Micro-strand DNA for production of adeno-associated viruses

Abstract

Provided herein are expression vectors, vector production systems, vectors free of bacterial sequences, and methods of producing adeno-associated viruses (AAVs). Also provided herein are AAVs as well as pharmaceutical compositions and uses thereof.

Description

[0001] Cross-reference to related applications

[0002] This PCT application claims priority to U.S. Provisional Application No. 63 / 382,070, filed on November 2, 2022, the entire contents of which are incorporated herein by reference.

[0003] Reference to electronically submitted sequence listings

[0004] The contents of the electronically submitted Sequence Listing (Name: 4471_003PC03_Seqlisting_ST26; Size: 155348 bytes; Creation Date: October 31, 2023) are incorporated herein by reference in their entirety. Field of the Invention

[0005] The present disclosure provides vectors and methods for producing adeno-associated virus (AAV) from ministring DNA. Background Art

[0006] AAV is a small, non-pathogenic virus containing a linear, single-stranded DNA genome packaged in a non-enveloped viral capsid. It is a parvovirus of the family Parvoviridae and a member of the genus Dependoparvovirus that requires the functions provided by a co-infected helper virus for efficient replication. See, for example, Daya and Berms, Clin. Microbiol. Rev. 21(4): 583-593 (2008); Lisowski et al., Curr. Opin. Pharmacol. 24: 59-67 (2015); Mary et al., Adeno-associated Virus Vectors in Gene Therapy, Gene and Cell Therapy: Biology and Applications (Jayandharan G. ed., 2018).

[0007] The wild-type AAV genome is approximately 5 kilobases long and encodes eight proteins from the replication (rep) gene and the capsid (cap) gene. Four Rep proteins (Rep40, Rep52, Rep68, and Rep78) and three Cap proteins (VP1, VP2, and VP3) and assembly activation protein (AAP) are expressed from the Rep and Cap genes via alternative promoters and as splice variants. Rep proteins are involved in replication and packaging, while Cap proteins and AAP are involved in the formation of the viral capsid. See, for example, Lisowski et al.; Salganik et al., Microbiol. Spectrum 3(4):MDNA3-0052-2014(2015).

[0008] Two inverted terminal repeats (ITRs) are located on either side of the AAV genome coding sequence and are required for replication and packaging. The end of each wild-type ITR contains a self-annealing palindromic region, resulting in a double-stranded T-shaped hairpin structure at each end of the AAV genome. This hairpin uses the DNA polymerase complex of the infected cell as the starting point for AAV DNA replication and complementary chain synthesis. Either the sense strand or the antisense strand of the double-stranded replication intermediate can be packaged into the viral capsid as a single-stranded genome. See, for example, Daya and Berns, Lisowski et al.; Ling et al., J. Mol. Genet. Med. 9(3): 175 (2015); Salganik et al.

[0009] Recombinant AAV can be produced by replacing the AAV genome between the ITRs with a nucleic acid sequence of interest and has been widely used in clinical and basic research. The standard method for producing recombinant AAV requires transfection of mammalian or insect AAV producer cells with a vector containing the nucleic acid sequence of interest flanked by ITRs and a separate helper vector or virus that provides the necessary AAV rep / cap and helper virus functions. For example, a typical production method involves transfecting human embryonic kidney 293 (HEK293) cells with three plasmids: one plasmid contains the nucleic acid sequence between the two ITRs, one plasmid contains the AAV rep gene and cap gene, and one plasmid contains the adenovirus helper gene. By providing the rep / cap sequence and helper sequence in trans, the nucleic acid of interest between the ITRs is packaged into the capsid to form recombinant AAV.

[0010] The production of AAV using existing methods is highly variable, inefficient, and often results in the packaging of unwanted DNA. See, e.g., Wright, JF, Gene Therapy 15:840-848 (2008). For example, 50-95% of AAV particles produced using standard methods are empty AAV capsids that do not contain any packaging DNA. See, e.g., Sommer et al., Mol. Ther. 7(1):122-128 (2003). Even when capsids are packaged, they may contain nucleic acids other than or in addition to the desired target nucleic acid sequence, such as auxiliary sequences, production cell sequences, and / or bacterial sequences from plasmids containing ITRs. These contaminating sequences can account for 1% to 8% of the total DNA in purified AAV particles and can lead to potential immunogenicity and / or carcinogenicity. See, e.g., Wright, JF. To date, prior art purification strategies have failed to remove these contaminating sequences from AAV vector preparations.

[0011] There is a need for improved AAV vectors and production systems. Summary of the Invention

[0012] The present disclosure relates to an expression vector comprising: (a) a first sequence comprising an inverted terminal repeat (ITR) and a multiple cloning site (MCS), wherein the ITR flanks at least one side of the MCS, and wherein the ITR comprises an adeno-associated virus (AAV) replication sequence and an AAV packaging signal, (b) a target sequence for a first recombinase flanking each side of the first sequence, and (c) one or more additional target sequences for one or more additional recombinases, the one or more additional target sequences being integrated within a non-binding region of the target sequence of the first recombinase, wherein the expression vector is used to generate a bacterial sequence-free vector (i.e., "expression vector A") having linear covalently closed ends. In some aspects, the ITR flanks only one side of the MCS. In some aspects, the ITR flanks each side of the MCS. In some aspects, the expression vector further comprises a spacer sequence between the target sequence of the first recombinase and the first sequence. In some aspects, the spacer sequence is 10 to 500 nucleotides. In some aspects, the expression vector further comprises an expression cassette comprising an AAV replication (rep) gene and an AAV capsid (cap) gene, flanked on one side by a target sequence for the first recombinase and on the other side by the first sequence.

[0013] The present disclosure relates to an expression vector comprising: (a) a first sequence comprising an ITR and an expression cassette comprising a nucleic acid sequence of interest, wherein the ITRs flank at least one side of the expression cassette comprising the nucleic acid sequence of interest, and wherein the ITRs comprise an AAV replication sequence and an AAV packaging signal, (b) a target sequence for a first recombinase flanking each side of the first sequence, and (c) one or more additional target sequences for one or more additional recombinases, the one or more additional target sequences being integrated within non-binding regions of the target sequence for the first recombinase, wherein the expression vector is used to generate a bacterial sequence-free vector having linear covalently closed ends (i.e., "expression vector B").

[0014] In some aspects, the ITR is located only on one side of the expression cassette containing the target nucleic acid sequence in expression vector B (i.e., "expression vector B1"). In some aspects, the expression vector further comprises a spacer sequence between the target sequence of the first recombinase and the first sequence. In some aspects, the spacer sequence is 10 to 500 nucleotides.

[0015] In some aspects, expression vector B1 further comprises an expression cassette comprising an AAV rep gene and an AAV cap gene, flanked on one side by the target sequence of the first recombinase and on the other side by the first sequence (ie, "expression vector B2").

[0016] In some aspects, the ITRs are located on each side of the expression cassette comprising the target nucleic acid sequence in expression vector B (i.e., "expression vector B3"). In some aspects, the expression vector further comprises a spacer sequence between the target sequence of the first recombinase and the first sequence. In some aspects, the spacer sequence is 10 to 500 nucleotides.

[0017] In some aspects, expression vector B3 further comprises an expression cassette comprising an AAV rep gene and an AAV cap gene, flanked on one side by the target sequence of the first recombinase and on the other side by the first sequence (ie, "expression vector B4").

[0018] The present disclosure relates to an expression vector comprising: (a) a first sequence comprising an ITR and a palindrome, wherein the ITRs flank each side of the palindrome, wherein the palindrome comprises an expression cassette containing a target nucleic acid sequence and a complementary sequence to the expression cassette, and wherein the ITRs comprise an AAV replication sequence and an AAV packaging signal, (b) a target sequence for a first recombinase flanking each side of the first sequence, and (c) one or more additional target sequences for one or more additional recombinases, wherein the one or more additional target sequences are integrated within a non-binding region of the target sequence of the first recombinase, wherein the expression vector is used to generate a bacterial sequence-free vector with linear covalent closed ends (i.e., "expression vector C"). In some aspects, the complementary sequence is separated from the expression cassette comprising the target nucleic acid sequence by a non-complementary spacer sequence. In some aspects, the expression vector further comprises a spacer sequence between the target sequence of the first recombinase and the first sequence. In some aspects, the spacer sequence is 10 to 500 nucleotides.

[0019] In some aspects, expression vector C further comprises an expression cassette comprising an AAV rep gene and an AAV cap gene, flanked on one side by the target sequence of the first recombinase and on the other side by the first sequence (ie, "expression vector C1").

[0020] The present disclosure relates to an expression vector comprising: (a) a first sequence comprising a portion of an expression cassette containing a nucleic acid sequence of interest flanked on one side by a splicing sequence, an ITR flanking each of the first sequence, wherein the ITR comprises an AAV replication sequence and an AAV packaging signal, (b) a target sequence for a first recombinase flanking each ITR, and (c) one or more additional target sequences for one or more additional recombinases, wherein the one or more additional target sequences are integrated within a non-binding region of the target sequence of the first recombinase, wherein the expression vector is used to produce a bacterial sequence-free vector having linear covalently closed ends (i.e., "expression vector D"). In some aspects, the portion of the expression cassette comprises a 5' portion that, in combination with the remainder of the expression cassette, provides the complete sequence of the expression cassette, and the splicing sequence is located 3' to the 5' portion. In some aspects, the portion of the expression cassette comprises a 3' portion that, in combination with the remainder of the expression cassette, provides the complete sequence of the expression cassette, and the splicing sequence is located 5' to the 3' portion. In some aspects, the expression vector further comprises a spacer sequence between the target sequence of the first recombinase and the first sequence. In some aspects, the spacer sequence is 10 to 500 nucleotides.

[0021] In some aspects, the AAV replication sequence in any of the above expression vectors comprises an AAV ITR replication (Rep) protein binding element (RBE) and a terminal dissociation site (TRS).

[0022] In some aspects, the AAV packaging signal in any of the above expression vectors comprises an AAV ITR D sequence.

[0023] The present disclosure relates to an expression vector comprising: (a) an expression cassette comprising an AAV rep gene and an AAV cap gene, (b) target sequences for a first recombinase flanking each side of the expression cassette, and (c) one or more additional target sequences for one or more additional recombinases, the one or more additional target sequences being integrated within non-binding regions of the target sequence for the first recombinase, wherein the expression vector is used to produce a bacterial sequence-free vector having linear covalently closed ends (i.e., "expression vector E").

[0024] The present disclosure relates to an expression vector comprising: (a) an expression cassette comprising one or more helper virus genes for producing AAV, (b) a target sequence for a first recombinase flanking each side of the expression cassette, and (c) one or more additional target sequences for one or more additional recombinases, the one or more additional target sequences being integrated within the non-binding region of the target sequence of the first recombinase, wherein the expression vector is used to produce a bacterial sequence-free vector with linear covalent closed ends (i.e., "expression vector F"). In some aspects, the one or more helper virus genes are from an adenovirus, a herpes virus, a retrovirus, a pox virus, and / or a lentivirus. In some aspects, the one or more helper virus genes comprise an adenovirus early 4 (E4) gene, an adenovirus early 2A (E2A) gene, and an adenovirus virus-associated (VA) gene.

[0025] In some aspects, the target sequence of the first recombinase and the one or more additional target sequences of the one or more additional recombinases in any of the above expression vectors are selected from the group consisting of the PY54 pal site, the N15 telRL site, and the In some aspects, any of the above expression vectors comprises each of these target sequences. In some aspects, any of the expression vectors comprises a Tel recombinase pal site and a telRL recombinase target binding sequence integrated into the pal site.

[0026] In some aspects, the target sequence for the first recombinase in any of the above expression vectors is the bacteriophage PY54 Tel 142 base pair target site.

[0027] The present disclosure relates to a vector production system comprising a recombinant cell designed to encode at least a first recombinase under the control of an inducible promoter, wherein the cell comprises any of the above-mentioned expression vectors BF. In some aspects, the inducible promoter is heat-regulated, chemically regulated, IPTG-regulated, glucose-regulated, arabinose-induced, T7 polymerase-regulated, cold-shock-induced, pH-induced, or a combination thereof. In some aspects, the first recombinase is selected from TelN and Tel, and the expression vector integrates the target sequence of at least the first recombinase. In some aspects, the recombinant cell has been further designed to encode a nuclease genome editing system, and wherein the expression vector further comprises a backbone sequence containing a cleavage site for the nuclease genome editing system. In some aspects, the nuclease genome editing system is a CRISPR nuclease system comprising a Cas nuclease and a gRNA, and the expression vector comprises the target sequence of the gRNA in the backbone sequence.

[0028] The present disclosure relates to a method of producing a bacterial sequence-free vector having linear covalently closed ends, comprising incubating any of the above-described vector production systems under conditions suitable for expression of a first recombinase.

[0029] The present disclosure relates to a method for producing a bacterial sequence-free vector having linear covalently closed ends, comprising incubating any of the above-described vector production systems under conditions suitable for expressing a first recombinase and a nuclease genome editing system. In some aspects, the method further comprises harvesting the bacterial sequence-free vector.

[0030] The present disclosure relates to a bacterial sequence-free vector produced by any of the above-mentioned methods for producing a bacterial sequence-free vector with linear covalently closed ends. In some aspects, the bacterial sequence-free vector is produced from expression vector B1. In some aspects, the bacterial sequence-free vector is produced from expression vector B2. In some aspects, the bacterial sequence-free vector is produced from expression vector B3. In some aspects, the bacterial sequence-free vector is produced from expression vector B4. In some aspects, the bacterial sequence-free vector is produced from expression vector C. In some aspects, the bacterial sequence-free vector is produced from expression vector C1. In some aspects, the bacterial sequence-free vector is produced from expression vector D. In some aspects, the bacterial sequence-free vector is produced from expression vector E. In some aspects, the bacterial sequence-free vector is produced from expression vector F.

[0031] The present disclosure relates to a method for producing single-chain AAV, wherein the method comprises: (a) transfecting cells capable of producing AAV with: (i) a bacterial sequence-free vector generated from expression vector B3, (ii) a bacterial sequence-free vector generated from expression vector E, or an expression vector comprising an expression cassette comprising an AAV rep gene and an AAV cap gene, and (iii) a bacterial sequence-free vector generated from expression vector F, or an expression vector comprising an expression cassette comprising one or more helper virus genes for producing AAV; and (b) incubating the cells under conditions suitable for producing AAV.

[0032] The present disclosure relates to a method for producing single-chain AAV, wherein the method comprises: (a) transfecting cells capable of producing AAV with: (i) a bacterial sequence-free vector generated from expression vector B4, (ii) a bacterial sequence-free vector generated from expression vector F, or an expression vector comprising an expression cassette containing one or more helper virus genes for producing AAV; and (b) incubating the cells under conditions suitable for producing AAV.

[0033] The present disclosure relates to a method for producing single-chain AAV, wherein the method comprises: (a) transfecting cells capable of producing AAV with a bacterial sequence-free vector generated from expression vector B3, wherein each AAV rep gene, AAV cap gene and one or more helper virus genes for producing AAV are encoded by the cells or by the vector; and (b) incubating the cells under conditions suitable for expressing the rep gene, cap gene and one or more helper virus genes and suitable for producing AAV.

[0034] The present disclosure relates to a method for producing self-complementary AAV, wherein the method comprises: (a) transfecting cells capable of producing AAV with: (i) a bacterial sequence-free vector generated from expression vector B1; (ii) a bacterial sequence-free vector generated from expression vector E, or an expression vector comprising an expression cassette comprising an AAV rep gene and an AAV cap gene; and (iii) a bacterial sequence-free vector generated from expression vector F, or an expression vector comprising an expression cassette comprising one or more helper virus genes for producing AAV, and (b) incubating the cells under conditions suitable for producing AAV.

[0035] The present disclosure relates to a method for producing self-complementary AAV, wherein the method comprises: (a) transfecting cells capable of producing AAV with: (i) a bacterial sequence-free vector generated from expression vector B2, (ii) a bacterial sequence-free vector generated from expression vector F, or an expression vector comprising an expression cassette containing one or more helper virus genes for producing AAV, and (b) incubating the cells under conditions suitable for producing AAV.

[0036] The present disclosure relates to a method for producing self-complementary AAV, wherein the method comprises: (a) transfecting cells capable of producing AAV with: (i) a bacterial sequence-free vector generated from expression vector C, (ii) a bacterial sequence-free vector generated from expression vector E, or an expression vector comprising an expression cassette comprising an AAV rep gene and an AAV cap gene, and (iii) a bacterial sequence-free vector generated from expression vector F, or an expression vector comprising an expression cassette comprising one or more helper virus genes for producing AAV, and (b) incubating the cells under conditions suitable for producing AAV.

[0037] The present disclosure relates to a method for producing self-complementary AAV, wherein the method comprises: (a) transfecting cells capable of producing AAV with: (i) a bacterial sequence-free vector generated from expression vector C1, (ii) a bacterial sequence-free vector generated from expression vector F, or an expression vector comprising an expression cassette containing one or more helper virus genes for producing AAV, and (b) incubating the cells under conditions suitable for producing AAV.

[0038] The present disclosure relates to a method for producing self-complementary AAV, wherein the method comprises: (a) transfecting cells capable of producing AAV with a bacterial sequence-free vector generated from expression vector B1 or C, wherein each AAV rep gene, AAV cap gene and one or more helper viral genes for producing AAV are encoded by the cells or by the vector, and (b) incubating the cells under conditions suitable for expressing the rep gene, cap gene and one or more helper viral genes and suitable for producing AAV.

[0039] In some aspects, the cells used in any of the above methods for producing single-chain AAV or self-complementary AAV are HEK293T cells.

[0040] In some aspects, any of the above methods for producing single-stranded AAV or self-complementary AAV further comprises harvesting the AAV.

[0041] The present disclosure relates to AAV produced by any of the above methods for producing single-chain AAV or self-complementary AAV.

[0042] The present disclosure relates to pharmaceutical compositions comprising the above-described AAV.

[0043] The present disclosure relates to methods of treating a disease or condition in a subject in need thereof, comprising administering to the subject the aforementioned AAV or the aforementioned pharmaceutical composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1Shown is a map of an exemplary expression vector comprising an inverted terminal repeat (ITR) sequence flanking each side of an expression cassette encoding green fluorescent protein (GFP), and specialized supersequence sites (interchangeably represented in the figure as SS or SSeq) containing a recombinase target sequence flanking each side of the ITR-cassette-ITR sequence.

[0045] Figure 2 Shown by Figure 1 Map of an exemplary ministranded DNA (msDNA) encoding GFP produced by the indicated expression vector.

[0046] Figure 3 A map of an exemplary msDNA is shown, in which only the 3' side of the expression cassette is flanked by ITRs.

[0047] Figure 4 Shown is a map of an exemplary expression vector comprising an SS flanked on each side by expression cassettes encoding AAV replication (Rep) and capsid (Cap) sequences.

[0048] Figure 5 Shown by Figure 4 Maps of exemplary msDNA encoding Rep and Cap sequences produced by the indicated expression vectors.

[0049] Figure 6 A map of an exemplary msDNA encoding helper sequences is shown.

