. Thus, the dosage regimen may vary widely, but can be determined routinely by a physician using standard methods.
  • the terms “therapeutic amount”, “therapeutically effective amounts” and “pharmaceutically effective amounts” include prophylactic or preventative amounts of the compositions of the described disclosure.
  • compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of, a disease, disorder or condition in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the onset of the disease, disorder or condition, including biochemical, histologic and/or behavioral symptoms of the disease, disorder or condition, its complications, and intermediate pathological phenotypes presenting during development of the disease, disorder or condition. It is generally preferred that a maximum dose be used, that is, the highest safe dose according to some medical judgment.
  • dose and “dosage” are used interchangeably herein.
  • therapeutic effect refers to a consequence of treatment, the results of which are judged to be desirable and beneficial.
  • a therapeutic effect can include, directly or indirectly, the arrest, reduction, or elimination of a disease manifestation.
  • a therapeutic effect can also include, directly or indirectly, the arrest reduction or elimination of the progression of a disease manifestation.
  • 31 ME148589913v.1 For any therapeutic agent described herein therapeutically effective amount may be initially determined from preliminary in vitro studies and/or animal models.
  • a therapeutically effective dose may also be determined from human data. The applied dose may be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other well-known methods is within the capabilities of the ordinarily skilled artisan.
  • Pharmacokinetic principles provide a basis for modifying a dosage regimen to obtain a desired degree of therapeutic efficacy with a minimum of unacceptable adverse effects. In situations where the drug's plasma concentration can be measured and related to therapeutic window, additional guidance for dosage modification can be obtained.
  • vector or “expression vector” are meant to refer to a replicon, such as plasmid, bacmid, phage, virus, virion, or cosmid, to which another DNA segment, i.e., an “insert” “transgene” or “transgene expression cassette”, may be attached so as to bring about the expression or replication of the attached segment (“transgene expression cassette”) in a cell.
  • a vector can be a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells.
  • a vector can be viral or non-viral in origin in the final form.
  • a “vector” generally refers to synthetic AAV vector or a nicked ceDNA vector. Accordingly, the term “vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells.
  • a vector can be a recombinant vector or an expression vector.
  • the phrase “recombinant vector” refers to a vector that includes a heterologous nucleic acid sequence, or “transgene” that is capable of expression in vivo. It is to be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal.
  • a suitable episomal vector provides a means of maintaining the nucleotide of interest in the subject in high copy number extra chromosomal DNA thereby eliminating potential effects of chromosomal integration.
  • the term “homologous recombination” means a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. Homologous recombination also produces new combinations of DNA sequences. These new combinations of DNA represent genetic variation. Homologous recombination is also used in horizontal gene transfer to exchange genetic material between different strains and species of viruses.
  • the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.
  • the term “consisting essentially of” refers to those 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.
  • the term “consisting of” refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • typical AAV ITR structures comprise a palindromic double-stranded T-shaped hairpin structure, in which the double-stranded A-A’ region forms the stem, and the double- stranded B-B’ and C-C’ regions form the cross-arms of the T-shaped structure (see, for example, Ling et al., J. Virology, 89(2):952-961, 2015).
  • the other nucleotides of the typical AAV ITR remain single stranded and are referred to as the single-stranded D(-) sequence (on the 3’ end of the ITR) and the single-stranded D(+) sequence (on the 5’ end of the ITR).
  • the single-stranded regions of the D(+) region and D(-) region undergo second-strand DNA synthesis to turn them into double- stranded D and D’ regions.
  • ITR sequence was required for the replication and packaging of both AAV and ceDNA vectors used in gene therapy applications. More specifically, it was believed that a ceDNA vector minimally required at least one Rep-binding element (RBE) and at least one terminal resolution sequence (trs).
  • RBE Rep-binding element
  • trs terminal resolution sequence
  • the ITR regions D and D contain sequences that act as binding sites for host transcription factors, including human transcription factors.
  • Wang et al. in 1996 J. Virol.70:1668-1677, 1996) reported that the D sequence plays a crucial role in high-efficiency rescue, selective replication, and encapsidation of the AAV genome and that a host cell protein, designated the D sequence-binding protein (D-BP), specifically interacts with this sequence.
  • D-BP D sequence-binding protein
  • ceDNA vectors comprising synthetic stem-loop structures comprising no viral ITR sequences showed comparable or even, in some embodiments, enhanced gene expression profiles for their respective transgenes.
  • the ceDNA vectors of the instant disclosure have advantages over ceDNA vectors comprising viral ITRs. Removing such sequences reduced the size of a ceDNA molecule, which may be advantageous for a variety of reasons. For example, it may allow for the inclusion of other desirable sequences or motifs (e.g., functional sequences such as aptamers) without increasing 34 ME148589913v.1 the size of the ceDNA molecule. It may also reduce the cost of manufacturing by reducing the amount of raw materials needed for ceDNA production.
  • the ceDNA vectors of the present disclosure also do not contain any of the following ITR sequences: a Rep binding element (RBE), RBE’, a terminal resolution site (trs), A, A’, B, B’, C, C’, D and/or D’ regions of a naturally occurring ITR sequence from, e.g., AAV, or any ITR sequences derived from a naturally occurring ITR sequence (e.g., an ITR sequence modified by an addition, deletion, or substitution in a naturally occurring ITR sequence).
  • a Rep binding element RBE
  • RBE terminal resolution site
  • the ceDNA vectors of the present disclosure do not contain any of the following ITR sequences: a Rep binding element (RBE), RBE’, a terminal resolution site (trs), an A- A’ region, B-B’ region, C-C’ region, or D-D’ region of a naturally occurring ITR sequence from, e.g., AAV, or any ITR sequences derived from a naturally occurring ITR sequence (e.g., an ITR sequence modified by an addition, deletion, or substitution in a naturally occurring ITR sequence).
  • the ceDNA vectors of the present disclosure do not comprise a B and B’ ITR region that would be present in a wild-type AAV ITR.
  • the ceDNA vectors of the present disclosure do not comprise a C and C’ ITR region that would be present in a wild-type AAV ITR. In one embodiment, the ceDNA vectors of the present disclosure do not comprise a B and B’ and C and C’ ITR regions that would be present in a wild-type AAV ITR. In one embodiment, the ceDNA vectors of the present disclosure do not comprise any of an A and A’, B and B’, C and C’ and D and D’ ITR region that would be present in a wild-type AAV ITR.
  • the disclosure provides non-viral, capsid-free ceDNA vectors with covalently-closed ends (ceDNA), wherein the ceDNA vector comprises at least one transgene expression cassette flanked by at least one stem-loop structure at the 3’ end, wherein the at least one stem-loop structure at the 3’ end does not comprise any viral ITR sequences.
  • the ceDNA vector may further comprise at least one stem-loop structure at the 5’ end, wherein the at least one stem-loop structure at the 5’ end does not comprise any viral ITR sequences.
  • the ceDNA vector may further comprise a lipid.
  • the at least one stem-loop structure at the 3’ end of the ceDNA vector does not comprise a B and B’ ITR region that would be present in a wild-type AAV ITR. In one embodiment, the at least one stem-loop structure at the 3’ end of the ceDNA vector does not comprise a C and C’ ITR region that would be present in a wild-type AAV ITR. In one embodiment, the at least one stem-loop structure at the 3’ end of the ceDNA vector does not comprise a B and B’ and C and C’ ITR regions that would be present in a wild-type AAV ITR.
  • the at least one stem-loop structure at the 3’ end of the ceDNA vector does not comprise any of an A and A’, B and B’, C and C’ and D and D’ ITR region that would be present in a wild-type AAV ITR.
  • the at least one stem-loop structure at the 3’ end does not comprise a rep binding element (RBE) that would be present in a wild-type ITR.
  • the at least one stem-loop structure at the 3’ end does not comprise a terminal resolution site (trs) that would be present in a wild-type ITR.
  • the at least one stem loop structure at the 3’ end is devoid of any viral capsid protein coding sequences.
  • the stem portion of the stem-loop is 4-500 nucleotides in length and the loop portion of the stem-loop is 3-500 nucleotides in length.
  • the stem portion of the stem-loop is 4-50 nucleotides in length and the loop portion of the stem-loop is 3-50 nucleotides in length.
  • the stem portion of the stem-loop is 4-20 nucleotides in length and the loop portion of the stem-loop is 3-20 nucleotides in length.
  • the stem portion of the stem-loop is 4-10 nucleotides in length and the loop portion of the stem-loop is 3-10 nucleotides in length.
  • the loop further comprises one or more nucleic acids or that are used to stabilize the ends.
  • the loop further comprises one or more nucleic acids that may be employed in therapeutic methods.
  • the loop further comprises one or more nucleic acids that may be employed in diagnostic methods.
  • the loop further comprises one or more nucleic acids that that may be employed for research purposes.
  • the minimal nucleic acid structure that is necessary at the 3’ end of the ceDNA is any structure that loops back on itself, i.e., a hairpin structure.
  • the ceDNA described herein may comprise at least one stem-loop structure at the 3’ end.
  • the ceDNA may comprise at least two stem-loop structures at the 3’ end.
  • the ceDNA may comprise at least three stem-loop structures at the 3’ end.
  • the ceDNA may comprise at least four stem-loop structures at the 3’ end.
  • the ceDNA may comprise at least five stem-loop structures at the 3’ end.
  • the nucleotides at the 3’ end form a cruciform DNA structure.
  • a DNA cruciform structure can be formed when both strands form a stem-loop structure at the same location in the molecule, and comprises a four-way junction and two closed hairpin-shaped points.
  • the nucleotides at the 3’ end form a hairpin DNA structure.
  • Hairpin loop structures in nucleic acids consist of a base-paired stem structure and a loop sequence with unpaired or non-Watson-Crick-paired nucleotides. 36 ME148589913v.1
  • the nucleotides at the 3’ end form a hammerhead DNA structure, made up of three base paired helices, separated by short linkers of conserved sequence.
  • the nucleotides at the 3’ end form a quadraplex DNA structure.
  • G- quadruplexes are four-stranded DNA secondary structures (G4s) that form from certain guanine-rich sequences.
  • the nucleotides at the 3’ end form a bulged DNA structure.
  • the nucleotides at the 3’ end form a multibranched loop.
  • the nucleotides at the 3’ end do not form a 2 stem-loop structure.
  • B. 5’ end In one embodiment, the at least one stem-loop structure at the 5’ end of the ceDNA vector does not comprise a B and B’ ITR region that would be present in a wild-type AAV ITR.
  • the at least one stem-loop structure at the 5’ end of the ceDNA vector does not comprise a C and C’ ITR region that would be present in a wild-type AAV ITR. In one embodiment, the at least one stem-loop structure at the 5’ end of the ceDNA vector does not comprise a B and B’ and C and C’ ITR regions that would be present in a wild-type AAV ITR. In one embodiment, the at least one stem-loop structure at the 5’ end of the ceDNA vector does not comprise any of an A and A’, B and B’, C and C’ and D and D’ ITR region that would be present in a wild-type AAV ITR.
  • the at least one stem-loop structure at the 5’ end does not comprise a rep binding element (RBE) that would be present in a wild-type ITR. In one embodiment, the at least one stem-loop structure at the 5’ end does not comprise a terminal resolution site (trs) that would be present in a wild-type ITR. In some embodiments, the at least one stem loop structure at the 5’ end is devoid of any viral capsid protein coding sequences. In one embodiment, the stem portion of the stem-loop is 4-500 nucleotides in length and the loop portion of the stem-loop is 3-500 nucleotides in length.
  • the stem portion of the stem-loop is 4-50 nucleotides in length and the loop portion of the stem-loop is 3-50 nucleotides in length. In one embodiment, the stem portion of the stem-loop is 4-20 nucleotides in length and the loop portion of the stem-loop is 3-20 nucleotides in length. In one embodiment, the stem portion of the stem-loop is 4-10 nucleotides in length and the loop portion of the stem-loop is 3-10 nucleotides in length. In one embodiment, the loop further comprises one or more nucleic acids or that are used to stabilize the ends. According to other embodiments, the loop further comprises one or more nucleic acids that may be employed in therapeutic methods.
  • the loop further comprises one or more nucleic acids that may be employed in diagnostic methods.
  • the loop further comprises one or more nucleic acids that that may be employed for research purposes.
  • the minimal nucleic acid structure that is necessary at the 5’ end of the ceDNA is any structure that loops back on itself, i.e., a hairpin structure.
  • a hairpin structure i.e., a hairpin structure.
  • the ceDNA described herein may comprise at least one stem-loop structure at the 5’ end.
  • the ceDNA may comprise at least two stem-loop structures at the 5’ end. In some embodiments, the ceDNA may comprise at least three stem-loop structures at the 5’ end. In some embodiments, the ceDNA may comprise at least four stem-loop structures at the 5’ end. In some embodiments, the ceDNA may comprise at least five stem-loop structures at the 5’ end. In one embodiment, the nucleotides at the 5’ end form a cruciform DNA structure. A DNA cruciform structure can be formed when both strands form a stem-loop structure at the same location in the molecule, and comprises a four-way junction and two closed hairpin-shaped points. In one embodiment, the nucleotides at the 5’ end form a hairpin DNA structure.
  • Hairpin loop structures in nucleic acids consist of a base-paired stem structure and a loop sequence with unpaired or non-Watson-Crick-paired nucleotides.
  • the nucleotides at the 5’ end form a hammerhead DNA structure, made up of three base paired helices, separated by short linkers of conserved sequence.
  • the nucleotides at the 5’ end form a quadraplex DNA structure.
  • G- quadruplexes are four-stranded DNA secondary structures (G4s) that form from certain guanine-rich sequences.
  • the nucleotides at the 5’ end form a bulged DNA structure.
  • the nucleotides at the 5’ end form a multibranched loop. In one embodiment, the nucleotides at the 5’ end do not form a 2 stem-loop structure. In one embodiment, the loop structure at either the 3’ or 5’ end further comprises one or more nucleic acids or that are used to stabilize the ends. According to other embodiments, the loop further comprises one or more nucleic acids that may be employed in therapeutic methods. According to other embodiments, the loop further comprises one or more nucleic acids that may be employed in diagnostic methods. According to other embodiments, the loop further comprises one or more nucleic acids that that may be employed for research purposes.
  • the nucleotides in the loop at either the 3’ or 5’ end are chemically modified with functional groups in order to alter their properties.
  • the loop at either the 3’ or 5’ end further comprises one or more aptamers.
  • the aptamer is identified from the Apta-index database of aptamers available to the public (aptagen.com/apta-index). 38 ME148589913v.1
  • the loop at either the 3’ or 5’ end comprises one or more synthetic ribozymes.
  • the loop at either the 3’ or 5’ end further comprises one or more antisense oligonucleotides (ASOs).
  • ASOs antisense oligonucleotides
  • the loop at either the 3’ or 5’ end further comprises one or more short- interfering RNAs (siRNAs). In one embodiment, the loop at either the 3’ or 5’ end further comprises one or more antiviral nucleoside analogues (ANAs). In one embodiment, the loop at either the 3’ or 5’ end further comprises one or more triplex forming oligonucleotides. In one embodiment, the loop at either the 3’ or 5’ end further comprises one or more gRNAs or gDNAs. In one embodiment, the loop at either the 3’ or 5’ end further comprises one or more molecular probes, for example nucleic acid based fluorescent probes.
  • siRNAs short- interfering RNAs
  • ANAs antiviral nucleoside analogues
  • the loop at either the 3’ or 5’ end further comprises one or more triplex forming oligonucleotides.
  • the loop at either the 3’ or 5’ end further comprises one or more gRNAs or gDNAs.
  • click azide-alkyne cycloaddition (Kolb et al., Angew. Chem. Int. Ed. Engl.2001, 40, 2004–2021) is used to modify the nucleotides in the loop. Click chemistry was developed to join together organic molecules under mild conditions in the presence of a diverse range of functional groups. Most click-mediated modifications are performed on the nitrogenous bases by introducing novel base analogues, attaching fluorophores or isotopic elements for molecular imaging, forming inter-strand linkages between oligonucleotides, and for the bioconjugation of molecules.
  • click chemistry is the Cu I catalyzed version of Huisgen’s [3 + 2] azide–alkyne cycloaddition reaction (Angew. Chem., Int. Ed.1963, 2, 633–645), discovered independently by Sharpless and Meldal (the CuAAC reaction) (Angew. Chem., Int. Ed.2002, 41, 2596–2599).
  • the introduction of active amino or thiol groups into synthesized oligonucleotides provides acceptors for, e.g., subsequent chemical fluorescent labeling.
  • the stem-loop structure may comprise alternative or modified nucleotides, including, but not limited to, ribonucleic acids (RNA), peptide-nucleic acids (PNA), locked nucleic acids (LNA).
  • the loop portion of the stem-loop structure may comprise a chemical structure that does not comprise nucleic acids.
  • the DNA structure at the 5’ end is the same as the DNA structure at the 3’ end. In one embodiment, the DNA structure at the 5’ end is different from the DNA structure at the 3’ end. In some embodiments, the nucleotides at the 3’ end of the ceDNA vector do not form an AAV ITR structure.
  • the nucleotides at the 5’ end of the ceDNA vector do not form an AAV ITR structure. 39 ME148589913v.1
  • the ceDNA vector comprises at least one stem-loop structure at the 3’ end of the ceDNA vector does not comprise a C and C’ ITR region that would be present in a wild- type AAV ITR.
  • the ceDNA vector is ceDNA14.
  • the ceDNA vector comprises at least one stem-loop structure at the 5’ end that does not comprise a B and B’ ITR region that would be present in a wild-type AAV ITR and at least one stem-loop structure at the 3’ end of the ceDNA vector does not comprise a C and C’ ITR region that would be present in a wild-type AAV ITR.
  • the ceDNA vector is ceDNA15.
  • the ceDNA vector comprises at least one stem-loop structure at the 5’ end of that does not comprise a B and B’ and a C and C’ ITR region that would be present in a wild- type AAV ITR and at least one stem-loop structure at the 3’ end of the ceDNA vector that does not comprise a B and B’ and a C and C’ ITR region that would be present in a wild-type AAV ITR.
  • the ceDNA vector is ceDNA16.
  • the ceDNA vector comprises at least one stem-loop structure at the 5’ end that does not comprise any of an A and A’, B and B’, C and C’ and D and D’ ITR region that would be present in a wild-type AAV ITR and at least one stem-loop structure at the 3’ end of the ceDNA vector that does not comprise any of an A and A’, B and B’, C and C’ and D and D’ ITR region that would be present in a wild-type AAV ITR.
  • the ceDNA vector is ceDNA17.
  • the ceDNA vector comprises at least one stem-loop structure at the 5’ end of that does not comprise a B and B’ and a C and C’ ITR region that would be present in a wild- type AAV ITR and at least one stem-loop structure at the 3’ end of the ceDNA vector that comprises wild-type AAV ITR.
  • the ceDNA vector is ceDNA18.
  • the ceDNA vector comprises at least one stem-loop structure at the 5’ end that does not comprise a B and B’ and a C and C’ ITR region that would be present in a wild-type AAV ITR and at least one stem-loop structure at the 3’ end of the ceDNA vector does not comprise a C and C’ ITR region that would be present in a wild-type AAV ITR.
  • the ceDNA vector is ceDNA19.
  • the ceDNA vector comprises at least one stem-loop structure at the 5’ end that does not comprise any of an A and A’, B and B’, C and C’ and D and D’ ITR region that would be present in a wild-type AAV ITR and at least one stem-loop structure at the 3’ end of the ceDNA vector that would be present in a wild-type AAV ITR.
  • the ceDNA vector is ceDNA20.
  • the ceDNA vector comprises at least one stem-loop structure at the 5’ end that would be present in a wild-type AAV ITR and at least one stem-loop structure at the 3’ end 40 ME148589913v.1 of the ceDNA vector that does not comprise any of an A and A’, B and B’, C and C’ and D and D’ ITR region that would be present in a wild-type AAV ITR.
  • the ceDNA vector is ceDNA35.
  • the ceDNA vectors disclosed herein have no packaging constraints imposed by the limiting space within the viral capsid.
  • ceDNA vectors represent a viable eukaryotically-produced alternative to prokaryote-produced plasmid DNA vectors, as opposed to encapsulated AAV genomes. This permits the insertion of control elements, e.g., regulatory switches as disclosed herein, large transgenes, multiple transgenes etc., and incorporation of the native genetic regulatory elements of the transgene, or a functional sequence like an aptamer, if desired.
  • ceDNA vectors preferably have a linear and continuous structure rather than a non-continuous structure, as determined by restriction enzyme digestion assay and electrophoretic analysis. The linear and continuous structure is believed to be more stable from attack by cellular endonucleases, as well as less likely to be recombined and cause mutagenesis.
  • the continuous, linear, single strand intramolecular duplex ceDNA vector can have covalently bound terminal ends (e.g., stem-loop hairpin structure), without viral ITR sequences or sequences encoding AAV capsid proteins.
  • These ceDNA vectors are structurally distinct from plasmids (including ceDNA plasmids described herein), which are circular duplex nucleic acid molecules of bacterial origin.
  • the complimentary strands of plasmids may be separated following denaturation to produce two nucleic acid molecules, whereas in contrast, ceDNA vectors, while having complimentary strands, are a single DNA molecule and, therefore, even if denatured, remain a single molecule.
  • ceDNA vectors as described herein can be produced without DNA base methylation of prokaryotic type, unlike plasmids. Therefore, the ceDNA vectors and ceDNA-plasmids are different both in terms of structure (in particular, linear versus circular) and also in view of the methods used for producing and purifying these different constructs (see below), and also in view of their DNA methylation which is of prokaryotic type for ceDNA-plasmids and of eukaryotic type for the ceDNA vector.
  • Exemplary viral ITRs that are contained in reference ceDNA vectors (useful for, e.g., comparison purposes) described herein are discussed below in the section entitled “ITRs”, and in Table 1 herein, or in Tables 2, 3, 4, 5, 6, 7, 8, 9 or 10A-10B of PCT/US18/49996 filed September 7, 2018, and incorporated by reference in its entirety herein, where the flanking ITR sequence is asymmetric, symmetric (e.g., inverse complement) thereof or substantially symmetric thereto.
  • the ceDNA vectors described herein do not comprise any viral ITR sequences.
  • a reference ceDNA vector described herein or a ceDNA vector of this disclosure comprises a transgene expression cassette with a transgene which is a therapeutic nucleic 41 ME148589913v.1 acid sequence, can be operatively linked to one or more regulatory sequence(s) that allows or controls expression of the transgene.
  • the polynucleotide comprises a first stem-loop sequence and a second stem-loop sequence, wherein the nucleotide sequence of interest is flanked by the first and second stem-loop sequences, and the first and second stem-loop sequences are asymmetric relative to each other or are symmetric relative to each other.
  • the vector comprises a first stem-loop region and a second stem-loop region, wherein both the first and second stem-loop regions contain only artificial sequences and no viral ITR sequences, wherein the nucleotide sequence of interest is flanked by the first and second stem-loop regions, and the first and second stem-loop regions are asymmetric relative to each other or are symmetric relative to each other.
  • an expression cassette comprises one or more of: a promoter operably linked to a transgene, a posttranscriptional regulatory element, and a polyadenylation and termination signal.
  • the promoter is regulatable (e.g., inducible or repressible).
  • the promoter can be any sequence that facilitates the transcription of the transgene.
  • the promoter is a CAG promoter, or variation thereof.
  • the posttranscriptional regulatory element is a sequence that modulates expression of the transgene, as a non-limiting example, any sequence that creates a tertiary structure that enhances expression of the transgene which is a therapeutic nucleic acid sequence.
  • the posttranscriptional regulatory element comprises a WPRE.
  • the polyadenylation and termination signal comprise a BGHpolyA.
  • any cis regulatory element known in the art, or combination thereof, can be additionally used e.g., SV40 late polyA signal upstream sequence element (USE), or other posttranscriptional processing elements including, but not limited to, the thymidine kinase gene of herpes simplex virus, or hepatitis B virus (HBV).
  • the expression cassette length in the 5’ to 3’ direction is greater than the maximum length known to be encapsidated in an AAV virion. In one embodiment, the length is greater than 4.6 kb, or greater than 5 kb, or greater than 6 kb, or greater than 7 kb.
  • Various expression cassettes are exemplified herein.
  • the expression cassette can 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.
  • the expression cassette can comprise a transgene (e.g., a therapeutic nucleic acid sequence) in the range of 500 to 50,000 nucleotides in length.
  • the expression cassette can comprise a transgene (e.g., a therapeutic nucleic acid sequence) in the range of 500 to 75,000 nucleotides in length.
  • the expression cassette can comprise a transgene (e.g., a therapeutic nucleic acid sequence) in the range of 500 to 10,000 nucleotides in length.
  • the expression cassette can comprise a transgene 42 ME148589913v.1 (e.g., a therapeutic nucleic acid sequence) in the range of 1000 to 10,000 nucleotides in length.
  • the expression cassette can comprise a transgene (e.g., a therapeutic nucleic acid sequence) in the range of 500 to 5,000 nucleotides in length.
  • the ceDNA vectors do not have the size limitations of encapsidated AAV vectors, thus enable delivery of a large-size expression cassette to provide efficient expression of transgenes.
  • the ceDNA vector is devoid of prokaryote-specific methylation.
  • the expression cassette can also comprise an internal ribosome entry site (IRES) and/or a 2A element.
  • the cis-regulatory elements include, but are not limited to, a promoter, a riboswitch, an insulator, a mir-regulatable element, a post-transcriptional regulatory element, a tissue- and cell type-specific promoter and an enhancer.
  • the ITR can act as the promoter for the transgene.
  • the ceDNA vector comprises additional components to regulate expression of the transgene, for example, a regulatory switch, described herein in the section entitled “Regulatory Switches,” for controlling and regulating the expression of the transgene, and can include if desired, a regulatory switch which is a kill switch to enable controlled cell death of a cell comprising a ceDNA vector.
  • a regulatory switch which is a kill switch to enable controlled cell death of a cell comprising a ceDNA vector.
  • the expression cassette can comprise 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 (e.g., for medical, diagnostic, or veterinary uses) or immunogenic (e.g., for vaccines) polypeptides.
  • the transgenes 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.
  • Illustrative therapeutic nucleic acids of the present disclosure can include, but are not limited to, minigenes, plasmids, minicircles, small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASO), ribozymes, closed ended double stranded DNA (e.g., ceDNA, CELiD, linear covalently closed DNA (“ministring”), doggyboneTM DNA, protelomere closed ended DNA, or dumbbell linear DNA), dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, gRNA, and DNA viral vectors, viral RNA vector, and any combination thereof.
  • minigenes plasmids, minicircles, small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASO), rib
  • 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 a mimetic or antibody, or antibody fragment, or antigen-binding fragment thereof, e.g., a neutralizing antibody or antibody fragment and the like. In some embodiments, the transgene encodes an antibody, including a full-length antibody or antibody fragment, as defined herein. In some embodiments, the antibody is an antigen-binding domain or an immunoglobulin variable domain sequence, as that is 43 ME148589913v.1 defined herein.
  • the transgene can encode one or more therapeutic agent(s), including, but not limited to, for example, protein(s), polypeptide(s), peptide(s), enzyme(s), antibodies, antigen binding fragments, as well as variants, and/or active fragments thereof, for use in the treatment, prophylaxis, and/or amelioration of one or more symptoms of a disease, dysfunction, injury, and/or disorder.
  • therapeutic agent(s) including, but not limited to, for example, protein(s), polypeptide(s), peptide(s), enzyme(s), antibodies, antigen binding fragments, as well as variants, and/or active fragments thereof.
  • Exemplary transgenes are described herein in the section entitled “Method of Treatment”.
  • ITRs Inverted Terminal Repeats
  • the ceDNA vectors described herein contain no viral ITR sequences (e.g., trs, RBE, A and A’, B and B’, C and C’ and/or D and D’ regions of an AAV ITR). Only the reference ceDNA vectors (e.g., ceDNA vectors that may be used for comparison purposes) that are described herein contain viral ITR sequences.
  • the reference ceDNA vectors are capsid-free, linear duplex DNA molecules formed from a continuous strand of complementary DNA with covalently-closed ends (linear, continuous and non-encapsidated structure), which comprise a synthetic 5’ inverted terminal repeat (ITR) sequence and a synthetic 3’ ITR sequence that are symmetric or asymmetric with respect to each other.
  • both of the ITR sequences of the ceDNA are hairpin (stem-loop) secondary structures.