[0050] Figure 7 Shown is a map of an exemplary expression vector comprising an expression cassette encoding GFP flanked on each side by ITR sequences and SSeqs flanked on each side by ITR-expression cassette-ITR sequences.

[0051] Figure 8 Shown by Figure 7 Maps of exemplary msDNAs produced by the indicated expression vectors.

[0052] Figures 9A-9D Shows the use Figure 7 expression vector (AAV ITR pDNA) or Figure 8 msDNA (AAV ITR msDNA) was transfected into HEK293 cells on the second day ( Figure 9A and Figure 9B ) and day 6 ( Figure 9C and Figure 9D ) of the transfection efficiency (TE) ( Figure 9A and Figure 9C ), median fluorescence intensity ( Figure 9B ) and cell survival rate ( Figure 9D ) histogram. Figures 9A-9C The arrow in indicates the highest value observed for msDNA. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, and ns = not significant.

[0053] Figure 10 Shows Figures 9A-9D Micrographs of GFP expression in the transfected cells. Cell nuclei were indicated by staining with diamidino-2-phenylindole (DAPI).

[0054] Figure 11 A map of an exemplary ITR-SacB-CmR-ITR expression cassette encoding SacB protein and chloramphenicol acetyltransferase is shown.

[0055] Figures 12A-12B Representative images of sucrose plates from cells transformed with ITR-sacB-CmR-ITRLCC DNA generated in vitro by PCR (Taq and Q5) or RCA (Phi29) or with msDNA generated in vivo in E. coli (MBI2) are shown ( Figure 12A ) and histograms of mutation rates ( Figure 12B ). ( Figure 9B ) The bar graph shows the mean of three biological replicates, and the error bars show one standard deviation. (Compared with MBI2) One-way ANOVA with Dunnett's test: **** = p < 0.0001.

[0056] Figure 13 Shown is a schematic diagram of AAV production in which the conventional GOI-containing plasmid is replaced by msDNA.

[0057] Figure 14 Shown is a map of an example plasmid without SSeq that contains ITR sequences flanking each side of an expression cassette encoding GFP.

[0058] Figure 15 The use of conventional helper plasmids, conventional Rep2 / Cap2 plasmids and Figure 8 msDNA ("msDNA") or Figure 14 Photomicrographs of GFP expression in samples of 35 mL cultures 72 hours after transfection with mixtures of the indicated plasmids ("pDNA") at a molar ratio of 1:2:1, 2:1.5:1, or 1.4:1.5:1.

[0059] Figure 16 For transfectants containing msDNA, conventional helper plasmids, conventional Rep2 / Cap1 plasmids (for AAV1 production) or conventional Rep2 / Cap2 plasmids (for AAV2 production), and Figure 8mixture of the indicated msDNA ("msDNA") at a molar ratio of 1.4:1.5:1; or for transfectants containing pDNA, a conventional helper plasmid, a conventional Rep2 / Cap1 plasmid (for AAV1 production) or a conventional Rep2 / Cap2 plasmid (for AAV2 production), and Figure 14 Photomicrographs of GFP expression in samples from 150 mL cultures used for AAV1 or AAV2 production 72 hours after transfection with a mixture of the indicated plasmids ("pDNA") at a molar ratio of 2:1.5:1.

[0060] Figure 17A and 17B Shows Figure 16 The cell viability in the sample is expressed as % of living cells ( Figure 17A ), and viable cell density (VCD), with a value of 10 6 The concentration is expressed as cells / mL ( Figure 17B ).

[0061] Figure 18 Shown are the results of a 72-hour harvest after transfection. Figure 15 After the described culture, the AAV2 vector genomes / mL (VG / mL) titer was determined by droplet digital PCR (ddPCR) of the GOI.

[0062] Figure 19A and 19B Shown 72 hours after harvest, as Figure 16 The method for producing AAV1 ( Figure 19A ) and AAV2( Figure 19B ) affinity chromatography chromatograms of cultures transfected with msDNA. The upper line in each graph is the absorbance at 280 nm, while the lower line is the absorbance at 260 nm. The amount "VP / mL" indicates the concentration of vector particles per milliliter of eluate, and the percentage indicates the proportion of particles packaged with DNA.

[0063] Figure 20A and 20B Shown 72 hours after harvest, as Figure 16 The method for producing AAV1 ( Figure 20A ) and AAV2( Figure 20B ) of the culture transfected with pDNA. The lines, "VP / mL" and percentages are as follows Figure 19A and 19B As stated.

[0064] Figure 21A A micrograph of an electrophoresis gel is shown, with the capsid proteins VP1, VP2, and VP3 shown as three corresponding bands at the top and bottom of each lane, respectively. Figure 19B and20B The indicated msDNA and pDNA AAV2 cultures were detected by ddPCR after affinity chromatography.

[0065] Figure 21B Shows Figures 19A-19B Bar graphs of AAV1 and AAV2 titers expressed in vector genomes / mL (VG / mL) after affinity chromatography of msDNA and pDNA AAV cultures as described in Figures 20A-20B.

[0066] Figure 22-23 Shown separately Figure 19A and 20A Chromatogram of anion exchange (AEX) chromatography of the affinity chromatography capture shown. VP / mL and percentage are as shown Figures 19A-19B Peak #1 in each figure includes particles that primarily package DNA, while peak #2 appears to include both empty particles and particles with packaged DNA.

[0067] Figure 24A Shown in Figure 22-23 After the AEX chromatography, Figure 21A Micrograph of electrophoresis gel of the bands, where pK#1 and pK#2 refer to peaks #1 and #2 of the AEX chromatogram, respectively.

[0068] Figure 24B and 24C The bar graphs show the titer of AAV1 (VG / mL) and the titer of Figure 22-23 Peaks #1 and #2 in the chromatogram of PCR products were identified as being associated with the GOI or backbone elements (origin of replication (Ori), kanamycin resistance gene (KanR), and ampicillin resistance gene (AmpR)) from conventional Rep / Cap and helper plasmids.

[0069] Figure 25 Shown are the 72-hour harvests after transfection with different ratios of msDNA or pDNA along with conventional Rep / Cap and helper plasmids. Figure 15 Bar graph of AAV2 titers (VG / mL) determined by ddPCR of GOI and backbone (Ori) sequences after the described cultures.

[0070] Figure 26 Shown in Figures 19A-19B Next generation sequencing (NGS) coverage maps of packaged genomes from msDNA and pDNA AAV cultures, respectively, relative to plasmid map positions after affinity chromatography capture as described in Figures 20A-20B.

[0071] Figure 27Shown is a map of an exemplary msDNA comprising expression cassettes encoding Rep2 and Cap2 and SSeq flanking each side of the expression cassettes.

[0072] Figure 28A and 28B It shows that the 1.4:1.5:1 molar ratio ( Figure 28A ) conventional helper plasmids ("pDNA-helper"), Figure 27 The msDNA ("msDNA-Rep2Cap2") shown and Figure 8 msDNA ("msDNA-cis") as shown; and 1:2:1 molar ratio ( Figure 29A ), a conventional helper plasmid, a conventional Rep2 / Cap2 plasmid ("pDNA-Rep2Cap2"), and Figure 14 Affinity chromatography chromatogram of a culture transfected with the indicated plasmid ("pDNA-cis"). Upper limit, lower limit, VP / mL and percentage are shown in Figure 2. Figures 19A-19B VG / mL represents the number of particles packed with DNA / mL of eluate.

[0073] Figures 29A-29C Chromatograms of affinity chromatography are shown. Figure 29A and 29C Shown are the Figure 28A and 28B Chromatograms of affinity chromatography of independent replicates of the cultures. Figure 29B The results show that the 1.4:1.5:1 molar ratio of Figure 28A and 28B Chromatogram of affinity chromatography of the culture transfected with pDNA-assisted:pDNA-RepCap2:msDNA-cis. Upper limit, lower limit, VP / mL and percentage are as follows Figures 19A-19B As described. VG / mL Figure 28A and 28B VP is the total number of viral particles calculated by multiplying the VP / mL value by the total volume of the eluate.

[0074] Figure 29D Shows the Figure 21A Micrograph of electrophoresis gel of the bands, wherein lanes (a)-(c) correspond to Figures 29A-29C The eluent of the chromatogram.

[0075] Figure 30-32 Shown separately Figures 29A-29C The percentages are shown in Figure 2. Figures 19A-19B As described. VG / mL Figure 28A and 28B As stated.

[0076] Figure 33A The titers of AAV2 vector genomes / mL (VG / mL) based on the presence of the GOI, as determined by ddPCR, are shown for samples from AAV2 initial harvest ("Harvest"), affinity chromatography ("Capture"), and AEX chromatography ("AEX") after lysis of cell culture. The two harvest values and two capture values for msDNA and pDNA samples are from independent replicates on different days, as shown in Figure 2. Figures 28A-28B msDNA = transfected with pDNA-helper, msDNA-Rep2Cap2, and msDNA-cis; pDNA = transfected with pDNA-helper, pDNA-Rep2Cap2, and pDNA-cis; mixed msDNA = transfected with pDNA-helper, pDNA-Rep2Cap2, and msDNA-cis.

[0077] Figure 33B Shows the Figure 33A The corresponding VG / mL titers in are the AAV2 titers determined for each sample (VG, mass balance).

[0078] Figure 34A Shows the A260 / A280 ratio Figure 33A A histogram of the percentage of intact particles determined for the samples, calculated from Figures 28A-28B Affinity chromatography captures shown (first "msDNA" and "pDNA" bars on the x-axis) and Figures 29A-29C Affinity chromatography capture and Figure 30-32 AEX chromatographic peaks are shown (subsequently "msDNA", mixed msDNA" and "pDNA" histograms).

[0079] Figure 34B The mass spectrometry of Figure 33A The sample from Figures 28A-28B Affinity chromatography capture and Figure 30-32 Histogram of the percentage of intact particles determined by AEX chromatographic peaks shown.

[0080] Figure 35 Shown is a diagram of AAV production in which all three conventional plasmids were replaced with msDNA.

[0081] Figure 36 Shown is a map of an exemplary msDNA containing an expression cassette encoding helper viral genes for AAV production.

[0082] Figures 37A-37D Shown are all msDNA ( Figures 37A-37C ) or all pDNA( Figure 37D) Affinity chromatography chromatogram of the transfected culture. Figure 8 、 27 msDNA cultures were transfected with 36% msDNA and a 1:1 ratio of transfection agent:total DNA ( Figure 37A ); using a 1:2:1 molar ratio Figure 8 、 27 msDNA cultures were transfected with 36% msDNA and a 2:1 ratio of transfection agent:total DNA ( Figure 37B ); using a 1:1:1 molar ratio Figure 8 、 27 msDNA cultures were transfected with 36% msDNA and a 2:1 ratio of transfection agent:total DNA ( Figure 37C ). Figure 37D Shows the Figure 29C The upper limit, lower limit, VP / mL and percentage are shown in Figure 2. Figures 19A-19B VG / L is the concentration of vector genomes in the culture.

[0083] Figure 38 include Figures 37A-37D The sample characteristics and data are summarized, and the capsid proteins VP1, VP2 and VP3 have Figure 21A Micrograph of electrophoresis gel of the bands.

[0084] Figures 39-42 Shown are the Figures 37A-37D The percentage and VG / mL are shown in Figure 2. Figures 19A-19B and 28A-28B. The micrographs inserted in each figure show that the capsid proteins VP1, VP2 and VP3 have Figure 21A Electrophoresis gel of the bands.

[0085] Figure 43A Shows the Figures 37A-37D The affinity chromatography capture and Figures 39-42 Histogram of the percentage of intact particles determined by the A260 / A280 ratio calculated from the AEX chromatographic peaks shown.

[0086] Figure 43B The mass spectrometry shows Figures 37A-37D The affinity chromatography capture and Figures 39-42 Histogram of the percentage of intact particles determined by the AEX chromatographic peaks shown.

[0087] Figure 44A Shows the Figures 37A-37D The initial harvest after culture lysis ("harvest"), Figures 37A-37D Affinity chromatography ("capture") and Figures 39-42 AEX chromatography peak #1 ("AEX") shown, AAV2 vector genomes / mL (VG / mL) titer based on the presence of GOI determined by ddPCR.

[0088] Figure 44B and 44C Shows the Figure 44A The corresponding VG / mL titer in is the AAV2 titer (VG, mass balance) determined for each sample, where Figure 44C Includes the sum of Peak #1 and 2 from the AEX chromatography.

[0089] Figure 45 Shown are NGS coverage plots of packaged genomes from AAV cultures containing one, two, or three msDNAs for AAV production as described in the previous figure, relative to their positions on the plasmid map, compared to AAV cultures containing all pDNAs used for AAV production.

[0090] Figure 46 Shown with a 1:1:1 molar ratio Figure 8 msDNA (msDNA) or Figure 14 Figure 2. Histogram of transfection efficiency 48 or 72 hours after transfection of a plasmid (AAV PP), a bacterial sequence-minimized / reduced Rep2 / Cap9 plasmid, and helper sequences for AAV9 production. msDNA1-9 represent different amounts of total transfected DNA and transfection agent (PEI):DNA ratios as follows: (msDNA1) 1.0 μg / mL DNA and 1.5:1 PEI:DNA, (msDNA2) 1.0 μg / mL DNA and 2:1 PEI:DNA, (msDNA3) 1.0 μg / mL DNA and 2.5:1 PEI:DNA, (msDNA4) 1.75 μg / mL DNA and 1.5:1 PEI:DNA, (msDNA5) 1.75 μg / mL DNA and 2:1 PEI:DNA, (msDNA6) 1.75 μg / mL DNA and 2.5:1 PEI:DNA, (msDNA7) 2.5 μg / mL DNA and 1.5:1 PEI:DNA, (msDNA8) 2.5 μg / mL DNA and 2:1 PEI:DNA, and (msDNA9) 2.5 μg / mL DNA and 2.5:1 PEI:DNA.

[0091] Figure 47 shows Figure 46 The cell viabilities histograms 48 or 72 hours after transfection are shown as viable cell density (VCD, cells / mL) and % viable cells.

[0092] Figure 48 Shows Figure 46 Bar graph of capsid titers of the samples, as determined by AAV9-specific ELISA 72 hours after transfection.

[0093] Figure 49 The expression of ITR region specific primers was determined by ddPCR 72 hours after transfection. Figure 46 Bar graph of AAV titers of the samples.

[0094] Figure 50 Shows Figure 46 Bar graph of total AAV9 titers determined by ddPCR after AEX chromatography from 10 L cultures of the transfectants msDNA5 ("msDNA") and AAV PP ("pDNA"). DETAILED DESCRIPTION

[0095] The present disclosure provides expression vectors, vector production systems, methods for producing bacterial sequence-free vectors, and bacterial sequence-free vectors for producing AAV, as well as methods for producing the AAV, the AAV, compositions comprising the AAV, and methods of using the AAV.

[0096] All publications cited herein are incorporated by reference in their entirety, including but not limited to all journal articles, books, manuals, patent applications, and patents cited herein, to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0097] I. Terminology

[0098] In order to make it easier to understand this disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the following meanings. Other definitions are set forth throughout this application.

[0099] It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a nucleotide sequence" should be understood to represent one or more nucleotide sequences. Therefore, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein.

[0100] As used herein, the term "and / or" should be considered to specifically disclose each of the two specified features or components, in combination with or without the other specified feature or component. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0101] It should be understood that wherever aspects of the invention are described herein using the expression "comprising," similar aspects described using the expression "consisting of" and / or "consisting essentially of" are also provided.

[0102] The term "approximately" or "substantially comprising" means, for a specific value or composition, a value or composition within an acceptable error range determined by a person of ordinary skill in the art, which depends in part on the manner used to measure or determine the value or composition, i.e., the limitations of the measurement system. For example, "approximately" or "substantially comprising" can mean within a range of 1 or more standard deviations according to the practice in the art. Alternatively, "approximately" or "substantially comprising" can mean a range of no more than 10%. In addition, particularly for biological systems or methods, these terms can mean values that are no more than an order of magnitude or no more than 5 times. When specific values or compositions are provided in the present application and claims, unless otherwise stated, "approximately" or "substantially comprising" should be presumed to mean within an acceptable error range for that specific value or composition.

[0103] In this document, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include the value of any integer within the range, as well as its fraction (e.g., one tenth and one hundredth of an integer) when appropriate. Numerical ranges include the numbers defining the range.

[0104] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure relates. For example, the Concise Dictionary of Biomedicine Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 5th edition, 2013, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, 2006, Oxford University Press, provide a general dictionary for those of skill in the art of many of the terms used in this disclosure.

[0105] Units, prefixes, and symbols are expressed in their form accepted by the International System of Units (SI).

[0106] Unless otherwise indicated, nucleotide sequences are written from left to right in 5' to 3' orientation. Amino acid sequences are written from left to right in amino to carboxyl orientation.

[0107] The headings provided herein are not limitations of the various aspects of the disclosure, which can be obtained by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0108] As used herein, "adeno-associated virus," or "AAV," refers to a parvovirus of the family Parvoviridae, a member of the genus Dependoparvovirus (formerly known as the genus Dependovirus). As disclosed herein, AAV containing a nucleic acid sequence of interest may be referred to interchangeably as "AAV," "recombinant AAV," "rAAV," or "AAV vector."

[0109] As used herein, "inverted terminal repeats," or "ITRs," refer to the sense or antisense strand (i.e., the + strand or - strand, respectively) of a single-stranded polynucleotide or double-stranded polynucleotide containing the AAV replication sequence and a non-palindromic packaging signal. "ITRs" disclosed herein include wild-type AAV 5' ITR and / or 3' ITR sequences, portions thereof, or artificial sequences.

[0110] As used herein, "AAV replication sequence" refers to sequences within the AAV ITR that are associated with AAV replication, including the Rep protein binding element (RBE), RBE', the terminal dissociation site (TRS), or any combination thereof. RBE may also be referred to herein interchangeably as the Rep protein binding site (RBS).

[0111] As used herein, "AAV packaging signal" refers to a non-palindromic sequence in the ITR that is involved in AAV encapsidation and comprises the "D region" of the 5' or 3' AAV ITR or a functional portion thereof.

[0112] "Protein" or "polypeptide" refers to any polymer formed by two or more (natural or non-natural) amino acids linked by peptide bonds, wherein the peptide bond is formed when the carboxyl carbon atom of the carboxylic acid group of one amino acid (or amino acid residue) bonded to the α-carbon is covalently bound to the amino nitrogen atom of the amino group bonded to the α-carbon of the adjacent amino acid. The term "protein" is understood to include within its meaning the terms "polypeptide" and "peptide" (which may sometimes be used interchangeably herein). In addition, proteins comprising multiple polypeptide subunits will also be understood to be included within the meaning of "protein" as used herein. Similarly, fragments of proteins and polypeptides are also within the scope of the present disclosure and may be referred to as "proteins" herein. In one aspect of the present disclosure, the polypeptide comprises a chimera of two or more parent peptide fragments. The term "polypeptide" also means and encompasses post-translational modification ("PTM") products of the polypeptide, including, but not limited to, disulfide bond formation, glycosylation, carbamylation, lipidation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, modification by non-naturally occurring amino acids, or any other manipulation or modification, such as conjugation to a labeling component. A polypeptide may be derived from a natural biological source or produced by recombinant techniques, and need not necessarily be translated from a designated nucleic acid sequence. A polypeptide may be produced in any manner, including by chemical synthesis. An "isolated" polypeptide or fragment, variant, or derivative thereof is one that is not in its natural environment. No specific level of purification is required. For example, an isolated polypeptide may simply be removed from its native or natural environment. For the purposes of this disclosure, recombinantly produced polypeptides and proteins expressed in a host cell are considered isolated, as are natural or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0113] " Polynucleotide " or " nucleic acid " used herein refers to the polymer form of nucleotide. In some cases, polynucleotide comprises following sequence, and described sequence is not directly adjacent to coding sequence or is directly adjacent to coding sequence (at 5 ' end or at 3 ' end) in the natural genome of its source organism. Therefore, this term includes, for example, being incorporated into vector, being incorporated into self-replicating plasmid or virus, being incorporated into recombinant DNA in the genomic DNA of prokaryotic or eukaryotic organism, or being independent of other sequences as recombinant DNA existing as separate molecule (such as cDNA). Nucleotide of the present disclosure can be ribonucleotide, deoxyribonucleotide or its modified form. Polynucleotide used herein especially refers to single-stranded and double-stranded DNA, DNA mixed with single-stranded region and double-stranded region, single-stranded and double-stranded RNA and RNA mixed with single-stranded region, hybrid molecule (it can be single-stranded or more typically double-stranded, or the mixture of single-stranded region and double-stranded region) comprising DNA and RNA. The term polynucleotide covers genomic DNA or RNA (depending on organism, i.e., the RNA genome of virus) and mRNA and cDNA encoded by genomic DNA. In certain embodiments, the polynucleotide comprises conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, as in peptide nucleic acids (PNA)). An "isolated" nucleic acid or polynucleotide refers to a nucleic acid molecule, such as DNA or RNA, that has been removed from its natural environment. For example, for the purposes of this disclosure, a nucleic acid molecule comprising a polynucleotide encoding a recombinant polypeptide in a vector is considered to be "isolated." Other examples of isolated polynucleotides include recombinant polynucleotides that have been maintained in heterologous host cells or purified (partially or substantially) from other polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides of the present disclosure. Isolated polynucleotides or nucleic acids according to the present disclosure also include synthetically produced polynucleotides and nucleic acids (e.g., nucleic acid molecules).

[0114] As used herein, an "expression cassette" comprises a nucleic acid sequence of interest (eg, a nucleic acid sequence for expressing a polypeptide, DNA, or RNA) and an expression control region.

[0115] As used herein, "transgene" is used interchangeably with "gene of interest" or "GOI" and refers to the portion of a polynucleotide containing codons that are translated into amino acids. While the "stop codon" (TAG, TGA, or TAA) is not typically translated into amino acids, it can be considered part of the transgene; however, any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of the transgene. The boundaries of the transgene are generally determined by a start codon at the 5' end, which encodes the amino terminus of the resulting polypeptide, and a translation stop codon at the 3' end, which encodes the carboxyl terminus of the resulting polypeptide.

[0116] As used herein, the term "expression control region" refers to a transcriptional control element that is operably linked to a nucleic acid sequence of interest to direct or control the expression of an expression product of the nucleic acid sequence of interest, including, for example, cis-regulatory elements (CRMs), promoters (e.g., tissue-specific promoters and / or inducible promoters), enhancers, operators, repressors, ribosome binding sites, translation leader sequences, introns, post-transcriptional elements, polyadenylation recognition sequences, RNA processing sites, effector binding sites, stem-loop structures, transcription termination signals, miRNA binding sites, and combinations thereof. An expression control region includes nucleotide sequences located upstream (5'), internally, or downstream (3') of a nucleic acid sequence of interest that can affect transcription, RNA processing, stability, or translation of the associated nucleic acid sequence of interest. If the transgene is intended to be expressed in eukaryotic cells, the polyadenylation signal and transcription termination sequence are typically located 3' of the transgene.

[0117] As used herein, the terms "host cell" and "cell" are used interchangeably and may refer to any type of cell or cell population that contains or is capable of containing a nucleic acid molecule (e.g., a recombinant nucleic acid molecule), such as a primary cell, a cultured cell, or a cell from a cell line. The host cell may be a prokaryotic cell, or alternatively, the host cell may be a eukaryotic cell, such as a fungal cell, such as a yeast cell, and various animal cells, such as an insect cell or a mammalian cell.

[0118] As used herein, "culture" and "cultured" refer to incubating cells under in vitro conditions that allow the cells to grow or divide or maintain the cells in a viable state. As used herein, "cultured cells" refer to cells that are propagated in vitro.

[0119] "Subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates, such as non-human primates, sheep, dogs, and rodents such as mice, rats, and guinea pigs. In preferred aspects, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0120] "Administering" refers to the physical introduction of a composition containing a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art.

[0121] "Treatment" or "therapy" of a subject refers to any type of intervention or procedure performed on a subject, or the administration of an active agent to a subject, to reverse, alleviate, ameliorate, inhibit, slow the progression, development, severity or recurrence of the symptoms, complications or conditions or biochemical markers associated with a disease, condition or disorder.

[0122] As used herein, "effective treatment" refers to treatment that produces a beneficial effect, such as an improvement in at least one symptom of a disease, condition, or disorder. A beneficial effect can take the form of an improvement relative to baseline, i.e., an improvement compared to measurements or observations taken before initiation of treatment according to the method. A beneficial effect can also take the form of preventing, slowing, delaying, or stabilizing adverse progression of a marker of a disease, condition, or disorder. Effective treatment can refer to alleviation of at least one symptom of a disease, condition, or disorder.

[0123] The term "effective amount" refers to the amount of an active agent that provides a desired biological, therapeutic, and / or preventive outcome. The outcome can be a reduction, improvement, mitigation, alleviation, delay, and / or alleviation of one or more signs, symptoms, or causes of a disease, or any other desired change in a biological system. In some aspects, an effective amount is an amount sufficient to prevent or delay the recurrence of symptoms of a disease, condition, or disorder. An effective amount can be administered in one or more ways.

[0124] Various aspects of the invention are described in further detail in the following subsections.

[0125] II. Production of AAV from msDNA

[0126] Ministring DNA vectors (interchangeably referred to herein as "msDNA vectors" or "msDNA") are bacterial sequence-free vectors with linear covalently closed (LCC) ends. See U.S. Patent Nos. 9,290,778 and 9,862,954 and International Publication No. WO 2022 / 264095; Nafissi and Slavcev, Microbial Cell Factories 11:154 (2012); and Nafissi et al., Nucleic Acids 3(6):e165 (2014), the entire contents of which are incorporated herein by reference. msDNA is a "bacterial sequence-free vector" because it lacks any bacterial backbone sequences, such as antibiotic resistance genes, bacterial origins of replication, or immunostimulatory non-methylated CpG motifs typical of plasmid-based vectors. Integration of msDNA into cellular chromosomes leads to chromosome breakage and elimination of the cell by programmed cell death. Thus, msDNA eliminates any risk of insertional mutagenesis and avoids the potential genotoxic and oncogenic events associated with integration when using other delivery vectors (see Nafissi et al.).

[0127] msDNA is produced from an expression vector (e.g., a plasmid) containing a specialized "Super Sequence" (e.g., "SS" or "SSeq") site that contains a target sequence for a recombinase. The SS site flanks an expression cassette containing a nucleic acid of interest. When the expression vector is present in a recombinant cell expressing a suitable recombinase, the msDNA containing the expression cassette is separated from the backbone DNA of the expression vector. The msDNA can then be purified and used directly as a delivery vector. See U.S. Patent Nos. 9,290,778 and 9,862,954, International Publication No. WO 2022 / 264095, Nafissi and Slavcev, and Nafissi et al.

[0128] A. Expression Vectors, Vector Production Systems, and msDNA

[0129] Provided herein are expression vectors for generating msDNA comprising sequences that can be used to produce AAV.

[0130] In one aspect, the expression vector comprises: (a) a first sequence comprising inverted terminal repeats (ITRs) flanking at least one side of a desired sequence, wherein the ITRs comprise an adeno-associated virus (AAV) replication sequence and an AAV packaging signal, (b) a target sequence for a first recombinase flanking each side of the first sequence, and (c) one or more additional target sequences for one or more additional recombinases, the one or more additional target sequences being integrated within a non-binding region of the target sequence for the first recombinase, wherein the expression vector is used to generate a bacterial sequence-free vector (i.e., msDNA) having linear covalently closed ends. In some aspects, the desired sequence is a portion of a multiple cloning site (MCS), an expression cassette comprising a nucleic acid sequence of interest, a palindromic sequence comprising an expression cassette comprising a nucleic acid sequence of interest and the complement of the expression cassette, or an expression cassette comprising a nucleic acid sequence of interest flanked on one side by a splicing sequence.

[0131] Any ITR sequence containing the AAV replication sequence and the AAV packaging signal as disclosed herein can be used in the aspects of the invention disclosed herein.

[0132] The end of each wild-type AAV ITR contains palindromic regions (A and A', B and B', and C and C'), which self-anneal to form a double-stranded T-shaped hairpin structure. The self-annealed B-B' and C-C' palindromic sequences form the cross arms of the hairpin, while the self-annealed A-A' palindromic sequence forms the handle of the hairpin. The hairpin is followed by a short non-palindromic region (D) in the ITR that provides a packaging signal. A, A', B, B', C, C' and D can be referred to interchangeably herein as "sequences" or "regions" (e.g., A sequences, sequence A, A regions, region A, etc.). Studies have shown that packaging and replication of AAV can occur in ITRs that lack B-B' and C-C' regions. See, for example, Zhou et al., Scientific Reports 7:5432 (2017).

[0133] ITR contains sequences related to Rep protein function. The 16-nucleotide tetramer repeat within A-A' is called the Rep protein binding element (RBE), which binds to Rep68 / Rep78, which has helicase activity and unwinds the RBE sequence. As used herein, RBE includes a double-stranded structure formed when the palindromic A-RBE and A'-RBE sequences self-anneal. A sequence located at one end of one of the internal palindromic B-B' regions, called RBE', directs Rep68 / Rep78 toward the terminal dissociation site (TRS). Rep68 / Rep78 endonuclease activity cuts the TRS during replication to dissociate the double-stranded sequence and produce a single-stranded genome for packaging. See, for example, Daya and Berns, Lisowski et al.; Ling et al., J. Mol. Genet. Med. 9(3):175(2015); Salganik et al., Microbiol. Spectrum 3(4):MDNA3-0052-2014.

[0134] Currently, more than 100 human and non-human primate AAVs have been identified, including 13 serotypes. See, for example, Daya and Berns; Lisowski et al.; and Mary et al. The ITR sequences for each serotype are known in the art or can be readily determined by one skilled in the art, including exemplary accession numbers from: AAV1 (NC_002077.1; AF063497.1), AAV2 (J01901.1; NC_001401.2), AAV3 (AAV3A, NC_001729.1; AAV3B, AF028705.1), AAV4 (NC_001829.1), AAV5 (NC_001902.1), AAV6 (NC_002077.1), AAV7 (NC_002077.1), AAV8 (NC_002077.1), AAV9 (NC_002077.1), AAV10 (NC_002077.1), AAV11 (NC_002077.1), AAV12 (NC_002077.1), AAV13 (NC_002077.1), AAV14 (NC_002077.1), AAV15 (NC_002077.1), AAV16 (NC_002077.1), AAV17 (NC_002077.1), AAV18 (NC_002077.1), AAV19 (NC_002077.1), AAV2 V5(NC_006152.1; AF085716.1), AAV6(AF028704.1), AAV7(NC_006260.1), AAV8(NC_006261.1), AAV9( AX753250.1), AAV10(AY631965.1), AAV11(AY631966.1), AAV12(DQ813647.1), and AAV13(EU285562.1).

[0135] In some aspects, the ITRs flank the MCS, expression cassette, palindromic sequence, or partial expression cassette in an expression vector disclosed herein on only one side (ie, the 5' ITR or the 3' ITR).

[0136] In some aspects, ITRs flank an MCS, expression cassette, palindromic sequence, or partial expression cassette in an expression vector disclosed herein on each side (ie, a 5' ITR and a 3' ITR).

[0137] In some aspects, the expression vectors, msDNA or AAV disclosed herein comprise wild-type AAV 5'ITR and / or 3'ITR sequences. In some aspects, the expression vectors, msDNA or AAV disclosed herein comprise a portion of the wild-type AAV ITR sequence or an artificial sequence containing the AAV replication sequence and the AAV packaging signal.

[0138] In some aspects, the ITR is a wild-type AAV ITR.

[0139] In some aspects, the ITR is a portion of a wild-type AAV ITR that includes the AAV replication sequence and the AAV packaging signal.

[0140] In some aspects, the ITR is an artificial ITR that comprises an AAV replication sequence and an AAV packaging signal.

[0141] In some aspects, the ITRs comprise A, A', and D sequences.

[0142] In some aspects, the ITR comprises the RBE and D sequences of an AAV ITR.

[0143] In some aspects, the ITRs are located on each side of the MCS, expression cassette, palindromic sequence, or portion of the expression cassette, and the ITRs on each side are identical.

[0144] In some aspects, the ITRs are located on each side of the MCS, expression cassette, palindromic sequence, or portion of the expression cassette, and the ITRs on each side are different.

[0145] In some aspects, any expression vector, msDNA, or AAV disclosed herein comprises a 5' ITR and a 3' ITR flanking the MCS, expression cassette, palindromic sequence, or partial expression cassette, and the 5' ITR and 3' ITR are from the same serotype or different serotypes.

[0146] In some aspects, the ITRs disclosed herein are chimeric ITRs comprising sequences from different AAV serotypes. Exemplary AAV ITR sequences are shown in Table 1.

[0147] Table 1. Exemplary AAV ITR sequences

[0148]

[0149] In some aspects, the ITRs of any expression vector, msDNA, or AAV disclosed herein comprise one or more sequences in Table 1.

[0150] In some aspects, the ITRs of any expression vector, msDNA, or AAV disclosed herein comprise one or more sequences from the negative strand (i.e., - strand or antisense strand) AAV genome corresponding to the positive strand (i.e., + strand or sense strand) AAV genome sequences in Table 1.

[0151] In some aspects, the ITR sequences of any expression vector, msDNA, or AAV disclosed herein comprise one or more 5'ITR, 3'ITR, A, A', B, B', C, C', D, A-RBE, A'-RBE, RBE', 5'ITR D, 3'ITR D, 5'ITR TRS, or 3'ITR TRS sequences that are at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the corresponding sequences in Table 1.

[0152] In some aspects, the ITR sequence of any expression vector, msDNA, or AAV disclosed herein comprises one or more 5'ITR, 3'ITR, A, A', B, B', C, C', D, A-RBE, A'-RBE, RBE', 5'ITR D, 3'ITR D, 5'ITR TRS, or 3'ITR TRS sequences that are at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the corresponding - strand (i.e., antisense strand) AAV genomic sequence of the + strand (i.e., sense strand) AAV genomic sequence in Table 1.

[0153] In some aspects, the ITRs of any expression vector, msDNA, or AAV disclosed herein comprise a 5' ITR having a polynucleotide sequence of SEQ ID NO: 16 and / or a 3' ITR having a polynucleotide sequence of SEQ ID NO: 17. In some aspects, the ITRs of any expression vector, msDNA, or AAV disclosed herein comprise a 5' ITR having a polynucleotide sequence of SEQ ID NO: 16 and a 3' ITR having a polynucleotide sequence of SEQ ID NO: 17.

[0154] In some aspects, the ITRs of any expression vector, msDNA, or AAV disclosed herein comprise a 5' ITR having a polynucleotide sequence of SEQ ID NO: 38 and / or a 3' ITR having a polynucleotide sequence of SEQ ID NO: 39. In some aspects, the ITRs of any expression vector, msDNA, or AAV disclosed herein comprise a 5' ITR having a polynucleotide sequence of SEQ ID NO: 38 and a 3' ITR having a polynucleotide sequence of SEQ ID NO: 39.

[0155] In some aspects, the expression vector disclosed herein further comprises a spacer sequence between the target sequence of the first recombinase and the first sequence (e.g., between the target sequence of the first recombinase and the ITR, e.g., between the 5' target sequence of the first recombinase and the 5' ITR and / or between the 3' target sequence of the first recombinase and the 3' ITR). In some aspects, the msDNA or AAV described herein further comprises a portion of the SSeq (e.g., the portion remaining after Tel recombination of the expression vector described herein comprising the SSeq, such as the portion provided in the polynucleotide sequence of SEQ ID NO: 37) and the ITR (i.e., 5' ITR and / or 3' ITR). In some aspects, the spacer sequence is about 10 to about 500 nucleotides. In some aspects, the spacer sequence is about 1 to about 10 nucleotides, about 10 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 250 nucleotides, or about 250 to about 500 nucleotides.

[0156] In some aspects, the 5' spacer sequence is the polynucleotide sequence of SEQ ID NO:40.

[0157] In some aspects, the 3' spacer sequence is the polynucleotide sequence of SEQ ID NO:41.

[0158] In some aspects, any expression vector, msDNA, or AAV disclosed herein comprises a 5' spacer sequence having the polynucleotide sequence of SEQ ID NO:40 and a 3' spacer sequence having the polynucleotide sequence of SEQ ID NO:41.

[0159] In some aspects, the 5' spacer sequence is the polynucleotide sequence of SEQ ID NO:40 and the 5' ITR is the polynucleotide sequence of SEQ ID NO:38, and / or the 3' spacer sequence is the polynucleotide sequence of SEQ ID NO:41 and the 3' ITR is the polynucleotide sequence of SEQ ID NO:39.

[0160] In some aspects, any expression vector, msDNA, or AAV disclosed herein comprises a 5' spacer sequence having the polynucleotide sequence of SEQ ID NO:40, a 5' ITR having the polynucleotide sequence of SEQ ID NO:38, a 3' spacer sequence having the polynucleotide sequence of SEQ ID NO:41, and a 3' ITR having the polynucleotide sequence of SEQ ID NO:39.

[0161] In some aspects, the expression vector does not have any spacer sequence between the target sequence of the first recombinase and the first sequence.

[0162] In some aspects, the expression vector further comprises an expression cassette comprising an AAV replication (rep) gene and / or an AAV capsid (cap) gene flanked on one side by a target sequence for the first recombinase and on the other side by the first sequence.

[0163] The AAV rep gene and cap gene in any expression vector or msDNA disclosed herein can be from any AAV disclosed herein, including any AAV serotype. The ITR, rep gene, and / or cap gene can be from the same AAV or different AAVs, including the same AAV serotype or different AAV serotypes. The rep gene and / or cap gene can also be a hybrid sequence containing sequences from different AAVs, such that any one or part of Rep40, Rep52, Rep68, Rep78, VP1, VP2, VP3, and AAP can be encoded by sequences from different AAVs.