  • the wild-type AAV ITR sequences are from e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV 5, AAV7, AAV8, AAV9, AAV10, AAV 11, AAV12, AAVrh8, AAVrh10, AAV-DJ, and AAV- DJ8 genome (NCBI: NC 002077; NC 001401; NC001729; NC001829; NC006152; NC 006260; NC 006261; see also Table 1, below)
  • an ITR of the ceDNA vector of the instant disclosure may be synthetic.
  • a synthetic ITR is based on ITR sequences that form the terminal loop portion of one of the ITR secondary structures (e.g., a hairpin (stem loop) secondary structure).
  • a synthetic ITR includes no AAV-based sequence lacking terminal resolution site (trs) and/or rep protein binding sequence (RBE).
  • trs terminal resolution site
  • RBE rep protein binding sequence
  • a synthetic ITR preserves the ITR structure described above although having no AAV-sourced sequence.
  • the flanking ITRs are substantially symmetric to each other. In one embodiment, the flanking ITRs are the same and symmetric to each other.
  • any synthetic ITR can be used as an ITR (e.g., hairpin stem-loop structure, aptamer forming a secondary structure) or as a base ITR for modification.
  • Table 1 Exemplary wt-ITRs from Different AAV Serotypes for Reference ceDNA Vectors 44 ME148589913v.1 45 ME148589913v.1
  • the ceDNA vector of the disclosure does not have a wt-ITR consisting of the nucleotide sequence selected from any of the sequences in Table 1.
  • a ceDNA vector of the instant disclosure comprises a stem-loop structure having no more than 50% identity, 45% identity, 40% identity, 35% identity, 30% identity, 25% identity, 20% identity, 15% identity, 10% identity, or 5% identity with, for example, a viral ITR sequence as described above, or with any other viral ITR sequences known in the art.
  • a viral ITR sequence as described above, or with any other viral ITR sequences known in the art.
  • the ceDNA vectors do not contain the trs and Rep protein binding element (RBE) and are defective with respect to Rep binding and Rep nicking.
  • the defect is completely lacking in function.
  • the host cells do not express viral capsid proteins and the ceDNA vector is devoid of any viral capsid coding sequences.
  • the ceDNA vector and host cells are devoid of AAV capsid genes and the resultant protein also do not encode or express capsid genes of other viruses.
  • the ceDNA vector is devoid of AAV Rep protein coding sequences.
  • the entire ceDNA vector is completely devoid of any AAV-derived sequence.
  • the structural element of the stem-loop structure is devoid of any structural element that is involved in the functional interaction of an ITR with a large Rep protein (e.g., Rep 78 or Rep 68).
  • the stem-loop structure can be modified structurally, for example, the stem-loop structure may be modified to include a functional sequence. In some embodiments, the stem-loop structure may be modified with an aptamer. In some embodiments, the compositions of the present disclosure comprise a non-viral, capsid-free DNA vector with covalently-closed ends (ceDNA vector), wherein the ceDNA vector comprises at least one transgene expression cassette flanked by a stem-loop structure, wherein the stem-loop structures do not comprise any viral ITR sequences.
  • ceDNA vector non-viral, capsid-free DNA vector with covalently-closed ends
  • each stem-loop structure independently comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1 (5’-CTCATCAGAATCACTTTGTGATTCTGA-3’), SEQ ID NO: 2 (5’- TCAGAATCACAAAGTGATTCTGATGAG-3’), and SEQ ID NO: 4 (5’- CACTCATCAGAATCACTTTGTGATTCTGATG-3’).
  • each stem-loop structure independently comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 46 ME148589913v.1 1 (5’-CTCATCAGAATCACTTTGTGATTCTGA-3’).
  • each IT stem-loop structure R independently consists of SEQ ID NO: 1.
  • each stem-loop structure independently comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2 (5’-TCAGAATCACAAAGTGATTCTGATGAG-3’).
  • each stem-loop structure independently consists of SEQ ID NO: 2.
  • each stem-loop structure independently comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3 (5’- TCAGAATCACAAAGTGATTCTGATGAG-3’).
  • each stem-loop structure independently consists of SEQ ID NO: 3.
  • each stem-loop structure independently comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4 (5’-CACTCATCAGAATCACTTTGTGATTCTGATG-3’). In some embodiments, each stem-loop structure independently consists of SEQ ID NO: 4.
  • each stem-loop structure independently comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 12 (5’- TGAGCCTTGATACCACACTTACCTTTCAAGCTTAATACCTTTAGGAGAGCAATTGCAT- 3’).
  • each stem-loop structure independently consists of SEQ ID NO: 12.
  • each stem-loop structure independently comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13 (5’- ATGCAATTGCTCTCCTAAAGGTATTAAGCTTGAAAGGTAAGTGTGGTATCAAGGCTCA- 3’).
  • each stem-loop structure independently consists of SEQ ID NO: 13.
  • at least one stem-loop structure comprises an aptamer.
  • each stem-loop structure independently comprises an aptamer.
  • the ceDNA vectors of the present disclosure can be produced from expression constructs that further comprise a specific combination of cis-regulatory elements.
  • the cis-regulatory elements include, but are not limited to, a promoter, an enhancer, a riboswitch, an insulator, a mir-regulatable element, a post-transcriptional regulatory element, a transcription factor binding site (or consensus 47 ME148589913v.1 site), including but not limited to a tissue- and cell type-specific promoter and an enhancer.
  • the ceDNA vectors of the present disclosure contain liver-specific regulatory elements.
  • the ceDNA vector comprises additional components to regulate expression of the transgene, for example, regulatory switches as described herein, to regulate the expression of the transgene, or a kill switch, which can kill a cell comprising the ceDNA vector.
  • the ceDNA vectors can be produced from expression constructs that further comprise a specific combination of cis-regulatory elements such as WHP posttranscriptional regulatory element (WPRE) and BGH polyA. Suitable expression cassettes for use in expression constructs are not limited by the packaging constraint imposed by the viral capsid.
  • WPRE WHP posttranscriptional regulatory element
  • BGH polyA BGH polyA
  • Suitable expression cassettes for use in expression constructs are not limited by the packaging constraint imposed by the viral capsid.
  • Promoters Expression cassettes of the present disclosure include a promoter, which can influence overall expression levels as well as cell-specificity. For transgene expression, they can include a highly active virus-derived immediate early promoter.
  • Expression cassettes can contain tissue-specific eukaryotic promoters to limit transgene expression to specific cell types and reduce toxic effects and immune responses resulting from unregulated, ectopic expression.
  • Suitable promoters including those described above, can be derived from viruses and can therefore be referred to as viral promoters, or they can be derived from any organism, including prokaryotic or eukaryotic organisms.
  • Suitable promoters can be used to drive expression by any RNA polymerase (e.g., pol I, pol II, pol III).
  • Exemplary promoters include, but are not limited to, the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, a human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497- 500 (2002)), an enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res.2003 Sep.1; 31(17)), a human H1 promoter (H1), a CAG promoter, a human alpha 1-antitypsin (hAAT) promoter, and the like.
  • LTR mouse mammary tumor virus long terminal repeat
  • Ad MLP adenovirus major late promoter
  • these promoters are altered at their downstream intron containing end to include one or more nuclease cleavage sites.
  • the DNA containing the nuclease cleavage site(s) is foreign to the promoter DNA.
  • a promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial expression and/or temporal expression of same.
  • a promoter may also comprise distal enhancer or repressor elements, which may be located as much as several thousand base pairs from the start site of transcription.
  • a promoter may be derived from sources including viral, bacterial, fungal, plants, insects, and animals.
  • a promoter may regulate the expression of a gene component constitutively, or differentially with respect to the cell, tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal 48 ME148589913v.1 ions, or inducing agents.
  • promoters include the 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 the CMV IE promoter, as well as the promoters listed below.
  • promoters and/or enhancers can be used for expression of any gene of interest, e.g., the gene editing molecules, donor sequence, therapeutic proteins etc.).
  • the vector may comprise a promoter that is operably linked to the nucleic acid sequence encoding a therapeutic protein.
  • the promoter operably linked to the therapeutic protein coding sequence may be a promoter from simian virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as the 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 the CMV immediate early promoter, Epstein Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter.
  • SV40 simian virus 40
  • MMTV mouse mammary tumor virus
  • HSV human immunodeficiency virus
  • HSV human immunodeficiency virus
  • BIV bovine immunodeficiency virus
  • LTR long terminal repeat
  • Moloney virus promoter an avian leukosis virus (ALV) promoter
  • CMV
  • the promoter may 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 metallothionein.
  • the promoter may also be a tissue specific promoter, such as a liver specific promoter, such as human alpha 1-antitypsin (hAAT), natural or synthetic.
  • delivery to the liver can be achieved using endogenous ApoE specific targeting of the composition comprising a ceDNA vector to hepatocytes via the low-density lipoprotein (LDL) receptor present on the surface of the hepatocyte.
  • the promoter used is the native promoter of the gene encoding the therapeutic protein.
  • the promoters and other regulatory sequences for the respective genes encoding the therapeutic proteins are known and have been characterized.
  • the promoter region used may further include one or more additional regulatory sequences (e.g., native), e.g., enhancers.
  • additional regulatory sequences e.g., native
  • suitable promoters for use in accordance with the present disclosure include the CAG promoter of, for example, the hAAT promoter, the human EF1- ⁇ promoter or a fragment of the EF1a promoter, IE2 promoter and the rat EF1- ⁇ promoter.
  • an expression cassette can contain a synthetic regulatory element, such as a CAG promoter.
  • the CAG promoter comprises (i) the cytomegalovirus (CMV) early enhancer element, (ii) the promoter, the first exon and the first intron of chicken beta-actin gene, and (iii) the splice acceptor of the rabbit beta-globin gene.
  • an expression cassette can contain an a hAAT promoter, Alpha-1-antitrypsin (AAT) promoter, a liver specific (LP1) promoter, a human elongation factor-1 alpha (EF1 ⁇ ) promoter, or a fragment thereof, a rat EF1 ⁇ promoter or an IE2 promoter.
  • the expression cassette includes one or more constitutive promoters, for example, a retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), or a cytomegalovirus (CMV) immediate early promoter (optionally with the CMV enhancer).
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus immediate early promoter
  • an inducible promoter, a native promoter for a transgene, a tissue-specific promoter, or various promoters known in the art can be used.
  • a sequence encoding a polyadenylation sequence can be included in the ceDNA vector to stabilize the mRNA expressed from the ceDNA vector, and to aid in nuclear export and translation.
  • the ceDNA vector does not include a polyadenylation sequence.
  • the vector includes at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, least 45, at least 50 or more adenine dinucleotides.
  • the polyadenylation sequence comprises about 43 nucleotides, about 40-50 nucleotides, about 40-55 nucleotides, about 45-50 nucleotides, about 35-50 nucleotides, or any range there between.
  • the expression cassettes can include a poly-adenylation sequence known in the art or a variation thereof, such as a naturally occurring sequence isolated from bovine BGHpA or a virus SV40pA, or a synthetic sequence. Some expression cassettes can also include SV40 late polyA signal upstream enhancer (USE) sequence. In some embodiments, the, USE can be used in combination with SV40pA or heterologous poly-A signal.
  • the expression cassettes can also include a post-transcriptional element to increase the expression of a transgene. In some embodiments, Woodchuck Hepatitis Virus (WHP) posttranscriptional regulatory element (WPRE) is used to increase the expression of a transgene.
  • WP Woodchuck Hepatitis Virus
  • WPRE posttranscriptional regulatory element
  • post-transcriptional processing elements such as the post-transcriptional element from the thymidine kinase gene of herpes simplex virus, or hepatitis B virus (HBV) can be used.
  • Secretory sequences can be linked to the transgenes, e.g., VH-02 and VK-A26 sequences.
  • the host cells do not express viral capsid proteins and the polynucleotide vector template is devoid of any viral capsid coding sequences.
  • the polynucleotide vector template is devoid of AAV capsid genes but also of capsid genes of other viruses).
  • the nucleic acid molecule is also devoid of AAV Rep protein coding sequences.
  • the nucleic acid molecule of the disclosure is devoid of both functional AAV cap and AAV rep genes.
  • Gene Editing Embodiments of the disclosure are based on methods and compositions comprising close ended linear duplexed (ceDNA) vectors as described herein that can express a transgene which is a gene editing molecule in a host cell (e.g., a transgene is a nuclease such as ZFN, TALEN, Cas; one or more guide RNA; CRISPR; a ribonucleoprotein (RNP), or any combination thereof) and result in more efficient genome editing.
  • a transgene is a nuclease such as ZFN, TALEN, Cas; one or more guide RNA; CRISPR; a ribonucleoprotein (RNP), or any combination thereof
  • ceDNA vectors described herein are not limited by size, thereby permitting, for example, expression of all of the components necessary for a gene editing system from a single vector (e.g., a CRISPR/Cas gene editing system (e.g., a Cas9 or modified Cas9 enzyme, a guide RNA and/or a homology directed repair template), or for a TALEN or Zinc Finger system).
  • a CRISPR/Cas gene editing system e.g., a Cas9 or modified Cas9 enzyme, a guide RNA and/or a homology directed repair template
  • TALEN or Zinc Finger system TALEN or Zinc Finger system
  • a ceDNA vector described herein for DNA knock-in method(s), e.g., for the introduction of one or more exogenous donor sequences into a specific target site on a cellular chromosome with high efficiency.
  • a ceDNA vector described herein comprising the expression cassette with a transgene which is a gene editing molecule, or a gene editing nucleic acid sequence, can be operatively linked to one or more regulatory sequence(s) that allows or controls expression of the transgene.
  • the expression cassette can comprise any transgene which is a gene editing molecule, or a gene editing nucleic acid sequence.
  • the gene editing ceDNA vector edit any gene of interest in the subject, which includes but are not limited to, nucleic acids encoding polypeptides, or non-coding nucleic acids (e.g., RNAi, miRs etc.), as well as exogenous genes and nucleotide sequences, including virus sequences in a subjects’ genome, e.g., HIV virus sequences and the like.
  • the gene editing ceDNA vector disclosed herein is used for therapeutic purposes (e.g., for medical, diagnostic, or veterinary uses) or immunogenic polypeptides.
  • the gene editing ceDNA vector can edit any gene of interest in the subject, which includes one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNAs, RNAis, antisense oligonucleotides, antisense polynucleotides, antibodies, antigen binding fragments, or any combination thereof.
  • ceDNA expression cassette can include, for example, an expressible exogenous sequence (e.g., open reading frame) that encodes a protein that is either absent, inactive, or insufficient activity in the recipient subject or a gene that encodes a protein having a desired biological or a therapeutic effect.
  • the exogenous sequence such as a donor sequence can encode a gene product that can function to correct the expression of a defective gene or transcript.
  • the expression cassette can also encode corrective DNA strands, encode polypeptides, sense or antisense oligonucleotides, or RNAs (coding or non-coding; e.g., siRNAs, shRNAs, micro-RNAs, and their antisense counterparts (e.g., antagoMiR)).
  • Expression cassettes can include an exogenous sequence that encodes a reporter protein to be used for experimental or diagnostic purposes, such as ⁇ -lactamase, ⁇ -galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art.
  • the expression cassette can include any gene that encodes a protein, polypeptide or RNA that is either reduced or absent due to a mutation or which conveys a therapeutic benefit when overexpressed is considered to be within the scope of the disclosure.
  • the ceDNA vector may comprise a template or donor nucleotide sequence used as a correcting DNA strand to be inserted after a double-strand break (or nick) provided by a nuclease.
  • the ceDNA vector may include a template nucleotide sequence used as a correcting DNA strand to be inserted after a double-strand break (or 51 ME148589913v.1 nick) provided by a guided RNA nuclease, meganuclease, or zinc finger nuclease.
  • non- inserted bacterial DNA is not present and preferably no bacterial DNA is present in the ceDNA compositions provided herein.
  • the protein can change a codon without a nick.
  • Sequences provided in the expression cassette, expression construct, or donor sequence of a ceDNA vector described herein can be codon optimized for the host cell.
  • the term “codon optimized” or “codon optimization” refers to the process of modifying a nucleic acid sequence for enhanced expression in the cells of the vertebrate of interest, e.g., mouse or human, by replacing at least one, more than one, or a significant number of codons of the native sequence (e.g., a prokaryotic sequence) with codons that are more frequently or most frequently used in the genes of that vertebrate.
  • Many organisms display a bias for use of particular codons to code for insertion of a particular amino acid in a growing peptide chain.
  • Codon preference or codon bias differences in codon usage between organisms, is afforded by degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules.
  • mRNA messenger RNA
  • tRNA transfer RNA
  • the gene editing gene (e.g., donor sequences) or guide RNA targets a therapeutic gene.
  • the guide RNA targets an antibody, or antibody fragment, or antigen-binding fragment thereof, e.g., a neutralizing antibody or antibody fragment and the like.
  • the gene editing gene e.g., donor sequences
  • guide RNA targets one or more therapeutic agent(s), including, but not limited to, for example, protein(s), polypeptide(s), peptide(s), enzyme(s), antibodies, antigen binding fragments, as well as variants, and/or active fragments thereof, for use in the treatment, prophylaxis, and/or amelioration of one or more symptoms of a disease, dysfunction, injury, and/or disorder.
  • therapeutic agent(s) including, but not limited to, for example, protein(s), polypeptide(s), peptide(s), enzyme(s), antibodies, antigen binding fragments, as well as variants, and/or active fragments thereof, for use in the treatment, prophylaxis, and/or amelioration of one or more symptoms of a disease, dysfunction, injury, and/or disorder.
  • Exemplary genes for targeting with the guide RNA are described herein in the section entitled “Method of Treatment”.
  • the components required for gene editing may include a nuclease, a guide RNA (if Cas9 or the like is utilized), a donor sequence and one or more homology arms included within a single ceDNA vector of the present disclosure.
  • a nuclease can be inactivated/diminished after gene editing, reducing or eliminating off-target editing, if any, that would otherwise occur with the persistence of an added nuclease within cells.
  • the methods and compositions described herein also provide for gene editing systems comprising a cellular switch.
  • DNA knock- in systems Such methods can be referred to as “DNA knock- in systems.”
  • the DNA knock-in system allows donor sequences to be inserted at any desired target site with high efficiency, making it feasible for many uses such as creation of transgenic animals expressing exogenous genes, preparing cell culture models of disease, preparing screening assay systems, modifying gene expression of engineered tissue constructs, modifying (e.g., mutating) a genomic locus, and gene editing, for example by adding an exogenous non-coding sequence (such as sequence tags or regulatory elements) into the genome.
  • the cells and animals produced using methods provided herein can find various applications, for example as cellular therapeutics, as disease models, as research tools, and as humanized animals useful for various purposes.
  • a ceDNA vector can comprise an endonuclease (e.g., Cas9) that is transcriptionally regulated by an inducible promoter.
  • the endonuclease is on a separate ceDNA vector, which can be administered to a subject with a ceDNA comprising homology arms and a donor sequence, which can optionally also comprise guide RNA (sgRNAs).
  • sgRNAs guide RNA
  • the endonuclease can be on an all-in-one ceDNA vector as described herein.
  • the ceDNA encodes an endonuclease as described herein under control of a promoter.
  • Non-limiting examples of inducible promoters include chemically-regulated promoters, which regulate transcriptional activity by the presence or absence of, for example, alcohols, tetracycline, steroids, metal, and pathogenesis-related proteins (e.g., salicylic acid, ethylene, and benzothiadiazole), and physically-regulated promoters, which regulate transcriptional activity by, for example, the presence or absence of light and low or high temperatures. Modulation of the inducible promoter allows for the turning off or on of gene-editing activity of a ceDNA vector. Inducible Cas9 promoters are further reviewed, for example in Cao J., et al. Nucleic Acids Research.44(19)2016, and Liu KI, et al.
  • the ceDNA vector described herein further comprises a second endonuclease that temporally targets and inhibits the activity of the first endonuclease (e.g., Cas9). Endonucleases that target and inhibit the activity of other endonucleases can be determined by those skilled in the art.
  • the ceDNA vector described herein further comprises 53 ME148589913v.1 temporal expression of an “anti-CRISPR gene” (e.g., L. monocytogenes ArcIIa).
  • anti-CRISPR gene refers to a gene shown to inhibit the commonly used S.
  • the second endonuclease that targets and inhibits the activity of the first endonuclease activity, or the anti-CRISPR gene is comprised in a second ceDNA vector that is administered after the desired gene-editing is complete.
  • the second endonuclease targets and inhibits a gene of interest, for example, a gene that has been transcriptionally enhanced by a ceDNA vector as described herein.
  • a ceDNA vector or composition thereof, as described herein can include a nucleotide sequence encoding a transcriptional activator that activates a target gene.
  • the transcriptional activator may be engineered.
  • an engineered transcriptional activator may be a CRISPR/Cas9-based system, a zinc finger fusion protein, or a TALE fusion protein.
  • the CRISPR/Cas9-based system as described above, may be used to activate transcription of a target gene with RNA.
  • the CRISPR/Cas9-based system may include a fusion protein, as described above, wherein the second polypeptide domain has transcription activation activity or histone modification activity.
  • the second polypeptide domain may include VP64 or p300.
  • the transcriptional activator may be a zinc finger fusion protein.
  • the zinc finger targeted DNA-binding domains as described above, can be combined with a domain that has transcription activation activity or histone modification activity.
  • the domain may include VP64 or p300.
  • TALE fusion proteins may be used to activate transcription of a target gene.
  • the TALE fusion protein may include a TALE DNA-binding domain and a domain that has transcription activation activity or histone modification activity.
  • the domain may include VP64 or p300.
  • ceDNA vectors as described herein can comprise nucleic acids encoding nuclease-dead DNA endonucleases, nickases, or other DNA endonucleases with modified function (e.g., unique PAM binding sequence) for enhanced production of a desired vector and/or delivery of the vector to a cell.
  • modified function e.g., unique PAM binding sequence
  • ceDNA vectors can also include promoter sequences and other regulatory or effector sequences as desired.
  • expression of a desired nuclease with modified function, and optionally, at least one guide RNA can be from nucleic acid sequences on the same vector and can be under the control of the same or different promoters.
  • At least two different modified endonucleases can be encoded in the same vector, for example, for multiplexed gene expression modulation (see “Multiplexed gene expression modulation” section herein) and under the control of the same or different promoters.
  • multiplexed gene expression modulation see “Multiplexed gene expression modulation” section herein
  • one of skill in the art could combine the desired functionality of at least two different Cas9 endonucleases (e.g., at least 3, at least 54 ME148589913v.1 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more) as desired including, for example, temporally regulated expression of at least two different modified endonucleases by one or more inducible promoters.
  • ceDNA vectors in accordance with the present disclosure are suitable for use in nuclease free HDR systems such as those described in Porro et al., Promoterless gene targeting without nucleases rescues lethality of a Crigler-Najjar syndrome mouse model, EMBO Molecular Medicine, July 27, 2017 (herein incorporated by reference in its entirety).
  • in vivo gene targeting approaches are suitable for ceDNA application based on the insertion of a donor sequence, without the use of nucleases.
  • the donor sequence may be promoterless.
  • nucleic Acid-guided Endonucleases Different types of nucleic acid-guided endonucleases can be used in the compositions and methods of the disclosure to facilitate ceDNA-mediated gene editing. Exemplary, nonlimiting, types of nucleic acid-guided endonucleases suited for the compositions and methods of the disclosure include RNA-guided endonucleases, DNA-guided endonucleases, and single-base editors. In some embodiments, the nuclease can be an RNA-guided endonuclease.
  • RNA-guided endonuclease refers to an endonuclease that forms a complex with an RNA molecule that comprises a region complementary to a selected target DNA sequence, such that the RNA molecule binds to the selected sequence to direct endonuclease activity to the selected target DNA sequence.
  • CRISPR/Cas systems As known in the art, a CRISPR-CAS9 system is a particular set of nucleic-acid guided- nuclease-based systems that includes a combination of protein and ribonucleic acid (“RNA”) that can alter the genetic sequence of an organism.
  • the CRISPR-CAS9 system continues to develop as a powerful tool to modify specific deoxyribonucleic acid (“DNA”) in the genomes of many organisms such as microbes, fungi, plants, and animals. For example, mouse models of human disease can be developed quickly to study individual genes much faster, and easily change multiple genes in cells at once to study their interactions.
  • CRISPR systems such as Type I, Type II, and Type III.
  • Type II CRISPR-CAS system has a well-known mechanism including three components: (1) a crDNA molecule, which is called a “guide sequence” or “targeter-RNA”; (2) a “tracr RNA” or “activator-RNA”; and (3) a protein called Cas9.
  • a number of interactions occur in the system including: (1) the guide sequence binding by specific base pairing to a specific sequence of DNA of interest (“target DNA”), (2) the guide sequence binds by specific base pairing at another sequence to an activator- RNA, and (3) activator-RNA interacts with the Cas protein (e.g., Cas9 protein), which then acts as a 55 ME148589913v.1 nuclease to cut the target DNA at a specific site.
  • the Cas protein e.g., Cas9 protein
  • ceDNA vectors in accordance with the present disclosure can be designed to include nucleotides encoding one or more components of these systems such as the guide sequence, tracr RNA, or Cas (e.g., Cas9).
  • a single promoter drives expression of a guide sequence and tracr RNA, and a separate promoter drives Cas (e.g., Cas9) expression.
  • PAM protospacer adjacent motif
  • the PAM may be adjacent to or within 1, 2, 3, or 4 nucleotides of the 3’ end of the target sequence.
  • the length and the sequence of the PAM can depend on the particular Cas protein.
  • Exemplary PAM sequences include NGG, NGGNG, NG, NAAAAN, NNAAAAAW, NNNNACA, GNNNCNNA, TTN and NNNNGATT (wherein N is defined as any nucleotide and W is defined as either A or T).
  • the PAM sequence can be on the guide RNA, for example, when editing RNA.
  • RNA-guided nucleases including Cas and Cas9 are suitable for use in ceDNA vectors designed to provide one or more components for genome engineering using the CRISPR-Cas9 system See e.g., US publication 2014/0170753 herein incorporated by reference in its entirety.
  • CRISPR-Cas 9 provides a set of tools for Cas9-mediated genome editing via non- homologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies.
  • NHEJ non- homologous end joining
  • HDR homology-directed repair
  • the CRISPR-Cas9 system may include a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs.
  • the ceDNA system includes a nuclease and guide RNAs that are directed to a ceDNA sequence.
  • a nicking CAS such as nCAS9 D10A can be used to increase the efficiency of gene editing.
  • the guide RNAs can direct nCAS nicking of the ceDNA thereby releasing torsional constraints of ceDNA for more efficient gene repair and/or expression. Using a nicking nuclease relieves the torsional constraints while retaining sequence and structural integrity allowing the nicked DNA can persist in the nucleus.
  • the guide RNAs can be directed to the same strand of DNA or the complementary strand.
  • the guide RNAs can be directed to e.g., the ITRS, or sequences proceeding promoters, or homology domains etc. 56 ME148589913v.1
  • the RNA-guided endonuclease is a CRISPR enzyme, such as a Cas protein.
  • Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (also known as Csn1 and Csx12), Cas10, Cas10d, Cas13, Cas13a, Cas13c, CasF, CasH, Csy1, Csy2, Csy3, Cse1, Cse2, Cse3, Cse4, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx11, Csx16, CsaX, Cs
  • the Cas protein is Cas9.
  • the Cas protein is nuclease-dead Cas9 (dCas9) or a Cas9 nickase.
  • the Cas protein is a nicking Cas enzyme (nCas).
  • the Cas9 nickase comprises nCas9 D10A.
  • D10A aspartate-to- alanine substitution
  • pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand).
  • a Cas9 nickase can be used in combination with guide sequence(s), e.g., two guide sequences, which target respectively sense and antisense strands of the DNA target. This combination allows both strands to be nicked and used to induce non-homologous end joining (NHEJ) repair.
  • the ceDNA vectors of the present disclosure are suitable for use in systems and methods based on RNA-programmed Cas9 having gene-targeting and genome editing functionality.
  • ceDNA vectors of the present disclosure are suitable for use with Clustered Regularly Interspaced Short Palindromic Repeats or the CRISPR associated (Cas) systems for gene targeting and gene editing.
  • CRISPR cas9 systems are known in the art and described, e.g., in U.S. Patent Application No.13/842,859 filed on March 2013, and U.S. Patent Nos.8,697,359, 8771,945, 8795,965, 8,865,406, 8,871,445 all of which are herein incorporated by reference in their entirety.
  • ceDNA vectors or compositions thereof can encode for modified DNA endonucleases as described in e.g., Fu et al.