[0164] The rep and cap gene sequences for each of the 13 identified serotypes are known in the art or can be readily determined by one skilled in the art, including from the exemplary accession numbers for the 13 serotypes disclosed herein. For example, the AAV2 rep and cap genes are provided herein as SEQ ID NOs: 21 and 22, respectively, or as SEQ ID NOs: 45 and 22, respectively. As used herein, a "serotype" refers to an AAV having a capsid that is serologically distinct from another AAV, as evidenced, for example, by a lack of cross-reactivity between antibodies raised against one AAV and antibodies raised against another AAV due to differences in the capsid proteins.

[0165] Serotypes differ in their tissue tropism (ie, the cell types they infect) based on their capsids. See, e.g., Likowski et al.; Daya and Berns.

[0166] In some aspects, AAV targets tissues or cells that contain a cell surface receptor for that AAV serotype. In some aspects, the cell surface receptor is a heparan sulfate proteoglycan (e.g., a cell surface receptor for AAV-3), an O-linked sialic acid (e.g., a cell surface receptor for AAV-4), a platelet-derived growth factor receptor (e.g., a cell surface receptor for AAV-5), or a 37-kDa / 67-kDa laminin receptor (e.g., a cell surface receptor for AAV-2, AAV-3, AAV-8, or AAV-9).

[0167] Mixing the genomic sequence from one AAV serotype with the capsid from another AAV serotype is called pseudotyping and is indicated herein by slashes. For example, a recombinant AAV with ITR sequences from AAV2 and capsids from AAV5 is indicated herein as AAV2 / 5.

[0168] In some aspects, the expression vectors, msDNA, combinations of expression vectors (i.e., ITR and cap sequences are located on separate expression vectors), combinations of msDNA, or AAV disclosed herein comprise an ITR / cap pseudotype of 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 11, 1 / 12, or 1 / 13. In some aspects, the pseudotyped AAV disclosed herein comprises an ITR / cap gene of 2 / 1, 2 / 3, 2 / 4, 2 / 5, 2 / 6, 2 / 7, 2 / 8, 2 / 9, 2 / 10, 2 / 11, 2 / 12, or 2 / 13. In some aspects, the pseudotyped AAV disclosed herein comprises an ITR / cap gene of 3 / 1, 3 / 2, 3 / 4, 3 / 5, 3 / 6, 3 / 7, 3 / 8, 3 / 9, 3 / 10, 3 / 11, 3 / 12, or 3 / 13. In some aspects, the pseudotyped AAV disclosed herein comprises an ITR / cap gene with 4 / 1, 4 / 2, 4 / 3, 4 / 5, 4 / 6, 4 / 7, 4 / 8, 4 / 9, 4 / 10, 4 / 11, 4 / 12, or 4 / 13. In some aspects, the pseudotyped AAV disclosed herein comprises an ITR / cap gene with 5 / 1, 5 / 2, 5 / 3, 5 / 4, 5 / 6, 5 / 7, 5 / 8, 5 / 9, 5 / 10, 5 / 11, 5 / 12, or 5 / 13. In some aspects, the pseudotyped AAV disclosed herein comprises an ITR / cap gene with 6 / 1, 6 / 2, 6 / 3, 6 / 4, 6 / 5, 6 / 7, 6 / 8, 6 / 9, 6 / 10, 6 / 11, 6 / 12, or 6 / 13. In some aspects, the pseudotyped AAV disclosed herein comprises 7 / 1, 7 / 2, 7 / 3, 7 / 4, 7 / 5, 7 / 6, 7 / 8, 7 / 9, 7 / 10, 7 / 11, 7 / 12, or 7 / 13 ITR / cap genes. In some aspects, the pseudotyped AAV disclosed herein comprises 8 / 1, 8 / 2, 8 / 3, 8 / 4, 8 / 5, 8 / 6, 8 / 7, 8 / 9, 8 / 10, 8 / 11, 8 / 12, or 8 / 13 ITR / cap genes. In some aspects, the pseudotyped AAV disclosed herein comprises 9 / 1, 9 / 2, 9 / 3, 9 / 4, 9 / 5, 9 / 6, 9 / 7, 9 / 8, 9 / 10, 9 / 11, 9 / 12, or 9 / 13 ITR / cap genes. In some aspects, the pseudotyped AAV disclosed herein comprises ITR / cap genes of 10 / 1, 10 / 2, 10 / 3, 10 / 4, 10 / 5, 10 / 6, 10 / 7, 10 / 8, 10 / 9, 10 / 11, 10 / 12, or 10 / 13. In some aspects, the pseudotyped AAV disclosed herein comprises ITR / cap genes of 11 / 1, 11 / 2, 11 / 3, 11 / 4, 11 / 5, 11 / 6, 11 / 7, 11 / 8, 11 / 9, 11 / 10, 11 / 12, or 11 / 13.In some aspects, the pseudotyped AAV disclosed herein comprises ITR / cap genes of 12 / 1, 12 / 2, 12 / 3, 12 / 4, 12 / 5, 12 / 6, 12 / 7, 12 / 8, 12 / 9, 12 / 10, 12 / 11, 12 / 12, or 12 / 13. In some aspects, the pseudotyped AAV disclosed herein comprises ITR / cap genes of 13 / 1, 13 / 2, 13 / 3, 13 / 4, 13 / 5, 13 / 6, 13 / 7, 13 / 8, 13 / 9, 13 / 10, 13 / 11, or 13 / 12.

[0169] The capsids disclosed herein can also be hybrid capsids produced using capsid proteins from multiple serotypes. For example, AAV-DJ has hybrid capsids derived from 8 serotypes. In some aspects, the expression vectors or msDNA disclosed herein encode hybrid capsids, or the AAV disclosed herein comprise hybrid capsids. In some aspects, the hybrid capsid comprises capsid proteins from any two or more of the serotypes AAV1-AAV13.

[0170] Table 2 provides an exemplary list of selected AAV serotypes, viral strains, and tissue tropisms of recombinant AAVs.

[0171] Table 2. Exemplary AAV tissue tropism

[0172]

[0173]

[0174] In some aspects, any expression vector, msDNA, or AAV disclosed herein comprises an ITR, rep gene, or cap gene from any AAV or any combination of AAVs listed in Table 2. In some aspects, any AAV disclosed herein comprises a capsid from any AAV listed in Table 2.

[0175] In some aspects, any expression vector, msDNA, or AAV disclosed herein comprising a rep gene and a cap gene comprises a rep2 gene from any AAV1-AAV13 serotype in combination with a cap gene for production of that serotype.

[0176] In some aspects, any expression vector, msDNA, or AAV disclosed herein comprising a rep gene and a cap gene comprises a rep2 gene and a cap1 gene for AAV1 production.

[0177] In some aspects, any expression vector, msDNA, or AAV disclosed herein comprising a rep gene and a cap gene comprises a rep2 gene and a cap2 gene for AAV2 production.

[0178] In some aspects, the msDNA disclosed herein for expressing rep and cap genes in AAV2 production comprises the polynucleotide sequence of SEQ ID NO: 24.

[0179] In some aspects, the msDNA disclosed herein for expressing rep and cap genes in AAV2 production comprises the polynucleotide sequence of SEQ ID NO:48.

[0180] In some aspects, the msDNA disclosed herein for expressing the rep gene and the cap gene in AAV5 production comprises the polynucleotide sequence of SEQ ID NO:49.

[0181] In some aspects, the msDNA disclosed herein for expressing the rep gene and the cap gene in AAV9 production comprises the polynucleotide sequence of SEQ ID NO: 50.

[0182] In some aspects, any expression vector, msDNA, or AAV disclosed herein comprising a rep gene and a cap gene comprises a rep2 gene and a cap5 gene for AAV5 production.

[0183] In some aspects, any expression vector, msDNA, or AAV disclosed herein comprising a rep gene and a cap gene comprises a rep2 gene and a cap9 gene for AAV9 production.

[0184] In some aspects, the cap gene comprises a sequence encoding a small peptide or ligand for targeting the AAV disclosed herein to cells and / or tissue types (i.e., the cap gene is a recombinant sequence). In some aspects, the sequence encoding a small peptide or ligand is used to target AAV to tumor cells or tumor tissue. In some aspects, the cap gene comprises a sequence for targeting AAV to tumor tissue. In some aspects, the cap gene comprises a sequence encoding an NGR peptide motif. In some aspects, the cap gene comprises a sequence encoding an RGD peptide motif (e.g., a 4C-RGD peptide). In some aspects, the cap gene comprises a sequence encoding a designed ankyrin repeat protein (DARPin). In some aspects, the cap gene comprises a mutation that enhances transduction efficiency. In some aspects, the cap gene is from AAV3 and encodes a capsid protein with Y701F, Y705F, Y731F, S663V, T492V, and / or K533R mutations. In some aspects, the cap gene is from any other serotype and encodes a capsid protein having Y701F, Y705F, Y731F, S663V, T492V and / or K533R mutations corresponding to the numbering of the AAV3 capsid proteins. In some aspects, the mutation comprises a combination of Y705F and Y731F. In a certain aspect, the mutation comprises S663V, T492V and K533R. In some aspects, the mutation comprises S663V and T492V. In some aspects, the cap gene comprises a sequence encoding a protease recognition sequence (e.g., a protease recognition sequence recognized by a matrix metalloproteinase (MMP)). See, for example, Santiago-Oritz et al., J. Control Release (2016), http: / / dx.doi.org / 10.1016 / j.jconrel.2016.01.001.

[0185] In some aspects, the expression vectors or msDNA disclosed herein comprise an MCS. The MCS comprises restriction sites for inserting a target nucleic acid sequence (e.g., a target gene) into the expression vector. The MCS can be effectively linked to any suitable expression control region known to those skilled in the art.

[0186] This article provides an expression vector comprising: (a) a first sequence comprising an inverted terminal repeat (ITR) and a multiple cloning site (MCS), wherein the ITR flanks at least one side of the MCS, and wherein the ITR comprises an adeno-associated virus (AAV) replication sequence and an AAV packaging signal, (b) a target sequence for a first recombinase on each side of the first sequence, and (c) one or more additional target sequences for one or more additional recombinases, wherein the one or more additional target sequences are integrated into the non-binding region of the target sequence of the first recombinase, wherein the expression vector is used to generate a vector (i.e., "expression vector A") having a linear covalently closed end and no bacterial sequence. In some aspects, the ITR is only located on one side of the MCS. In some aspects, the ITR is located on each side of the MCS. In some aspects, the expression vector further comprises a spacer sequence between the target sequence of the first recombinase and the first sequence. In some aspects, the spacer sequence is 10 to 500 nucleotides. In some aspects, the expression vector further comprises an expression cassette comprising an AAV replication (rep) gene and an AAV capsid (cap) gene, flanked on one side by a target sequence for the first recombinase and on the other side by the first sequence.

[0187] Provided herein is an expression vector comprising: (a) a first sequence comprising an ITR and an expression cassette comprising a nucleic acid sequence of interest, wherein the ITRs flank at least one side of the expression cassette comprising the nucleic acid sequence of interest, and wherein the ITRs comprise an AAV replication sequence and an AAV packaging signal, (b) a target sequence for a first recombinase flanking each side of the first sequence, and (c) one or more additional target sequences for one or more additional recombinases, the one or more additional target sequences being integrated within the non-binding region of the target sequence of the first recombinase, wherein the expression vector is used to generate a bacterial sequence-free vector having linear covalently closed ends (i.e., "expression vector B").

[0188] In some aspects, the ITR is located only on one side of the expression cassette containing the target nucleic acid sequence in expression vector B (i.e., "expression vector B1"). In some aspects, the expression vector further comprises a spacer sequence between the target sequence of the first recombinase and the first sequence. In some aspects, the spacer sequence is 10 to 500 nucleotides.

[0189] In some aspects, expression vector B1 further comprises an expression cassette comprising an AAV rep gene and an AAV cap gene, flanked on one side by the target sequence of the first recombinase and on the other side by the first sequence (ie, "expression vector B2").

[0190] In some aspects, the ITRs are located on each side of the expression cassette comprising the target nucleic acid sequence in expression vector B (i.e., "expression vector B3"). In some aspects, the expression vector further comprises a spacer sequence between the target sequence of the first recombinase and the first sequence. In some aspects, the spacer sequence is 10 to 500 nucleotides.

[0191] In some aspects, expression vector B3 further comprises an expression cassette comprising an AAV rep gene and an AAV cap gene, flanked on one side by the target sequence of the first recombinase and on the other side by the first sequence (ie, "expression vector B4").

[0192] Provided herein is an expression vector comprising: (a) a first sequence comprising an ITR and a palindrome, wherein the ITRs flank each side of the palindrome, wherein the palindrome comprises an expression cassette containing a target nucleic acid sequence and a complementary sequence to the expression cassette, and wherein the ITRs comprise an AAV replication sequence and an AAV packaging signal, (b) a target sequence for a first recombinase flanking each side of the first sequence, and (c) one or more additional target sequences for one or more additional recombinases, wherein the one or more additional target sequences are integrated within a non-binding region of the target sequence of the first recombinase, wherein the expression vector is used to generate a bacterial sequence-free vector (i.e., "expression vector C") having linear covalently closed ends. In some aspects, the complementary sequence is separated from the expression cassette comprising the target nucleic acid sequence by a non-complementary spacer sequence. In some aspects, the expression vector further comprises a spacer sequence between the target sequence of the first recombinase and the first sequence. In some aspects, the spacer sequence is 10 to 500 nucleotides.

[0193] In some aspects, expression vector C further comprises an expression cassette comprising an AAV rep gene and an AAV cap gene, flanked on one side by the target sequence of the first recombinase and on the other side by the first sequence (ie, "expression vector C1").

[0194] Provided herein is an expression vector comprising: (a) a first sequence comprising a portion of an expression cassette containing a nucleic acid sequence of interest flanked on one side by a splicing sequence, ITRs flanking each side of the first sequence, wherein the ITRs comprise an AAV replication sequence and an AAV packaging signal, (b) a target sequence for a first recombinase flanking each ITR, and (c) one or more additional target sequences for one or more additional recombinases, the one or more additional target sequences being integrated within a non-binding region of the target sequence of the first recombinase, wherein the expression vector is used to produce a bacterial sequence-free vector having linear covalently closed ends (i.e., "expression vector D"). In some aspects, the partial expression cassette comprises a 5' portion that, in combination with the remainder of the expression cassette, provides the complete sequence of the expression cassette, and the splicing sequence is located at the 3' end of the 5' portion. In some aspects, the partial expression cassette comprises a 3' portion that, in combination with the remainder of the expression cassette, provides the complete sequence of the expression cassette, and the splicing sequence is located at the 5' end of the 3' portion. In some aspects, the expression vector further comprises a spacer sequence between the target sequence of the first recombinase and the first sequence. In some aspects, the spacer sequence is 10 to 500 nucleotides.

[0195] The expression cassette in any expression vector disclosed herein can include any suitable expression control region known to those skilled in the art.

[0196] In some aspects, the expression control region is a cis-regulatory module (CRM), a promoter, an enhancer, an operator, a repressor, a ribosome binding site, a translation leader sequence, an intron, a post-transcriptional element, a polyadenylation recognition sequence, an RNA processing site, an effector binding site, a stem-loop structure, a transcription termination signal, a miRNA binding site, or a combination thereof. See, for example, Domenger and Grimm, Hum. Mol. Genet. 28(R1):R3-R14(2019).

[0197] In some aspects, the promoter is a mammalian, viral, wild-type promoter, or a synthetic promoter, including, for example, tissue- and / or cell-specific promoters.

[0198] In some aspects, the post-transcriptional element is a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE).

[0199] In some aspects, the 3' end of the expression cassette includes a miRNA binding site for controlling expression (eg, tissue and / or cell-specific expression) from the vector.

[0200] The nucleic acid sequence of interest in any expression vector disclosed herein can be any desired sequence and is not limited by any particular requirements other than the packaging constraints imposed by the AAV capsid. In particular, single-stranded DNA sequences of no more than about 5 kilobases can be packaged into a single AAV capsid, including any ITR sequences, and double-stranded DNA sequences of no more than about half that size can be packaged.

[0201] In some aspects, the nucleic acid sequence of interest comprises a sequence encoding a polypeptide, RNA (messenger RNA (mRNA), microRNA (miRNA), small interfering RNA (siRNA)), small hairpin RNA (shRNA), ribozyme or antisense RNA) or non-coding DNA (e.g., antisense oligonucleotide). In some aspects, the nucleic acid sequence of interest comprises a sequence encoding an anticancer agent, a tumor suppressor, an apoptosis agent, an anti-angiogenic agent, an enzyme, a cytotoxic agent, a suicide gene, a cytokine, an interferon, an interleukin, an immunomodulator, an immunostimulatory agent, an immunosuppressant, a chemokine, an antigen for stimulating antigen-presenting cells, an antibody (e.g., a monoclonal, chimeric, humanized or human antibody, or an antigen-binding fragment thereof), or an immunogenic agent (e.g., as a vaccine).

[0202] Exemplary nucleic acid sequences of interest and exemplary related therapies include: surfactant protein B (SP-B, for the treatment of surfactant dysfunction), surfactant protein C (SP-C, for the treatment of surfactant dysfunction), ATP-binding cassette subfamily A member 3 (ABCA3, for the treatment of surfactant dysfunction), solute carrier family 34 member 2 (SLC34A2, for the treatment of alveolar microlithiasis and / or testicular microlithiasis), cystic fibrosis transmembrane conductance regulator (CFTR, for the treatment of cystic fibrosis), glutamate decarboxylase (GAD, for example, GAD65 or GAD67, for the treatment of Parkinson's disease), aspartate acylase gene (ASPA, also known as aminoacylase (AAC), for the treatment of Canavan disease), aromatic L-amino acid decarboxylase (AADC, for the treatment of Parkinson's disease and / or for the treatment of AADC deficiency), Neurturin (NRTN, for the treatment of Parkinson's disease), glial cell line-derived neurotrophic factor (GDNF, for the treatment of Parkinson's disease), and mitochondrial isomerase (MIT) gene. Alzheimer's disease), nerve growth factor (NGF, used to treat Alzheimer's disease), tripeptidyl peptidase I (TPP1, also known as neuronal ceroid lipofuscinosis type 2 (CLN2), used to treat Batten disease), arylsulfatase A (ARSA, used to treat metachromatic leukodystrophy), N-sulfoglucosamine sulfohydrolase (SGSH, used to treat Sanfilippo syndrome, type A), sulfatase modifying factor 1 (SUMF1, used to treat Sanfilippo syndrome), and sulfatase inhibitory factor 2 (IGF-1) (used to treat Alzheimer's disease). syndrome, type A), N-acetyl-α-glucosaminidase (NAGLU, used to treat Sanfilippo syndrome, type B), survival of motor neuron 1 (SMN1, used to treat spinal muscular atrophy 1), retinal pigment epithelium-specific 65 kDa protein (RPE65, also known as retinoid isomerohydrolase, used to treat Leber congenital amaurosis), Rab escort protein 1 (REP1, used to treat choroideremia), retinoschisin 1 (Retinoschisin 1, Rs1, for the treatment of X-linked juvenile retinoschisis), alpha-1 antitrypsin (AAT, for the treatment of hereditary emphysema or AAT deficiency), mini-dystrophin (for the treatment of Duchenne muscular dystrophy), alpha-sarcoglycan (αSG, for the treatment of Duchenne muscular dystrophy or limb-girdle muscular dystrophy type 2), beta-sarcoglycan (βSG), gamma-sarcoglycan (γSG, for the treatment of limb-girdle muscular dystrophy type 2), delta-sarcoglycan (γSG), lipoprotein lipase (LPL, for the treatment of familial LPL deficiency), acid alpha-glucosidase (GAA, for the treatment of Pompe disease), tumor necrosis factor receptor: Fc (TNFR:Fc, for the treatment of arthritis, such as inflammatory arthritis), sarcoplasmic / endoplasmic reticulum Ca(2+) ATPase 2a (SERCA2a,for the treatment of congestive heart failure), factor VIII, factor IX (FIX, for the treatment of hemophilia B), porphobilinogen deaminase gene (PBGD, for the treatment of acute intermittent porphyria), soluble FMS-like tyrosine kinase-1 (sFlt1, for the treatment of age-related macular degeneration or cancer, such as ovarian cancer), soluble chimeric vascular endothelial growth factor (VEGF) receptor comprising VEGFR-1 and VEGF-R2 domains (for the treatment of cancer, such as melanoma or colon cancer), soluble VEGFR3 (for the treatment of cancer, such as endometrial cancer), soluble VEGF-C decoy receptor (sVEGFR3-Fc, for the treatment of cancer, such as melanoma, renal cell carcinoma or prostate cancer), pigment epithelium-derived growth factor (PEDF, for the treatment of cancer, such as Lewis lung cancer), neutralizing mAbs to VEGFR2 (e.g., DC101, for treating cancers such as melanoma or glioblastoma), endostatin (for treating cancers such as bladder cancer or pancreatic cancer), angiostatin (for treating cancers such as liver cancer), both endostatin and angiostatin (i.e., as a bicistronic sequence, for treating cancers such as ovarian cancer or prostate cancer), endostatin mutants (i.e., P1254A-endostatin, for treating cancers such as ovarian cancer), the anti-angiogenic domain of TSP-1 (3TSR, for treating cancers such as pancreatic cancer), tissue factor pathway inhibitor-2 (TFPI-2, for treating cancers such as glioblastoma), plasminogen fragments (e.g., Kringle 5, for treating cancers such as ovarian cancer), plasminogen kringle 1-5 (for treating cancers such as melanoma or lung cancer), siRNA against unfolded protein response proteins (UPR; such as IRE1α, XBP-1 or ATF6, for treating cancers such as breast cancer), Vasostatin (for treating cancers such as lung cancer), herpes simplex virus type 1 thymidine kinase (HSV-TK, for treating cancers such as breast cancer), sc39TK (for treating cancers such as cervical cancer), diphtheria toxin A (DTA, for treating cancers such as cervical cancer or myeloma), p53 upregulated apoptosis modulator (PUMA, for treating cancers , such as cervical cancer or myeloma), tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL, for treating cancers such as lymphoma, hepatocellular carcinoma, head and neck squamous cell carcinoma (i.e., head and neck cancer), or glioblastoma), soluble TRAIL (for treating cancers such as liver cancer or lung adenocarcinoma), IFN-β (for treating cancers such as colorectal cancer, lung cancer, neuroblastoma, or glioblastoma multiforme), IFN-α (for treating cancers such as metastatic melanoma), CD-40 ligand (CD40L) or CD40L mutants (for treating cancers such as leukemia, leukemia, and scleroderma).for treating cancers such as lung cancer), melanoma differentiation-associated gene-7 and interleukin-24 (MDA-7 and IL24, for treating cancers such as Ehrlich ascites), apoptotin and IL24 (for treating cancers such as liver cancer), IL24 (for treating cancers such as mixed lineage leukemia (MLL) / AF4-positive acute lymphoblastic leukemia (ALL)), IL15 (for treating cancers such as metastatic hepatocellular carcinoma), secondary lymphoid tissue chemokine (SLC, for treating cancers such as liver cancer), Nk4 (the N-terminal hairpin followed by four kringle domains of hepatocyte growth factor (HGF), for treating cancers such as metastatic Lewis lung cancer), tumor necrosis factor superfamily member 14 (TNFSF14, also known as LIGHT, for treating cancers such as cervical cancer), granulocyte-macrophage colony-stimulating factor (GM-CSF, for treating cancers), TNF-α (e.g., for treating cancers such as gliomas), dominant negative mutants of survivin (e.g., C84A or T34A, for treating cancers such as colon cancer or gastric cancer), C-terminal fragment of human telomerase reverse transcriptase (hTERTC27, for treating cancers such as glioblastoma multiforme), maspin (e.g., for treating cancers such as prostate cancer), nm23H1 (e.g., for treating cancers such as metastatic ovarian cancer), human hepatocyte thymocyte necrosis factor (HTN-1), HTN-23H1 (e.g., for treating cancers such as prostate cancer), and human hepatocyte thymocyte necrosis factor (HTN-1). Kringle 1 domain of growth factor (HGFK1, for treating cancers such as colorectal cancer), anti-calcitonin ribozyme (for treating cancers such as prostate cancer), eukaryotic translation initiation factor 4E binding protein 1 (4EBP1, for treating cancers such as lung cancer), CXC motif chemokine receptor 2 (CXCR2) C-tail sequence (for treating cancers such as pancreatic cancer), α-tocopherol-associated protein (TAP, for treating cancers such as prostate cancer), trichosanthin (for treating cancers such as hepatocellular carcinoma), decorin (for treating cancers such as glioblastoma multiforme), Catheli cidin (for the treatment of cancer, for example, colon cancer), Niemann-Pcik type C2 (NPC2, for the treatment of cancer, for example, hepatocellular carcinoma), Mullerian inhibitory substances (MISs, for the treatment of cancer, for example, ovarian cancer), P53 (for the treatment of cancer, for example, bronchioloalveolar carcinoma), shRNA against cancer highly expressed protein 1 (Hec1, for the treatment of cancer, for example, glioma), shRNA against Epstein-Barr virus latent membrane protein-1 (EBVLMP-1, for the treatment of cancer, for example, nasopharyngeal carcinoma), antisense RNA against human papillomavirus 16E7 oncogene (HPV16-E7, for the treatment of cancer, for example, cervical cancer),shRNAs targeting androgen receptor (AR, for treating cancers such as prostate cancer), siRNAs targeting Snail (also known as SNA1, for treating cancers such as pancreatic cancer), siRNAs targeting Slug (i.e., the protein product of SNAI2, for treating cancers such as bile duct cancer (liver cancer)), shRNAs targeting four and a half LIM-Only protein 2 (FHL2, for treating cancers such as colon cancer), miR-26a (for treating cancers such as hepatocellular carcinoma), HPV16 structural protein L1 (HPV16-L1, for treating cancers such as cervical cancer), HPV16 E5, E6 and E7 oncogenes (HPV16 E5 / E6 / E7, for treating cancers such as cervical cancer), B cell leukemia / lymphoma 1 (BLC1) idiotype (for treating cancers such as B cell leukemia / lymphoma 1), EBV LMP1 and LMP2 fused to heat shock protein (EBV LMP2 / 1-HSP, for treating cancers such as nasopharyngeal carcinoma), carcinoembryonic antigen (CEA, for treating cancers such as colon cancer), a soluble form of B / T lymphocyte attenuating factor combined with heat shock protein (BTLA and HSP70, for treating cancers such as melanoma lung metastasis), HPV16-L1 / E7 (for treating cancers such as cervical cancer), HPV16-L1 (for treating cancers such as cervical cancer), anti-EGFR antibodies (e.g., 14D1, for treating cancers such as vulvar cancer), anti-death receptor 5 (DR5) antibodies (e.g., , adximab, for treating cancer, e.g., liver or colon cancer), anti-enolase 1 (ENOI1) antibodies (for treating cancer, e.g., pancreatic ductal adenocarcinoma), anti-VEGFA antibodies (e.g., bevacizumab, for treating cancer, e.g., metastatic lung or ovarian cancer), mucin 1 (MUC1) antigen (for treating cancer (e.g., gastric cancer), or aquaporins (e.g., hAQP1, for treating radiation-induced parotid salivary gland hypofunction, i.e., xerostomia). See, e.g., Lisowski et al., Santiago-Oritz et al.,

[0203] In some aspects, the AAV replication sequence in any of the above expression vectors comprises an AAV ITR replication (Rep) protein binding element (RBE) and a terminal dissociation site (TRS).

[0204] In some aspects, the AAV packaging signal in any of the above expression vectors comprises an AAV ITR D sequence.

[0205] Provided herein is an expression vector for producing a bacterial sequence-free vector (i.e., msDNA) with linear covalently closed ends comprising sequences encoding AAV rep, AAV cap, and / or helper virus genes required for producing AAV. In some aspects, the expression vector comprises AAV rep. In some aspects, the expression vector comprises AAV cap. In some aspects, the expression vector comprises AAV rep and AAV cap. In some aspects, the expression vector comprises helper virus genes. In some aspects, the expression vector comprises AAV rep and helper virus genes. In some aspects, the expression vector comprises AAV cap and helper virus genes. In some aspects, the expression vector comprises AAV rep, AAV cap, and helper virus genes. The expression vector comprising AAV rep, AAV cap, and / or helper virus genes lacks (i.e., does not include) ITRs at both ends of AAV rep, AAV cap, and / or helper virus genes to avoid packaging AAV rep and AAV cap and / or helper virus genes into AAV capsids. In some aspects, the coding sequence of the expression vector disclosed herein is essentially composed of AAV rep, AAV cap, and / or helper virus genes.

[0206] Helper virus genes disclosed herein include one or more genes from a virus that provides the functions required for AAV replication. In some aspects, the helper virus genes include one or more genes from adenovirus, herpes virus (e.g., herpes simplex virus (HSV), Epstein-Barr virus (EBV), cytomegalovirus (CMV) or pseudorabies virus (PRV)), retrovirus, pox virus (e.g., vaccinia virus) and / or lentivirus. In some aspects, the helper virus genes include one or more genes from adenovirus early 4 (E4) gene, adenovirus early 2A (E2A) gene or adenovirus virus-associated (VA) gene. In some aspects, the helper virus genes include adenovirus E4, E2A and VA genes.

[0207] In some aspects, the msDNA disclosed herein for expressing an auxiliary gene comprises the polynucleotide sequence of SEQ ID NO:25.

[0208] In some aspects, the msDNA disclosed herein for expressing an auxiliary gene comprises the polynucleotide sequence of SEQ ID NO:51.

[0209] Provided herein is an expression vector comprising: (a) an expression cassette comprising an AAV rep gene and an AAV cap gene, (b) target sequences for a first recombinase flanking each side of the expression cassette, and (c) one or more additional target sequences for one or more additional recombinases, the one or more additional target sequences being integrated within the non-binding region of the target sequence for the first recombinase, wherein the expression vector is used to produce a bacterial sequence-free vector having linear covalently closed ends (i.e., "expression vector E").

[0210] Provided herein is an expression vector comprising: (a) an expression cassette comprising one or more helper virus genes for producing AAV, (b) a target sequence for a first recombinase flanking each side of the expression cassette, and (c) one or more additional target sequences for one or more additional recombinases, the one or more additional target sequences being integrated within the non-binding region of the target sequence for the first recombinase, wherein the expression vector is used to generate a bacterial sequence-free vector with linear covalently closed ends (i.e., "expression vector F"). In some aspects, the one or more helper virus genes are from an adenovirus, a herpes virus, a retrovirus, a pox virus, and / or a lentivirus. In some aspects, the one or more helper virus genes comprise an adenovirus E4 gene, an adenovirus E2A gene, and an adenovirus VA gene.

[0211] In some aspects, the target sequence of the first recombinase and the one or more additional target sequences of the one or more additional recombinases in any expression vector disclosed herein are selected from the group consisting of the PY54 pal site, the N15 telRL site, and the In some aspects, any expression vector disclosed herein comprises each of these target sequences. In some aspects, any expression vector disclosed herein comprises a Tel recombinase pal site and a telRL recombinase target binding sequence integrated into the pal site.

[0212] In some aspects, the target sequence for the first recombinase in any of the expression vectors disclosed herein is the bacteriophage PY54 Tel 142 base pair target site.

[0213] This paper provides a vector production system, which includes a recombinant cell designed to encode at least the first recombinase under the control of an inducible promoter, wherein the cell includes any expression vector disclosed herein. In some aspects, the cell includes any one of the expression vectors BF disclosed herein. In some aspects, the recombinant cell is an Escherichia coli cell, a yeast cell such as Saccharomyces cerevisiae (Saccharomyces cerevisiae) or a mammalian cell, as disclosed in U.S. Patent No. 9862954. In some aspects, the inducible promoter is heat-regulated, chemically regulated, IPTG-regulated, glucose-regulated, arabinose-induced, T7 polymerase-regulated, cold shock-induced, pH-induced, or a combination thereof. In some aspects, the first recombinase is selected from TelN and Tel, and the expression vector incorporates the target sequence of at least the first recombinase. In some aspects, the recombinant cell has been further designed to encode a nuclease genome editing system, and wherein the expression vector further includes a backbone sequence containing a cleavage site for the nuclease genome editing system. In some aspects, the nuclease genome editing system is a CRISPR nuclease system comprising a Cas nuclease and a gRNA, and the expression vector comprises a target sequence for the gRNA in the backbone sequence.

[0214] Provided herein is a method for producing a bacterial sequence-free vector having linear covalently closed ends (ie, msDNA or msDNA vector), comprising incubating any of the vector production systems disclosed herein under conditions suitable for expression of a first recombinase.

[0215] Provided herein is a method for producing a bacterial sequence-free vector having linear covalently closed ends, comprising incubating any vector production system disclosed herein under conditions suitable for expressing a first recombinase and a nuclease genome editing system. In some aspects, the method further comprises harvesting the bacterial sequence-free vector.

[0216] Provided herein is a method for producing a vector free of bacterial sequences having linear covalently closed ends, comprising incubating any expression vector disclosed herein in vitro with a Tel / Pal recombination system derived from bacteriophage PY54. In some aspects, the method further comprises harvesting the vector free of bacterial sequences.

[0217] Provided herein is a vector without bacterial sequences, which is produced by any method disclosed herein for producing a vector without bacterial sequences having linear covalently closed ends. In some aspects, the vector without bacterial sequences is produced from expression vector B1. In some aspects, the vector without bacterial sequences is produced from expression vector B2. In some aspects, the vector without bacterial sequences is produced from expression vector B3. In some aspects, the vector without bacterial sequences is produced from expression vector B4. In some aspects, the vector without bacterial sequences is produced from expression vector C. In some aspects, the vector without bacterial sequences is produced from expression vector C1. In some aspects, the vector without bacterial sequences is produced from expression vector D. In some aspects, the vector without bacterial sequences is produced from expression vector E. In some aspects, the vector without bacterial sequences is produced from expression vector F.

[0218] B. Methods for producing AAV from msDNA

[0219] Wild-type AAV is packaged as a single-stranded genome (ie, single-stranded AAV, "ssAAV"). The msDNA disclosed herein comprising an expression cassette flanked by ITRs can be used to produce ssAAV in the presence of rep, cap, and helper viral genes.

[0220] The packaging capacity of the AAV capsid is approximately 5 kb. A nucleic acid sequence of interest larger than about 5 kb and no larger than about 10 kb can be delivered by co-infecting cells with two separate ssAAVs, each carrying a portion of the nucleic acid sequence of interest. These portions can be joined together in the co-infected cells by trans-splicing or homologous recombination to reconstitute the entire nucleic acid sequence of interest.

[0221] Trans-splicing utilizes the ability of the AAV genome to form head-to-tail concatemers by recombination in the ITRs after infection of cells. See, for example, Daya and Berns; Yan et al., PNAS 97(12):6716-6721(2000). The separated 5' and 3' portions can be joined by transcription from recombinant AAV and then splicing the mRNA transcripts. Specifically, in the first AAV, the 5' portion of the target nucleic acid sequence with a splicing sequence (e.g., a 3' splice donor) is flanked by ITRs on both sides; meanwhile, in the second AAV, the remaining 3' portion of the target nucleic acid sequence with a splicing sequence (e.g., a 5' splice acceptor) is flanked by ITRs on each side. After infection, the 5' and 3' portions from each AAV are spliced together to form the complete target nucleic acid sequence.

[0222] Alternatively, the nucleic acid sequence of interest can be split into two parts with substantial sequence overlap between the two separate ssAAVs. Co-expression in infected cells induces homologous recombination and forms the complete nucleic acid sequence of interest.

[0223] The msDNA disclosed herein can be used to produce ssAAV comprising portions of a nucleic acid sequence of interest for delivery of sequences up to approximately 10 kb to target cells and tissues by co-infection and trans-splicing or homologous recombination.

[0224] Because AAV relies on the cellular DNA replication machinery to synthesize the complementary strand of the ssAAV genome, expression of the target nucleic acid sequence (e.g., a transgene) from ssAAV may be delayed after infection of cells or tissues. To overcome the delayed expression of the target nucleic acid sequence, self-complementary AAV (scAAV) can be generated, which contains complementary sequences that can spontaneously anneal to form double-stranded DNA capable of transcription after infection. See, for example, Daya and Berns. In the presence of rep, cap, and helper viral genes, the msDNA disclosed herein comprising a palindromic sequence (e.g., an expression cassette comprising a target nucleic acid sequence and a complementary sequence to the expression cassette) can be packaged as scAAV, wherein the palindromic sequence is flanked on each side by ITRs.

[0225] scAAV can also be formed using the msDNA disclosed herein containing only a single ITR on one side of the expression cassette. Without both ITRs, the msDNA does not replicate as an ssAAV intermediate. Instead, in the presence of rep, cap, and helper viral genes, the sequence is directly packaged as double-stranded DNA due to the packaging signals in the ITRs. Then, upon infection, the nucleic acid sequence of interest can be obtained as double-stranded DNA that can be transcribed.

[0226] The AAV rep and cap sequences can be provided in standard plasmids used for AAV production, or can be provided in one or more of the msDNAs disclosed herein.

[0227] Helper viral genes can be provided in a standard plasmid, as a helper virus, or in one or more of the msDNAs disclosed herein.

[0228] In some aspects, the production of AAV2 disclosed herein comprises: msDNA encoding a GOI disclosed herein, msDNA comprising the rep2 and cap2 genes comprising the polynucleotide sequence of SEQ ID NO: 24, and / or msDNA comprising the helper virus genes comprising the polynucleotide sequence of SEQ ID NO: 25.

[0229] In some aspects, the production of AAV2 disclosed herein comprises: msDNA encoding a GOI disclosed herein, msDNA comprising the rep2 and cap2 genes comprising the polynucleotide sequence of SEQ ID NO: 48, and / or msDNA comprising the helper virus genes comprising the polynucleotide sequence of SEQ ID NO: 51.

[0230] In some aspects, the production of AAV5 disclosed herein comprises: msDNA encoding a GOI disclosed herein, msDNA comprising the rep2 and cap5 genes comprising the polynucleotide sequence of SEQ ID NO: 49, and / or msDNA comprising a helper virus gene comprising the polynucleotide sequence of SEQ ID NO: 51.