  • the endonuclease described herein can be a megaTAL.
  • MegaTALs are engineered fusion proteins which comprise a transcription activator-like (TAL) effector domain and a 57 ME148589913v.1 meganuclease domain. MegaTALs retain the ease of target specificity engineering of TALs while reducing off-target effects and overall enzyme size and increasing activity.
  • MegaTAL construction and use is described in more detail in, e.g., Boissel et al.2014 Nucleic Acids Research 42(4):2591- 601 and Boissel 2015 Methods Mol Biol 1239:171-196; each of which is incorporated by reference herein in its entirety. Protocols for megaTAL-mediated gene knockout and gene editing are known in the art, see, e.g., Sather et al. Science Translational Medicine 20157(307):ra156 and Boissel et al. 2014 Nucleic Acids Research 42(4):2591-601; each of which is incorporated by reference herein in its entirety. MegaTALs can be used as an alternative endonuclease in any of the methods and compositions described herein. F.
  • a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific targeting of an RNA-guided endonuclease complex to the selected genomic target sequence.
  • a guide RNA binds and e.g., a Cas protein can form a ribonucleoprotein (RNP), for example, a CRISPR/Cas complex.
  • RNP ribonucleoprotein
  • the guide RNA (gRNA) sequence comprises a targeting sequence that directs the gRNA sequence to a desired site in the genome, fused to a crRNA and/or tracrRNA sequence that permit association of the guide sequence with the RNA-guided endonuclease.
  • the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm is at least 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.
  • Optimal alignment can be determined with the use of any suitable algorithm for aligning sequences, such as the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP, and Maq.
  • a guide sequence is 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length.
  • the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches.
  • the guide RNA sequence comprises a palindromic sequence, for example, the self-targeting sequence comprises a palindrome.
  • the targeting sequence of the guide RNA is typically 19-21 base pairs long and directly precedes the hairpin that binds the entire guide RNA (targeting sequence + hairpin) to a Cas such as Cas9.
  • the inverted repeat element can be e.g., 9, 10, 11, 12, or more nucleotides in length.
  • a palindromic inverted repeat element of 9 or 10 nucleotides provides a targeting sequence of desirable length.
  • the Cas9-guide RNA hairpin complex can then recognize and cut any 58 ME148589913v.1 nucleotide sequence (DNA or RNA) e.g., a DNA sequence that matches the 19-21 base pair sequence and is followed by a “PAM” sequence e.g., NGG or NGA, or other PAM.
  • AAM e.g., NGG or NGA, or other PAM.
  • the ability of a guide sequence to direct sequence-specific binding of an RNA-guided endonuclease complex to a target sequence can be assessed by any suitable assay.
  • the components of an RNA-guided endonuclease system sufficient to form an RNA-guided endonuclease complex can be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of the RNA-guided endonuclease sequence, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay (TransgenomicTM, New Haven, CT).
  • a host cell having the corresponding target sequence such as by transfection with vectors encoding the components of the RNA-guided endonuclease sequence, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay (TransgenomicTM, New Haven, CT).
  • cleavage of a target polynucleotide sequence can be evaluated in a test tube by providing the target sequence, components of an RNA-guided endonuclease complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions.
  • a guide sequence can be selected to target any target sequence.
  • the target sequence is a sequence within a genome of a cell.
  • the target sequence is the sequence encoding a first guide RNA in a self-cloning plasmid, as described herein.
  • the target sequence in the genome will include a protospacer adjacent (PAM) sequence for binding of the RNA-guided endonuclease.
  • PAM protospacer adjacent
  • the PAM sequence and the RNA-guided endonuclease should be selected from the same (bacterial) species to permit proper association of the endonuclease with the targeting sequence.
  • the PAM sequence for CAS9 is different than the PAM sequence for cpF1. Design is based on the appropriate PAM sequence.
  • the sequence of the guide RNA should not contain the PAM sequence.
  • the length of the targeting sequence in the guide RNA is 12 nucleotides; in other embodiments, the length of the targeting sequence in the guide RNA is 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35 or 40 nucleotides.
  • the guide RNA can be complementary to either strand of the targeted DNA sequence.
  • the gRNA when modifying the genome to include an insertion or deletion, can be targeted closer to the N-terminus of a protein coding region.
  • V. Regulatory Switches A molecular regulatory switch is one which generates a measurable change in state in response to a signal. Such regulatory switches can be usefully combined with the ceDNA vectors described herein to control the output of the ceDNA vector.
  • the ceDNA vector comprises a regulatory switch that serves to fine tune expression of the transgene.
  • the switch can serve as a biocontainment function of the ceDNA vector.
  • the switch is an “ON/OFF” switch that is designed to start or stop (i.e., shut down) expression of the gene of interest 59 ME148589913v.1 in the ceDNA in a controllable and regulatable fashion.
  • the switch can include a “kill switch” that can instruct the cell comprising the ceDNA vector to undergo cell programmed death once the switch is activated.
  • the ceDNA vector comprises a regulatory switch that can serve to controllably modulate expression of the transgene.
  • the expression cassette located between the 5’ and 3’ stem-loop regions of the ceDNA vector may additionally comprise a regulatory region, e.g., a promoter, cis-element, repressor, enhancer etc., that is operatively linked to the gene of interest, where the regulatory region is regulated by one or more cofactors or exogenous agents.
  • a regulatory region e.g., a promoter, cis-element, repressor, enhancer etc.
  • the regulatory region is regulated by one or more cofactors or exogenous agents.
  • cofactor(s) or exogenous agents may be used to de-repress the transcription and expression of the gene of interest.
  • nucleic acid regulatory regions known by a person of ordinary skill in the art can be employed in a ceDNA vector designed to include a regulatory switch.
  • regulatory regions can be modulated by small molecule switches or inducible or repressible promoters.
  • inducible promoters are hormone-inducible or metal-inducible promoters.
  • Other exemplary inducible promoters/enhancer elements include, but are not limited to, an RU486-inducible promoter, an ecdysone-inducible promoter, a rapamycin-inducible promoter, and a metallothionein promoter.
  • the regulatory switch can be selected from any one or a combination of: an orthogonal ligand/nuclear receptor pair, for example retinoid receptor variant/LG335 and GRQCIMFI, along with an artificial promoter controlling expression of the operatively linked transgene, such as that as disclosed in Taylor, et al.
  • an orthogonal ligand/nuclear receptor pair for example retinoid receptor variant/LG335 and GRQCIMFI
  • an artificial promoter controlling expression of the operatively linked transgene such as that as disclosed in Taylor, et al.
  • the regulatory switch to control the transgene or expressed by the ceDNA vector is a pro-drug activation switch, such as that disclosed in US patents 8,771,679, and 6,339,070.
  • Exemplary regulatory switches for use in the ceDNA vectors include, but are not limited to, those in Table 2.
  • C. “Passcode” Regulatory Switches In some embodiments the regulatory switch can be a “passcode switch” or “passcode circuit”. Passcode switches allow fine tuning of the control of the expression of the transgene from the ceDNA vector when specific conditions occur – that is, a combination of conditions need to be present for transgene expression and/or repression to occur. For example, for expression of a transgene to occur at least conditions A and B must occur.
  • a passcode regulatory switch can be any number of conditions, e.g., at least 2, or at least 3, or at least 4, or at least 5, or at least 6 or at least 7 or more conditions to be present for transgene expression to occur. In some embodiments, at least 2 conditions (e.g., A, B conditions) need to occur, and in some embodiments, at least 3 conditions need to occur (e.g., A, B and C, or A, B and D).
  • conditions A, B and C must be present.
  • Conditions A, B and C could be as follows; condition A is the presence of a condition or disease, condition B is a hormonal response, and condition C is a response to the transgene expression.
  • condition A is the presence of a condition or disease
  • condition B is a hormonal response
  • condition C is a response to the transgene expression.
  • the transgene is insulin
  • Condition A occurs if the subject has diabetes
  • Condition B is if the sugar level in the blood is high
  • Condition C is the level of endogenous insulin not being expressed at required amounts.
  • the transgene e.g., insulin
  • Condition A is the presence of 61 ME148589913v.1 Chronic Kidney Disease (CKD)
  • Condition B occurs if the subject has hypoxic conditions in the kidney
  • Condition C is that Erythropoietin-producing cells (EPC) recruitment in the kidney is impaired; or alternatively, HIF-2 activation is impaired.
  • EPC Erythropoietin-producing cells
  • the passcode regulatory switch can be modular in that it comprises multiple switches, e.g., a tissue specific, inducible promoter that is turned on only in the presence of a certain level of a metabolite.
  • the inducible agent must be present (condition A), in the desired cell type (condition B) and the metabolite is at, or above or below a certain threshold (Condition C).
  • the passcode regulatory switch can be designed such that the transgene expression is on when conditions A and B are present, but will turn off when condition C is present.
  • a passcode regulatory switch encompassed for use in the ceDNA vector is disclosed in International Patent Application Publication No. WO2017/059245, which describes a switch referred to as a “Passcode switch” or a “Passcode circuit” or “Passcode kill switch” which is a synthetic biological circuit that uses hybrid transcription factors (TFs) to construct complex environmental requirements for cell survival.
  • WO2017/059245 are particularly useful for use in the ceDNA vectors, as they are modular and customizable, both in terms of the environmental conditions that control circuit activation and in the output modules that control cell fate.
  • the Passcode circuit has particular utility to be used in ceDNA vectors, since without the appropriate “passcode” molecules it will allow transgene expression only in the presence of the required predetermined conditions. If something goes wrong with a cell or no further transgene expression is desired for any reason, then the related kill switch (i.e., deadman switch) can be triggered.
  • a passcode regulatory switch or “Passcode circuit” encompassed for use in the ceDNA vector comprises hybrid transcription factors (TFs) to expand the range and complexity of environmental signals used to define biocontainment conditions.
  • the “passcode circuit” allows cell survival or transgene expression in the presence of a particular “passcode”, and can be easily reprogrammed to allow transgene expression and/or cell survival only when the predetermined environmental condition or passcode is present.
  • a “passcode” system that restricts cell growth to the presence of a predetermined set of at least two selected agents, includes one or more nucleic acid constructs 62 ME148589913v.1 encoding expression modules comprising: i) a toxin expression module that encodes a toxin that is toxic to a host cell, wherein sequence encoding the toxin is operably linked to a promoter P1 that is repressed by the binding of a first hybrid repressor protein hRP1; ii) a first hybrid repressor protein expression module that encodes the first hybrid repressor protein hRP1, wherein expression of hRP1 is controlled by an AND gate formed by two hybrid transcription factors hTF1 and hTF2, the binding or activity of which is responsive to agents A1 and A2, respectively, such that both agents A1 and A2 are required for expression of hRP1, wherein in the absence of either A1 or A2, hRP1 expression is insufficient to repress toxin promoter module
  • hybrid factors hTF1, hTF2 and hRP1 each comprise an environmental sensing module from one transcription factor and a DNA recognition module from a different transcription factor that renders the binding of the respective passcode regulatory switch sensitive to the presence of an environmental agent, A1, or A2, that is different from that which the respective subunits would typically bind in nature.
  • a ceDNA vector can comprise a ‘Passcode regulatory circuit” that requires the presence and/or absence of specific molecules to activate the output module.
  • this passcode regulatory circuit can not only be used to regulate transgene expression, but also can be used to create a kill switch mechanism in which the circuit kills the cell if the cell behaves in an undesired fashion (e.g., it leaves the specific environment defined by the sensor domains, or differentiates into a different cell type).
  • the modularity of the hybrid transcription factors, the circuit architecture, and the output module allows the circuit to be reconfigured to sense other environmental signals, to react to the environmental signals in other ways, and to control other functions in the cell in addition to induced cell death, as is understood in the art.
  • a regulatory switch for use in a passcode system can be selected from any or a combination of the switches in Table 2.
  • Nucleic acid-based regulatory switches to control transgene expression is based on a nucleic-acid based control mechanism.
  • exemplary nucleic acid control mechanisms are known in the art and are envisioned for use.
  • such mechanisms include riboswitches, 63 ME148589913v.1 such as those disclosed in, e.g., US2009/0305253, US2008/0269258, US2017/0204477, WO2018026762A1, US patent 9,222,093 and EP application EP288071, and also disclosed in the review by Villa JK et al., Microbiol Spectr.2018 May;6(3).
  • metabolite-responsive transcription biosensors such as those disclosed in WO2018/075486 and WO2017/147585.
  • Other art- known mechanisms envisioned for use include silencing of the transgene with an siRNA or RNAi molecule (e.g., miR, shRNA).
  • the ceDNA vector can comprise a regulatory switch that encodes an RNAi molecule that is complementary to the transgene expressed by the ceDNA vector. When such RNAi is expressed even if the transgene is expressed by the ceDNA vector, it will be silenced by the complementary RNAi molecule, and when the RNAi is not expressed when the transgene is expressed by the ceDNA vector the transgene is not silenced by the RNAi.
  • RNAi molecule controlling gene expression or as a regulatory switch is disclosed in US2017/0183664.
  • the regulatory switch comprises a repressor that blocks expression of the transgene from the ceDNA vector.
  • the on/off switch is a Small transcription activating RNA (STAR)-based switch, for example, such as the one disclosed in Chappell J. et al., Nat Chem Biol.2015 Mar;11(3):214-20; and Chappell et al., Microbiol Spectr. 2018 May;6(3.
  • STAR Small transcription activating RNA
  • the regulatory switch is a toehold switch, such as that disclosed in US2009/0191546, US2016/0076083, WO2017/087530, US2017/0204477, WO2017/075486 and in Green et al, Cell, 2014; 159(4); 925-939.
  • the regulatory switch is a tissue-specific self-inactivating regulatory switch, for example as disclosed in US2002/0022018, whereby the regulatory switch deliberately switches transgene expression off at a site where transgene expression might otherwise be disadvantageous.
  • the regulatory switch is a recombinase reversible gene expression system, for example as disclosed in US2014/0127162 and US Patent 8,324,436.
  • the regulatory switch to control the transgene or gene of interest expressed by the ceDNA vector is a hybrid of a nucleic acid-based control mechanism and a small molecule regulator system.
  • a nucleic acid-based control mechanism and a small molecule regulator system.
  • Such systems are well known to persons of ordinary skill in the art and are envisioned for use herein.
  • Examples of such regulatory switches include, but are not limited to, an LTRi system or “Lac-Tet-RNAi” system, e.g., as disclosed in US2010/0175141 and in Deans T. et al., Cell., 2007, 130(2); 363-372, WO2008/051854 and US patent 9,388,425.
  • the regulatory switch to control the transgene or gene of interest expressed by the ceDNA vector involves circular permutation, as disclosed in US patent 8,338,138.
  • the molecular switch is multistable, i.e., able to switch between at least two states, or alternatively, bistable, i.e., a state is either “ON” or “OFF,” for example, able to emit light or not, able to bind or not, able to catalyze or not, able to transfer electrons or not, and so forth.
  • the molecular switch uses a fusion molecule, therefore the switch is able to switch between more than two states.
  • the respective other sequence of the fusion may exhibit a 64 ME148589913v.1 range of states (e.g., a range of binding activity, a range of enzyme catalysis, etc.).
  • a nucleic acid based regulatory switch can be selected from any or a combination of the switches in Table 2.
  • the regulatory switch to control the transgene or gene of interest expressed by the ceDNA vector is a post-transcriptional modification system.
  • such a regulatory switch can be an aptazyme riboswitch that is sensitive to tetracycline or theophylline, as disclosed in US2018/0119156, GB201107768, WO2001/064956A3, EP Patent 2707487 and Beilstein et al., ACS Synth. Biol., 2015, 4 (5), pp 526–534; Zhong et al., Elife.2016 Nov 2;5. Pii: e18858.
  • the regulatory switch to control the transgene or gene of interest expressed by the ceDNA vector is a post-translational modification system.
  • the gene of interest or protein is expressed as pro-protein or pre-proprotein, or has a signal response element (SRE) or a destabilizing domain (DD) attached to the expressed protein, thereby preventing correct protein folding and/or activity until post-translation modification has occurred.
  • SRE signal response element
  • DD destabilizing domain
  • DD destabilizing domain
  • SRE destabilizing domain
  • the de-stabilization domain is post- translationally cleaved in the presence of an exogenous agent or small molecule.
  • One of ordinary skill in the art can utilize such control methods as disclosed in US patent 8,173,792 and PCT application WO2017180587.
  • Other post-transcriptional control switches envisioned for use in the ceDNA vector for controlling functional transgene activity are disclosed in Rakhit et al., Chem Biol. 2014;21(9):1238-52 and Navarro et al., ACS Chem Biol.2016; 19; 11(8): 2101–2104A.
  • a regulatory switch to control the transgene or gene of interest expressed by the ceDNA vector is a post-translational modification system that incorporates ligand sensitive inteins into the transgene coding sequence, such that the transgene or expressed protein is inhibited prior to splicing.
  • a post-transcriptional based regulatory switch can be selected from any or a combination of the switches in Table 2.
  • Any known regulatory switch can be used in the ceDNA vector to control the gene expression of the transgene expressed by the ceDNA vector, including those triggered by environmental changes.
  • 65 ME148589913v.1 Additional examples include, but are not limited to; the BOC method of Suzuki et al., Scientific Reports 8; 10051 (2016); genetic code expansion and a non-physiologic amino acid; radiation- controlled or ultra-sound controlled on/off switches (see, e.g., Scott S et al., Gene Ther.2000 Jul;7(13):1121-5; US patents 5,612,318; 5,571,797; 5,770,581; 5,817,636; and WO1999/025385A1.
  • the regulatory switch is controlled by an implantable system, e.g., as disclosed in US patent 7,840,263; US2007/0190028A1 where gene expression is controlled by one or more forms of energy, including electromagnetic energy, that activates promoters operatively linked to the transgene in the ceDNA vector.
  • a regulatory switch envisioned for use in the ceDNA vector is a hypoxia-mediated or stress-activated switch, e.g., such as those disclosed in WO1999060142A2, US patent 5,834,306; 6,218,179; 6,709,858; US2015/0322410; Greco et al., (2004) Targeted Cancer Therapies 9, S368, as well as FROG, TOAD and NRSE elements and conditionally inducible silence elements, including hypoxia response elements (HREs), inflammatory response elements (IREs) and shear-stress activated elements (SSAEs), e.g., as disclosed in U.S. Patent 9,394,526.
  • HREs hypoxia response elements
  • IREs inflammatory response elements
  • SSAEs shear-stress activated elements
  • a regulatory switch envisioned for use in the ceDNA vector is an optogenetic (e.g., light controlled) regulatory switch, e.g., such as one of the switches reviewed in Polesskaya et al., BMC Neurosci.2018; 19(Suppl 1): 12, and are also envisioned for use herein.
  • a ceDNA vector can comprise genetic elements are light sensitive and can regulate transgene expression in response to visible wavelengths (e.g., blue, near IR).
  • ceDNA vectors comprising optogenetic regulatory switches are useful when expressing the transgene in locations of the body that can receive such light sources, e.g., the skin, eye, muscle etc., and can also be used when ceDNA vectors are expressing transgenes in internal organs and tissues, where the light signal can be provided by a suitable means (e.g., implantable device as disclosed herein).
  • Such optogenetic regulatory switches include use of the light responsive elements, or light-inducible transcriptional effector (LITE) (e.g., disclosed in 2014/0287938), a Light-On system (e.g., disclosed in Wang et al., Nat Methods.2012 Feb 12;9(3):266-9; which has reported to enable in vivo control of expression of an insulin transgene, the Cry2/CIB1 system (e.g., disclosed on Kennedy et al., Nature Methods; 7, 973–975 (2010); and the FKF1/GIGANTEA system (e.g., disclosed in Yazawa et al., Nat Biotechnol. 2009 Oct;27(10):941-5).
  • LITE light-inducible transcriptional effector
  • Kill Switches Other embodiments of the disclosure relate to a ceDNA vector comprising a kill switch.
  • a kill switch as disclosed herein enables a cell comprising the ceDNA vector to be killed or undergo programmed cell death as a means to permanently remove an introduced ceDNA vector from the subject’s system. It will be appreciated by one of ordinary skill in the art that use of kill switches in 66 ME148589913v.1 the ceDNA vectors of the disclosure would be typically coupled with targeting of the ceDNA vector to a limited number of cells that the subject can acceptably lose or to a cell type where apoptosis is desirable (e.g., cancer cells).
  • a “kill switch” as disclosed herein is designed to provide rapid and robust cell killing of the cell comprising the ceDNA vector in the absence of an input survival signal or other specified condition.
  • a kill switch encoded by a ceDNA vector herein can restrict cell survival of a cell comprising a ceDNA vector to an environment defined by specific input signals.
  • kill switches serve as a biological biocontainment function should it be desirable to remove the ceDNA vector from a subject or to ensure that it will not express the encoded transgene.
  • kill switches are synthetic biological circuits in the ceDNA vector that couple environmental signals with conditional survival of the cell comprising the ceDNA vector.
  • different ceDNA vectors can be designed to have different kill switches.
  • a ceDNA vector can comprise a kill switch which is a modular biological containment circuit.
  • a kill switch encompassed for use in the ceDNA vector is disclosed in WO2017/059245, which describes a switch referred to as a “Deadman kill switch” that comprises a mutually inhibitory arrangement of at least two repressible sequences, such that an environmental signal represses the activity of a second molecule in the construct (e.g., a small molecule-binding transcription factor is used to produce a ‘survival’ state due to repression of toxin production).
  • a ceDNA vector comprising a deadman kill switch upon loss of the environmental signal, the circuit switches permanently to the ‘death’ state, where the toxin is now derepressed, resulting in toxin production which kills the cell.
  • a synthetic biological circuit referred to as a “Passcode circuit” or “Passcode kill switch” that uses hybrid transcription factors (TFs) to construct complex environmental requirements for cell survival.
  • the Deadman and Passcode kill switches described in WO2017/059245 are particularly useful for use in ceDNA vectors, as they are modular and customizable, both in terms of the environmental conditions that control circuit activation and in the output modules that control cell fate.
  • toxins including, but not limited to an endonuclease, e.g., an EcoRI
  • Passcode circuits present in the ceDNA vector can be used to not only kill the host cell comprising the ceDNA vector, but also to degrade its genome and accompanying plasmids.
  • kill switches known to a person of ordinary skill in the art are encompassed for use in the ceDNA vector as disclosed herein, e.g., as disclosed in US2010/0175141; US2013/0009799; US2011/0172826; US2013/0109568, as well as kill switches disclosed in Jusiak et al, Reviews in Cell Biology and molecular Medicine; 2014; 1-56; Kobayashi et al., PNAS, 2004; 101; 8419-9; Marchisio et al., Int. Journal of Biochem and Cell Biol., 2011; 43; 310-319; and in Reinshagen et al., Science Translational Medicine, 2018, 11.
  • the ceDNA vector can comprise a kill switch nucleic acid construct, which comprises the nucleic acid encoding an effector toxin or reporter protein, where the expression of the effector toxin (e.g., a death protein) or reporter protein is controlled by a predetermined condition.
  • a predetermined condition can be the presence of an environmental agent, such as, e.g., an exogenous agent, without which the cell will default to expression of the effector toxin (e.g., a death protein) and be killed.
  • a predetermined condition is the presence of two or more environmental agents, e.g., the cell will only survive when two or more necessary exogenous agents are supplied, and without either of which, the cell comprising the ceDNA vector is killed.
  • the ceDNA vector is modified to incorporate a kill-switch to destroy the cells comprising the ceDNA vector to effectively terminate the in vivo expression of the transgene being expressed by the ceDNA vector (e.g., therapeutic gene, protein or peptide etc.).
  • the ceDNA vector is further genetically engineered to express a switch-protein that is not functional in mammalian cells under normal physiological conditions.
  • HSV-thymidine kinase Only upon administration of a drug or environmental condition that specifically targets this switch-protein, the cells expressing the switch- protein will be destroyed thereby terminating the expression of the therapeutic protein or peptide. For instance, it was reported that cells expressing HSV-thymidine kinase can be killed upon administration of drugs, such as ganciclovir and cytosine deaminase. See, for example, Dey and Evans, Suicide Gene Therapy by Herpes Simplex Virus-1 Thymidine Kinase (HSV-TK), in Targets in Gene Therapy, edited by You (2011); and Beltinger et al., Proc. Natl. Acad. Sci. USA 96(15):8699- 8704 (1999).
  • drugs such as ganciclovir and cytosine deaminase
  • the ceDNA vector can comprise a siRNA kill switch referred to as DISE (Death Induced by Survival gene Elimination) (Murmann et al., Oncotarget.2017; 8:84643- 84658. Induction of DISE in ovarian cancer cells in vivo).
  • a deadman kill switch is a biological circuit or system rendering a cellular response sensitive to a predetermined condition, such as the lack of an agent in the cell growth environment, e.g., an exogenous agent.
  • Such a circuit or system can comprise a nucleic acid construct comprising expression modules that form a deadman regulatory circuit sensitive to the predetermined condition, the construct comprising expression modules that form a regulatory circuit, the construct including: i) a first repressor protein expression module, wherein the first repressor protein binds a first repressor protein nucleic acid binding element and represses transcription from a coding sequence comprising the first repressor protein binding element, and wherein repression activity of the first repressor protein is sensitive to inhibition by a first exogenous agent, the presence or absence of the first exogenous agent establishing a predetermined condition; ii) a second repressor protein expression module, wherein the second repressor protein binds a second repressor protein nucleic acid binding element and represses transcription from a coding 68 ME148589913v.1 sequence comprising the second repressor protein binding element, wherein the second repressor protein is different from the first
  • the effector is a toxin or a protein that induces a cell death program. Any protein that is toxic to the host cell can be used. In some embodiments the toxin only kills those cells in which it is expressed. In other embodiments, the toxin kills other cells of the same host organism. Any of a large number of products that will lead to cell death can be employed in a deadman kill switch. Agents that inhibit DNA replication, protein translation or other processes or, e.g., that degrade the host cell’s nucleic acid, are of particular usefulness. To identify an efficient mechanism to kill the host cells upon circuit activation, several toxin genes were tested that directly damage the host cell’s DNA or RNA.
  • the endonuclease ecoRI 21 , the DNA gyrase inhibitor ccdB 22 and the ribonuclease-type toxin mazF 23 were tested because they are well-characterized, are native to E. coli, and provide a range of killing mechanisms.
  • the system can be further adapted to express, e.g., a targeted protease or nuclease that further interferes with the repressor that maintains the death gene in the “off” state. Upon loss or withdrawal of the survival signal, death gene repression is even more efficiently removed by, e.g., active degradation of the repressor protein or its message.
  • mf-Lon protease was used to not only degrade LacI but also target essential proteins for degradation.
  • the mf-Lon degradation tag pdt#1 can be attached to the 3’ end of five essential genes whose protein products are particularly sensitive to mf-Lon degradation 20 , and cell viability was measured following removal of Atc.
  • the peptidoglycan biosynthesis gene murC provided the strongest and fastest cell death phenotype (survival ratio ⁇ 1 x 10 -4 within 6 hours).
  • the term “predetermined input” refers to an agent or condition that influences the activity of a transcription factor polypeptide in a known manner.
  • predetermined inputs include, but are not limited to, environmental input agents that are not required for the survival of a given host organism (i.e., in the absence of a synthetic biological circuit as described herein).
  • Conditions that can provide a predetermined input include, for example temperature, e.g., where the activity of one or more factors is temperature-sensitive, the presence or absence of light, including light of a given spectrum of wavelengths, and the concentration of a gas, salt, metal or mineral.
  • Environmental input agents include, for example, a small molecule, biological agents such as pheromones, hormones, growth factors, metabolites, nutrients, and the like and analogs thereof; concentrations of chemicals, environmental byproducts, metal ions, and other such molecules or agents; light levels; temperature; mechanical stress or pressure; or electrical signals, such as currents and voltages.
  • reporters are used to quantify the strength or activity of the signal received by the modules or programmable synthetic biological circuits of the disclosure.
  • reporters can be fused in-frame to other protein coding sequences to identify where a protein is located in a cell or organism.
  • Luciferases can be used as effector proteins for various embodiments described herein, for example, measuring low levels of gene expression, because cells tend to have little to no background luminescence in the absence of a luciferase.
  • enzymes that produce colored substrates can be quantified using spectrophotometers or other instruments that can take absorbance measurements including plate readers.
  • enzymes like ⁇ -galactosidase can be used for measuring low levels of gene expression because they tend to amplify low signals.