[0231] In some aspects, the production of AAV9 disclosed herein comprises: msDNA encoding a GOI disclosed herein, msDNA comprising the rep2 and cap9 genes comprising the polynucleotide sequence of SEQ ID NO: 50, and / or msDNA comprising a helper virus gene comprising the polynucleotide sequence of SEQ ID NO: 51.

[0232] Alternatively, according to the methods disclosed herein, AAV producer cell lines containing integrated rep, cap and / or helper viral genes in the genome of the producer cells can be used to produce AAV to provide consistent and stable expression of AAV replication and packaging proteins. AAV producer cell lines can be generated to contain integrated rep, cap and / or helper viral genes by homologous recombination with the corresponding msDNA disclosed herein containing homology arms for recombination.

[0233] The AAV producer cell can be any cell capable of producing AAV. In some aspects, the producer cell is a mammalian cell (e.g., HEK293, COS, HeLa, or KB). In some aspects, the producer cell is HEK293. In some aspects, the producer cell is an insect cell (e.g., Drosophila Schneider 2 (S2), Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, 5Tn368, HzAml, Ha2302, or Hz2E5). In some aspects, when the producer cell is an insect cell, the expression vector disclosed herein for producing msDNA is a baculovirus vector.

[0234] Provided herein is a method for producing single-chain AAV, wherein the method comprises: (a) transfecting cells capable of producing AAV with: (i) a bacterial sequence-free vector generated from expression vector B3, (ii) a bacterial sequence-free vector generated from expression vector E, or an expression vector comprising an expression cassette comprising an AAV rep gene and an AAV cap gene, and (iii) a bacterial sequence-free vector generated from expression vector F, or an expression vector comprising an expression cassette comprising one or more helper virus genes for producing AAV; and (b) incubating the cells under conditions suitable for producing AAV.

[0235] Provided herein is a method for producing single-chain AAV, wherein the method comprises: (a) transfecting cells capable of producing AAV with: (i) a bacterial sequence-free vector generated from expression vector B4, (ii) a bacterial sequence-free vector generated from expression vector F, or an expression vector comprising an expression cassette containing one or more helper virus genes for producing AAV; and (b) incubating the cells under conditions suitable for producing AAV.

[0236] Provided herein is a method for producing single-chain AAV, comprising: (a) transfecting cells capable of producing AAV with a bacterial sequence-free vector generated from expression vector B3, wherein each AAV rep gene, AAV cap gene, and one or more helper virus genes for producing AAV are encoded by the cells or by the vector; and (b) incubating the cells under conditions suitable for expressing the rep gene, cap gene, and one or more helper virus genes and for producing AAV.

[0237] Provided herein is a method for producing self-complementary AAV, wherein the method comprises: (a) transfecting cells capable of producing AAV with: (i) a bacterial sequence-free vector generated from expression vector B1; (ii) a bacterial sequence-free vector generated from expression vector E, or an expression vector comprising an expression cassette comprising an AAV rep gene and an AAV cap gene; and (iii) a bacterial sequence-free vector generated from expression vector F, an expression vector comprising an expression cassette comprising one or more helper virus genes for producing AAV, and (b) incubating the cells under conditions suitable for producing AAV.

[0238] Provided herein is a method for producing self-complementary AAV, wherein the method comprises: (a) transfecting cells capable of producing AAV with: (i) a bacterial sequence-free vector generated from expression vector B2, (ii) a bacterial sequence-free vector generated from expression vector F, or an expression vector comprising an expression cassette containing one or more helper viral genes for producing AAV, and (b) incubating the cells under conditions suitable for producing AAV.

[0239] Provided herein is a method for producing self-complementary AAV, wherein the method comprises: (a) transfecting cells capable of producing AAV with: (i) a bacterial sequence-free vector generated from expression vector C, (ii) a bacterial sequence-free vector generated from expression vector E, or an expression vector comprising an expression cassette comprising an AAV rep gene and an AAV cap gene, and (iii) a bacterial sequence-free vector generated from expression vector F, or an expression vector comprising an expression cassette comprising one or more helper virus genes for producing AAV, and (b) incubating the cells under conditions suitable for producing AAV.

[0240] Provided herein is a method for producing self-complementary AAV, wherein the method comprises: (a) transfecting cells capable of producing AAV with: (i) a bacterial sequence-free vector generated from expression vector C1, (ii) a bacterial sequence-free vector generated from expression vector F, or an expression vector comprising an expression cassette containing one or more helper virus genes for producing AAV, and (b) incubating the cells under conditions suitable for producing AAV.

[0241] Provided herein is a method for producing self-complementary AAV, wherein the method comprises: (a) transfecting cells capable of producing AAV with a bacterial sequence-free vector generated from expression vector B1 or C, wherein each AAV rep gene, AAV cap gene, and one or more helper viral genes for producing AAV are encoded by the cells or by the vector, and (b) incubating the cells under conditions suitable for expression of the rep gene, cap gene, and one or more helper viral genes and suitable for production of AAV.

[0242] In some aspects, the cells used in any of the methods disclosed herein for producing single-chain AAV or self-complementary AAV are HEK293T cells.

[0243] In some aspects, any of the methods disclosed herein for producing single-stranded AAV or self-complementary AAV further comprise harvesting the AAV.

[0244] In one aspect, the present disclosure relates to AAV produced by any of the methods disclosed herein for producing single-chain AAV or self-complementary AAV.

[0245] In some aspects, the AAV produced according to the methods disclosed herein has a reduction in the number of contaminating bacterial sequences compared to AAV produced using another method (e.g., a system in which three plasmids respectively contain a nucleic acid sequence of interest, a rep / cap, and a helper viral gene (i.e., a three-plasmid system). In some aspects, the number of contaminating bacterial sequences is reduced to below the limit of detection (i.e., the contaminating bacterial sequences are not detectable in the AAV). In some aspects, the reduction in the number of contaminating bacterial sequences is determined by real-time quantitative PCR for bacterial backbones and / or other plasmid-associated impurities, such as ampicillin resistance (ampR) genes, origins of replication (ori, e.g., F1 ori), kanamycin resistance (kanR) genes, or sequences for chromatographic affinity recombination (SCAR).

[0246] In some aspects, AAV produced according to the methods disclosed herein provides higher quantities of AAV comprising a nucleic acid sequence of interest compared to AAV produced using another method.

[0247] In some aspects, the AAV produced according to the methods disclosed herein comprises a reduced number of empty capsids compared to AAV produced using another method.

[0248] The number of empty capsids, including the ratio of intact AAV particles to empty AAV particles, can be assessed by any method known in the art, including, for example, analytical ultracentrifugation, transmission electron microscopy, anion exchange high performance liquid chromatography, and / or capillary isoelectric focusing. See, for example, Burnham et al., Hum. Gene Ther. Methods 26(6):228-242 (2015); Chen, Microsc. Microanal. 13(5), 384-389 (2007); Fu et al., Hum. Gene Ther. Methods 30(4):144-152 (2019); Li et al., Curr. Mol. Med. doi:10.2174 / 1566524020666200915105456 (2020).

[0249] In some aspects, AAV produced according to the methods disclosed herein provides higher transfection efficiency compared to AAV produced using another method.

[0250] In some aspects, AAV produced according to the methods disclosed herein provides a higher copy number per unit transfection compared to AAV produced using another method.

[0251] In some aspects, AAV produced according to the methods disclosed herein provides for greater nuclear localization of AAV compared to AAV produced using another method.

[0252] In some aspects, AAV produced according to the methods disclosed herein results in a reduced immune response, fewer neutralizing antibodies, lower risk of genomic integration, lower silencing of the nucleic acid of interest, and / or lower risk of antibiotic resistance following administration to a subject or as measured in vitro, compared to AAV produced using another method.

[0253] III. Pharmaceutical Compositions and Therapeutic Uses

[0254] Provided herein are pharmaceutical compositions comprising the AAV disclosed herein.

[0255] In some aspects, the composition further comprises a physiologically acceptable carrier, excipient or stabilizer. See, for example, Remington: The Science and Practice of Pharmacy, 22nd ed. (2013). Acceptable carriers, excipients or stabilizers can include those that are nontoxic to the subject. In certain embodiments, the composition or one or more components of the composition are sterile. Sterile components can be prepared, for example, by filtering (for example, by sterile filtration membrane) or by radiation (for example, by gamma radiation).

[0256] When added to a pharmaceutical composition, the excipient of the present invention can be described as a "pharmaceutically acceptable" excipient, which means that the excipient is a compound, material, composition, salt and / or dosage form that is suitable for contact with the tissues of humans and animals within the scope of reasonable medical judgment, and does not cause excessive toxicity, irritation, allergic reactions or other problematic complications during the expected contact period consistent with a reasonable benefit / risk ratio. In some embodiments, the term "pharmaceutically acceptable" refers to, for animals and especially human applications, obtaining approval from the regulatory agencies of the federal or state governments or being recorded in the U.S. Pharmacopoeia or other generally recognized international pharmacopoeias. Various excipients can be used. In some embodiments, the excipient can be, but is not limited to, an alkaline agent, a stabilizer, an antioxidant, a binding agent, a separating agent, a coating agent, an external phase component, a controlled release component, a solvent, a surfactant, a wetting agent, a buffer, a filler, a lubricant or a combination thereof. In addition to the excipients discussed herein, excipients may also include, but are not limited to, those listed in Remington: The Science and Practice of Pharmacy, 22nd ed. (2013). In this article, inclusion of an excipient in a particular classification (e.g., "solvent") is intended to illustrate, rather than limit, the role of the excipient. A specific excipient may belong to multiple categories.

[0257] Pharmaceutical composition of the present disclosure is formulated to be compatible with its expected route of administration.The example of route of administration includes: the route of administration of composition disclosed herein includes intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as, by injection or infusion.Phrase " parenteral administration " used herein refers to the mode of administration except enteral and topical application, usually by injection, and includes but is not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous (subcuticular), intraarticular, subcapsular, subarachnoid space, spinal, epidural and intrasternal injection and infusion, and electroporation in vivo.In some aspects, composition is used by non-parenteral route, in some aspects, by oral administration.Other non-parenteral routes include local, epidermal or mucosal routes of administration, such as intranasal, sublingual or topical application.Use can be for example once, repeatedly and / or in one or more extended time periods. In some aspects, the pharmaceutical composition comprising an AAV disclosed herein further comprises a delivery agent. In some aspects, the delivery agent comprises a nanoparticle. In some aspects, the delivery agent is selected from liposomes, non-lipid polymer molecules, endosomes, and any combination thereof. In some aspects, the delivery agent (e.g., nanoparticle) comprises a targeting ligand.

[0258] Provided herein is a method of treating a disease or condition in a subject in need thereof, comprising administering to the subject an AAV or pharmaceutical composition disclosed herein.

[0259] As long as clinical benefit is observed, or before unacceptable toxicity or disease progression occurs, treatment can continue. The dosage and frequency of administration can vary depending on whether the treatment is preventive or therapeutic. In preventive applications, relatively low dosage is usually administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, it is sometimes necessary to use relatively high dosage at relatively short intervals until the progression of the disease reduces or terminates, and preferably until the patient shows partial or complete improvement of disease symptoms. After this, a preventive regimen can be performed on the patient.

[0260] For a particular patient, composition and mode of administration, actual dosage levels can vary to obtain the amount of the nucleic acid sequence of interest disclosed herein to effectively achieve the desired therapeutic response without causing excessive toxicity to the patient. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition of the present disclosure used, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical field. The compositions of the present disclosure can be administered by one or more routes of administration using one or more of the various methods well known in the art. It will be understood by those skilled in the art that the route of administration and / or mode will vary depending on the desired result.

[0261] All references cited above and all references cited herein are incorporated by reference in their entirety.

[0262] The following examples are offered by way of illustration only and not limitation.

[0263] Example

[0264] Example 1

[0265] Generation of expression vectors and ministranded DNA

[0266] According to the methods disclosed in U.S. Patent Nos. 9,290,778 and 9,862,954 and International Publication No. WO 2022 / 264095, which are incorporated herein by reference in their entireties, ministranded DNA (msDNA) containing the gene of interest, Rep / Cap sequences for AAV replication / packaging, or adenoviral helper sequences is generated.

[0267] A. Generate an expression vector containing GFP

[0268] An expression vector containing green fluorescent protein (GFP) as an exemplary target gene was prepared, wherein the target gene was flanked by 5' ITR and 3' ITR ("ITR-GFP-ITR", see e.g. Figure 1 ) or the gene of interest is only flanked by a 3' ITR ("GFP-ITR").

[0269] ITR-GFP-ITR sequences and GFP-ITR sequences were obtained from AAV-GFP vectors by restriction digestion or polymerase chain reaction (PCR) amplification, or the GFP sequence was cloned into a plasmid carrying appropriate ITRs.

[0270] ITR-GPP-ITR and GFP-ITR were each inserted into the multiple cloning site between two specialized super sequence ("SS" or "SSeq") sites in separate expression vectors (pMinistring, Mediphage Bioceuticals, Inc., Toronto, CA; U.S. Patent Nos. 9,290,778 and 9,862,954, and International Publication No. WO2022 / 264095).

[0271] A map of an exemplary expression vector encoding ITR-GFP-ITR msDNA is shown in FIG. Figure 1 The nucleic acid sequence of the vector is shown as “ITR-CAG-GFP-ITR plasmid” in FIG. 18.

[0272] The map and sequence of an exemplary expression vector encoding GFP-ITR are identical to those of the ITR-CAG-GFP-ITR plasmid, except that the expression cassette encoding GFP-ITR in the expression vector is only flanked by a 3' ITR and not a 5' ITR.

[0273] Additional expression vectors were prepared using ITR-GFP-ITR and GFP-ITR, wherein spacer sequences of varying lengths were included between the 5' SS and 5' ITR and between the 3' ITR and 3' SS for ITR-GFP-ITR, or between the 3' ITR and 3' SS for GFP-ITR. Exemplary spacer sequence lengths are 0-10, 10-15, 50-100, 100-250, and 250-500 nucleotides.

[0274] Expression vectors were also prepared in which the ITR in ITR-GFP-ITR and GFP-ITR was a minimal ITR lacking the B-B' and C-C' palindromic sequences (ie, the ITR contained only the A-A' palindromic sequence and the D sequence).

[0275] B. Generate expression vectors containing rep and cap sequences

[0276] The sequence containing rep and cap is obtained from a plasmid containing the gene by restriction digestion or PCR amplification and inserted between the two SS sites in the expression vector.

[0277] A map of an exemplary expression vector encoding Rep-Cap msDNA is provided in Figure 4 The vector is represented by “PGL2-SS-CMV-Rep-Cap-SS”, and the nucleic acid sequence of the vector is shown in SEQ ID NO: 23.

[0278] Additional expression vectors were prepared in which the rep sequence and the cap sequence were combined with the GOI into a single expression vector, for example, expression vectors in which the rep and cap sequences were flanked by SS on one side and ITR-GFP-ITR or GFP-ITR on the other side (e.g., SS-Rep-Cap-ITR-GFP-ITR-SS and SS-Rep-Cap-GFP-ITR-SS).

[0279] C. Generation of expression vectors containing adenoviral helper sequences

[0280] Adenoviral helper sequences for AAV replication are obtained from the AAV helper plasmid by restriction digestion or PCR amplification and inserted into the multiple cloning site between the two SS sites in the ministrand vector.

[0281] D. Generation of msDNA from Expression Vectors

[0282] DNA ministrands (msDNA) were produced in inducible E. coli cells according to the methods described in U.S. Patent Nos. 9,290,778 and 9,862,954 and International Publication No. WO 2022 / 264095, the entire contents of which are incorporated herein by reference.

[0283] Figure 2 Shown by Figure 1 A map of an exemplary ITR-GFP-ITR msDNA, "ITR-CAG-GFP-ITR msDNA," produced by the expression vector shown. The nucleic acid sequence of ITR-CAG-GFP-ITR msDNA is shown in SEQ ID NO: 19.

[0284] Figure 3 A map of an exemplary GFP-ITR msDNA, "CAG-GFP-ITR msDNA," is shown. The nucleic acid sequence of CAG-GFP-ITR msDNA is shown in SEQ ID NO: 20.

[0285] Figure 5 Shows the Figure 4 A map of an exemplary Rep-Cap msDNA, "PGL2-SS-CMV-Rep-Cap-SS msDNA," produced by the expression vector shown. The nucleic acid sequence of PGL2-SS-CMV-Rep-Cap-SS msDNA is shown in SEQ ID NO: 24.

[0286] Figure 6 A map of an exemplary "helper sequence msDNA" is shown. The nucleic acid sequence of the helper sequence msDNA is shown in SEQ ID NO: 25.

[0287] Example 2

[0288] Production and characterization of AAV produced with msDNA

[0289] One aim of this study was to evaluate AAV produced using the msDNA described in Example 1 in comparison to AAV produced using conventional plasmids.

[0290] A. AAV Production

[0291] AAV was produced using conventional plasmids and the msDNA of Example 1. Conventional plasmids include: a plasmid containing GFP flanked by ITRs (i.e., pITR-GFP-ITR), a plasmid containing the AAV rep and cap genes (e.g., pRep-Cap, such as pRep2-Cap1 for production of AAV1, pRep2-Cap2 for production of AAV2, pRep2-Cap5 for production of AAV5, pRep2-Cap9 for production of AAV9, etc.), and a plasmid containing adenovirus helper genes (i.e., pHelper).

[0292] A combination of three conventional plasmids used for AAV production (i.e., pITR-GFP-ITR, pRep-Cap, and pHelper, as shown in combination 1 in Table 3 below) was used as a baseline and compared to AAV produced using either msDNA as the GOI vector and a combination of conventional plasmids as the vector for Rep / Cap and helper sequences or msDNA as the vector for Rep / Cap and helper sequences.

[0293] Table 3. Combinations of msDNA and plasmids used for AAV production

[0294]

[0295]

[0296] Combinations also included ITR-GFP-ITR msDNA and GFP-ITR msDNA having spacer sequences of varying lengths between the SS and ITR, as well as the minimal ITR described in Example 1.

[0297] The combinations in Table 3 were transfected into mammalian production cells (e.g., HEK293T, CRL-3216 TM ), cells were cultured to produce AAV and purified according to standard procedures.

[0298] B. Generation of Stable AAV Producer Cell Lines Using msDNA

[0299] Stable mammalian AAV producer cell lines (e.g., HEK-293) are generated by integrating the Rep / Cap gene and / or the helper gene, respectively, into the cell line genome via nuclease-mediated homologous recombination using Rep-Cap msDNA and / or helper msDNA as described in Example 1. This results in a producer cell line that stably and continuously expresses AAV replication and packaging proteins, allowing AAV production by transfection with a single vector carrying the GOI.

[0300] AAV producer cells with stably integrated Rep / Cap genes and / or helper genes were transfected with ITR-GFP-ITR msDNA or GFP-ITR msDNA from Example 1, and the cells were incubated to produce AAV according to standard procedures, and the AAV was purified.

[0301] C. Characterization of AAV

[0302] AAV produced by each combination in Table 3 as well as AAV produced by producer cells with stably integrated Rep / Cap genes and helper genes were characterized.