  • an effector protein can be an enzyme that can degrade or otherwise destroy a given toxin.
  • an effector protein can be an odorant enzyme that converts a substrate to an odorant product.
  • an effector protein can be an enzyme that phosphorylates or dephosphorylates either small molecules or other proteins, or an enzyme that methylates or demethylates other proteins or DNA.
  • an effector protein can be a receptor, ligand, or lytic protein. Receptors tend to have three domains: an extracellular domain for binding ligands such as proteins, peptides or small molecules, a transmembrane domain, and an intracellular or cytoplasmic domain which frequently can participate in some sort of signal transduction event such as phosphorylation.
  • transporter, channel, or pump gene sequences are used as effector proteins.
  • a “modulator protein” is a protein that modulates the expression from a target nucleic acid sequence.
  • Modulator proteins include, for example, transcription factors, including transcriptional activators and repressors, among others, and proteins that bind to or modify a 70 ME148589913v.1 transcription factor and influence its activity.
  • a modulator protein includes, for example, a protease that degrades a protein factor involved in the regulation of expression from a target nucleic acid sequence.
  • Preferred modulator proteins include modular proteins in which, for example, DNA-binding and input agent-binding or responsive elements or domains are separable and transferrable, such that, for example, the fusion of the DNA binding domain of a first modulator protein to the input agent-responsive domain of a second results in a new protein that binds the DNA sequence recognized by the first protein, yet is sensitive to the input agent to which the second protein normally responds.
  • the term “modulator polypeptide,” and the more specific “repressor polypeptide” include, in addition to the specified polypeptides, e.g., “a LacI (repressor) polypeptide,” variants, or derivatives of such polypeptides that responds to a different or variant input agent.
  • LacI mutants or variants that bind to agents other than lactose or IPTG.
  • agents other than lactose or IPTG.
  • Table 2 Exemplary regulatory switches .
  • d A ligand or other physical stimuli (e.g., temperature, electromagnetic radiation, electricity) which stabilizes the switch either in its ON or OFF state.
  • the resulting vectors have fewer impurities than comparable vectors made using conventional cell-based production methodologies, which may translate into better in vivo expression that is sustained a longer duration of time after administration (see e.g., FIG.16B of International Patent Application No. PCT/US2022/053868, filed December 22, 2022, incorporated herein by reference in its entirety).
  • this cell-free synthesis is also scalable, e.g., from small reactions ( ⁇ 1 mL) and up to a large scale (>40, 100, 200, 500, 1,000 mL) and further without compromising the purity (see e.g., FIG.15 of International Patent Application No.
  • the cell-free method described herein involves rolling circle and multiple strand displacement (MSD) amplification of DNA plasmid template by >1000 fold and subsequent conversion of the resultant products into ceDNA molecules using type II endonuclease, ligase, ITR oligonucleotides (for reference ceDNA vectors) or 5’ and 3’ oligonucleotides containing no viral ITR sequences (for ceDNA vectors of the present disclosure), and exonuclease enzymes.
  • MSD rolling circle and multiple strand displacement
  • the disclosure provides a method of producing a closed-ended DNA (ceDNA) vector, the method comprising (a) contacting a double-stranded DNA construct having a sense strand and an antisense strand with at least a first restriction endonuclease and at least a second restriction endonuclease, wherein the construct comprises a transgene expression cassette, a first non-palindromic restriction endonuclease recognition site and a corresponding first cleavage site upstream of the transgene expression cassette, and a second non-palindromic restriction endonuclease recognition site and a corresponding second cleavage site downstream of the transgene expression cassette; and wherein the first restriction endonuclease is capable of cleaving the double-stranded DNA construct at the first cleavage site, and wherein the second restriction endonuclease is capable of cleaving the double-stranded DNA construct at the second cleavage site, and wherein
  • the first oligonucleotide comprises a stem-loop structure (e.g., a stem- loop structure devoid of viral ITR sequences).
  • the second oligonucleotide comprises a stem-loop structure (e.g., a stem-loop structure devoid of viral ITR sequences).
  • the first oligonucleotide does not comprise a viral inverted terminal repeat (ITR).
  • the second oligonucleotide does not comprise a viral ITR.
  • the first oligonucleotide comprises an artificial sequence.
  • the second oligonucleotide comprises an artificial sequence.
  • Oligonucleotides containing no viral ITR sequences are designed for the synthetic production of ceDNA vectors of the present disclosure that contain no viral ITRs. According to other embodiments, the first oligonucleotide and the second oligonucleotide are different. According to some other embodiments, the first oligonucleotide and the second oligonucleotide are the same. In some embodiments, reference ceDNA vectors comprising viral ITR sequences may be produced synthetically using similar oligonucleotides. 75 ME148589913v.1 An overview of an exemplary embodiment of cell-free synthetic method of preparing a ceDNA vector is illustrated in FIG.4 of International Patent Application No.
  • the transgene expression cassette (in diagonal stripes in FIG.4 of International Patent Application No. PCT/US2022/053868) is excised from a double-stranded DNA construct using with at least one restriction endonuclease, followed up by ligation of the insert with inverted terminal repeat (ITR) oligonucleotides (e.g., 5’ and 3’ oligonucleotides that contain no viral ITR sequences) to form ceDNA.
  • ITR oligonucleotides are single-stranded oligonucleotides that self-anneal to form an ITR-like three-dimensional configuration.
  • the first and second oligonucleotides or the 5’ and 3’ oligonucleotides are single-stranded oligonucleotides that self-anneal to form a stem-loop region in the ceDNA vector that, In one embodiment, comprises a primary stem region and one or more loop structures, e.g., a hairpin loop structure (see FIGS.1A and 1B).
  • restriction endonucleases used in the cell-free synthetic methods disclosed herein also recognize non- palindromic nucleotide sequences such that the recognition sequence (which is also the binding site) for the enzyme is only encoded on one strand (see e.g., FIG.5 of International Patent Application No. PCT/US2022/053868). Therefore, cleavage by this class of restriction endonucleases is directional, occurring either upstream or downstream of the recognition site, but not within the recognition site itself, unlike other restriction endonucleases that are most heavily used in molecular biology such as EcoRI (see FIG.5 of International Patent Application No. PCT/US2022/053868).
  • the strand that encodes the recognition sequence dictates which side (i.e., downstream or upstream) of the sequence is cleaved.
  • side i.e., downstream or upstream
  • the unique activity of the restriction endonucleases used in the methods described herein allows any sequence within a pre-determined distance from a specific recognition site to be cleaved by the restriction endonuclease and consequently, any overhang sequence to be generated. Digestion with the special restriction endonuclease(s) creates cohesive overhangs at both 5’ and 3’ ends of the excised insert that are compatible with the overhangs of the oligonucleotide.
  • the design of the oligonucleotides and insert overhangs drives the high specificity of the ligation process such that the oligonucleotide overhangs and the insert overhangs are compatible with each other.
  • the desired ceDNA product is not susceptible to digestion with the restriction endonuclease because the recognition site is not re-generated.
  • the recognition sites are re-generated and therefore allow the construct to be cleaved.
  • the digestion and ligation can take place in a single reaction vessel without a need to purify the 76 ME148589913v.1 digestion products prior to ligation.
  • the digestion/ligation is ensued by treatment with an exonuclease to degrade open-ended DNA fragments and intermediates, as shown in FIG.4 of International Patent Application No.
  • restriction endonuclease digestion, ligation, and exonuclease degradation take place in a single reaction vessel and all the reactions can occur simultaneously.
  • unique activity of the restriction endonucleases used in the cell-free synthetic methods described herein allow the directionality of ligation reactions that utilize more than one oligonucleotides, thereby enabling preparation of reference ceDNA having asymmetric ITRs or ceDNA having asymmetric stem-loop regions.
  • Cell-free synthetic production method in general Disclosed herein is a process for synthesis of closed-ended DNA vectors which does not require use of any microbiological steps.
  • the process allows for synthesis of closed-ended DNA vectors in a system using enzymatic cleavage steps using restriction endonucleases and ligation steps to generate the closed-ended DNA vectors.
  • the synthetic system for DNA vector production is a cell-free system. It will be appreciated by one of ordinary skill in the art that one or more enzymes used in the synthetic production method or one or more of the oligonucleotide components can be produced from a cell and used in the methods of the disclosure in purified form. Accordingly, in some embodiments, the procedures themselves in synthetic production method are cell-free.
  • the raw materials i.e., the starting materials
  • enzymes such as restriction endonucleases and ligases
  • a restriction endonuclease and/or a ligation-competent protein can be expressed or provided from an expression vector in a cell, e.g., bacterial cell.
  • a cell such as a bacterial cell, comprising an expression vector expressing one or more of the restriction endonucleases or the ligase enzymes can be present.
  • the methods disclosed herein are primarily directed to cell-free synthetic methods to generate the DNA vectors disclosed herein, also encompassed in one embodiment are synthetic production methods where a cell, e.g., bacterial cell but not an insect cell is present and can be used to express one or more of the enzymes required in the method.
  • the cell expressing a restriction endonuclease and/or ligation- competent protein is not an insect cell.
  • the cell does not replicate the close- ended DNA vector. Stated differently, the intracellular machinery of the cell does not replicate, or is not involved in the replication of the DNA vector.
  • synthesis of DNA vectors is carried out in an in vitro cell-free process starting from either a double-stranded DNA construct or one or more oligonucleotides.
  • the double-stranded DNA construct or one or more oligonucleotides are 77 ME148589913v.1 cleaved with restriction endonucleases and ligated to form the DNA molecules.
  • the oligonucleotides can be synthesized chemically, thus avoiding use of large starting templates encoding the entirety of the desired sequence which would typically need to be propagated in bacteria.
  • a desired DNA sequence can be cleaved and ligated with other oligonucleotides as disclosed herein.
  • the use of multiple oligonucleotides in the generation of closed- ended DNA vectors using the methods disclosed herein allows for a modular approach to DNA vector generation, enabling tailoring and/or specific selection of the terminal repeats (e.g., ITRs) or stem- loop regions, as well as the spacing of the terminal repeats or stem-loop regions, and also selection of the heterologous nucleic acid sequence in the synthetically produced closed-ended DNA vectors.
  • the terminal repeats e.g., ITRs
  • stem- loop regions as well as the spacing of the terminal repeats or stem-loop regions
  • a closed-ended DNA vector is generated by excising a transgene expression cassette from a double-stranded DNA construct, followed by ligation of the ends of the insert to a first oligonucleotide comprising one or more hairpin structures and a second oligonucleotide comprising one or more hairpin structures to form the ceDNA.
  • each of oligonucleotides independently includes 1, 2, 3, 4, or more stem-loop regions.
  • each of the oligonucleotides independently includes 2 or 3 stem-loop regions.
  • the first oligonucleotide comprising one or more hairpin structures and the second oligonucleotide comprising one or more hairpin structures are each a single-stranded oligonucleotide that self-anneals to form a three-dimensional configuration.
  • the three-dimensional configuration is a T- or Y-shaped stem-loop structure.
  • a closed-ended DNA vector is generated by excising a transgene expression cassette from a double-stranded DNA construct, followed by ligation of the ends of the insert to stem- loop oligonucleotides to form the ceDNA.
  • the ligation may be effected by a ligase (e.g., T4 ligase).
  • the reaction mixture is not purified prior to ligation.
  • the excision of the transgene expression cassette (e.g., with one or more restriction endonucleases) and ligation take place simultaneously in a single reaction vessel.
  • the reaction mixture is purified prior to ligation.
  • the resultant closed-ended DNA vector as prepared by the cell-free synthetic methods described herein comprises at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the monomeric species of the vector.
  • the resultant closed-ended DNA vector as prepared by the cell-free synthetic methods described herein comprises less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the sub-monomeric species of the vector.
  • the double-stranded DNA construct is selected from a bacmid, a plasmid, a minicircle, and a linear double-stranded DNA molecule.
  • the double- stranded DNA construct is provided with at least, in 5’ to 3’ order: a first non-palindromic restriction endonuclease recognition site and a corresponding first cleavage site upstream of the transgene expression cassette; a transgene expression cassette; and a second non-palindromic restriction endonuclease recognition site and a corresponding second cleavage site downstream of the transgene expression cassette.
  • the first restriction endonuclease is capable of cleaving the double-stranded DNA construct at the first cleavage site
  • the second restriction endonuclease is capable of cleaving the double-stranded DNA construct at the second cleavage site
  • contacting the double-stranded DNA construct with the first restriction endonuclease and the second restriction endonuclease releases an insert having a first end comprising a first single- stranded overhang and a second end comprising a second single-stranded overhang.
  • the first restriction endonuclease and the second restriction endonuclease are different restriction endonucleases.
  • the first restriction endonuclease and the second restriction endonuclease are the same restriction endonuclease.
  • the double- stranded DNA construct for the synthetic production of a reference ceDNA is provided with at least, in 5’ to 3’ order: a first non-palindromic restriction endonuclease recognition site and a corresponding first cleavage site upstream of the transgene expression cassette; a first partial stem-loop structure; a transgene expression cassette; a second partial stem-loop structure; and a second non-palindromic restriction endonuclease recognition site and a corresponding second cleavage site downstream of the transgene expression cassette.
  • the double-stranded DNA construct for the synthetic production of a ceDNA vector of the present disclosure is provided with at least, in 5’ to 3’ order: a first non-palindromic restriction endonuclease recognition site and a corresponding first cleavage site upstream of the transgene expression cassette; a 5’ secondary stem region; a transgene expression cassette; a 3’ secondary stem region; and a second non-palindromic restriction endonuclease recognition site and a corresponding second cleavage site downstream of the transgene expression cassette.
  • the double-stranded DNA construct for the synthetic production of a ceDNA vector of the present disclosure is provided with at least, in 5’ to 3’ order: a first non-palindromic restriction endonuclease recognition site and a corresponding first cleavage site upstream of the transgene expression cassette; a 5’ secondary stem region; 5’ spacer; a transgene expression cassette; a 3’ spacer; a 3’ secondary stem region; and a second non-palindromic restriction endonuclease recognition site and a corresponding second cleavage site downstream of the transgene expression cassette (see e.g., ceDNA 384 in FIG.1A).
  • the double-stranded DNA construct for the synthetic production of a ceDNA vector of the present disclosure is provided with at least, in 5’ to 3’ order: a first non-palindromic restriction endonuclease recognition site and a corresponding first cleavage site upstream of the transgene expression cassette; a 5’ spacer; a transgene expression cassette; a 3’ spacer; and a second non-palindromic restriction endonuclease 79 ME148589913v.1 recognition site and a corresponding second cleavage site downstream of the transgene expression cassette (see e.g., ceDNA 558 in FIG.1B).
  • the double-stranded DNA construct is contacted with at least one restriction endonuclease is capable of cleaving the construct at the first and second cleavage sites to release an insert having single-stranded overhangs at the 5’ and 3’ ends (i.e., cohesive ends) of the insert. These ends of the insert are then ligated to a first inverted terminal repeat oligonucleotide and a second oligonucleotide to form the ceDNA vector.
  • one or both of the single-stranded overhangs at the 5’ and 3’ ends of the inserts is a 5’ overhang.
  • one or both of the single-stranded overhangs at the 5’ and 3’ ends of the inserts is a 3’ overhang.
  • these overhangs are about 1 to about 30 nucleotides in length, e.g., about 1 to about 25 nucleotides, or about 1 to about 20 nucleotides, or about 1 to about 18 nucleotides, or about 1 to about 15 nucleotides, or about 1 to about 12 nucleotides, or about 1 to about 10 nucleotides, or about 1 to about 8 nucleotides, or about 2 to about 8 nucleotides, or about 2 to about 7 nucleotides, or about 2 to about 6 nucleotides, or about 1 nucleotide, or about 2 nucleotides, or about 3 nucleotides, or about 4 nucleotides, or about 5 nucleotides, or about 6 nucleotides, or about 7 nucleotides, or about 8 nucleotides
  • restriction endonucleases that recognize non-palindromic nucleotide sequences and have cleavage sites distinct from their recognition and binding sites
  • the restriction endonuclease(s) used in the synthetic methods provided herein recognizes non-palindromic nucleotide sequences.
  • non-palindromic when referring to a double-stranded polynucleotide or oligonucleotide having different 5’ ⁇ 3’ nucleotide sequences between the sense strand and the anti-sense strand; whereas the term “palindromic” when referring to a double-stranded polynucleotide or oligonucleotide having identical 5’ ⁇ 3’ nucleotide sequences between the sense strand and the anti-sense strand. Accordingly, as illustrated in FIG.5 of International Patent Application No.
  • restriction endonuclease(s) used in the synthetic methods described herein is that the enzyme cleaves the DNA at a cleavage site that is either upstream or downstream of the recognition sequence, but not within the recognition site itself.
  • the strand that encodes the recognition site dictates which side (i.e., downstream or upstream) of the recognition sequence is cleaved.
  • the first non-palindromic restriction endonuclease recognition site and the corresponding first cleavage site are separate and distinct sites from each other that are located upstream of the transgene expression cassette.
  • the first cleavage site is about 1 to 35 nucleotides away from the first non-palindromic restriction endonuclease recognition site in at least one of the sense and the antisense strands of the construct, e.g., about 1 to about 22 nucleotides away, or about 1 to about 20 nucleotides away, or about 1 to about 15 nucleotides away, or about 1 to about 12 nucleotides away, or about 1 to about 10 nucleotides away, or about 1 to about 8 nucleotides away, or about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10 nucleotides away from the first non-palindromic restriction endonuclease recognition site in at least one of the sense and the antisense strands of the construct.
  • the second non-palindromic restriction endonuclease recognition site and the corresponding second cleavage site are separate and distinct sites from each other that are located downstream of the expression cassette.
  • the second cleavage site is about 1 to 35 nucleotides away from the second non-palindromic restriction endonuclease recognition site in at least one of the sense and the antisense strands of the construct, e.g., about 1 to about 22 nucleotides away, or about 1 to about 20 nucleotides away, or about 1 to about 15 nucleotides away, or about 1 to about 12 nucleotides away, or about 1 to about 10 nucleotides away, or about 1 to about 8 nucleotides away, or about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10 nucleotides away from the second non-palindromic restriction endonuclease recognition site in at least one of the sense and the antis
  • a single restriction endonuclease can target both the first and second non-palindromic restriction endonuclease recognition sites and their corresponding cleavage sites.
  • two different restriction endonucleases target both the first and second non-palindromic restriction endonuclease recognition sites and their corresponding cleavage sites.
  • Type IIS restriction endonucleases In one embodiment, the restriction endonuclease(s) used in the synthetic methods provided herein, which recognizes non-palindromic nucleotide sequences and cleaves a DNA outside of the recognition site is a Type IIS restriction endonuclease.
  • Type IIS restriction endonucleases include AcuI, AlwI, Alw26I, BasI, BbsI, BbvI, BceAI, BcgI, BCiVI, BcoDI, BruAI, BmrI, BpiI, BpuEI, BsaI, BsaXI, BseGI, BseRI, BsgI, BsmAI, BsmBI, BsmFI, BsmI, BspCNI, BspMI, BspQI, BsrDI, BsrI, BtgZI, BtsCI, BtsI, MutI, CspCI, EarI, EciI, Eco31I, Esp3I, FauI, FokI, HgaI, HphI, HpyAV, LguI, MboII, MlyI, MmeI, MnlI, Mva1269I, NmeAIII, PaqCI,
  • Isoschizomers are pairs of restriction endonucleases that are specific to the same recognition sequence.
  • BcoDI and BsmAI are isoschizomers of each other, both being specific to the recognition sequence of 5’-GTCTC-3’.
  • the Type IIS endonuclease(s) is selected from BbsI, BsaI, BsmBI, Esp3I, and SapI, and an isoschizomer thereof.
  • the Type IIS endonuclease is BbsI or an isoschizomer thereof.
  • the Type IIS endonuclease is BsaI or an isoschizomer thereof.
  • the Type IIS endonuclease is BsmBI or an isoschizomer thereof. In one embodiment, the Type IIS endonuclease is BbsI or an isoschizomer thereof. In one embodiment, the Type IIS endonuclease is Esp3I or an isoschizomer thereof. In one embodiment, the Type IIS endonuclease is SapI or an isoschizomer thereof.
  • First and second oligonucleotides (or 5’ and 3’ oligonucleotides)
  • the first and second oligonucleotides to which the 5’ and 3’ ends of the insert are ligated to in the cell-free synthetic methods disclosed herein are each a single-stranded oligonucleotide that self- anneals to form a three-dimensional configuration, such has a three-dimensional structure like a hairpin structure or a T- or Y-shaped stem-loop structure.
  • one or both of the first and second oligonucleotides are synthetic or synthesized.
  • the oligonucleotides each self-anneal to further form a single-stranded overhang at either the 5’ or the 3’ end of the oligonucleotide.
  • the single-stranded overhangs of the insert are ligated to the single-stranded overhangs of the oligonucleotides.
  • the stem-loop structureoligonucleotide overhangs are about 1 to about 30 nucleotides in length, e.g., about 1 to about 25 nucleotides, or about 1 to about 20 nucleotides, or about 1 to about 18 nucleotides, or about 1 to about 15 nucleotides, or about 1 to about 12 nucleotides, or about 1 to about 10 nucleotides, or about 1 to about 8 nucleotides, or about 2 to about 8 nucleotides, or about 2 to about 7 nucleotides, or about 2 to about 6 nucleotides, or about 1 nucleotide, or about 2 nucleotides, or about 3 nucleotides, or about 4 nucleotides, or about 5 nucleotides, or about 6 nucleotides, or about 7 nucleotides, or about 8 nucleotides, or about 9 nucleotides, or about 10 nucleotides in length.
  • the oligonucleotide overhang comprises a 5’ ⁇ 3’ nucleotide sequence of CTCT, CTCA, CACT, CTC, or GCT.
  • the overhangs of the first and second oligonucleotides comprise non-complementary 5’ ⁇ 3’ nucleotide sequences to each other.
  • overhangs of the first and second oligonucleotides comprise or have identical sequences, i.e., the same 5’ ⁇ 3’ nucleotide sequence and in a further embodiment, the first and second oligonucleotides are the same oligonucleotide.
  • the overhangs at the 5’ and 3’ ends of the insert comprise or have the same 5’ ⁇ 3’ nucleotide sequence.
  • the overhangs of the oligonucleotides are complementary to either and both of the overhangs of the insert.
  • the term “stem-loop region” when referring to ceDNA vector of the present disclosure refers to a region of a ceDNA vector of the present disclosure, which may be found upstream of the 5’ end of the at least one transgene expression cassette in the ceDNA vector and/or a second stem-loop region downstream of the 3’ end of the at least one transgene expression cassette.
  • the stem-loop region comprises an artificial nucleic acid sequence and does not comprise a viral ITR sequence.
  • each stem-loop region comprises a double-stranded primary stem region and a single-stranded region.
  • each stem-loop region is formed from a 5’ oligonucleotide or a 3’ oligonucleotide, or a first oligonucleotide or a second oligonucleotide, or a left oligonucleotide or a right oligonucleotide, that self-anneals to form the double-stranded primary stem region (where there is complimentarity in the nucleotides) and the single-stranded region (where there is no complimentarity in the nucleotides).
  • each double-stranded primary stem region comprises about 1 to about 50 bp, or about 1 to about 40 bp, or about 1 to about 30 bp, or about 1 to about 25 bp, or about 1 to about 20 bp, or about 1 to about 15 bp, or about 2 to about 20 bp, or about 2 to about 15 bp, or about 3 to about 20 bp, or about 3 to about 15 bp, or about 4 to about 20 bp, or about 4 to about 15 bp, or about 5 to about 20 bp, or about 5 to about 15 bp, or about 1 bp, or about 2 bp, or about 3 bp, or about 4 bp, or about 5 bp, or about 6 bp, or about 7 bp, or about 8 bp, or about 9 bp, or about 10 bp, or about 11 bp, or about 12 bp, or about 13 bp,
  • each single-stranded region comprises about 2 to about 100 nt, about 2 to about 90 nt, about 2 to about 80 nt, about 2 to about 70 nt, about 2 to about 60 nt, about 2 to about 50 nt, about 2 to about 40 nt, about 2 to about 30 nt, about 2 to about 20 nt, about 2 to about 15 nt, about 2 to about 12 nt, about 2 to about 10 nt, about 2 to about 8 nt, about 3 to about 20 nt, about 3 to about 15 nt, about 3 to about 12 nt, about 3 to about 10 nt, about 3 to about 8 nt, or about 2 nt, or about 3 nt, or about 4 nt, or about 5 nt, or about 6 nt, or about 7 nt, or about 8 nt, or about 9 nt, or about 10 nt, or about 11 nt,
  • each single-stranded region comprises at least one loop structure. In some embodiments of a ceDNA vector of the present disclosure, each single-stranded region comprises an aptamer.
  • overhangs of the first and second oligonucleotides comprise or have different sequences, i.e., different 5’ ⁇ 3’ nucleotide sequences and hence, the first and second oligonucleotides are different oligonucleotides. In such an embodiment, the overhangs at the 5’ and 3’ ends of the insert comprise or have different 5’ ⁇ 3’ nucleotide sequences.
  • the overhangs of the oligonucleotides are each complementary to only one of the overhangs of the insert.
  • the first stem-loop region and the second stem-loop region each independently comprise a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence similarity to any one of SEQ ID NO: 1 (5’-CTCATCAGAATCACTTTGTGATTCTGA-3’), SEQ ID NO: 2 (5’-TCAGAATCACAAAGTGATTCTGATGAG-3’), and SEQ ID NO: 4 (5’- CACTCATCAGAATCACTTTGTGATTCTGATG-3’).
  • the first stem-loop region and the second stem-loop region are each independently formed from an oligonucleotide comprising a nucleic acid sequence having at least 80%, at least 85%, at least 90%, 95%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any one of SEQ ID NO: 1 (5’-CTCATCAGAATCACTTTGTGATTCTGA-3’), SEQ ID NO: 2 (5’- TCAGAATCACAAAGTGATTCTGATGAG-3’), and SEQ ID NO: 4 (5’- CACTCATCAGAATCACTTTGTGATTCTGATG-3’).
  • the 5’ ends of the oligonucleotide may be phosphorylated.
  • the first ITR and the second ITR in a reference ceDNA vector each comprise a hairpin structure and/or a T- or Y-shaped stem-loop structure.
  • the first ITR and the second ITR each comprise a T- or Y-shaped stem-loop structure.
  • the first ITR and the second ITR are symmetric or substantially symmetric to each other.
  • the first ITR and the second ITR are asymmetric ITRs.
  • the T- or Y-shaped stem-loop structure (e.g., single stem + two loops or single stem + two loops) of the first ITR oligonucleotide and the second ITR oligonucleotide comprises a stem region that is at least about 4 base pairs (nucleotides) in length, e.g., about 4 base pairs to about 30 base pairs, or 4 base pairs to about 25 base pairs, or 4 base pairs to about 22 base pairs, or 4 base pairs to about 20 base pairs, or 4 base pairs to about 18 base pairs, or 4 base pairs to about 15 base pairs, or 4 base pairs to about 12 base pairs, or 4 base pairs to about 10 base pairs, or 4 base pairs to about 8 base pairs, or 4 base pairs to about 7 base pairs, or 4 base pairs to about 6 base pairs or 6 base pairs to about 8 base pairs, or about 4 base pairs, or about 5 base pairs, or about 6 base pairs, or about 7 base pairs, or about 8 base pairs, or about 9 base pairs, or about 10 base pairs,
  • this stem region length does not include the overhang length.
  • Partial ITRs in reference ceDNA, secondary stem regions in ceDNA of the present disclosure, and spacers in some embodiments, the ceDNA vectors of the present disclosure, such as ceDNA 384 (see FIG.1A), contain one or more “secondary stem regions” such as 5’ secondary stem region and 3’ secondary stem region that flank the transgene expression cassette.
  • 5’ secondary stem region is adjacent to the primary stem region of the 5’ stem-loop region and the 3’ secondary stem region is adjacent to the primary stem region of the 3’ stem-loop region.
  • Both of the secondary stem regions if incorporated in a ceDNA vector of the present disclosure such as in ceDNA 384, contain only artificial sequences and no viral ITR sequences, and the length of each of these artificial secondary stem regions is corresponds to the A, A’, D, and D’ regions of a reference ceDNA having 5’ and 3’ AAV derived wt-ITRs such as reference ceDNA 382 or the A, A’ and D regions of an AAV derived wt-ITRs.
  • the 5’ secondary stem region (SEQ ID NO: 12) and a 3’ secondary stem region (SEQ ID NO: 13), which are reverse complement of each other, 84 ME148589913v.1 each contains 58 bp.
  • the presence of the artificial secondary stem regions may provide some beneficial properties to the vector, such as improving or enhancing the genetic stability of the vector.
  • the double-stranded primary stem region in the stem-loop accounts partially for the entire stem region of a ceDNA of the present disclosure, while the remaining and continuing sequence of the stem region is found in the secondary stem regions on the insert.
  • the double-stranded DNA construct e.g., a plasmid or a linear double-stranded DNA molecule
  • the transgene expression cassette is excised in the cell-free synthetic methods disclosed herein further comprises a least a first secondary stem region and a second secondary stem region each flanking the transgene expression cassette.
  • the first secondary stem region is upstream of the transgene expression cassette and downstream of the first non-palindromic restriction endonuclease recognition site and the corresponding first cleavage site.
  • the second secondary stem region is downstream of the transgene expression cassette and upstream of the second non-palindromic restriction endonuclease recognition site and the corresponding second cleavage site.
  • the first cleavage site is adjacent to the first secondary stem region and the second cleavage site is adjacent to the second secondary stem region (i.e., no spacer between the cleavage site and the secondary stem region).
  • the first secondary stem region and the second secondary stem region each comprise about 1 to about 100 bp, or about 1 to about 90 bp, or about 1 to about 80 bp, or about 1 to about 70 bp, or about 2 to about 70 bp, or about 3 to about 70 bp, or about 5 to about 70 bp, or about 10 to about 70 bp, or about 15 to about 70 bp, or about 20 to about 70 bp, or about 25 to about 70 bp, or about 30 to about 70 bp, or about 35 to about 70 bp, or about 40 to about 70 bp, or about 45 to about 70 bp, or about 50 to about 70 bp, or about 50 bp, or about 50 bp, or about 51 bp, or about 52 bp, or about 53 bp, or about 54 bp, or about 55 bp, or about 56 bp, or about 57 bp, or about 58 bp, or about 59 bp, or about
  • the double-stranded DNA construct or the excised insert further comprises one or more spacer regions.
  • the double-stranded DNA construct e.g., a plasmid or a linear double-stranded DNA molecule
  • the insert further comprises a first spacer between the first restriction endonuclease cleavage site and the transgene expression cassette.
  • the double-stranded DNA construct further comprises a second spacer between the second restriction endonuclease cleavage site and the transgene expression cassette.
  • Each spacer region or sequence is about 1-50 nucleotides in length, e.g., about 2, 5, 7, 8, 10, 11, 12, 13, 15, 17, 18, 20, 22, 23, 25, 27, 28, 30, 32, 33, 35, 37, 38, 40, 42, 43, 45, 47, 48, or 50 nucleotides.
  • the spacer between a first (or left) restriction endonuclease cleavage site or the second (or right) restriction endonuclease cleavage site and the transgene expression cassette is selected from the spacers comprising the sequences as shown in Table 2 of International Patent Application No. 85 ME148589913v.1 PCT/US2022/053868, which are incorporated by reference in their entireties herein.
  • the spacer comprises a nucleic acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40 of International Patent Application No. PCT/US2022/053868; SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11 in Table 3 below.
  • the spacer consists of a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40 of International Patent Application No. PCT/US2022/053868; SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 in Table 3 below. Table 3.
  • the disclosure provides a method of producing a double-stranded DNA construct from a plasmid template via rolling-circle amplification, comprising the steps of (a) contacting the plasmid template with a thermostable polymerase having strand-displacement activity, wherein the ratio of plasmid template concentration (in ng/ ⁇ l) to polymerase concentration (in U/ ⁇ l) is greater than about 1; (b) contacting the plasmid template with an oligonucleotide primer and dNTPs; (c) incubating the plasmid template, the polymerase, the oligonucleotide primer, and the dNTPs at a temperature of about 40°C or less, for a time period of at least about 5 hours; thereby producing a double-stranded DNA construct.
  • the ratio of plasmid template concentration (in ng/ ⁇ l) to polymerase concentration (in U/ ⁇ l) is greater than about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20.
  • the plasmid template concentration is about 0.01 ng/ ⁇ l, about 0.05 ng/ ⁇ l, about 0.1 ng/ ⁇ l, about 0.15 ng/ ⁇ l, about 0.2 ng/ ⁇ l, about 0.21 ng/ ⁇ l, about 0.22 ng/ ⁇ l, about 0.23 ng/ ⁇ l, about 0.24 ng/ ⁇ l, about 0.2 ng/ ⁇ l 5, about 0.26 ng/ ⁇ l, about 0.27 ng/ ⁇ l, about 0.28 ng/ ⁇ l, about 0.29 ng/ ⁇ l, about 0.3 ng/ ⁇ l, about 0.35 ng/ ⁇ l, about 0.4 ng/ ⁇ l, about 0.45 ng/ ⁇ l, about 0.5 ng/ ⁇ l, about 0.6 ng/ ⁇ l, about 0.7 ng/ ⁇ l, about 0.8 ng/ ⁇ l, about 0.9 ng/ ⁇ l, or about 1.0 ng/ ⁇ l.
  • the polymerase concentration is about 0.01 U/ ⁇ l, 86 ME148589913v.1 about 0.02 U/ ⁇ l, about 0.03 U/ ⁇ l, about 0.04 U/ ⁇ l, about 0.05 U/ ⁇ l, about 0.06 U/ ⁇ l, about 0.07 U/ ⁇ l, about 0.08 U/ ⁇ l, about 0.09 U/ ⁇ l, about 0.1 U/ ⁇ l, about 0.15 U/ ⁇ l, about 0.2 U/ ⁇ l, about 0.25 U/ ⁇ l, about 0.3 U/ ⁇ l, about 0.35 U/ ⁇ l, about 0.4 U/ ⁇ l, or about 0.45 U/ ⁇ l.
  • the temperature in step (c) is less than about 40°C, about 39°C, about 38°C, about 37°C, about 36°C, about 35°C, about 34°C, about 33°C, about 32°C, about 31°C, about 30°C, about 29°C, about 28°C, about 27°C, about 26°C, about 25°C, about 24°C, about 23°C, about 22°C, or about 21°C.
  • the time period is at least about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 21 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, or about 40 hours.
  • the time period is less than about 6 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 21 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, or about 40 hours.
  • the plasmid template concentration is about 0.25 ng/ ⁇ l
  • the temperature is about 30°C
  • the polymerase concentration is about 0.05 U/ ⁇ l
  • the time period is about 18-26 hours.
  • the oligonucleotide primer concentration is less than about 50 ⁇ M.
  • the oligonucleotide primer concentration is at least about 10 ⁇ M.
  • the oligonucleotide primer concentration is at least about 10 ⁇ M and less than about 50 ⁇ M.
  • the thermostable polymerase is Phi29 DNA polymerase or a derivative or variant thereof.
  • the thermostable polymerase is EQUIPHI29 TM .
  • the method is performed in a total reaction volume of at least about 100 ⁇ l. In other further embodiments, the method is performed in a total reaction volume of at least about 100 ⁇ l, about 200 ⁇ l, about 300 ⁇ l, about 400 ⁇ l, about 500 ⁇ l, about 600 ⁇ l, about 700 ⁇ l, about 800 ⁇ l, about 900 ⁇ l, about 1 ml, about 2 ml, about 3 ml, about 4 ml, about 5 ml, about 6 ml, about 7 ml, about 8 ml, about 9 ml, about 10 ml, about 15 ml, about 20 ml, about 25 ml, about 30 ml, about 35 ml, about 40 ml, about 45 ml, about 50 ml, about 55 ml, about 60 ml, about 65 ml, about 70 ml, about 75 ml, about 80 ml, about 85 ml, about 90 ml, about
  • the method is performed in a reaction vessel that has a capacity of at least twice the total reaction volume.
  • the oligonucleotide primer hybridizes to a backbone sequence in the plasmid template.
  • the oligonucleotide primer is a universal primer.
  • the dNTP concentration is about 4 mM. In some embodiments, the dNTP concentration is between about 3.8-4.2 mM. Isolation and Purification Methods to generate and isolate a ceDNA vector, which is an exemplary closed-ended DNA vector, are described herein.
  • a closed-ended DNA vector e.g., ceDNA vector produced by the synthetic methods described herein can be harvested or collected at an appropriate time after the last ligation reaction and can be optimized to achieve a high-yield production of the ceDNA vectors.
  • the closed-ended DNA vector e.g., ceDNA vectors can be purified by any means known to those of skill in the art for purification of DNA.
  • ceDNA vectors are purified as DNA molecules.
  • any art-known nucleic acid purification methods can be adopted, as well as commercially available DNA extraction kits.
  • purification can be implemented by subjecting a reaction mixture to chromatographic separation.
  • the process can be performed by loading the reaction mixture on an ion exchange column (e.g., SARTOBIND Q®) which retains nucleic acids, and then eluting (e.g., with a 1.2 M NaCl solution) and performing a further chromatographic purification on a gel filtration column (e.g., 6 fast flow GE).
  • an ion exchange column e.g., SARTOBIND Q®
  • eluting e.g., with a 1.2 M NaCl solution
  • a gel filtration column e.g., 6 fast flow GE
  • the DNA vector e.g., ceDNA vector is then recovered by, e.g., precipitation.
  • the presence of the ceDNA vector can be confirmed by digesting the vector DNA isolated from the cells with a restriction enzyme having a single recognition site on the DNA vector and analyzing both digested and undigested DNA material using gel electrophoresis to confirm the presence of characteristic bands of linear and continuous DNA as compared to linear and non- continuous DNA.
  • the closed-ended DNA vectors produced by the synthetic production methods disclosed herein can be delivered to a target cell in vitro or in vivo by various suitable methods as discussed herein. Vectors alone can be applied or injected. Vectors can be delivered to a cell without the help of a transfection reagent or other physical means.
  • vectors can be delivered using a transfection reagent or other physical means that facilitates entry of DNA into a cell, e.g., liposomes, alcohols, polylysine- rich compounds, arginine-rich compounds calcium phosphate, microvesicles, microinjection, and the like.
  • a transfection reagent or other physical means that facilitates entry of DNA into a cell, e.g., liposomes, alcohols, polylysine- rich compounds, arginine-rich compounds calcium phosphate, microvesicles, microinjection, and the like.
  • 88 ME148589913v.1 Circular DNA vectors produced using the synthetic production method Provided herein are various methods of in vitro production of DNA molecules and closed- ended DNA vectors.
  • the closed-ended DNA vector is a ceDNA vector, as described herein.
  • the closed-ended DNA vector is, e.g., a dumbbell DNA vector or a doggybone DNA vector (see e.g., International Patent Application Publication No. WO2010/0086626, incorporated by reference in its entirety herein).
  • pharmaceutical compositions are provided.
  • the pharmaceutical composition comprises a ceDNA vector as disclosed herein and a pharmaceutically acceptable carrier or diluent.
  • the DNA-vectors disclosed herein can be incorporated into pharmaceutical compositions suitable for administration to a subject for in vivo delivery to cells, tissues, or organs of the subject.
  • the pharmaceutical composition comprises a ceDNA-vector as disclosed herein and a pharmaceutically acceptable carrier.
  • the ceDNA vectors described herein can be incorporated into a pharmaceutical composition suitable for a desired route of therapeutic administration (e.g., parenteral administration).
  • a desired route of therapeutic administration e.g., parenteral administration
  • Passive tissue transduction via high pressure intravenous or intraarterial infusion, as well as intracellular injection, such as intranuclear microinjection or intracytoplasmic injection, are also contemplated.
  • Pharmaceutical compositions for therapeutic purposes can be formulated as a solution, microemulsion, dispersion, liposomes, or other ordered structure suitable to high ceDNA vector concentration.
  • Sterile injectable solutions can be prepared by incorporating the ceDNA vector compound in the required amount in an appropriate buffer with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • compositions comprising a ceDNA vector can be formulated to deliver a transgene in the nucleic acid to the cells of a recipient, resulting in the therapeutic expression of the transgene therein.
  • the composition can also include a pharmaceutically acceptable carrier.
  • a ceDNA vector as disclosed herein can be incorporated into a pharmaceutical composition suitable for topical, systemic, intra-amniotic, intrathecal, intracranial, intraarterial, intravenous, intralymphatic, intraperitoneal, subcutaneous, tracheal, intra-tissue (e.g., intramuscular, intracardiac, intrahepatic, intrarenal, intracerebral), intrathecal, intravesical, conjunctival (e.g., extra-orbital, intraorbital, retroorbital, intraretinal, subretinal, choroidal, sub-choroidal, intrastromal, intracameral and intravitreal), intracochlear, and mucosal (e.g., oral, rectal,
  • compositions for therapeutic purposes typically must be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, dispersion, liposomes, or other ordered structure suitable to high ceDNA vector concentration.
  • Sterile injectable solutions can be prepared by incorporating the ceDNA vector compound in the required amount in an appropriate buffer with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • Various techniques and methods are known in the art for delivering nucleic acids to cells.
  • nucleic acids such as ceDNA can be formulated into lipid nanoparticles (LNPs), lipidoids, liposomes, lipid nanoparticles, lipoplexes, or core-shell nanoparticles.
  • LNPs are composed of nucleic acid (e.g., ceDNA) molecules, one or more ionizable or cationic lipids (or salts thereof), one or more non-ionic or neutral lipids (e.g., a phospholipid), a molecule that prevents aggregation (e.g., PEG or a PEG-lipid conjugate), and optionally a sterol (e.g., cholesterol).
  • nucleic acid e.g., ceDNA
  • ionizable or cationic lipids or salts thereof
  • non-ionic or neutral lipids e.g., a phospholipid
  • a sterol e.g., cholesterol
  • nucleic acids such as ceDNA to a cell
  • Another method for delivering nucleic acids, such as ceDNA to a cell is by conjugating the nucleic acid with a ligand that is internalized by the cell.
  • the ligand can bind a receptor on the cell surface and internalized via endocytosis.
  • the ligand can be covalently linked to a nucleotide in the nucleic acid.
  • Exemplary conjugates for delivering nucleic acids into a cell are described, example, in International Patent Application Publication Nos.
  • Nucleic acids such as ceDNA
  • Useful transfection methods include, but are not limited to, lipid-mediated transfection, cationic polymer- mediated transfection, or calcium phosphate precipitation.
  • Transfection reagents are well known in the art and include, but are not limited to, TurboFect Transfection Reagent (Thermo Fisher Scientific), Pro-Ject Reagent (Thermo Fisher Scientific), TRANSPASSTM P Protein Transfection Reagent (New England Biolabs), CHARIOTTM Protein Delivery Reagent (Active Motif), PROTEOJUICETM Protein Transfection Reagent (EMD Millipore), 293fectin, LIPOFECTAMINETM 2000, LIPOFECTAMINETM 3000 (Thermo Fisher Scientific), LIPOFECTAMINETM (Thermo Fisher Scientific), LIPOFECTINTM (Thermo Fisher Scientific), DMRIE-C, CELLFECTINTM (Thermo Fisher Scientific), OLIGOFECTAMINETM (Thermo Fisher Scientific), LIPOFECTACETM, FUGENETM (Roche, Basel, Switzerland), FUGENETM HD (Roche), TRANSFECTAMTM(Transfectam, Promega, Madison, Wis.),
  • Nucleic acids such as ceDNA
  • Methods of non-viral delivery of nucleic acids in vivo or ex vivo include electroporation, lipofection (see, U.S. Pat.
  • ceDNA vectors as described herein can also be administered directly to an organism for transduction of cells in vivo. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells including, but not limited to, injection, infusion, topical application and electroporation. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route. Methods for introduction of a nucleic acid vector ceDNA vector as disclosed herein can be delivered into hematopoietic stem cells, for example, by the methods as described, for example, in U.S. Pat. No.5,928,638.
  • ceDNA vectors are delivered by making transient penetration in cell membrane by mechanical, electrical, ultrasonic, hydrodynamic, or laser-based energy so that DNA entrance into the targeted cells is facilitated.
  • a ceDNA vector can be delivered by transiently disrupting cell membrane by squeezing the cell through a size-restricted channel or by other means known in the art.
  • a ceDNA vector alone is directly injected as naked DNA into skin, thymus, cardiac muscle, skeletal muscle, or liver cells.
  • a ceDNA vector is delivered by gene gun.
  • Gold or tungsten spherical particles (1–3 ⁇ m diameter) coated with capsid-free AAV vectors can be accelerated to high speed by pressurized gas to penetrate into target tissue cells.
  • electroporation is used to deliver ceDNA vectors. Electroporation causes temporary destabilization of the cell membrane target cell tissue by insertion of a pair of electrodes into the tissue so that DNA molecules in the surrounding media of the destabilized 91 ME148589913v.1 membrane would be able to penetrate into cytoplasm and nucleoplasm of the cell. Electroporation has been used in vivo for many types of tissues, such as skin, lung, and muscle.
  • a ceDNA vector is delivered by hydrodynamic injection, which is a simple and highly efficient method for direct intracellular delivery of any water-soluble compounds and particles into internal organs and skeletal muscle in an entire limb.
  • ceDNA vectors are delivered by ultrasound by making nanoscopic pores in membrane to facilitate intracellular delivery of DNA particles into cells of internal organs or tumors, so the size and concentration of plasmid DNA have great role in efficiency of the system.
  • ceDNA vectors are delivered by magnetofection by using magnetic fields to concentrate particles containing nucleic acid into the target cells.
  • chemical delivery systems can be used, for example, by using nanomeric complexes, which include compaction of negatively charged nucleic acid by polycationic nanomeric particles, belonging to cationic liposome/micelle or cationic polymers.
  • Cationic lipids used for the delivery method includes, but not limited to monovalent cationic lipids, polyvalent cationic lipids, guanidine containing compounds, cholesterol derivative compounds, cationic polymers, (e.g., poly(ethylenimine), poly-L-lysine, protamine, other cationic polymers), and lipid-polymer hybrid.
  • cationic polymers e.g., poly(ethylenimine), poly-L-lysine, protamine, other cationic polymers
  • lipid-polymer hybrid e.g., lipid-polymer hybrid.
  • Exosomes are small membrane vesicles of endocytic origin that are released into the extracellular environment following fusion of multivesicular bodies with the plasma membrane. Their surface consists of a lipid bilayer from the donor cell's cell membrane, they contain cytosol from the cell that produced the exosome, and exhibit membrane proteins from the parental cell on the surface. Exosomes are produced by various cell types including epithelial cells, B and T lymphocytes, mast cells (MC) as well as dendritic cells (DC). Some embodiments, exosomes with a diameter between 10nm and 1 ⁇ m, between 20nm and 500nm, between 30nm and 250nm, between 50nm and 100nm are envisioned for use.
  • Exosomes can be isolated for delivery to target cells using either their donor cells or by introducing specific nucleic acids into them.
  • Various approaches known in the art can be used to produce exosomes containing capsid-free AAV vectors of the present disclosure.
  • B. Microparticle/Nanoparticles In some embodiments, a ceDNA vector as disclosed herein is delivered by a lipid nanoparticle.
  • lipid nanoparticles comprise an ionizable amino lipid (e.g., heptatriaconta- 6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, DLin-MC3-DMA, a phosphatidylcholine (1,2- distearoyl-sn-glycero-3-phosphocholine, DSPC), cholesterol and a coat lipid (polyethylene glycol- 92 ME148589913v.1 dimyristolglycerol, PEG-DMG), for example as disclosed by Tam et al. (2013). Advances in Lipid Nanoparticles for siRNA delivery. Pharmaceuticals 5(3): 498-507.
  • an ionizable amino lipid e.g., heptatriaconta- 6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, DLin-MC3-DMA, a phosphatid
  • the LNPs of the disclosure have a mean diameter selected to provide an intended therapeutic effect.
  • the LNPs of the disclosure have a mean diameter that is compatible with a target organ, such that the LNPs of the disclosure are able to diffuse through the fenestrations of a target organ (e.g., liver) or a target cell subpopulation (e.g., hepatocytes).
  • a target organ e.g., liver
  • a target cell subpopulation e.g., hepatocytes.
  • Lipid particles of the disclosure are characterized by an average diameter of about 70-80 nm or less, making them particularly useful for therapeutic administration.
  • lipid particles of the disclosure typically have a mean diameter of from about 20 nm to about 75 nm, about 20 nm to about 70 nm, about 25 nm to about 75 nm, about 25 nm to about 70 nm, from about 30 nm to about 75 nm, from about 30 nm to about 70 nm, from about 35 nm to about 75 nm, from about 35 nm to about 70 nm, from about 40 nm to about 75 nm, from about 40 nm to about 70 nm, from about 45 nm to about 75 nm, from about 50 nm to about 75 nm, from about 50 nm to about 70 nm, from about 60 nm to about 75 nm, from about 60 nm to about 70 nm, from about 65 nm to about 75 nm, from about 65 nm to about 70 nm, or about 20 nm, about 25 nm, about 30 nm, about 20 n
  • lipid nanoparticles known in the art can be used to deliver ceDNA vector disclosed herein.
  • various delivery methods using lipid nanoparticles are described in U.S. Patent Nos.9,404,127, 9,006,417 and 9,518,272.
  • a ceDNA vector disclosed herein is delivered by a gold nanoparticle.
  • a nucleic acid can be covalently bound to a gold nanoparticle or non-covalently bound to a gold nanoparticle (e.g., bound by a charge-charge interaction), for example as described by Ding et al. (2014). Gold Nanoparticles for Nucleic Acid Delivery. Mol. Ther.22(6); 1075-1083.
  • gold nanoparticle-nucleic acid conjugates are produced using methods described, for example, in U.S. Patent No.6,812,334.
  • C. Conjugates In some embodiments, a ceDNA vector as disclosed herein is conjugated (e.g., covalently bound to an agent that increases cellular uptake.
  • An “agent that increases cellular uptake” is a molecule that facilitates transport of a nucleic acid across a lipid membrane.
  • a nucleic acid can be conjugated to a lipophilic compound (e.g., cholesterol, tocopherol, etc.), a cell penetrating peptide (CPP) (e.g., penetratin, TAT, Syn1B, etc.), and polyamines (e.g., spermine).
  • a lipophilic compound e.g., cholesterol, tocopherol, etc.
  • CPP cell penetrating peptide
  • PEP cell penetrating peptide
  • polyamines e.g., spermine
  • a ceDNA vector as disclosed herein is conjugated to a polymer (e.g., a polymeric molecule) or a folate molecule (e.g., folic acid molecule).
  • a polymer e.g., a polymeric molecule
  • a folate molecule e.g., folic acid molecule
  • delivery of nucleic acids conjugated to polymers is known in the art, for example as described in WO2000/34343 and WO2008/022309.
  • a ceDNA vector as disclosed herein is conjugated to a poly(amide) polymer, for example as described by U.S. Patent No.8,987,377.
  • a nucleic acid described by the disclosure is conjugated to a folic acid molecule as described in U.S.
  • a ceDNA vector as disclosed herein is conjugated to a carbohydrate, for example as described in U.S. Patent No.8,450,467.
  • D. Nanocapsule Alternatively, nanocapsule formulations of a ceDNA vector as disclosed herein can be used. Nanocapsules can generally entrap substances in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 ⁇ m) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use. E.
  • Liposomes The ceDNA vectors in accordance with the present disclosure can be added to liposomes for delivery to a cell or target organ in a subject.
  • Liposomes are vesicles that possess at least one lipid bilayer. Liposomes are typical used as carriers for drug/ therapeutic delivery in the context of pharmaceutical development. They work by fusing with a cellular membrane and repositioning its lipid structure to deliver a drug or active pharmaceutical ingredient (API).
  • API active pharmaceutical ingredient
  • Liposome compositions for such delivery are composed of phospholipids, especially compounds having a phosphatidylcholine group, however these compositions may also include other lipids. The formation and use of liposomes is generally known to those of skill in the art.
  • Liposomes have been developed with improved serum stability and circulation half-times (U.S. Pat. No. 5,741,516). Further, various methods of liposome and liposome like preparations as potential drug carriers have been described (U.S. Pat. Nos.5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587). Liposomes have been used successfully with a number of cell types that are normally resistant to transfection by other procedures. In addition, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems. Liposomes have been used effectively to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors and allosteric effectors into a variety of cultured cell lines and animals.
  • Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs). MLVs generally have diameters of from 25 nm to 4 ⁇ m. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 ANG, containing an aqueous solution in the core.
  • a liposome comprises cationic lipids.
  • cationic lipid includes lipids and synthetic lipids having both polar and non-polar domains and which are capable of being positively charged at or around physiological pH and which bind to polyanions, such as nucleic acids, and facilitate the delivery of nucleic acids into cells.
  • cationic lipids include saturated and unsaturated alkyl and alicyclic ethers and esters of amines, amides, or derivatives thereof.
  • cationic lipids comprise straight-chain, branched alkyl, alkenyl groups, or any combination of the foregoing.
  • cationic lipids contain from 1 to about 25 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 carbon atoms. In some embodiments, cationic lipids contain more than 25 carbon atoms. In some embodiments, straight chain or branched alkyl or alkene groups have six or more carbon atoms.
  • a cationic lipid can also comprise, in some embodiments, one or more alicyclic groups. Non-limiting examples of alicyclic groups include cholesterol and other steroid groups. In some embodiments, cationic lipids are prepared with one or more counter ions.
  • counter ions examples include, but are not limited to, Cl ⁇ , Br ⁇ , I ⁇ , F ⁇ , acetate, trifluoroacetate, sulfate, nitrite, and nitrate.
  • the disclosure provides for a liposome formulation that includes one or more compounds with a polyethylene glycol (PEG) functional group (so-called “PEG-ylated compounds”) which can reduce the immunogenicity/ antigenicity of, provide hydrophilicity and hydrophobicity to the compound(s) and reduce dosage frequency.
  • the liposome formulation simply includes polyethylene glycol (PEG) polymer as an additional component.
  • the molecular weight of the PEG or PEG functional group can be from 62 Da to about 5,000 Da.
  • the disclosure provides for a liposome formulation that will deliver an API with extended release or controlled release profile over a period of hours to weeks.
  • the liposome formulation may comprise aqueous chambers that are bound by lipid bilayers.
  • the liposome formulation encapsulates an API with components that undergo a physical transition at elevated temperature which releases the API over a period of hours to weeks.
  • the liposome formulation comprises sphingomyelin and one or more lipids disclosed herein.
  • the liposome formulation comprises optisomes.
  • the disclosure provides for a liposome formulation that includes one or more lipids selected from: N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3- phosphoethanolamine sodium salt, (distearoyl-sn-glycero-phosphoethanolamine), MPEG (methoxy polyethylene glycol)-conjugated lipid, HSPC (hydrogenated soy phosphatidylcholine); PEG 95 ME148589913v.1 (polyethylene glycol); DSG (distearoyl-rac-glycerol); DSPE (distearoyl-sn-glycero- phosphoethanolamine); DSPC (distearoylphosphatidylcholine); DOPC (dioleoylphosphatidylcholine); DPPG (dipalmitoylphosphatidylglycerol); EPC (egg phosphatidylcholine); DO
  • the disclosure provides for a liposome formulation comprising phospholipid, cholesterol and a PEG-ylated lipid in a molar ratio of 56:38:5. In some aspects, the liposome formulation’s overall lipid content is from 2-16 mg/mL. In some aspects, the disclosure provides for a liposome formulation comprising a lipid containing a phosphatidylcholine functional group, a lipid containing an ethanolamine functional group and a PEG-ylated lipid.
  • the disclosure provides for a liposome formulation comprising a lipid containing a phosphatidylcholine functional group, a lipid containing an ethanolamine functional group and a PEG-ylated lipid in a molar ratio of 3:0.015:2 respectively.
  • the disclosure provides for a liposome formulation comprising a lipid containing a phosphatidylcholine functional group, cholesterol and a PEG-ylated lipid.
  • the disclosure provides for a liposome formulation comprising a lipid containing a phosphatidylcholine functional group and cholesterol.
  • the PEG-ylated lipid is PEG-2000-DSPE.
  • the disclosure provides for a liposome formulation comprising DPPG, soy PC, MPEG-DSPE lipid conjugate and cholesterol. In some aspects, the disclosure provides for a liposome formulation comprising one or more lipids containing a phosphatidylcholine functional group and one or more lipids containing an ethanolamine functional group. In some aspects, the disclosure provides for a liposome formulation comprising one or more: lipids containing a phosphatidylcholine functional group, lipids containing an ethanolamine functional group, and sterols, e.g., cholesterol. In some aspects, the liposome formulation comprises DOPC/ DEPC; and DOPE.