[0303] Capsid components were analyzed by Western blotting using a capsid protein-specific mouse primary antibody and a peroxidase-conjugated donkey anti-mouse IgG secondary antibody.

[0304] Transduction titers were determined by transducing HeLa cells with serial dilutions of the vector.

[0305] Transduction efficiency was assessed by applying the same viral titer to cells and evaluating transgene expression by flow cytometry 72 hours after transduction. For packaging efficiency, the number of fully loaded AAV particles relative to empty capsids was determined. For transfection efficiency, the level and persistence of GFP expression was determined.

[0306] Viral vector genome particles (genomic titer) were assessed by real-time quantitative PCR (qPCR). Total DNA was isolated from AAV preparations and then subjected to real-time qPCR using transgene-specific primers.

[0307] The bacterial backbone and other plasmid-associated impurities were quantified by real-time qPCR using the primers listed in Table 4.

[0308] Table 4. Primers used for qPCR analysis

[0309]

[0310] Multiple t-test or ANOVA was used for statistical analysis, followed by Tukey's test, and P values less than 0.05 were considered statistically significant.

[0311] Example 3

[0312] Generation of ITR-GOI-ITR msDNA

[0313] As described in Example 1, U.S. Patent Nos. 9,290,778 and 9,862,954, and International Publication No. WO 2022 / 264095, expression vectors were prepared for generating msDNA encoding enhanced green fluorescent protein (eGFP) as the gene of interest (GOI) flanked by artificial AAV2 ITRs. Figure 7 A map of the expression vector (pITR2Cis (precursor plasmid)) is shown, which includes a specialized supersequence site ("SSeq*" (SEQ ID NO: 36)) with target sequences for the recombinase (telL, FRT (minimal), and loxP) flanking artificial AAV2 ITR sequences (5' "AAV ITR2" (SEQ ID NO: 38) and 3' "AAV ITR2" (SEQ ID NO: 39), which in turn flank an expression cassette containing a synthetic promoter comprising a cytomegalovirus (CMV) enhancer, a promoter from chicken β-actin, and a chimeric intron, a sequence encoding enhanced green fluorescent protein (eGFP), and a bovine growth hormone polyadenylation signal (bGHpA). The SSeq is separated from each of the 5' and 3' ITRs by artificial spacer sequences (SEQ ID NOs: 40 and 41, respectively). The nucleic acid sequence of pITR2Cis (precursor plasmid) is shown in SEQ ID NO:42.

[0314] As described in Example 1, U.S. Patent Nos. 9290778 and 9862954, and International Publication No. WO 2022 / 264095, Figure 7 The indicated precursor plasmids produce msDNA. Figure 8 The map of the msDNA (ITR2Cis msDNA) is shown, which includes a portion of the Tel recombinant SSeq at the 5' and 3' ends (SEQ ID NO: 37). The nucleic acid sequence of the ITR2Cis msDNA is shown in SEQ ID NO: 43.

[0315] Generation of ITR-GOI-ITR msDNA

[0316] The transfection efficiency of this msDNA (ITR2Cis msDNA) was compared with that of its parental plasmid (pITR2Cis).

[0317] Briefly, equimolar concentrations of ITR2Cis msDNA and pITR2Cis were prepared with LIPOFECTAMINE 3000 and transfected into adherent HEK293 cells. Transfection efficiency (TE, percentage of GFP-positive cells) and median fluorescence intensity (MFI) were assessed by flow cytometry on days 2 and 6 after transfection. Figure 9A and Figure 9B The results of TE and MFI on day 2 are shown respectively. Figure 9C and Figure 9D Results for TE and MFI at day 6 are shown, respectively.

[0318] The results showed that lower doses of msDNA provided greater levels of TE and GFP expression, with a 0.125 pMol dose of msDNA (0.38 μg) showing the highest TE and MFI. Maximum transfection efficiency and transgene expression were observed 2 days after transfection. The results also showed that AAV ITR-GOI-ITR msDNA significantly outperformed an equivalent molar amount of ITR-GOI-ITR precursor plasmid DNA.

[0319] ITR-GOI-ITR msDNA did not affect cell viability ( Figure 9D ).

[0320] In addition, live cell imaging was performed on day 3 after HEK293 cells were transfected with 0.25 pMol msDNA (0.58 μg) or 0.25 pMol precursor plasmid DNA (1.05 μg) and LIPOFECTAMINE 3000 at a ratio of 1:4. Figure 10 Micrographs showing GFP expression in transfected cells. Cell nuclei are indicated by staining with diaminobenzyl-2-phenylindole (DAPI). msDNA exhibits much stronger TE than the parental plasmid.

[0321] Example 4

[0322] Sucrose toxicity assay

[0323] The sucrose toxicity (SuTox) fidelity assay, based on a conditional loss-of-function (LOF) mutation in the toxic sacB gene, is used to assess the fidelity of msDNA synthesis in vivo and in vitro. Specifically, the SacB protein is toxic to bacteria in the presence of sucrose, allowing for positive selection of mutants. Faithful replication of the sacB gene leads to bacterial cell death, whereas if a LOF mutation occurs in sacB during DNA synthesis, the colonies will grow on sucrose.

[0324] A multigene expression vector for msDNA was prepared, comprising an expression cassette with the sacB gene and a chloramphenicol resistance gene (encoding chloramphenicol acetyltransferase), flanked by 5' and 3' AAV ITR2 sequences, followed by 5' and 3' SSeq sequences. See Examples 1 and 3, U.S. Patent Nos. 9,290,778 and 9,862,954, and International Publication No. WO 2022 / 264095. Figure 11 The ITR-sacB-CmR-ITR cassette contained in the expression vector is shown.

[0325] msDNA was expressed in vivo from expression vectors in E. coli cells (MB12 and MB13 strains, Mediphage Bioceuticals, Inc., Toronto, CA) using the methods described in Example 1, U.S. Patent Nos. 9,290,778 and 9,862,954, and International Publication No. WO 2022 / 264095.

[0326] For in vitro replication, primers containing SacI or SalI restriction enzyme sites are designed to just bind to the outside of the ITR-sacB-cmR-ITR box in the expression vector. Using these primers, Taq (FroggaBio T-500) or Q5 (New England Biolabs M0491) polymerase is used, and the buffer and thermal cycling conditions recommended by its respective manufacturer are applied to amplify the box by PCR. The same primers are used to guide rolling circle amplification (RCA) with Phi29 polymerase (New England Biolabs M0269). After PCR or RCA, enzymes and buffer reagents are removed using a commercial PCR purification kit (Thermo Fisher K0702) to obtain in vitro synthesized DNA. Because Phi29 produces polymers that are difficult to purify, the completed Phi29 reaction is digested with SacI and SalI restriction enzymes before the PCR purification scheme. DNA input is calculated as: the amount of the starting plasmid added to the reaction as a template, multiplied by the fraction of the amplified region length accounting for the total plasmid size. DNA output was calculated as the concentration of the PCR purification (obtained using a nanodrop spectrophotometer) multiplied by the elution volume.

[0327] DNA synthesized in vivo and in vitro, as well as the pUC19 vector with ampicillin resistance, were digested with SacI and SalI restriction enzymes (New England Biolabs R3156, R3138). The sacB-CmR fragment was isolated using a commercial gel extraction kit (ThermoFisher K0691), and each insert:vector combination was ligated overnight using T4 ligase (New England Biolabs M0202). A no-insert reaction was also included as a negative control. The ligations were transformed into a high-efficiency (1-3 x 10 9 CFU / μg pUC19DNA) competent cells (New England Biolabs C3040). The transformants were serially diluted and plated on LB with ampicillin (100 μg / ml), chloramphenicol (25 μg / ml) and 1% NaCl (standard MillerLB) or 6% sucrose. The plates were grown at 37°C for 16-24 hours. For each sample, colony forming units (CFU) were counted with and without sucrose. Any CFU counted from the negative control transformation was subtracted, and any CFU counted from the negative control transformation was used as the background for all samples. After subtracting the background, samples without CFU on sucrose were considered to be below the detection limit (BDL) and were calculated as 0.5 CFU on the sucrose plate. The CFU from the sucrose plate was counted as the sacB mutant. Figure 12A Representative images of sucrose plates transformed with ITR-sacB-CmR-ITR LCC DNA generated by PCR (Taq and Q5), RCA (Phi29), or in vivo in E. coli (MBI2) are shown. Taq is plated at a quarter volume relative to the other images.

[0328] The CFU counts on the standard LB plate were used as the total transformants. The DNA multiplication number was calculated as Log2 (DNA output / input). Figure 12B As shown, the mutation rate is calculated as sacB mutants / total transformants / number of DNA doublings. In other words, this calculates the proportion of DNA molecules containing LOF sacB mutations, where this proportion is normalized to the number of times the original template has been replicated. The mutation rate calculated from the SuTox method describes the LOF mutation in the sacB gene.

[0329] To obtain an estimate of the number of mutations per bp replicate, the mutation rate was divided by the length of the sacB ORF and promoter (1533 bp) and then multiplied by 1000 bp to obtain the number of mutations per kb replicate, which was then multiplied by 100% to express the value as a percentage. The mutation rates are shown in Table 5 below.

[0330] Table 5. Estimated LOF mutation rate per kilobase of synthesis

[0331]

[0332]

[0333] Figure 12B The data in Table 5 show that in vitro replication of Taq, Phi29, or Q5 results in approximately 3000-fold, 750-fold, and 85-fold more errors, respectively, compared to in vivo replication of msDNA. Thus, the data show that DNA generated in vivo by E. coli cells significantly exceeds the accuracy of PCR or RCA methods. Using DNA generated in vivo by E. coli, such as msDNA, can effectively reduce mutations, thereby reducing risk and improving the overall quality of the final product.

[0334] Example 5

[0335] AAV production using msDNA

[0336] AAV is produced by replacing one, two, or all three components of the conventional plasmid AAV production system with msDNA.

[0337] Test 1 - one msDNA, two conventional plasmids

[0338] Figure 13 Shown is a schematic diagram of AAV production in which the conventional GOI-containing plasmid is replaced by msDNA.

[0339] The ITR-GOI-ITR msDNA described in Example 3 (i.e., ITR2Cis msDNA) or a plasmid encoding GFP and not containing SSeq (i.e., ITR2Cis no SSeq plasmid control, such as Figure 14 The ITR2Cis no SSeq plasmid control is shown in SEQ ID NO: 44.

[0340] pDNA-helper, pDNA-Rep / Cap, and ITR2Cis msDNA or ITR2Cis no SSeq plasmid control were mixed at a molar ratio of 1:2:1, 2:1.5:1, or 1.4:1.5:1. Each mixture was then complexed with FECTOVIR-AAV transfection reagent at a 1:1 ratio in 5% high glucose DMEM medium for 15 minutes. After complexation, the total concentration was 1 μg DNA / 1x10 62 μg / mL of each mixture was transfected into 2×10 6 The DNA was selected for AAV2 production in 35 mL cultures or 150 mL cultures of GIBCO VCP2.0 cells (ThermoFisher Scientific), a clonal cell line derived from the HEK293F parental cell line, at a density of 10 cells / mL. DNA was selected for AAV2 production in 35 mL cultures, while DNA was selected for production of both AAV1 and AAV2 serotypes in 150 mL cultures.

[0341] Figure 15 GFP expression is shown for samples from 35 mL cultures at all three ratios.

[0342] Figure 16 GFP expression is shown for 150 mL culture samples of AAV1 and AAV2 serotypes generated with a 1.4:1.5:1 molar ratio of pDNA-helper:pDNA-Rep / Cap:ITR2Cis msDNA ("msDNA") or a 2:1.5:1 molar ratio of pDNA-helper:pDNA-Rep / Cap:ITR2Cis no SSeq plasmid control ("pDNA"). Cell viability associated with these transfections is shown. Figure 17A (% live cells) and Figure 17B (viable cell density (VCD), x10 6 cells / mL).

[0343] After 72 hours, cells in the 35 mL and 150 mL cultures were lysed in lysis buffer containing 1% Tween, 500 mM NaCl, 2 mM MgCl2 buffer and 20 U / mL DENARASE at 37°C on a shaker for 2 hours.

[0344] Droplet digital PCR (ddPCR) was performed to determine the AAV2 vector genomes / mL (VG / mL) titer based on the presence of GOI in 35 mL culture using ITR2Cis msDNA ("msDNA") or ITR2Cis no SSeq plasmid control ("pDNA") at different ratios. Figure 18 and as shown in Table 6 below.

[0345] Table 6. ddPCR titers of bulk AAV2 harvest (30 mL)

[0346] Sample (carrier ratio) AAV2 titer (VG / mL) msDNA (1:2:1) <![CDATA[3.00x 10 10 ]]> msDNA (2:1.5:1) <![CDATA[1.85x 10 10 ]]> msDNA (1.4:1.5:1) <![CDATA[3.55x 10 10 ]]> pDNA (1:2:1) <![CDATA[1.65x 10 10 ]]> pDNA (2:1.5:1) <![CDATA[2.34x 10 10 ]]> pDNA (1.4:1.5:1) <![CDATA[1.38x 10 10 ]]>

[0347] msDNA = AAV generated from msDNA at the ratio pDNA-helper:pDNA-Rep / Cap:ITR2Cis

[0348] pDNA = AAV generated from the stated ratio pDNA-helper:pDNA-Rep / Cap:ITR2Cis no SSeq plasmid control

[0349] AAV1 and AAV2 were purified from 150 mL shake flask cultures using affinity chromatography. Figures 19A-19B and Figure 20A-Figure 20B It is shown that, using ITR2Cis msDNA (msDNA (1.4:1.5:1), Figure 19A Affinity chromatography chromatograms of cultures generated using ITR2Cis without SSeq plasmid control (pDNA (2:1.5:1), Figure 20A Figure 2 shows affinity chromatography chromatograms of cultures produced with AAV1 (AAV1) and 20B (AAV2). The upper line in each graph is the absorbance at 280 nm, representing empty AAV1 or AAV2 capsids (i.e., they do not include encapsulated DNA), while the lower line is the absorbance at 260 nm, representing AAV1 or AAA2 capsids containing encapsulated DNA. The amount of "VP / mL" indicates the concentration of vector particles per milliliter of eluate, and the percentage indicates the proportion of particles that are packaged with DNA (i.e., % intact load calculated by A260 / A280 (see, e.g., Werle et al., Mol. Ther. Methods Clin. Dev. 23:254-262 (Dec 2021)), where reported directly from the AKTA chromatogram; or calculated by mass photometry (REFEYN)). The percentage of intact particles can include particles packaged with the GOI as well as particles with unusually packaged DNA, such as conventional plasmid backbones. Figure 21A Shown is a micrograph of an electrophoresis gel, with capsid proteins VP1, VP2, and VP3 shown as three corresponding bands per lane from top to bottom, detected by ddPCR after affinity chromatography of msDNA (1.4:1.5:1) and pDNA (2:1.5:1) samples from AAV2 harvest. Figure 21B Shown are vector genome titers of AAV1 and AAV2 harvests from msDNA (1.4:1.5:1) and pDNA (2:1.5:1) samples, as determined by ddPCR of the GOI.

[0350] AAV1 purified from 150 mL shake flask cultures by affinity chromatography was further purified using MUSTANG Q anion exchange (AEX) chromatography. Figure 22 and 23The results show that the ITR2Cis msDNA (msDNA (1.4:1.5:1), Figure 22 ) or ITR2Cis no SSeq plasmid control (pDNA (2:1.5:1), Figure 23 ) were generated from cultures obtained from the same culture medium. The upper and lower lines, VP / mL, and percentages are as described in Figures 19-20. Peak #1 in each figure includes particles primarily packaged with DNA, while Peak #2 appears to include both empty particles and particles with packaged DNA. Figure 24A Shown is a micrograph of an electrophoresis gel with capsid proteins VP1, VP2, and VP3 detected by ddPCR from peaks #1 and #2 of the MUSTANG QAEX chromatogram shown as three corresponding bands from the top to the bottom of each lane. Figure 24B and 24C The titers of peaks #1 and #2 are shown, respectively, associated with packaged DNA containing the gene of interest (GOI) or conventional plasmid DNA such as the origin of replication (Ori), KanR gene, or AmpR gene. These data indicate that msDNA produced a 1.9-fold higher yield and a 3.5% higher full load: empty load ratio.

[0351] Figure 25 Comparison of AAV2 titers for 35 mL cultures generated from different msDNA and pDNA ratios as described above, containing either encapsulated conventional plasmid DNA ("Ori(backbone)") or a GOI-containing expression cassette ("CMV(GOI)"). The figure shows that even with a single msDNA, packaging of the plasmid backbone sequence is much lower.

[0352] Figure 26 Next generation sequencing (NGS) coverage plots of AAV1 and AAV2 packaged genomes generated using ITR2Cis msDNA (msDNA (1.4:1.5:1) as described above) or ITR2Cis no SSeq plasmid control (pDNA (2:1.5:1) as described above) relative to plasmid map positions are shown. Based on the lack of backbone sequences in msDNA vectors, Figure 26 The results showed that when using a single msDNA, aberrant packaging of sequences outside the ITR-GOI-ITR region was almost negligible. Therefore, when using msDNA as a GOI vector in AAV production, aberrant backbone plasmid sequences can be eliminated compared to conventional plasmids.

[0353] Test 2 - Two msDNAs, one regular plasmid

[0354] The ITR-GOI-ITR msDNA described in Example 3 (ie, ITR2Cis msDNA) or the conventional plasmid encoding GFP and not containing SSeq described in Test 1 (ie, ITR2Cis no SSeq plasmid control) was used. Figure 27 AAV2 is produced by combining the Rep2 / Cap2 msDNA shown or a conventional Rep2 / Cap2 plasmid with a conventional helper plasmid (i.e., pDNA-helper from Applied Viromics). The nucleic acid sequence of Rep2 / Cap2 msDNA is shown in SEQ ID NO: 48.

[0355] pDNA-Helper, Rep2 / Cap2 msDNA and ITR2Cis msDNA were mixed at a molar ratio of 1.4:1.5:1 ("msDNA"). pDNA-Helper, pDNA-Rep2 / Cap2 and ITR2Cis msDNA were mixed at a molar ratio of 1.4:1.5:1 ("mixed msDNA"). pDNA-Helper, pDNA-Rep / Cap and ITR2Cis no SSeq plasmid control were mixed at a molar ratio of 1:2:1 ("pDNA"). Each mixture was then complexed with FECTOVIR-AAV transfection reagent at a 1:1 ratio in 5% high glucose DMEM medium for 15 minutes. After complexation, each mixture was transfected into a 150 mL culture of GIBCO VCP2.0 cells (passage 37). 2% Glutamax was added to the VPC medium before use, and all medium components were warmed before culture. Cells were grown at 37°C, 7% CO₂, and shaken at 130 rpm and harvested 72 hours after transfection. Cells were then lysed by adding 1% Tween, 500 mM NaCl, and 25 U / mL DENARASE and mixing for 2 hours. The lysate was then clarified by centrifugation at 4000 rpm for 40 minutes and then purified. Table 7 below shows the viability of samples after 72 hours of culture.