  • the disclosure provides for a liposome formulation further comprising one or more pharmaceutical excipients, e.g., sucrose and/or glycine.
  • the disclosure provides for a liposome formulation that is wither unilamellar or multilamellar in structure.
  • the disclosure provides for a liposome formulation that comprises multi-vesicular particles and/or foam-based particles.
  • the disclosure provides for a liposome formulation that are larger in relative size to common nanoparticles and about 150 to 250 nm in size.
  • the liposome formulation is a lyophilized powder.
  • the disclosure provides for a liposome formulation that is made and loaded with ceDNA vectors disclosed or described herein, by adding a weak base to a mixture having the 96 ME148589913v.1 isolated ceDNA outside the liposome. This addition increases the pH outside the liposomes to approximately 7.3 and drives the API into the liposome.
  • the disclosure provides for a liposome formulation having a pH that is acidic on the inside of the liposome. In such cases the inside of the liposome can be at pH 4-6.9, and more preferably pH 6.5.
  • the disclosure provides for a liposome formulation made by using intra-liposomal drug stabilization technology.
  • polymeric or non-polymeric highly charged anions and intra-liposomal trapping agents are utilized, e.g., polyphosphate or sucrose octasulfate.
  • the disclosure provides for a liposome formulation comprising phospholipids, lecithin, phosphatidylcholine and phosphatidylethanolamine.
  • Non-limiting examples of cationic lipids include polyethylenimine, polyamidoamine (PAMAM) starburst dendrimers, Lipofectin (a combination of DOTMA and DOPE), Lipofectase, LIPOFECTAMINETM (e.g., LIPOFECTAMINETM 2000), DOPE, Cytofectin (Gilead Sciences, Foster City, Calif.), and Eufectins (JBL, San Luis Obispo, Calif.).
  • Exemplary cationic liposomes can be made from N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-[1 - (2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP), 3 ⁇ -[N-(N′,N′- dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), 2,3,-dioleyloxy-N- [2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2- dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide; and dimethyldioctadecylammonium bromide (DDAB).
  • DOTMA N-[1-(2,
  • Nucleic acids can also be complexed with, e.g., poly (L-lysine) or avidin and lipids can, or cannot, be included in this mixture, e.g., steryl-poly (L-lysine).
  • a ceDNA vector as disclosed herein is delivered using a cationic lipid described in U.S. Patent No.8,158,601, or a polyamine compound or lipid as described in U.S. Patent No.8,034,376. F.
  • Liposomes and Lipid Nanoparticle (LNP) Compositions The ceDNA vectors in accordance with the present disclosure can be added to liposomes for delivery to a cell in need of gene editing, e.g., in need of a donor sequence.
  • Liposomes are vesicles that possess at least one lipid bilayer. Liposomes are typical used as carriers for drug/ therapeutic delivery in the context of pharmaceutical development. They work by fusing with a cellular membrane and repositioning its lipid structure to deliver a drug or active pharmaceutical ingredient (API).
  • Liposome compositions for such delivery are composed of phospholipids, especially compounds having a phosphatidylcholine group, however these compositions may also include other lipids.
  • the disclosure provides for a liposome formulation that includes one or more compounds with a polyethylene glycol (PEG) functional group (so-called “PEG-ylated compounds”) which can reduce the immunogenicity/ antigenicity of, provide hydrophilicity and hydrophobicity to 97 ME148589913v.1 the compound(s) and reduce dosage frequency.
  • the liposome formulation simply includes polyethylene glycol (PEG) polymer as an additional component.
  • the molecular weight of the PEG or PEG functional group can be from 62 Da to about 5,000 Da.
  • the disclosure provides for a liposome formulation that will deliver an API with extended release or controlled release profile over a period of hours to weeks.
  • the liposome formulation may comprise aqueous chambers that are bound by lipid bilayers. In other related aspects, the liposome formulation encapsulates an API with components that undergo a physical transition at elevated temperature which releases the API over a period of hours to weeks. In some aspects, the liposome formulation comprises sphingomyelin and one or more lipids disclosed herein. In some aspects, the liposome formulation comprises optisomes.
  • the disclosure provides for a liposome formulation that includes one or more lipids selected from: N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3- phosphoethanolamine sodium salt, (distearoyl-sn-glycero-phosphoethanolamine), MPEG (methoxy polyethylene glycol)-conjugated lipid, HSPC (hydrogenated soy phosphatidylcholine); PEG (polyethylene glycol); DSPE (distearoyl-sn-glycero-phosphoethanolamine); DSPC (distearoylphosphatidylcholine); DOPC (dioleoylphosphatidylcholine); DPPG (dipalmitoylphosphatidylglycerol); EPC (egg phosphatidylcholine); DOPS (dioleoylphosphatidylserine); POPC (palmitoylo
  • the disclosure provides for a liposome formulation comprising phospholipid, cholesterol and a PEG-ylated lipid in a molar ratio of 56:38:5. In some aspects, the liposome formulation’s overall lipid content is from 2-16 mg/mL. In some aspects, the disclosure provides for a liposome formulation comprising a lipid containing a phosphatidylcholine functional group, a lipid containing an ethanolamine functional group and a PEG-ylated lipid.
  • the disclosure provides for a liposome formulation comprising a lipid containing a phosphatidylcholine functional group, a lipid containing an ethanolamine functional group and a PEG-ylated lipid in a molar ratio of 3:0.015:2 respectively.
  • the disclosure provides for a liposome formulation comprising a lipid containing a phosphatidylcholine functional group, cholesterol and a PEG-ylated lipid.
  • the disclosure provides for a liposome formulation comprising a lipid containing a phosphatidylcholine functional group and cholesterol.
  • the PEG-ylated lipid is PEG-2000-DSPE.
  • the disclosure provides for a liposome formulation comprising DPPG, soy PC, MPEG-DSPE lipid conjugate and cholesterol.
  • a liposome formulation comprising DPPG, soy PC, MPEG-DSPE lipid conjugate and cholesterol.
  • 98 ME148589913v.1 the disclosure provides for a liposome formulation comprising one or more lipids containing a phosphatidylcholine functional group and one or more lipids containing an ethanolamine functional group.
  • the disclosure provides for a liposome formulation comprising one or more: lipids containing a phosphatidylcholine functional group, lipids containing an ethanolamine functional group, and sterols, e.g., cholesterol.
  • the liposome formulation comprises DOPC/ DEPC; and DOPE.
  • the disclosure provides for a liposome formulation further comprising one or more pharmaceutical excipients, e.g., sucrose and/or glycine.
  • the disclosure provides for a liposome formulation that is either unilamellar or multilamellar in structure.
  • the disclosure provides for a liposome formulation that comprises multi-vesicular particles and/or foam-based particles.
  • the disclosure provides for a liposome formulation that are larger in relative size to common nanoparticles and about 150 to 250 nm in size.
  • the liposome formulation is a lyophilized powder.
  • the disclosure provides for a liposome formulation that is made and loaded with ceDNA vectors disclosed or described herein, by adding a weak base to a mixture having the isolated ceDNA outside the liposome. This addition increases the pH outside the liposomes to approximately 7.3 and drives the API into the liposome.
  • the disclosure provides for a liposome formulation having a pH that is acidic on the inside of the liposome. In such cases the inside of the liposome can be at pH 4-6.9, and more preferably pH 6.5.
  • the disclosure provides for a liposome formulation made by using intra-liposomal drug stabilization technology.
  • the disclosure provides for a liposome formulation comprising phospholipids, lecithin, phosphatidylcholine and phosphatidylethanolamine.
  • the liposomal formulation is a formulation described in Table 6 of International Patent Application Publication No. WO2020/097417, which is incorporated herein by reference in its entirety.
  • the disclosure provides for a lipid nanoparticle comprising ceDNA and an ionizable lipid.
  • lipid nanoparticle formulation that is made and loaded with ceDNA obtained by the process as disclosed in International Application PCT/US2018/050042, filed on September 7, 2018, which is incorporated herein.
  • This can be accomplished by high energy mixing of ethanolic lipids with aqueous ceDNA at low pH which protonates the ionizable lipid and provides favorable energetics for ceDNA/lipid association and nucleation of particles.
  • the particles can be further stabilized through aqueous dilution and removal of the organic solvent.
  • the particles can be concentrated to the desired level.
  • the ionizable lipid is present in the LNP provided by the present disclosure in an amount of about 20 mol% to about 70 mol%, about 20 mol% to about 65 mol%, about 20 mol% to about 60 mol%, about 20 mol% to about 55 mol%, about 20 mol% to about 50 mol%, about 25 mol% to about 70 mol%, about 25 mol% to about 65 mol%, about 25 mol% to about 60 mol%, about 25 mol% to about 55 mol%, about 25 mol% to about 50 mol%, about 30 mol% to about 70 mol%, about 30 mol% to about 65 mol%, about 30 mol% to about 60 mol%, about 30 mol% to about 55 mol%, about 30 mol% to about 50 mol%, about 35 mol% to about 70 mol%, about 35 mol% to about 70 mol%, about 35 mol% to about 65 mol%, about 35 mol% to about 60 mol%, about 35
  • the lipid particles are prepared at a total lipid to ceDNA (mass or weight) ratio of from about 10:1 to 30:1.
  • the lipid to ceDNA ratio (mass/mass ratio; w/w ratio) can be in the range of from about 1:1 to about 25:1, from about 10:1 to about 14:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1.
  • the amounts of lipids and ceDNA can be adjusted to provide a desired N/P ratio, for example, N/P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or higher.
  • the lipid particle formulation can range from about 5 mg/ml to about 30 mg/mL.
  • the ionizable lipid is typically employed to condense the nucleic acid cargo, e.g., ceDNA at low pH and to drive membrane association and fusogenicity.
  • ionizable lipids are lipids comprising at least one amino group that is positively charged or becomes protonated under acidic conditions, for example at pH of 6.5 or lower. Ionizable lipids are also referred to as cationic lipids herein.
  • Exemplary ionizable lipids are described in PCT patent publications WO2015/095340, WO2015/199952, WO2018/011633, WO2017/049245, WO2015/061467, WO2012/040184, WO2012/000104, WO2015/074085, WO2016/081029, WO2017/004143, WO2017/075531, WO2017/117528, WO2011/022460, WO2013/148541, WO2013/116126, WO2011/153120, WO2012/044638, WO2012/054365, WO2011/090965, WO2013/016058, WO2012/162210, WO2008/042973, WO2010/129709, WO2010/144740 , WO2012/099755, WO2013/049328, WO2013/086322, WO2013/086373, WO2011/071860, WO2009/132131, WO2010/048536, WO2010/
  • the ionizable lipid is MC3 (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- tetraen-19-yl-4-(dimethylamino) butanoate (DLin-MC3-DMA or MC3) having the following structure: .
  • the lipid Dlin-MC3-DMA is described in Jayaraman et al., Angew. Chem. Int. Ed Engl.
  • the ionizable lipid is the lipid ATX-002 having the following structure: .
  • the lipid ATX-002 is described in WO2015/074085, content of which is incorporated herein by reference in its entirety.
  • the ionizable lipid is (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16- dien-1-amine (Compound 32) having the following structure: . 101 ME148589913v.1 Compound 32 is described in WO2012/040184, content of which is incorporated herein by reference in its entirety.
  • the ionizable lipid is Compound 6 or Compound 22 having the following structure: Compounds 6 and 22 are described in WO2015/199952, content of which is incorporated herein by reference in its entirety.
  • ionizable lipid can comprise 20-90% (mol) of the total lipid present in the lipid nanoparticle.
  • ionizable lipid molar content can be 20-70% (mol), 30-60% (mol) or 40-50% (mol) of the total lipid present in the lipid nanoparticle.
  • ionizable lipid comprises from about 50 mol % to about 90 mol % of the total lipid present in the lipid nanoparticle.
  • the lipid nanoparticle can further comprise a non-cationic lipid.
  • Non-ionic lipids include amphipathic lipids, neutral lipids and anionic lipids. Accordingly, the non-cationic lipid can be a neutral uncharged, zwitterionic, or anionic lipid. Non-cationic lipids are typically employed to enhance fusogenicity.
  • non-cationic lipids include, but are not limited to, distearoyl-sn-glycero- phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (
  • acyl groups in these lipids are preferably acyl groups derived from fatty acids having C 10 -C 24 carbon chains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.
  • non-cationic lipids suitable for use in the lipid nanoparticles include nonphosphorous lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethyl ammonium bromide, ceramide, sphingomyelin, and the like.
  • nonphosphorous lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stereate,
  • the non-cationic lipid is a phospholipid. In some embodiments, the non-cationic lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some preferred embodiments, the non-cationic lipid is DPSC. Exemplary non-cationic lipids are described in PCT Publication WO2017/099823 and US patent publication US2018/0028664, the contents of both of which are incorporated herein by reference in their entirety. In some examples, the non-cationic lipid is oleic acid or a compound of by reference in its entirety. The non-cationic lipid can comprise 0-30% (mol) of the total lipid present in the lipid nanoparticle.
  • the non-cationic lipid content is 5-20% (mol) or 10-15% (mol) of the total lipid present in the lipid nanoparticle.
  • the molar ratio of ionizable lipid to the neutral lipid ranges from about 2:1 to about 8:1.
  • the lipid nanoparticles do not comprise any phospholipids.
  • the lipid nanoparticle can further comprise a component, such as a sterol, to provide membrane integrity.
  • a component such as a sterol
  • One exemplary sterol that can be used in the lipid nanoparticle is cholesterol and derivatives thereof.
  • Non-limiting examples of cholesterol derivatives include polar analogues such as 5 ⁇ -cholestanol, 5 ⁇ -coprostanol, cholesteryl-(2′-hydroxy)-ethyl ether, cholesteryl-(4′-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as 5 ⁇ -cholestane, cholestenone, 5 ⁇ - cholestanone, 5 ⁇ -cholestanone, and cholesteryl decanoate; and mixtures thereof.
  • the cholesterol derivative is a polar analogue such as cholesteryl-(4′-hydroxy)-butyl ether.
  • the component providing membrane integrity can comprise 0-50% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, such a component is 20-50% (mol) 30-40% (mol) of the total lipid content of the lipid nanoparticle.
  • the lipid nanoparticle can further comprise a polyethylene glycol (PEG) or a conjugated lipid molecule or a lipid-anchored polymer. Generally, these are used to inhibit aggregation of lipid nanoparticles and/or provide steric stabilization.
  • 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.
  • the conjugated lipid molecule is a PEG- lipid conjugate, for example, a (methoxy polyethylene glycol)-conjugated lipid.
  • Exemplary lipid-anchored polymer that are PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-O- (2’,3’-di(tetradecanoyloxy)propyl-1-O-(w-methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-
  • lipid-anchored polymer that is a PEG-lipid is a compound of Formula , Formula (III-a-I), 104 ME148589913v.1 , as defined in US2018/0028664, the content of which is incorporated herein by reference in its entirety.
  • a PEG-lipid is of Formula (II), , as defined in US20150376115 or in US2016/0376224, the content of both of which is incorporated herein by reference in its entirety.
  • the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG- dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl.
  • the PEG-lipid can be one or more of PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG- disterylglycamide, PEG-cholesterol (1-[8’-(Cholest-5-en-3[beta]-oxy)carboxamido-3’,6’- dioxaoctanyl] carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4- Ditetradecoxylbenzyl- [omega]-methyl-poly(ethylene glycol) ether), and 1,2-dimyristoyl-sn-glycero- 3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000].
  • the PEG-lipid can be selected from the group consisting of PEG-DMG, 1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], , 105 ME148589913v.1 .
  • Lipid-anchored polymers whereby the lipids are conjugated with a molecule other than a PEG can also be used in place of PEG-lipid.
  • polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic-polymer lipid (CPL) conjugates can be used in place of or in addition to the PEG-lipid.
  • lipid-anchored polymers or conjugated lipids i.e., PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates and cationic polymer-lipids are described in the PCT patent application publications WO1996/010392, WO1998/051278, WO2002/087541, WO2005/026372, WO2008/147438, WO2009/086558, WO2012/000104, WO2017/117528, WO2017/099823, WO2015/199952, WO2017/004143, WO2015/095346, WO2012/000104, WO2012/000104, and WO2010/006282, US patent application publications US2003/0077829, US2005/0175682, US2008/0020058, US2011/0117125, US2013/0303587, US2018/0028664, US2015/0376115, US2016/0376224, US2016/0317458, US2013/0303587, US2013
  • the PEG or the conjugated lipid or the lipid-anchored polymer can comprise 0-20% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, PEG or the conjugated lipid content is 0.5-10% or 2-5% (mol) of the total lipid present in the lipid nanoparticle.
  • Further exemplary lipid-anchored polymers are described in International Patent Application Publication No. WO2021/046265; the contents of all of which are incorporated herein by reference in their entirety. Molar ratios of the ionizable lipid, non-cationic lipid, sterol, and PEG/conjugated lipid can be varied as needed.
  • the lipid particle can comprise 30-70% ionizable lipid by mole or by total weight of the composition, 0-60% cholesterol by mole or by total weight of the composition, 0- 30% non-cationic lipid by mole or by total weight of the composition and 1-10% conjugated lipid by mole or by total weight of the composition.
  • the composition comprises 30-40% ionizable lipid by mole or by total weight of the composition, 40-50% cholesterol by mole or by total weight of the composition, and 10-20% non-cationic lipid by mole or by total weight of the composition.
  • the composition is 50-75% ionizable lipid by mole or by total weight of the composition, 20-40% cholesterol by mole or by total weight of the composition, and 5 to 10% non- 106 ME148589913v.1 cationic lipid, by mole or by total weight of the composition and 1-10% conjugated lipid by mole or by total weight of the composition.
  • the composition may contain 60-70% ionizable lipid by mole or by total weight of the composition, 25-35% cholesterol by mole or by total weight of the composition, and 5-10% non-cationic lipid by mole or by total weight of the composition.
  • the composition may also contain up to 90% ionizable lipid by mole or by total weight of the composition and 2 to 15% non-cationic lipid by mole or by total weight of the composition.
  • the formulation may also be a lipid nanoparticle formulation, for example comprising 8-30% ionizable lipid by mole or by total weight of the composition, 5-30% non-cationic lipid by mole or by total weight of the composition, and 0-20% cholesterol by mole or by total weight of the composition; 4-25% ionizable lipid by mole or by total weight of the composition, 4-25% non-cationic lipid by mole or by total weight of the composition, 2 to 25% cholesterol by mole or by total weight of the composition, 10 to 35% conjugate lipid by mole or by total weight of the composition, and 5% cholesterol by mole or by total weight of the composition; or 2-30% ionizable lipid by mole or by total weight of the composition, 2-30% non- cationic lipid by mole or by total weight of the composition
  • the lipid particle formulation comprises ionizable lipid, phospholipid, cholesterol and a PEG-ylated lipid in a molar ratio of 50:10:38.5:1.5. In some other embodiments, the lipid particle formulation comprises ionizable lipid, cholesterol and a PEG-ylated lipid in a molar ratio of 60:38.5:1.5.
  • the lipid particle comprises ionizable lipid, non-cationic lipid (e.g., phospholipid), a sterol (e.g., cholesterol) and a PEG-ylated lipid, where the molar ratio of lipids ranges from 20 to 70 mole percent for the ionizable lipid, with a target of 40-60, the mole percent of non-cationic lipid ranges from 0 to 30, with a target of 0 to 15, the mole percent of sterol ranges from 20 to 70, with a target of 30 to 50, and the mole percent of PEG-ylated lipid ranges from 1 to 6, with a target of 2 to 5.
  • non-cationic lipid e.g., phospholipid
  • a sterol e.g., cholesterol
  • PEG-ylated lipid e.g., PEG-ylated lipid
  • Lipid nanoparticles comprising ceDNA are disclosed in International Application PCT/US2018/050042, filed on September 7, 2018, which is incorporated herein in its entirety and envisioned for use in the methods and compositions as disclosed herein.
  • Lipid nanoparticle particle size can be determined by quasi-elastic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK) and is approximately 50-150 nm diameter, approximately 55-95 nm diameter, or approximately 70-90 nm diameter.
  • the pKa of formulated cationic lipids can be correlated with the effectiveness 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 (20 l 0), both of which are 107 ME148589913v.1 incorporated by reference in their entirety).
  • the preferred range of pKa is ⁇ 5 to ⁇ 7.
  • the pKa of each cationic lipid is determined in lipid nanoparticles using an assay based on fluorescence of 2-(p- toluidino)-6-napthalene sulfonic acid (TNS).
  • Lipid nanoparticles comprising 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 as described herein and elsewhere.
  • TNS can be prepared as a 100 ⁇ M stock solution in distilled water.
  • Vesicles can be diluted to 24 ⁇ M lipid in 2 mL of buffered solutions containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, 130 mM NaCl, where the pH ranges from 2.5 to 11.
  • TNS solution An aliquot of the TNS solution can be added to give a final concentration of 1 ⁇ M and following vortex mixing fluorescence intensity is measured at room temperature in a SLM Aminco Series 2 Luminescence Spectrophotometer using excitation and emission wavelengths of 321 nm and 445 nm. A sigmoidal best fit analysis can be applied to the fluorescence data and the pKa is measured as the pH giving rise to half-maximal fluorescence intensity. Relative activity can be determined by measuring luciferase expression in the liver 4 hours following administration via tail vein injection. The activity is compared at a dose of 0.3 and 1.0 mg ceDNA/kg and expressed as ng luciferase/g liver measured 4 hours after administration.
  • a lipid nanoparticle of the disclosure includes a lipid formulation that can be used to deliver a capsid-free, non-viral DNA vector to a target site of interest (e.g., cell, tissue, organ, and the like).
  • a target site of interest e.g., cell, tissue, organ, and the like.
  • the lipid nanoparticle comprises capsid-free, non-viral DNA vector and an ionizable lipid or a salt thereof.
  • the lipid particle comprises ionizable lipid / non-cationic lipid / sterol / conjugated lipid at a molar ratio of approximately 50:10:38.5:1.5.
  • the lipid particle comprises ionizable lipid / non-cationic lipid / sterol / conjugated lipid at a molar ratio of approximately 50.0:7.0:40.0:3.0.
  • the disclosure provides for a lipid nanoparticle formulation comprising phospholipids, lecithin, phosphatidylcholine and phosphatidylethanolamine. Further exemplary lipid nanoparticle compositions are described in International Patent Application Publication No. WO2021/046265; the contents of all of which are incorporated herein by reference in their entirety.
  • one or more additional compounds can also be included. Those compounds can be administered separately or the additional compounds can be included in the lipid nanoparticles of the disclosure.
  • the lipid nanoparticles can contain other compounds in addition to the ceDNA or at least a second ceDNA, different than the first.
  • additional compounds can be selected from the group consisting of small or large organic or inorganic molecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and derivatives thereof, peptidomimetics, nucleic 108 ME148589913v.1 acids, nucleic acid analogs and derivatives, an extract made from biological materials, or any combinations thereof.
  • the one or more additional compound can be a therapeutic agent.
  • the therapeutic agent can be selected from any class suitable for the therapeutic objective.
  • the therapeutic agent can be selected from any class suitable for the therapeutic objective.
  • the therapeutic agent can be selected according to the treatment objective and biological action desired.
  • the additional compound can 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).
  • the additional compound can be an antimicrobial agent (e.g., an antibiotic or antiviral compound).
  • the additional compound can be a compound that modulates an immune response (e.g., an immunosuppressant, immunostimulatory compound, or compound modulating one or more specific immune pathways).
  • an immunosuppressant e.g., an immunosuppressant, immunostimulatory compound, or compound modulating one or more specific immune pathways.
  • different cocktails of different lipid nanoparticles containing different compounds, such as a ceDNA encoding a different protein or a different compound, such as a therapeutic may be used in the compositions and methods of the disclosure.
  • the additional compound is an immune modulating agent.
  • the additional compound is an immunosuppressant.
  • the additional compound is immunostimulatory.
  • a pharmaceutical composition comprising the lipid nanoparticle and a pharmaceutically acceptable carrier or excipient.
  • the disclosure provides for a lipid nanoparticle formulation further comprising one or more pharmaceutical excipients.
  • the lipid nanoparticle formulation further comprises sucrose, tris, trehalose and/or glycine.
  • the lipid nanoparticles of the disclosure have a mean diameter selected to provide an intended therapeutic effect.
  • the lipid nanoparticle has a mean diameter from about 30 nm to about 150 nm, more typically from 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.
  • the disclosure provides for lipid particles that are larger in relative size to common nanoparticles and about 150 to 250 nm in size. Lipid nanoparticle particle size can be determined by quasi-elastic light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, UK) system.
  • the proportions of the components can vary and the delivery efficiency of a particular formulation can be measured using, for example, an endosomal release parameter (ERP) assay.
  • ERP endosomal release parameter
  • the ceDNA can be complexed with the lipid portion of the particle or encapsulated in the lipid position of the lipid nanoparticle.
  • the ceDNA can be fully encapsulated in the lipid position of the lipid nanoparticle, thereby protecting it from degradation by a nuclease, e.g., in an aqueous solution.
  • the ceDNA in the lipid nanoparticle is not substantially degraded after exposure of the lipid nanoparticle to a nuclease at 37°C. for at least about 20, 30, 45, or 60 minutes. In some embodiments, the ceDNA in the lipid nanoparticle is not substantially degraded after incubation of the particle in serum at 37 o C. for at least about 30, 45, or 60 minutes or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In certain embodiments, the lipid nanoparticles are substantially non-toxic to a subject, e.g., to a mammal such as a human. In some aspects, the lipid nanoparticle formulation is a lyophilized powder.
  • lipid nanoparticles are solid core particles that possess at least one lipid bilayer.
  • the lipid nanoparticles have a non-bilayer structure, i.e., a non- lamellar (i.e., non-bilayer) morphology.
  • the non-bilayer morphology can include, for example, three dimensional tubes, rods, cubic symmetries, etc.
  • the non-lamellar morphology (i.e., non-bilayer structure) of the lipid particles can be determined using analytical techniques known to and used by those of skill in the art.
  • Such techniques include, but are not limited to, Cryo- Transmission Electron Microscopy (“Cryo-TEM”), Differential Scanning calorimetry (“DSC”), X- Ray Diffraction, and the like.
  • Cryo-TEM Cryo- Transmission Electron Microscopy
  • DSC Differential Scanning calorimetry
  • X- Ray Diffraction X- Ray Diffraction
  • the morphology of the lipid nanoparticles (lamellar vs. non-lamellar) can readily be assessed and characterized using, e.g., Cryo-TEM analysis as described in US2010/0130588, the content of which is incorporated herein by reference in its entirety.
  • the lipid nanoparticles having a non-lamellar morphology are electron dense.
  • the disclosure provides for a lipid nanoparticle that is either unilamellar or multilamellar in structure.
  • the disclosure provides for a lipid nanoparticle formulation that comprises multi-vesicular particles and/or foam-based particles.
  • a lipid nanoparticle formulation that comprises multi-vesicular particles and/or foam-based particles.
  • lipid particle size can be controlled by controlling the composition and concentration of the lipid conjugate.
  • the pKa of formulated cationic lipids can be correlated with the effectiveness of the LNPs for delivery of nucleic acids (see Jayaraman et al, Angewandte Chemie, International Edition (2012), 110 ME148589913v.1 51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176 (20 l 0), both of which are incorporated by reference in their entirety).
  • the preferred range of pKa is ⁇ 5 to ⁇ 7.
  • the pKa of the cationic lipid can be determined in lipid nanoparticles using an assay based on fluorescence of 2-(p- toluidino)-6-napthalene sulfonic acid (TNS).
  • Encapsulation of ceDNA in lipid particles 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, for example, an Oligreen ® assay or PicoGreen ® assay.
  • 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 nonionic detergent.
  • a ceDNA vector can be delivered to a target cell in vitro or in vivo by various suitable methods. ceDNA vectors alone can be applied or injected. CeDNA vectors can be delivered to a cell without the help of a transfection reagent or other physical means.
  • ceDNA vectors can be delivered using any art-known transfection reagent or other art-known physical means that facilitates entry of DNA into a cell, e.g., liposomes, alcohols, polylysine- rich compounds, arginine-rich compounds, calcium phosphate, microvesicles, microinjection, electroporation and the like.
  • transductions with capsid-free AAV vectors disclosed herein can efficiently target cell and tissue-types that are difficult to transduce with conventional AAV virions using various delivery reagents.
  • a ceDNA vector is administered to the CNS (e.g., to the brain or to the 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 (corpus striatum, cerebrum including the occipital, temporal, parietal and frontal lobes, cortex, basal ganglia, hippocampus and portaamygdala), limbic system, neocortex, corpus 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 further be administered intravascularly to the CNS in situations in which the blood-brain barrier has been perturbed (e.g., brain tumor or cerebral infarct).
  • the ceDNA vector can be administered to the desired region(s) of the CNS by any route known in the art, including but not limited to, intrathecal, intra-ocular, 111 ME148589913v.1 intracerebral, intraventricular, intravenous (e.g., in the presence of a sugar such as mannitol), intranasal, intra-aural, intra-ocular (e.g., intra-vitreous, sub-retinal, anterior chamber) and peri-ocular (e.g., sub-Tenon's region) delivery as well as intramuscular delivery with retrograde delivery to motor neurons.
  • intrathecal intra-ocular, 111 ME148589913v.1 intracerebral
  • intraventricular intravenous (e.g., in the presence of a sugar such as mannitol)
  • intravenous e.g., in the presence of a sugar such as mannitol
  • intra-aural e.g., intra-vitreous, sub
  • the ceDNA vector is administered in a liquid formulation by direct injection (e.g., stereotactic injection) to the desired region or compartment in the CNS.
  • the ceDNA vector can be provided by topical application to the desired region or by intra-nasal administration of an aerosol formulation. Administration to the eye may be by topical application of liquid droplets.
  • the ceDNA vector can be administered as a solid, slow-release formulation (see, e.g., U.S. Pat. No.7,201,898).
  • 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.).
  • the ceDNA vector can be delivered to muscle tissue from which it can migrate into neurons.
  • IX. Methods of Use The ceDNA vector of the disclosure can be used in a method for the delivery of a nucleotide sequence of interest to a target cell. The method may in particular be a method for delivering a therapeutic gene of interest to a cell of a subject in need thereof.
  • a method for the delivery of a nucleic acid of interest in a cell of a subject can comprise the administration to said subject of a ceDNA vector of the disclosure comprising said nucleic acid of interest.
  • the disclosure provides a method for the delivery of a nucleic acid of interest in a cell of a subject in need thereof, comprising multiple administrations of the ceDNA vector of the disclosure comprising said nucleic acid of interest. Since the ceDNA vector of the disclosure does not induce an immune response, such a multiple administration strategy will not be impaired by the host immune system response against the ceDNA vector of the disclosure, contrary to what is observed with encapsidated vectors.
  • the ceDNA vector nucleic acid(s) are administered in sufficient amounts to transfect the cells of a desired tissue and to provide sufficient levels of gene transfer and expression without undue adverse effects.
  • routes of administration include, but are not limited to, intravenous (e.g., in a liposome formulation), direct delivery to the selected organ (e.g., intraportal delivery to the liver), intramuscular, and other parental routes of administration. Routes of administration may be combined, if desired.
  • 112 ME148589913v.1 ceDNA vector delivery is not limited to one species of ceDNA vector. As such, in another aspect, multiple ceDNA vectors comprising different exogenous DNA sequences can be delivered simultaneously or sequentially to the target cell, tissue, organ, or subject. Therefore, this strategy can allow for the expression of multiple genes.
  • the disclosure also provides for a method of treating a disease in a subject comprising introducing into a target cell in need thereof (in particular a muscle cell or tissue) of the subject a therapeutically effective amount of a ceDNA vector, optionally with a pharmaceutically acceptable carrier. While the ceDNA vector can be introduced in the presence of a carrier, such a carrier is not required.
  • the ceDNA vector implemented comprises a nucleotide sequence of interest useful for treating the disease.
  • the ceDNA vector may comprise a desired exogenous DNA sequence operably linked to control elements capable of directing transcription of the desired polypeptide, protein, or oligonucleotide encoded by the exogenous DNA sequence when introduced into the subject.
  • the ceDNA vector can be administered via any suitable route as provided above, and elsewhere herein.
  • XI. Methods of Treatment The technology described herein also demonstrates methods for making, as well as methods of using the disclosed ceDNA vectors in a variety of ways, including, for example, ex situ, in vitro and in vivo applications, methodologies, diagnostic procedures, and/or gene therapy regimens.
  • a method of treating a disease or disorder in a subject comprising introducing into a target cell in need thereof (for example, a muscle cell or tissue, or other affected cell type) of the subject a therapeutically effective amount of a ceDNA vector, optionally with a pharmaceutically acceptable carrier.
  • a target cell in need thereof for example, a muscle cell or tissue, or other affected cell type
  • a pharmaceutically acceptable carrier for example, a pharmaceutically acceptable carrier.
  • the ceDNA vector can be introduced in the presence of a carrier, such a carrier is not required.
  • the ceDNA vector implemented comprises a nucleotide sequence of interest useful for treating the disease.
  • the ceDNA vector may comprise a desired exogenous DNA sequence operably linked to control elements capable of directing transcription of the desired polypeptide, protein, or oligonucleotide encoded by the exogenous DNA sequence when introduced into the subject.
  • the ceDNA vector can be administered via any suitable route as provided above, and elsewhere herein. Any transgene may be delivered by the ceDNA vectors as disclosed herein.
  • Transgenes of interest include nucleic acids encoding polypeptides, or non-coding nucleic acids (e.g., RNAi, miRs etc.) preferably therapeutic (e.g., for medical, diagnostic, or veterinary uses) or immunogenic (e.g., for vaccines) polypeptides.
  • the transgenes to be expressed by the ceDNA vectors described herein will express or encode one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNAs, 113 ME148589913v.1 RNAis, antisense oligonucleotides, antisense polynucleotides, antibodies, antigen binding fragments, or any combination thereof.
  • the transgene can encode one or more therapeutic agent(s), including, but not limited to, for example, protein(s), polypeptide(s), peptide(s), enzyme(s), antibodies, antigen binding fragments, as well as variants, and/or active fragments thereof, agonists, antagonists, mimetics for use in the treatment, prophylaxis, and/or amelioration of one or more symptoms of a disease, dysfunction, injury, and/or disorder.
  • the disease, dysfunction, trauma, injury and/or disorder is a human disease, dysfunction, trauma, injury, and/or disorder.
  • the transgene can encode a therapeutic protein or peptide, or therapeutic nucleic acid sequence or therapeutic agent, including but not limited to one or more agonists, antagonists, anti-apoptosis factors, inhibitors, receptors, cytokines, cytotoxins, erythropoietic agents, glycoproteins, growth factors, growth factor receptors, hormones, hormone receptors, interferons, interleukins, interleukin receptors, nerve growth factors, neuroactive peptides, neuroactive peptide receptors, proteases, protease inhibitors, protein decarboxylases, protein kinases, protein kinase inhibitors, enzymes, receptor binding proteins, transport proteins or one or more inhibitors thereof, serotonin receptors, or one or more uptake inhibitors thereof, serpins, serpin receptors, tumor suppressors, diagnostic molecules, chemotherapeutic agents, cytotoxins, or any combination thereof.
  • a transgene in the expression cassette, expression construct, or ceDNA vector described herein can be codon optimized for the host cell.
  • the term “codon optimized” or “codon optimization” refers to the process of modifying a nucleic acid sequence for enhanced expression in the cells of the vertebrate of interest, e.g., mouse or human (e.g., humanized), by replacing at least one, more than one, or a significant number of codons of the native sequence (e.g., a prokaryotic sequence) with codons that are more frequently or most frequently used in the genes of that vertebrate.
  • Various species exhibit particular bias for certain codons of a particular amino acid.
  • codon optimization does not alter the amino acid sequence of the original translated protein.
  • Optimized codons can be determined using e.g., Aptagen's Gene Forge® codon optimization and custom gene synthesis platform (Aptagen, Inc.) or another publicly available database.
  • the ceDNA vector expresses the transgene in a subject host cell.
  • the subject host cell is a human host cell, including, for example blood cells, stem cells, hematopoietic cells, CD34 + cells, liver cells, cancer cells, vascular cells, muscle cells, pancreatic cells, neural cells, ocular or retinal cells, epithelial or endothelial cells, dendritic cells, fibroblasts, or any other cell of mammalian origin, including, without limitation, hepatic (i.e., liver) cells, lung cells, cardiac cells, pancreatic cells, intestinal cells, diaphragmatic cells, renal (i.e., kidney) cells, neural cells, blood cells, bone marrow cells, or any one or more selected tissues of a subject for which gene therapy is contemplated.
  • the subject host cell is a human host cell.
  • 114 ME148589913v.1 Disclosed herein are ceDNA vector compositions and formulations that include one or more of the ceDNA vectors of the present disclosure together with one or more pharmaceutically- acceptable buffers, diluents, or excipients. Such compositions may be included in one or more diagnostic or therapeutic kits, for diagnosing, preventing, treating or ameliorating one or more symptoms of a disease, injury, disorder, trauma or dysfunction.
  • the disease, injury, disorder, trauma or dysfunction is a human disease, injury, disorder, trauma or dysfunction.
  • Another aspect of the technology described herein provides a method for providing a subject in need thereof with a diagnostically- or therapeutically-effective amount of a ceDNA vector, the method comprising providing to a cell, tissue or organ of a subject in need thereof, an amount of the ceDNA vector as disclosed herein; and for a time effective to enable expression of the transgene from the ceDNA vector thereby providing the subject with a diagnostically- or a therapeutically-effective amount of the protein, peptide, nucleic acid expressed by the ceDNA vector.
  • the subject is human.
  • Another aspect of the technology described herein provides a method for diagnosing, preventing, treating, or ameliorating at least one or more symptoms of a disease, a disorder, a dysfunction, an injury, an abnormal condition, or trauma in a subject.
  • the method includes at least the step of administering to a subject in need thereof one or more of the disclosed ceDNA vectors, in an amount and for a time sufficient to diagnose, prevent, treat or ameliorate the one or more symptoms of the disease, disorder, dysfunction, injury, abnormal condition, or trauma in the subject.
  • the subject is human.
  • Another aspect is use of the ceDNA vector as a tool for treating or reducing one or more symptoms of a disease or disease states.
  • deficiency states usually of enzymes, which are generally inherited in a recessive manner
  • unbalanced states which may involve regulatory or structural proteins, and which are typically but not always inherited in a dominant manner.
  • ceDNA vectors can be used to deliver transgenes to bring a normal gene into affected tissues for replacement therapy, as well, in some embodiments, to create animal models for the disease using antisense mutations.
  • ceDNA vectors can be used to create a disease state in a model system, which could then be used in efforts to counteract the disease state.
  • ceDNA vectors and methods disclosed herein permit the treatment of genetic diseases.
  • a disease state is treated by partially or wholly remedying the deficiency or imbalance that causes the disease or makes it more severe.
  • the ceDNA vector as disclosed herein can be used to deliver any transgene to treat, prevent, or ameliorate the symptoms associated with any disorder related to gene expression.
  • Illustrative disease states include, but are not-limited to: cystic fibrosis (and other diseases of the lung), hemophilia A, hemophilia B, thalassemia, anemia and other blood disorders, AIDS, Alzheimer’s disease, Parkinson’s disease, Huntington's disease, amyotrophic lateral sclerosis, 115 ME148589913v.1 epilepsy, and other neurological disorders, cancer, diabetes mellitus, muscular dystrophies (e.g., Duchenne, Becker), Hurler’s disease, adenosine deaminase deficiency, metabolic defects, retinal degenerative diseases (and other diseases of the eye), mitochondriopathies (e.g., Leber’s hereditary optic neuropathy (LHON), Leigh syndrome, and subacute sclerosing encephalopathy), myopathies (e.g., facioscapulohumeral myopathy (FSHD) and cardiomyopathies), diseases of solid organs (e.g.
  • the ceDNA vectors as disclosed herein can be advantageously used in the treatment of individuals with metabolic disorders (e.g., omithine transcarbamylase deficiency).
  • the ceDNA vector described herein can be used to treat, ameliorate, and/or prevent a disease or disorder caused by mutation in a gene or gene product.
  • Exemplary diseases or disorders that can be treated with a ceDNA vectors include, but are not limited to, metabolic diseases or disorders (e.g., Fabry disease, Gaucher disease, phenylketonuria (PKU), glycogen storage disease); urea cycle diseases or disorders (e.g., ornithine transcarbamylase (OTC) deficiency); lysosomal storage diseases or disorders (e.g., metachromatic leukodystrophy (MLD), mucopolysaccharidosis Type II (MPSII; Hunter syndrome)); liver diseases or disorders (e.g., progressive familial intrahepatic cholestasis (PFIC); blood diseases or disorders (e.g., hemophilia (A and B), thalassemia, and anemia); cancers and tumors, and genetic diseases or disorders (e.g., cystic fibrosis).
  • metabolic diseases or disorders e.g., Fabry disease, Gaucher disease, phenylketonuria (PKU
  • a ceDNA vector as disclosed herein may be employed to deliver a heterologous nucleotide sequence in situations in which it is desirable to regulate the level of transgene expression (e.g., transgenes encoding hormones or growth factors, as described herein).
  • the transgene may inhibit a pathway that controls the expression or activity of a target gene.
  • the transgene may enhance the activity of a pathway that controls the expression or activity of a target gene.
  • the ceDNA vector described herein can be used to correct an abnormal level and/or function of a gene product (e.g., an absence of, or a defect in, a protein) that results in the disease or disorder.
  • the ceDNA vector can produce a functional protein and/or modify levels of the protein to alleviate or reduce symptoms resulting from, or confer benefit to, a particular disease or disorder caused by the absence or a defect in the protein.
  • treatment of OTC deficiency can be achieved by producing functional OTC enzyme;
  • treatment of hemophilia A and B can be achieved by modifying levels of Factor VIII, Factor IX, and Factor X;
  • treatment of PKU can be achieved by modifying levels of phenylalanine hydroxylase enzyme;
  • treatment of Fabry or Gaucher disease can be achieved by producing functional alpha galactosidase or beta glucocerebrosidase, respectively;
  • treatment of MLD or MPSII can be achieved by producing functional arylsulfatase A or iduronate-2-sulfatase, respectively;
  • treatment of cystic fibrosis can be achieved by producing functional cystic fibrosis transmembrane conductance regulator;
  • treatment of glycogen storage disease
  • the ceDNA vectors as disclosed herein can be used to provide an antisense nucleic acid to a cell in vitro or in vivo.
  • the transgene is a RNAi molecule
  • expression of the antisense nucleic acid or RNAi in the target cell diminishes expression of a particular protein by the cell.
  • transgenes which are RNAi molecules or antisense nucleic acids may be administered to decrease expression of a particular protein in a subject in need thereof.
  • Antisense nucleic acids may also be administered to cells in vitro to regulate cell physiology, e.g., to optimize cell or tissue culture systems.
  • exemplary transgenes encoded by the ceDNA vector include, but are not limited to: lysosomal enzymes (e.g., hexosaminidase A, associated with Tay-Sachs disease, or iduronate sulfatase, associated, with Hunter Syndrome/MPS II), erythropoietin, angiostatin, endostatin, superoxide dismutase, globin, leptin, catalase, tyrosine hydroxylase, as well as cytokines (e.g., a interferon, ⁇ -interferon, interferon- ⁇ , interleukin-2, interleukin-4, interleukin 12, granulocyte- macrophage colony stimulating factor, lymphotoxin, and the like), peptide growth factors and hormones (e.g., somatotropin, insulin, insulin-like growth factors 1 and 2, platelet derived growth factor (PDGF), epidermal growth factor (EG), epidermal
  • the transgene encodes a monoclonal antibody specific for one or more desired targets. In some exemplary embodiments, more than one transgene is encoded by the ceDNA vector. In some exemplary embodiments, the transgene encodes a fusion protein comprising two different polypeptides of interest. In some embodiments, the transgene encodes an antibody, including a full-length antibody or antibody fragment, as defined herein. In some embodiments, the antibody is an antigen-binding domain or an immunoglobulin variable domain sequence, as that is defined herein.
  • transgene sequences encode suicide gene products (thymdine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, and tumor necrosis factor), proteins conferring resistance to a drug used in cancer therapy, and tumor suppressor gene products.
  • suicide gene products thymdine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, and tumor necrosis factor
  • the transgene expressed by the ceDNA vector can be used for the treatment of muscular dystrophy in a subject in need thereof, the method comprising: administering a treatment-, amelioration- or prevention-effective amount of ceDNA vector described herein, wherein the ceDNA vector comprises a heterologous nucleic acid encoding dystrophin, a mini- dystrophin, a micro-dystrophin, myostatin propeptide, follistatin, activin type II soluble receptor, IGF- 1, anti-inflammatory polypeptides such as the Ikappa B dominant mutant, sarcospan, utrophin, a micro-dystrophin, laminin- ⁇ 2, ⁇ -sarcoglycan, ⁇ -sarcoglycan, ⁇ -sarcoglycan, ⁇ -sarcoglycan, IGF-1, an antibody or antibody fragment against myostatin or myostatin propeptide, and/or RNAi against 117 ME148589913v.
  • the ceDNA vector can be administered to skeletal, diaphragm and/or cardiac muscle as described elsewhere herein.
  • the ceDNA vector can be used to deliver a transgene to skeletal, cardiac or diaphragm muscle, for production of a polypeptide (e.g., an enzyme) or functional RNA (e.g., RNAi, microRNA, antisense RNA) that normally circulates in the blood or for systemic delivery to other tissues to treat, ameliorate, and/or prevent a disorder (e.g., a metabolic disorder, such as diabetes (e.g., insulin), hemophilia (e.g., VIII), a mucopolysaccharide disorder (e.g., Sly syndrome, Hurler Syndrome, Scheie Syndrome, Hurler-Scheie Syndrome, Hunter’s Syndrome, Sanfilippo Syndrome A, B, C, D, Morquio Syndrome, Maroteaux-Lamy Syndrome, etc.) or a lysosomal storage disorder (such as Gaucher’s disease [
  • a metabolic disorder such
  • the ceDNA vector as disclosed herein can be used to deliver a transgene in a method of treating, ameliorating, and/or preventing a metabolic disorder in a subject in need thereof.
  • Illustrative metabolic disorders and transgenes encoding polypeptides are described herein.
  • the polypeptide is secreted (e.g., a polypeptide that is a secreted polypeptide in its native state or that has been engineered to be secreted, for example, by operable association with a secretory signal sequence as is known in the art).
  • Another aspect of the disclosure relates to a method of treating, ameliorating, and/or preventing congenital heart failure or PAD in a subject in need thereof, the method comprising administering a ceDNA vector as described herein to a mammalian subject, wherein the ceDNA vector comprises a transgene encoding, for example, a sarcoplasmic endoreticulum Ca 2+ -ATPase (SERCA2a), an angiogenic factor, phosphatase inhibitor I (I-1), RNAi against phospholamban; a phospholamban inhibitory or dominant-negative molecule such as phospholamban S16E, a zinc finger protein that regulates the phospholamban gene, ⁇ 2-adrenergic receptor, .beta.2-adrenergic receptor kinase (BARK), PI3 kinase, calsarcan, a .beta.-adrenergic receptor kinase inhibitor ( ⁇ ARKct), inhibitor
  • the ceDNA vectors as disclosed herein can be administered to the lungs of a subject by any suitable means, optionally by administering an aerosol suspension of respirable particles comprising the ceDNA vectors, which the subject inhales.
  • the respirable particles can be liquid or solid. Aerosols of liquid particles comprising the ceDNA vectors may be produced by any suitable means, such as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as is known to those of skill in the art. See, e.g., U.S. Pat. No.4,501,729. Aerosols of solid particles comprising the ceDNA vectors may 118 ME148589913v.1 likewise be produced with any solid particulate medicament aerosol generator, by techniques known in the pharmaceutical art.
  • the ceDNA vectors can be administered to tissues of the CNS (e.g., brain, eye).
  • the ceDNA vectors as disclosed herein may be administered to treat, ameliorate, or prevent diseases of the CNS, including genetic disorders, neurodegenerative disorders, psychiatric disorders and tumors.
  • Illustrative diseases of the CNS include, but are not limited to, Alzheimer’s disease, Parkinson’s disease, Huntington's disease, Canavan disease, Leigh’s disease, Refsum disease, Tourette syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, Pick’s disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger's disease, trauma due to spinal cord or head injury, Tay Sachs disease, Lesch- Nyan disease, epilepsy, cerebral infarcts, psychiatric disorders including mood disorders (e.g., depression, bipolar affective disorder, persistent affective disorder, secondary mood disorder), schizophrenia, drug dependency (e.g., alcoholism and other substance dependencies), neuroses (e.g., anxiety, obsessional disorder, somatoform disorder, dissociative disorder, grief, post-partum depression), psychosis (e.g., hallucinations and delusions), dementia, paranoia, attention deficit disorder, psychosexual
  • Ocular disorders that may be treated, ameliorated, or prevented with the ceDNA vectors of the disclosure include ophthalmic disorders involving the retina, posterior tract, and optic nerve (e.g., retinitis pigmentosa, diabetic retinopathy and other retinal degenerative diseases, uveitis, age-related macular degeneration, glaucoma). Many ophthalmic diseases and disorders are associated with one or more of three types of indications: (1) angiogenesis, (2) inflammation, and (3) degeneration.
  • the ceDNA vector as disclosed herein can be employed to deliver anti-angiogenic factors; anti-inflammatory factors; factors that retard cell degeneration, promote cell sparing, or promote cell growth and combinations of the foregoing.
  • Diabetic retinopathy for example, is characterized by angiogenesis. Diabetic retinopathy can be treated by delivering one or more anti- angiogenic factors either intraocularly (e.g., in the vitreous) or periocularly (e.g., in the sub-Tenon’s region). One or more neurotrophic factors may also be co-delivered, either intraocularly (e.g., intravitreally) or periocularly.
  • Additional ocular diseases that may be treated, ameliorated, or prevented with the ceDNA vectors of the disclosure include geographic atrophy, vascular or “wet” macular degeneration, Stargardt disease, Leber Congenital Amaurosis (LCA), Usher syndrome, pseudoxanthoma elasticum (PXE), x-linked retinitis pigmentosa (XLRP), x-linked retinoschisis (XLRS), Choroideremia, Leber hereditary optic neuropathy (LHON), Archomatopsia, cone-rod dystrophy, Fuchs endothelial corneal dystrophy, diabetic macular edema and ocular cancer and tumors.
  • geographic atrophy vascular or “wet” macular degeneration
  • Stargardt disease Leber Congenital Amaurosis (LCA), Usher syndrome, pseudoxanthoma elasticum (PXE), x-linked retinitis pigmentosa (XLRP), x-linked retinoschisis (XLRS
  • inflammatory ocular diseases or disorders can be treated, ameliorated, or prevented by the ceDNA vectors of the disclosure.
  • One or more anti-inflammatory factors can be expressed by intraocular (e.g., vitreous or anterior chamber) administration of the ceDNA vector as disclosed herein.
  • ocular diseases or disorders characterized by retinal degeneration e.g., retinitis pigmentosa
  • retinal degeneration e.g., retinitis pigmentosa
  • Intraocular e.g., vitreal administration
  • the ceDNA vector as disclosed herein encoding one or more neurotrophic factors can be used to treat such retinal degeneration-based diseases.
  • diseases or disorders that involve both angiogenesis and retinal degeneration e.g., age-related macular degeneration
  • Age-related macular degeneration can be treated by administering the ceDNA vector as disclosed herein encoding one or more neurotrophic factors intraocularly (e.g., vitreous) and/or one or more anti-angiogenic factors intraocularly or periocularly (e.g., in the sub- Tenon's region).
  • Glaucoma is characterized by increased ocular pressure and loss of retinal ganglion cells.
  • Treatments for glaucoma include administration of one or more neuroprotective agents that protect cells from excitotoxic damage using the ceDNA vector as disclosed herein. Accordingly, such agents include N-methyl-D-aspartate (NMDA) antagonists, cytokines, and neurotrophic factors, can be delivered intraocularly, optionally intravitreally using the ceDNA vector as disclosed herein.
  • the ceDNA vector as disclosed herein may be used to treat seizures, e.g., to reduce the onset, incidence or severity of seizures.
  • the efficacy of a therapeutic treatment for seizures can be assessed by behavioral (e.g., shaking, ticks of the eye or mouth) and/or electrographic means (most seizures have signature electrographic abnormalities).
  • the ceDNA vector as disclosed herein can also be used to treat epilepsy, which is marked by multiple seizures over time.
  • somatostatin (or an active fragment thereof) is administered to the brain using the ceDNA vector as disclosed herein to treat a pituitary tumor.
  • the ceDNA vector as disclosed herein encoding somatostatin (or an active fragment thereof) is administered by microinfusion into the pituitary.
  • such treatment can be used to treat acromegaly (abnormal growth hormone secretion from the pituitary).
  • the ceDNA vector can encode a transgene that comprises a secretory signal as described in U.S. Pat. No.7,071,172.
  • Another aspect of the disclosure relates to the use of a ceDNA vector as described herein to produce antisense RNA, RNAi or other functional RNA (e.g., a ribozyme) for systemic delivery to a subject in vivo.
  • the ceDNA vector can comprise a transgene that encodes an antisense nucleic acid, a ribozyme (e.g., as described in U.S. Pat. No.5,877,022), RNAs that affect spliceosome-mediated trans-splicing (see, Puttaraju et al., (1999) Nature Biotech.17:246; U.S. Pat. No.6,013,487; U.S. Pat.
  • a transgene that encodes an antisense nucleic acid
  • a ribozyme e.g., as described in U.S. Pat. No.5,877,022
  • RNAs that affect spliceosome-mediated trans-splicing see, Puttaraju et al., (1999) Nature Biotech.17:246; U.S. Pat. No.6,013,487; U.S. Pat.
  • the ceDNA vector can further also comprise a transgene that encodes a reporter polypeptide (e.g., an enzyme such as Green Fluorescent Protein, or alkaline phosphatase).
  • a reporter polypeptide e.g., an enzyme such as Green Fluorescent Protein, or alkaline phosphatase
  • a transgene that encodes a reporter protein useful for experimental or diagnostic purposes is selected from any of: ⁇ -lactamase, ⁇ -galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art.
  • ceDNA vectors comprising a transgene encoding a reporter polypeptide may be used for diagnostic purposes or as markers of the ceDNA vector’s activity in the subject to which they are administered.
  • the ceDNA vector can comprise a transgene or a heterologous nucleotide sequence that shares homology with, and recombines with, a locus on the host chromosome. This approach may be utilized to correct a genetic defect in the host cell.
  • the ceDNA vector can comprise a transgene that can be used to express an immunogenic polypeptide in a subject, e.g., for vaccination.
  • the transgene may encode any immunogen of interest known in the art including, but not limited to, immunogens from human immunodeficiency virus, influenza virus, gag proteins, tumor antigens, cancer antigens, bacterial antigens, viral antigens, and the like.
  • more than one administration may be employed to achieve the desired level of gene expression over a period of various intervals, e.g., daily, weekly, monthly, yearly, etc.
  • exemplary modes of administration of the ceDNA vector disclosed herein includes oral, rectal, transmucosal, intranasal, inhalation (e.g., via an aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intraendothelial, in utero (or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intracranial, intramuscular [including administration to skeletal, diaphragm and/or cardiac muscle], intrapleural, intracerebral, and intraarticular), topical (e.g., to both skin and mucosal surfaces, including airway surfaces, and transdermal administration), intralymphatic, and the like, as well as
  • Administration of the ceDNA vector can be to any site in a subject, including, without limitation, a site selected from the group consisting of the brain, a skeletal muscle, a smooth muscle, the heart, the diaphragm, the airway epithelium, the liver, the kidney, the spleen, the pancreas, the skin, and the eye.
  • Administration of the ceDNA vector can also be to a tumor (e.g., in or near a tumor or a lymph node). The most suitable route in any given case will depend on the nature and severity of 121 ME148589913v.1 the condition being treated, ameliorated, and/or prevented and on the nature of the particular ceDNA vector that is being used.
  • ceDNA permits one to administer more than one transgene in a single vector, or multiple ceDNA vectors (e.g., a ceDNA cocktail).
  • Administration of the ceDNA vector disclosed herein to skeletal muscle according to the present disclosure includes but is not limited to administration to skeletal muscle in the limbs (e.g., upper arm, lower arm, upper leg, and/or lower leg), back, neck, head (e.g., tongue), thorax, abdomen, pelvis/perineum, and/or digits.
  • the ceDNA as disclosed herein vector can be delivered to skeletal muscle by intravenous administration, intra-arterial administration, intraperitoneal administration, limb perfusion, (optionally, isolated limb perfusion of a leg and/or arm; see, e.g., Arruda et al., (2005) Blood 105: 3458-3464), and/or direct intramuscular injection.
  • the ceDNA vector as disclosed herein is administered to a limb (arm and/or leg) of a subject (e.g., a subject with muscular dystrophy such as DMD) by limb perfusion, optionally isolated limb perfusion (e.g., by intravenous or intra-articular administration.
  • the ceDNA vector as disclosed herein can be administered without employing "hydrodynamic" techniques.
  • Administration of the ceDNA vector as disclosed herein to cardiac muscle includes administration to the left atrium, right atrium, left ventricle, right ventricle and/or septum.
  • the ceDNA vector as described herein can be delivered to cardiac muscle by intravenous administration, intra- arterial administration such as intra-aortic administration, direct cardiac injection (e.g., into left atrium, right atrium, left ventricle, right ventricle), and/or coronary artery perfusion.
  • Administration to diaphragm muscle can be by any suitable method including intravenous administration, intra-arterial administration, and/or intra-peritoneal administration.
  • Administration to smooth muscle can be by any suitable method including intravenous administration, intra-arterial administration, and/or intra- peritoneal administration.
  • administration can be to endothelial cells present in, near, and/or on smooth muscle.
  • a ceDNA vector according to the present disclosure is administered to skeletal muscle, diaphragm muscle and/or cardiac muscle (e.g., to treat, ameliorate and/or prevent muscular dystrophy or heart disease (e.g., PAD or congestive heart failure).
  • A. Ex vivo treatment In some embodiments, cells are removed from a subject, a ceDNA vector is introduced therein, and the cells are then replaced back into the subject.
  • a ceDNA vector is introduced into cells from another subject, into cultured cells, or into cells from any other suitable source, and the cells are administered to a subject in need thereof.
  • Cells transduced with a ceDNA vector are preferably administered to the subject in a “therapeutically-effective amount” in combination with a pharmaceutical carrier.
  • the ceDNA vector can encode a transgene (sometimes called a heterologous nucleotide sequence) that is any polypeptide that is desirably produced in a cell in vitro, ex vivo, or in vivo.
  • a transgene sometimes called a heterologous nucleotide sequence
  • the ceDNA vectors may be introduced into cultured cells and the expressed gene product isolated therefrom, e.g., for the production of antigens or vaccines.
  • the ceDNA vectors can be used in both veterinary and medical applications.
  • Suitable subjects for ex vivo gene delivery methods as described above include both avians (e.g., chickens, ducks, geese, quail, turkeys and pheasants) and mammals (e.g., humans, bovines, ovines, caprines, equines, felines, canines, and lagomorphs), with mammals being preferred.
  • Human subjects are preferred. Human subjects include neonates, infants, juveniles, and adults. Human subjects may also include fetuses.
  • One aspect of the technology described herein relates to a method of delivering a transgene to a cell.
  • the ceDNA vector may be introduced into the cell using the methods as disclosed herein, as well as other methods known in the art.
  • ceDNA vectors disclosed herein are preferably administered to the cell in a biologically-effective amount. If the ceDNA vector is administered to a cell in vivo (e.g., to a subject), a biologically-effective amount of the ceDNA vector is an amount that is sufficient to result in transduction and expression of the transgene in a target cell.
  • a biologically-effective amount of the ceDNA vector is an amount that is sufficient to result in transduction and expression of the transgene in a target cell.
  • B. Dose ranges In vivo and/or in vitro assays can optionally be employed to help identify optimal dosage ranges for use. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the condition, and should be decided according to the judgment of the person of ordinary skill in the art and each subject's circumstances.
  • Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • a ceDNA vector is administered in sufficient amounts to transfect the cells of a desired tissue and to provide sufficient levels of gene transfer and expression without undue adverse effects.
  • Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, those described above in the “Administration” section, such as direct delivery to the selected organ (e.g., intraportal delivery to the liver), oral, inhalation (including intranasal and intratracheal delivery), intraocular, intravenous, intramuscular, subcutaneous, intradermal, intratumoral, and other parental routes of administration. Routes of administration can be combined, if desired.
  • the dose of the amount of a ceDNA vector required to achieve a particular “therapeutic effect,” will vary 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 a therapeutic effect, the 123 ME148589913v.1 specific 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 a ceDNA vector dose range to treat a patient having a particular disease or disorder based on the aforementioned factors, as well as other factors that are well known in the art. [0001] Dosage regime can be adjusted to provide the optimum therapeutic response.
  • the oligonucleotide can be repeatedly administered, e.g., several doses can be administered daily or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • One of ordinary skill in the art will readily be able to determine appropriate doses and schedules of administration of the subject oligonucleotides, whether the oligonucleotides are to be administered to cells or to subjects.
  • a “therapeutically effective dose” will fall in a relatively broad range that can be determined through clinical trials and will depend on the particular application (neural cells will require very small amounts, while systemic injection would require large amounts).
  • a therapeutically effective dose will be on the order of from about 1 ⁇ g to 100 g of the ceDNA vector. If exosomes or microparticles are used to deliver the ceDNA vector, then a therapeutically effective dose can be determined experimentally, but is expected to deliver from 1 ⁇ g to about 100 g of vector.
  • Formulation of pharmaceutically-acceptable excipients and carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens.
  • an effective amount of a ceDNA vector to be delivered to cells will be on the order of 0.1 to 100 ⁇ g ceDNA vector, preferably 1 to 20 ⁇ g, and more preferably 1 to 15 ⁇ g or 8 to 10 ⁇ g. Larger ceDNA vectors will require higher doses. If exosomes or microparticles are used, an effective in vitro dose can be determined experimentally but would be intended to deliver generally the same amount of the ceDNA vector. Treatment can involve administration of a single dose or multiple doses.
  • more than one dose can be administered to a subject; in fact multiple doses can be administered as needed, because the ceDNA vector elicits does not elicit an anti-capsid host immune response due to the absence of a viral capsid.
  • the number of doses administered can, for example, be on the order of 1-100, preferably 2-20 doses.
  • the lack of typical anti-viral immune response elicited by administration of a ceDNA vector as described by the disclosure i.e., the absence of capsid components
  • the number of occasions in which a heterologous nucleic acid is delivered to a subject is in a range of 2 to 10 times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 times).
  • a ceDNA vector is delivered to a subject more than 10 times.
  • a dose of a ceDNA vector is administered to a subject no more than once per calendar day (e.g., a 24-hour period).
  • a dose of a ceDNA vector is administered to a subject no more than once per 2, 3, 4, 5, 6, or 7 calendar days.
  • a dose of a ceDNA vector is administered to a subject no more than once per calendar week (e.g., 7 calendar days). In some embodiments, a dose of a ceDNA vector is administered to a subject no more than bi-weekly (e.g., once in a two calendar week period). In some embodiments, a dose of a ceDNA vector is administered to a subject no more than once per calendar month (e.g., once in 30 calendar days). In some embodiments, a dose of a ceDNA vector is administered to a subject no more than once per six calendar months.
  • a dose of a ceDNA vector is administered to a subject no more than once per calendar year (e.g., 365 days or 366 days in a leap year).
  • the pharmaceutical compositions can be presented in unit dosage form.
  • a unit dosage form will typically be adapted to one or more specific routes of administration of the pharmaceutical composition.
  • the unit dosage form is adapted for administration by inhalation.
  • the unit dosage form is adapted for administration by a vaporizer.
  • the unit dosage form is adapted for administration by a nebulizer.
  • the unit dosage form is adapted for administration by an aerosolizer.
  • the unit dosage form is adapted for oral administration, for buccal administration, or for 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 intracerebroventricular administration. In some embodiments, the pharmaceutical composition is formulated for topical administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.
  • the double-stranded DNA constructs for the preparation of ceDNA 384 and ceDNA 558 are double- stranded DNA constructs having SEQ ID NO: 5 and SEQ ID NO: 6, shown below.
  • ceDNA 384 contains a 5’ secondary stem region and a 3’ secondary stem region that flank the transgene expression cassette.
  • ceDNA 384 contains 5’ and 3’ stem-loop regions at the 5’ and 3’ ends of the vector that are symmetric with respect to each other with each other (SEQ ID NO: 1 for 5’ stem-loop region and SEQ ID NO 2 for 3’ stem-loop region).
  • ceDNA 558 contains 5’ and 3’ stem-loop regions at the 5’ and 3’ ends of the vector that are asymmetric with respect to each other (SEQ ID NO: 1 for 5’ stem-loop region and SEQ ID NO: 4 for 3’ stem-loop region).
  • the 5’ and 3’ stem-loop regions at the 5’ and 3’ ends of a ceDNA vector of the present disclosure contain one or more loop structures, such as the hairpin loop structure in ceDNA 384 and ceDNA 558. These 5’ and 3’ stem-loop regions contain only artificial sequences and no viral ITR sequences.
  • Both of the secondary stem regions if incorporated in a ceDNA vector of the present disclosure such as in ceDNA 384, contain only artificial sequences and no viral ITR sequences, and the length of each of these secondary stem regions is equivalent to the A, A’, D, and D’ regions of a ceDNA having 5’ and 3’ AAV wt-ITRs such as reference ceDNA 382, or the A, A’ and D regions of an AAV wt-ITRs.
  • the presence of the secondary stem regions may provide some beneficial properties to the vector, such as improving or enhancing the genetic stability of the vector.
  • a base vector was digested with XbaI and NotI to release the spacer (filler or placeholder) region. The digest was then run on an agarose gel where two bands representing two fragments should be present: the placeholder region and the base vector backbone. The base vector backbone was gel extracted.
  • the transgene was PCR-amplified from a template vector carrying the cassette, for example, using a specifically designed primer with Q5 High Fidelity polymerase.
  • the PCR reaction was then treated with fast digest DpnI to eliminate the plasmid template. After that, the PCR reaction was purified using the Zymo Research DNA Clean and Concentration Kit. Through homology cloning, the base vector backbone and PCR-amplified transgene expression cassette were ligated and the ligation mixture was used to transform in E. coli and plated on selection medium. Several colonies were selected from the plate and the plasmid DNA purified from the colonies was sequenced for verification. The double-stranded construct serves as the template for the cell-free synthesis of ceDNA. Exemplary synthetic methods of preparing ceDNA 384 and ceDNA 558 are illustrated in FIGS.2A and 2B, respectively.
  • the transgene expression cassette was excised from a double- stranded DNA construct using at least one Type IIS restriction endonuclease, e.g., BsmBI. This is then 129 ME148589913v.1 followed by ligation (e.g., with a ligase such as T4 ligase or an AAV Rep protein) of the excised insert (containing the transgene expression cassette) into the 5’ and 3’ oligonucleotides (SEQ ID NO: 1 for ceDNA 384; SEQ ID NO: 1 and SEQ ID NO: 2 for ceDNA 558, which are each a single- stranded oligonucleotide that self-anneals to form a hairpin loop structure.
  • a ligase such as T4 ligase or an AAV Rep protein
  • SEQ ID NO: 8 5’- CTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGG CCTCAGG – 3’ Digestion with the Type IIS restriction endonuclease(s) such as BsmBI created cohesive overhangs at both 5’ and 3’ ends of the excised insert that are compatible with the overhangs of the 5’ and 3’ oligonucleotides, thereby facilitating ligation.
  • Type IIS restriction endonuclease(s) such as BsmBI created cohesive overhangs at both 5’ and 3’ ends of the excised insert that are compatible with the overhangs of the 5’ and 3’ oligonucleotides
  • ceDNA having no viral ITR sequences exhibited equivalent or higher in vitro GFP expression than ceDNA having AAV2 wt-ITRs 30,000 human fibroblast (HFF1) cells (with DNA sensing capability such as cGAS and STING pathways) or human embryonic kidney 293T cells (without DNA sensing capability were seeded per well of a collaged-I coated 96-well plate in 100 ⁇ L DMEM + 10% FBS.
  • DNA exonuclease treatment e.g., T5 exonuclease treatment
  • HFF1 cells were then incubated for ⁇ 24 hours at 5% CO 2 / 37°C.
  • Expression data in HFF1 cells can be a useful tool to correlate innate immune response and transgene expression; whereas 293T cells can be used to inform transgene expression independently of the innate immune response.
  • transfection reactions were set up with VIAFECT transfection reagent according to manufacturer’s instructions. 0.4 ⁇ L VIAFECT reagent was used per 100 ng of DNA for HFF1 and per 30 ngm or 90 ng, or 180 ng of 293T cells. After all transfection mixtures were normalized to molecule number at the top dose, 10-fold serial dilutions of all mixes were made using OPTIMEM media.
  • FIG.3A it was surprisingly shown that in 293T cells transfected with ceDNA 558A having no viral ITR sequences, there was a clear dose responsiveness and at a high ceDNA concentration of 180 ng, the GFP expression was higher in ceDNA 558A than reference ceDNA 382 having 5’ and 3’ AAV2 wt-ITRs.
  • FIG.3B it can be seen that the GFP expression in HFF1 cells transfected with ceDNA 558A having no viral ITR sequences was about two times higher in intensity as compared to the GFP expression in HFF1 cells transfected with reference ceDNA 382 having 5’ and 3’ AAV2 wt- ITRs.
  • Example 3 it was surprisingly shown that in 293T cells transfected with ceDNA 558A having no viral ITR sequences, there was a clear dose responsiveness and at a high ceDNA concentration of 180 ng, the GFP expression was higher in ceDNA 558A than reference ceDNA 382 having 5’ and 3’ AAV2 wt-
  • ceDNA having no viral ITR sequences exhibited equivalent or higher in vivo luciferase expression than ceDNA having AAV2 wt-ITRs ceDNA 558 and ceDNA 384 each containing no viral ITR sequences and reference ceDNA 382 were further tested for in vivo expression in mice as lipid nanoparticle (LNP) formulations.
  • LNPs lipid nanoparticles
  • Lipid nanoparticles (LNPs) were prepared at a total lipid to ceDNA weight ratio of approximately 10:1 to 30:1.
  • a cationic lipid of the present disclosure an optional non- cationic lipid (e.g., distearoylphosphatidylcholine (DSPC)), a component to provide membrane integrity (such as a sterol, e.g., cholesterol), a conjugated lipid molecule (such as a PEGylated lipid conjugate) e.g., 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol, with an average PEG molecular weight of 2000 (“DMG-PEG2000”)) and optionally a conjugate lipid molecule with a targeting moiety (e.g., 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) with an average PEG molecular weight of 2000 and conjugated to a tri-antennary or tri-valent N-acetylgalactosamine targeting ligand (DSPE-PEG2000-GalNAc3), were so
  • the ceDNA was diluted to a desired concentration in buffer solution.
  • the ceDNA was diluted to a concentration of 0.1 mg/ml to 0.25 mg/ml in a buffer solution comprising sodium acetate, sodium acetate and magnesium chloride, citrate, malic acid, or malic acid and sodium chloride.
  • the ceDNA was diluted to 0.2 mg/mL in 10 to 50 mM citrate buffer, pH 4.
  • the alcoholic lipid solution was mixed with ceDNA aqueous solution using, for example, syringe pumps or an impinging jet mixer, at a ratio of about 1:5 to 1:3 (vol/vol) with total flow rates above 10 ml/min.
  • the alcoholic lipid solution was mixed with ceDNA aqueous at a ratio of about 1:3 (vol/vol) with a flow rate of 12 ml/min.
  • the alcohol was removed, and the buffer was replaced with PBS by dialysis.
  • the buffers were replaced with PBS using centrifugal tubes. Alcohol removal and simultaneous buffer exchange were accomplished by, for example, dialysis or tangential flow filtration.
  • the obtained lipid nanoparticles are filtered through a 0.2 ⁇ m pore sterile filter.
  • Additional or alternative method of preparing LNPs are described in detail, e.g., in International Patent Application Publication Nos.
  • FIGS.5A-5L which are fluorescent images of the mice indicated that the fluorescence which resulted from luciferase transgene expression was detected in the torso area of all mice, the location of the liver.
  • FIG.4A and FIG.4B show the amount of total fluorescence measured (IVIS) for both Reference LNP and LNP1 at respectively, Day 7 and Day 14 post-dosing, for both groups of mice.
  • the results shown in FIG.4A and FIG.4B indicated that the expression was higher on both Day 7 and Day 14 in C57 BL/6 mice and compared to their immunodeficient counterparts, Rag2KO mice.
  • LNP1 containing ceDNA 558 consistently exhibited at least equivalent luciferase transgene expression with both groups of mice and on both Day 7 and Day 14, as compared to Reference LNP containing reference ceDNA 382.
  • 132 ME148589913v.1 at Day 7 the luciferase transgene expression in C57 BL/6 mice dosed with LNP1 containing ceDNA 558 was slightly higher than it was in the same type of mice dosed with Reference LNP.
  • the tolerability of Reference LNP and LNP1 was also evaluated and compared.
  • FIGS.6A and 6B indicate that for both C57 BL/6 mice and Rag2KO mice, body weight recovery was achieved by Day 28 whether the mice were dosed with Reference LNP or LNP1, with the immunodeficient Rag2KO mice recovering at a slower rate. In other words, there was no significant difference between the tolerability of Reference LNP and the tolerability of LNP1 in terms of body weight recovery in mice. Furthermore, the immunogenicity of Reference LNP and LNP1 was also evaluated and compared.
  • FIGS.7A-7E indicate that the blood serum levels of the various cytokines were largely equal across both strains of mice and both reference ceDNA 382 and ceDNA 558 constructs, with TNF- ⁇ being slightly higher in both strains of mice injected with ceDNA 558 (FIG.7D) but with IL-6 being slightly lower in both strains of mice injected with ceDNA 558 (FIG.7E).
  • Example 4
  • FIG.8 provides a schematic diagram of the ITR structures of various synthetic ceDNA constructs with one or more complete sections of the ITR regions A and A’, B and B’, C and C’, or D and D’ removed.
  • the vector “Mu” designates a mutant ITR structure with a deletion of the complete B or C regions ( ⁇ B and B’ or ⁇ C and C’).
  • “Blunt” designates a mutant ITR structure with a deletion of both the B and C arms ( ⁇ B and B’ and ⁇ C and C’), but retaining structures corresponding to A and A’, and D and D’.
  • the term “hpin” designates a mutant ceDNA with no A and A’, B and B’, C and C’, or D and D’ regions.
  • the hpin construct was represented by ceDNA558 (as shown in FIG 1B) and had minimal residual ITR comprising of 5’ and 3’ asymmetric stem loops and none of the A and A’, B and B’, C and C’, or D and D’ regions.
  • the structural ITR variant constructs were designated as follows: ceDNA178 (wt- wt); ceDNA4 (wt-wt); ceDNA14 (wt-mu); ceDNA15 (mu-mu); ceDNA16 (blunt-blunt); ceDNA17 (hpin-hpin); ceDNA18 (blunt-wt); ceDNA19 (blunt-mu); ceDNA20 (hpin-wt); ceDNA35 (wt-hpin).
  • ITR constructs were also used to help to determine whether non-viral secondary structures in ceDNA (ITRs) played a role in influencing detection by cellular proteins, and 133 ME148589913v.1 whether any ceDNA:host interactions impacted transgene expression, either in a positive or negative fashion.
  • ITRs ceDNA
  • ME148589913v.1 whether any ceDNA:host interactions impacted transgene expression, either in a positive or negative fashion.
  • a further objective of the work described herein was to evaluate the impact of the various ITR structural regions on activation of the innate immune response.
  • FIG.8 A qualitative analysis was made by Bioanalyzer and eGel of the various structural variant ITR constructs shown in FIG.8. Bioanalyzer and eGel data for the synthetic constructs showed the presence of different submonomeric bands (Data not shown). Based on these results, it was thought that the DNA in these submonomeric bands could influence the activation of the innate immune response.
  • the results of transfection and expression of ceDNA variants in human leukemia monocytic cells (THP-1) and human fibroblast cell line (HFF1) cells is shown in FIG.9A and FIG.9B. THP-1 cells robustly responded to DNA (FIG.9B), but the transfection efficiency with LNPs was very low ( ⁇ 1-5%).
  • FIG.9A shows the results of transfection of THP-1 human leukemia monocytic cells with LNPs (i) and transfection of adherent HFF1 cells with LNPs.
  • FIG.9B shows the results of Western blot that demonstrated both THP-1 cells and HFF1 cells responded to ceDNA, as shown by the expression of DNA sensing proteins, cGAS and STING. 293T human embryonic kidney cells, with no DNA sensing pathway, were used as a control. HFF1 cells showed high expression of cGAS and STING (DNA sensing pathway). As shown in FIG.9A and FIG.9B, THP1 cells robustly responded to DNA, but the transfection efficiency with LNPs is very low ( ⁇ 1-5%). These results demonstrated that HFF1 fibroblasts can be a useful tool to correlate innate immune response and transgene expression.
  • FIG.10 The performance of the various structural variant ITR constructs tested in FIG.9 was assessed in an expression assay and the results are shown in FIG.10 and FIG.11.
  • FIG.10 In the absence of DNA sensing pathway, all of the structural variant ITR constructs expressed some protein in 293T cells FIG.10.
  • cGAS and STING pathway all of the structural variant ITR constructs showed some capacity to express protein as shown in HFF1 cells, but ceDNA17 (hpin-hpin) and ceDNA20 (hpin-wt) showed the highest levels of expression in HFF1 cells (FIG.11).
  • IFN bioassay is an in vitro assay used to quantify the presence of active type IFN-I in the supernatant of cells. This assay can help to quantitatively evaluate the innate immune response observed in different cell types from the same species without the need to perform an ELISA.
  • FIG.12 is a graph that shows the results of analysis of different ceDNA structural variant ITR constructs shown in FIG.8 by IFN bioassay in THP1-ISG reporter cells (1E5 THP1 cells/well).
  • ISG promoter activation was determined by transferring supernatant from 293T cells and supernatant transferred from HFF1 cells. As shown in FIG.12, 293T cells did not show secretion of IFN-I after DNA transfection. This correlated with the high GFP expression across all constructs (compared to HFF1 cells). In HFF cells, ceDNA20 (hpin-wt), ceDNA16 (blunt-blunt), ceDNA 19 (blunt-mut) showed significantly lower activation of the THP1-ISG cells (FIG.12). Supernatant from cells transfected with the same DNA mass of ceDNA178 and pceDNA178 (plasmid ceDNA) induced the lowest activation in THP1-ISG cells.
  • FIG.13A and FIG.13B show the results of analysis of ceDNA structural variant ITR constructs shown in FIG.8 by Bioassay, and a correlation of transgene expression and IFN response.
  • FIG.13A is a graph that shows transgene expression in human HFF1 fibroblasts. In this example, 150 ng/DNA was used per well.
  • ceDNA4 wt-wt
  • ceDNA15 mu-mu
  • the low GFP expression observed for ceDNA178 can be due to lower presence of monomer in the total DNA preparation (approximately 1/4 of total DNA).
  • ceDNA16 (blunt-blunt) showed the highest expression level and this suggests there is an inherent ability to be expressed for even a minimal expression cassette.
  • the information contained in the 5’ and 3’ ITRs may be necessary for modulating expression under various conditions but the initial expression cassette seems to contain an inherent capacity for expression.
  • ceDNA20 (hpin-wt) with no ITR in the 5’ end and a WT ITR in the 3’ end showed the highest transgene expression which correlated with a mild activation of the innate immune response.
  • FIG.14A and FIG.14B are graphs that show the correlation of transgene expression and innate immune response in ceDNA4 (wt-wt) and ceDNA20 (hpin-wt) constructs.
  • ceDNA20 which lacks the left ITR (5’end), expressed higher levels of transgene (GFP), and as shown in FIG.14B, ceDNA20 was also less immunogenic than ceDNA4 (wt-wt) according to IFN bioassay-THP1 ISG. Based on the results from this comparison, a ceDNA having one ITR seemed to be the more desirable vector.
  • Example 5 Effect of ITR Position on Transgene Expression
  • the effect of ITR position on a ceDNA and whether an ITR is needed at all were investigated.
  • ceDNA35 structural variant ITR as the 136 ME148589913v.1 opposite of ceDNA20 structural variant ITR (hpin -wt) where they each have one complete ITR.
  • the vector ceDNA35 has a 5’ left ITR and ceDNA20 has a complete 3’ right ITR.
  • the comparison also includes ceDNA17 without either ITR and also known as “hpin- hpin”.
  • ceDNA4 was included as a fully “wild-type” version with both 5’ left ITR and 3’ right ITRs.
  • Human fibroblast (HFF1) or human retinal epithelial cells (RPE) were transfected with different ceDNA constructs (150ng/well).
  • FIG.15A is a graph that shows the expression of ceDNA20 (hpin -wt) having 3’ left, wt-ITR and ceDNA35 with opposing ITR (wt- hpin) having 5’ right wt-ITR configurations in HFF1 cells.
  • the ceDNA20 (hpin -wt) 3’ left ITR expresses significantly more transgene (GFP) than the rest of the construct designs.
  • FIG.15B is a graph that shows the results obtained in the interferon bioassay in THP1 -ISG reporter cells using these 4 vector designs.
  • GFP expression in retinal pigment epithelial (RPE) was conducted for ceDNA4 (wt-wt), ceDNA17 (hpin- hpin), ceDNA20 (hpin -wt) and ceDNA35 (wt- hpin) and is shown in FIG.16A and FIG.16B.
  • FIG.16A and FIG.16B the higher level of transgene expression that was observed for ceDNA20 in HFF1 cells was also reproduced in human RPE cells.
  • GFP expression from ceDNA4, ceDNA17, ceDNA20 and ceDNA35 was conducted in RAW-ISG murine macrophage cell line and is shown in FIG.17A and FIG.17B.
  • FIG.17A no significant difference in immunogenicity was seen across the constructs.
  • ceDNA4 WT/WT
  • ceDNA17 hpin-hpin
  • ceDNA had more potency for the GFP construct, and there was a higher presence of monomer (3/4).
  • Synthetic ceDNA production in insect cells was further confirmed by transient transfection and GFP detection (not shown).
  • the next step was to test the concept of ceDNA amplification in Sf9 cells using ceDNA as substrate.
  • the purpose of the study was to be a proof of concept for ceDNA amplification in Sf9 cells 137 ME148589913v.1 and to test the requirement of ITRs for amplification.
  • a hypothesis was that using ceDNA will decrease the presence of non-ceDNA species.
  • Sf9 cells were transiently transfected with synthetic ceDNA constructs as follows: Lane 1- ceDNA1-Luc (wt-wt) Lane 2- ceDNA36-Luc (hpin-wt) Lane 3- ceDNA4-GFP (wt-wt) Lane 4- ceDNA20-GFP (hpin-wt) Plasmid DNA (pDNA) was used as a control for the conventional production method.
  • FIG. 19 shows the results of synthetic ceDNA production in insect cells by transient transfection and the absence of detectable non-ceDNA species. A second experiment was performed as a proof of concept for production of ceDNA in Sf9 by transient transfection, using synthetic constructs as substrate.
  • FIG.20 shows the results of Western blot. These results showed that Sf9 cells can amplify ceDNA constructs by transient transfection independently of ORF (GFP and Luc).
  • ceDNA with one ITR (right) can still be amplified in SF9 cells (no ceDNA dimer was being produced under this experimental condition). Furthermore, ceDNA produced from synthetic ceDNA starting materials showed significantly less DNA bands than ceDNA produced from plasmid (lanes 1 and 2 vs lanes 3-6), particularly for the sub-monomeric Exo5 resistant species.

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Abstract

Sont décrits dans la présente invention des vecteurs d'ADN non viraux et exempts de capside comportant des extrémités fermées de manière covalente (vecteurs d'ADNce) présentant une structure linéaire et continue qui peuvent être produits avec des rendements élevés et utilisés aux fins d'un transfert et d'une expression efficaces d'un transgène. Les vecteurs d'ADNce décrits dans la présente invention comprennent au moins une cassette d'expression transgénique et ne comprennent pas de séquences de répétition terminale inversée (ITR) virales. Certains vecteurs d'ADNce proposés dans la présente invention comprennent en outre des éléments cis-régulateurs et assurent des efficacités d'expression génique élevées. La présente invention concerne en outre des compositions et des compositions pharmaceutiques comprenant des nanoparticules lipidiques (LNP). La présente invention concerne en outre des procédés et des lignées cellulaires permettant une production fiable et efficace des vecteurs d'ADN linéaires, continus et exempts de capside.
PCT/US2024/030986 2023-05-26 2024-05-24 Vecteurs d'adn à extrémité fermée (adnce) modifiés, compositions et utilisations associées Pending WO2024249298A2 (fr)

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