[0356] Table 7. Survival rate of test 2 samples

[0357]

[0358] msDNA = pDNA-Helper, Rep2 / Cap2 msDNA, and ITR2Cis msDNA

[0359] Mixed msDNA = pDNA-Helper, pDNA-Rep2 / Cap2, and ITR2Cis msDNA

[0360] pDNA = pDNA-Helper, pDNA-Rep / Cap, and ITR2Cis no SSeq plasmid control

[0361] AAV2 was purified from the culture using affinity chromatography. Figure 28A and Figure 28B Chromatograms of AAV produced from msDNA and pDNA samples are shown, respectively. The upper and lower lines, VP / mL, and percentages are as described for Figures 19-20. VP / mL represents the number of DNA-packaged particles / mL in the eluate. Consistent production was observed from independent replicates. Figures 29A-29C Chromatograms of AAV generated from the replicate msDNA, mixed msDNA, and pDNA samples are shown, respectively. Figure 29D Micrographs of electrophoresis gels are shown, with capsid proteins VP1, VP2, and VP3 shown as three corresponding bands from the top to the bottom of each lane, detected by ddPCR after affinity chromatography of msDNA samples in lane (a), mixed msDNA samples in lane (b), and pDNA samples in lane (c).

[0362] Figures 29A-29C Approximately 87.5% of the affinity chromatography captures from this replicate were used for further AEX purification, yielding Figure 30-32 The chromatograms shown are as follows. As described above, the peaks labeled VG / ml and % full amounts in each graph indicate the number of vector particles containing encapsulated DNA and the percentage of vector particles containing encapsulated DNA, respectively. These peaks represent the eluate with the highest proportion of particles containing encapsulated DNA.

[0363] Figure 33A Shown are the titers of AAV2 vector genomes / mL (VG / mL) based on the presence of the GOI, as determined by ddPCR, for samples from the initial AAV2 harvest ("Harvest"), affinity chromatography ("Capture"), and AEX chromatography ("AEX") following lysis of cell culture. Two harvest values and two capture values for msDNA and pDNA samples, respectively, are from independent replicates on different days, as shown in Figures 28 and 29.

[0364] Figure 33B Shows that by Figure 33A The AAV2 titer (VG, mass balance) was determined for each sample by multiplying the corresponding VG / mL titer in by the total volume of the sample.

[0365] Figure 34A Displayed by A260 / A280 ratio Figure 33A The intact particles of the sample are estimated, and Figure 34BIntact particles were estimated by mass photometry for samples shown in independent replicates as described above.

[0366] The data showed that using two msDNAs instead of two of the three conventional plasmids typically used in AAV manufacturing resulted in higher intact particle counts and a large improvement in avoiding aberrant packaging of the backbone plasmid DNA.

[0367] Test 2 - Three msDNA

[0368] Figure 35 Shown is a diagram of AAV production in which all three conventional plasmids were replaced with msDNA.

[0369] All msDNAs used were ITR-GOI-ITR msDNA (i.e., ITR2Cis msDNA) described in Example 3, Rep2 / Cap2 msDNA described in Test 2, and Figure 36 AAV was produced by using Helpers DNA with the nucleic acid sequence shown in SEQ ID NO: 51. Figure 29C AAV generated from an independent replicate of assay 2 as shown) was used as a comparison.

[0370] ITR2Cis msDNA, Rep2 / Cap2 msDNA, and Helper msDNA were mixed at a molar ratio of 1:2:1 or 1:1:1. The 1:2:1 mixture was complexed at a FECTOVIR-AAV transfection reagent:DNA ratio of 1:1 or 2:1, while the 1:1:1 mixture was complexed at a FECTOVIR-AAV transfection reagent:DNA ratio of 2:1. Complexation was allowed to proceed for 15 minutes in 5% high-glucose DMEM medium. After complexation, each mixture was transfected into a 150 mL culture of GIBCO VCP2.0 cells. 2% Glutamax was added to the VPC medium before use, and all medium components were warmed before incubation. Cells were grown at 37°C, 7% CO2, shaken at 130 rpm, and harvested 72 hours after transfection. Cells were then lysed by adding 1% Tween, 500 mM NaCl, and 25 U / mL DENARASE for 2 hours. The lysate was then clarified by centrifugation at 4000 rpm for 40 minutes before purification.

[0371] AAV2 was purified from the culture using affinity chromatography. Figures 37A-37DChromatograms of AAV produced from a FECTOVIR:DNA sample with a 1:2:1 molar ratio of msDNA (A), a FECTOVIR:DNA sample with a 1:21:1 molar ratio of msDNA (B), and a FECTOVIR:DNA sample with a 1:1:1 molar ratio of msDNA (C) are shown. Figure 37D Shows Figure 29C Figure 7. Chromatogram of a complete pDNA sample (pDNA-Helper, pDNA-Rep / Cap, and ITR2Cis no SSeq plasmid control at a molar ratio of 1:2:1, and FECTOVIR:DNA at a 1:1 ratio). Upper and lower limits, VP / mL, VG / mL, and % intact values are as described above. VG / L is the concentration of vector genomes in the culture. Figure 38 include Figures 37A-37D Summary of sample characteristics, including micrographs of electrophoresis gels, with capsid proteins VP1, VP2, and VP3 shown as three corresponding bands per lane from top to bottom, as detected by ddPCR after affinity chromatography of the samples.

[0372] Figures 37A-37D About 87.5% of the affinity chromatography capture was used for further AEX purification to give Figures 39-42 Chromatograms are shown. As described above, the peaks labeled VG / ml and % intact in each graph indicate the number of vector particles containing encapsulated DNA and the percentage of vector particles containing encapsulated DNA, respectively. These peaks represent the eluate with the highest proportion of particles containing encapsulated DNA.

[0373] Figure 43A Shown are estimates of intact particles by the A260 / A280 ratio of affinity chromatography ("Capture") and AEX chromatography ("AEX Peak #1" and "AEX Peak #2") samples. Figure 43B An estimate of the intact particles of the sample by mass photometry is shown.

[0374] Figure 44A Shown are AAV2 vector genomes / mL (VG / mL) titers based on GOI presence, as determined by ddPCR on samples from the initial AAV2 harvest (“Harvest”), affinity chromatography (“Capture”), and AEX chromatography peak #1 (“AEX”) after lysis of cell culture. Figure 44B Shows that by Figure 44A The AAV2 titer (VG, mass balance) was determined for each sample by multiplying the corresponding VG / mL titer in by the total volume of the sample. Figure 44C Shown are the AAV2 titers (VG, mass balance) determined for each sample, including peaks #1 and 2 from the AEX chromatography.

[0375] The data showed that using msDNA instead of all three conventional plasmids significantly improved the intact load:empty capsid ratio under semi-optimized conditions, from 5.4% intact load (transfection with three pDNA conventional plasmids) to >20% intact load. Culture and other process-specific parameters were not optimized for msDNA and were operated based on existing plasmid SOPs.

[0376] Figure 45 Shown are NGS coverage plots of AAV2 packaged genomes generated using one, two, or three msDNAs instead of three conventional plasmids.

[0377] Figure 45 It was also demonstrated that replacing the conventional plasmid encoding the GOI with msDNA resulted in a 100-fold improvement in abnormal packaging compared to using all three plasmids. Replacing the conventional plasmid encoding the GOI and Rep2 / Cap2 with msDNA resulted in a 1,000-fold improvement. Furthermore, replacing all conventional plasmids with msDNA resulted in a 10,000-fold improvement.

[0378] Example 6

[0379] Production of AAV9 using msDNA

[0380] Test 1

[0381] AAV9 was generated using the ITR-GOI-ITR msDNA described in Example 3 (i.e., ITR2Cis msDNA) or the plasmid encoding GFP and not containing SSeq described in Example 5 (i.e., ITR2Cis no SSeq plasmid control), respectively, in combination with bacterial sequence minimization / reduction plasmids providing Rep2 / Cap9 and Helper sequences.

[0382] ITR2Cis msDNA or ITR2Cis no SSeq plasmid control was mixed with Rep2 / Cap9 plasmid and Helper plasmid at a molar ratio of 1:1:1, and each mixture was complexed with polyethyleneimine (PEI) at PEI:DNA ratios of 1.5:1, 2:1, and 2.5:1.

[0383] For combinations with msDNA, 150 mL of HEK-293 cell culture was transfected with total DNA concentrations of 1.0 μg / mL (0.156 μg / mL msDNA, 0.379 μg / mL Rep2 / Cap9 plasmid, and 0.465 μg / mL helper plasmid), 1.75 μg / mL (0.273 μg / mL msDNA, 0.663 μg / mL Rep2 / Cap9 plasmid, and 0.813 μg / mL helper plasmid), and 2.5 μg / mL (0.39 μg / mL msDNA, 0.948 μg / mL Rep2 / Cap9 plasmid, and 1.162 μg / mL helper plasmid) at each ratio to generate nine different msDNA transfectants: 1.0 μg / mL msDNA (msDNA1) DNA and 1.5:1 PEI:DNA, (msDNA2) 1.0 μg / mL DNA and 2:1 PEI:DNA, (msDNA3) 1.0 μg / mL DNA and 2.5:1 PEI:DNA, (msDNA4) 1.75 μg / mL DNA and 1.5:1 PEI:DNA, (msDNA5) 1.75 μg / mL DNA and 2:1 PEI:DNA, (msDNA6) 1.75 μg / mL DNA and 2.5:1 PEI:DNA, (msDNA7) 2.5 μg / mL DNA and 1.5:1 PEI:DNA, (msDNA8) 2.5 μg / mL DNA and 2:1 PEI:DNA, and (msDNA9) 2.5 μg / mL DNA and 2.5:1 PEI:DNA. This is summarized.

[0384] The ITR2Cis no SSeq plasmid control, as well as the Rep2 / Cap9 and Helper bacterial sequence minimization / reduction plasmids, were complexed with PEI at a 2:1 PEI:DNA ratio and a total DNA concentration of 2 μg / mL (0.36 μg / mL ITR2Cis plasmid control, 0.74 μg / mL Rep2 / Cap9 plasmid, and 0.9 μg / mL helper plasmid).

[0385] Table 8 provides a summary of the DNA concentrations and PEI:DNA ratios of the transfectants.

[0386] Table 8. Summary of transfectant DNA concentration and PEI:DNA ratio

[0387]

[0388] Figure 46 Shown are the transfection efficiencies of three plasmids ("AAV-PP") and nine different msDNA transfectants 48 and 72 hours post-transfection, assessed using flow cytometry for the GFP GOI.

[0389] Figure 47 shows the viable cell density (VCD, viable cells / mL) and viability (% viable cells) of samples 48 hours and 72 hours post-transfection analyzed using Vi-CELL XR HEK293.

[0390] Figure 48 The capsid titer of samples measured by AAV9-specific ELISA 72 hours after transfection is shown. The capsid ELISA does not distinguish between empty and full capsids. The figure shows that by replacing the conventional plasmid carrying the GOI with msDNA, 2.5-3 times higher AAV / mL was obtained.

[0391] Figure 49 Shown are the AAV titers of samples measured 72 hours after transfection by ddPCR using primers specific for the ITR region. Compared to standard plasmid conditions (2 μg / mL total DNA and a 2:1 ratio of PEI:DNA), replacing the GOI-containing plasmid with msDNA resulted in similar VG / mL with half the starting mass, while similar starting mass yielded twice as much VG / mL.

[0392] Test 2

[0393] msDNA5 (1.75 μg / mL total DNA and a 2:1 ratio of PEI:DNA) and standard plasmid conditions (2 μg / mL total DNA and a 2:1 ratio of PEI:DNA) were scaled up into 10 L AAV culture.

[0394] Figure 50 showed that similar mass of transfection DNA including msDNA instead of conventional plasmid containing the GOI produced higher AAV titers in 10 L of culture as determined by ddPCR after AEX chromatography.

[0395] Full capsids were also measured by mass spectrometry, yielding 46.1% complete capsids using msDNA5 compared to 40.2% under standard plasmid conditions.

[0396] Sequence SEQ ID NO: 1 AAV-2 wild type 5'ITR (+ strand)

[0397]

[0398] SEQ ID NO: 2 AAV-2 wild-type 3' ITR (+ strand)

[0399]

[0400] SEQ ID NO: 3 AAV-2 wild-type ITR A-sequence (+ strand)

[0401]

[0402] SEQ ID NO:4 AAV-2 wild-type ITR A'-sequence (+ strand)

[0403]

[0404] SEQ ID NO:5 AAV-2 wild-type ITR B-sequence (+ strand)

[0405]

[0406] SEQ ID NO:6 AAV-2 wild-type ITR B'-sequence (+ strand)

[0407]

[0408] SEQ ID NO:7 AAV-2 wild-type ITR C-sequence (+ strand)

[0409]

[0410] SEQ ID NO:8 AAV-2 wild-type ITR C'-sequence (+ strand)

[0411]

[0412] SEQ ID NO:9 AAV-2 wild-type 5' ITR D-sequence (+ strand)

[0413]

[0414] SEQ ID NO: 10 AAV-2 wild-type 3'ITR D-sequence (+ strand)

[0415]

[0416] SEQ ID NO: 11 AAV-2 wild-type ITR A-RBS (+ chain)

[0417]

[0418] SEQ ID NO: 12 AAV-2 wild-type ITR A'-RBS (+ strand)

[0419]

[0420] SEQ ID NO: 13 AAV-2 wild-type ITR RBE' (+ strand)

[0421]

[0422] SEQ ID NO: 14 AAV-2 wild-type 5' ITR TRS (+ strand)

[0423]

[0424] SEQ ID NO: 15 AAV-2 wild-type 3' ITR TRS (+ strand)

[0425]

[0426] SEQ ID NO: 16 AAV artificial 5' ITR (+ strand)

[0427]

[0428] SEQ ID NO: 17 AAV artificial 3' ITR (+ strand)

[0429]

[0430] SEQ ID NO: 18 ITR-CAG-GFP-ITR plasmid

[0431]

[0432]

[0433] SEQ ID NO:19 ITR-CAG-GFP-ITR msDNA

[0434]

[0435]

[0436] SEQ ID NO:20 CAG-GFP-ITR msDNA

[0437]

[0438]

[0439] SEQ ID NO:21AAV-2 rep

[0440]

[0441] SEQ ID NO:22AAV-2cap

[0442]

[0443] SEQ ID NO:23PGL2-SS-CMV-Rep-Cap-SS plasmid

[0444]

[0445]

[0446] SEQ ID NO:24 PGL2-SS-CMV-Rep-Cap-SS msDNA

[0447]

[0448]

[0449] SEQ ID NO:25 Helper sequence msDNA

[0450]

[0451]

[0452]

[0453]

[0454] SEQ ID NO: 26 ampR forward primer

[0455] SEQ ID NO: 27ampR reverse primer

[0456] SEQ ID NO:28F1 ori forward primer

[0457] SEQ ID NO:29F1 ori reverse primer

[0458] SEQ ID NO:30ori forward primer

[0459] SEQ ID NO:31ori reverse primer

[0460] SEQ ID NO: 32 SCAR forward primer

[0461] SEQ ID NO: 33 SCAR reverse primer

[0462] SEQ ID NO:34kanR forward primer

[0463] SEQ ID NO:35kanR reverse primer

[0464] SEQ ID NO:36SSeq

[0465]

[0466] SEQ ID NO: 37 SSeq after Tel recombinant

[0467]

[0468] SEQ ID NO: 38 AAV artificial 5' ITR (+ strand)

[0469]

[0470] SEQ ID NO: 39 AAV artificial 3' ITR (+ strand)

[0471]

[0472] SEQ ID NO: 40 5' spacer sequence between SSeq and ITR

[0473] SEQ ID NO:41 3' spacer sequence between SSeq and ITR

[0474] SEQ ID NO:42 pITR2Cis msDNA precursor plasmid

[0475]

[0476]

[0477] SEQ ID NO:43ITR2Cis msDNA

[0478]

[0479] SEQ ID NO:44 pITR2Cis no SSeq plasmid control

[0480]

[0481] SEQ ID NO:45 AAV-2 rep

[0482]

[0483]

[0484] SEQ ID NO:46 AAV-5 cap

[0485]

[0486] SEQ ID NO:47 AAV-9 cap

[0487]

[0488]

[0489] SEQ ID NO:48 Rep2 / Cap2 msDNA

[0490]

[0491] SEQ ID NO:49 Rep2 / Cap5 msDNA

[0492]

[0493] SEQ ID NO:50 Rep2 / Cap9 msDNA

[0494]

[0495] SEQ ID NO:51 Helper msDNA

[0496]

[0497]

[0498]

[0499]

[0500]

Claims

1. An expression vector comprising: (a) a first sequence comprising an inverted terminal repeat (ITR) and a multiple cloning site (MCS), wherein the ITRs are flanked on at least one side of the MCS, and wherein the ITRs comprise an adeno-associated virus (AAV) replication sequence and an AAV packaging signal, (b) a target sequence for a first recombinase flanking each side of said first sequence, and (c) one or more additional target sequences for one or more additional recombinases, said one or more additional target sequences being integrated within a non-binding region of the target sequence of said first recombinase, The expression vector is used to produce a bacterial sequence-free vector with linear covalently closed ends.

2. The expression vector of claim 1, wherein the ITR flanks the MCS on only one side.

3. The expression vector of claim 1, wherein the ITRs flank the MCS on each side. The expression vector according to any one of claims 1 to 3, further comprising a spacer sequence between the target sequence of the first recombinase and the first sequence. The expression vector according to claim 4 , wherein the spacer sequence is 10 to 500 nucleotides.

6. The expression vector according to any one of claims 1 to 3, further comprising an expression cassette containing an AAV replication (rep) gene and an AAV capsid (cap) gene, with a target sequence for the first recombinase on one side and the first sequence on the other side.

7. An expression vector comprising: (a) a first sequence comprising an ITR and an expression cassette comprising a nucleic acid sequence of interest, wherein the ITR flanks the expression cassette comprising the nucleic acid sequence of interest on at least one side, and wherein the ITR comprises an AAV replication sequence and an AAV packaging signal, (b) a target sequence for a first recombinase flanking each side of said first sequence, and (c) one or more additional target sequences for one or more additional recombinases, said one or more additional target sequences being integrated within a non-binding region of the target sequence of said first recombinase, The expression vector is used to produce a bacterial sequence-free vector with linear covalently closed ends.

8. The expression vector according to claim 7, wherein the ITR is located only on one side of the expression cassette containing the target nucleic acid sequence. The expression vector according to claim 8 , further comprising a spacer sequence between the target sequence of the first recombinase and the first sequence.

10. The expression vector according to claim 9, wherein the spacer sequence is 10 to 500 nucleotides.

11. The expression vector according to any one of claims 8 to 10, further comprising an expression cassette comprising an AAV rep gene and an AAVcap gene, with a target sequence for the first recombinase on one side and the first sequence on the other side.

12. The expression vector of claim 7, wherein the ITRs flank the expression cassette containing the nucleic acid sequence of interest on each side. The expression vector according to claim 12 , further comprising a spacer sequence between the target sequence of the first recombinase and the first sequence. The expression vector according to claim 13 , wherein the spacer sequence is 10 to 500 nucleotides.

15. The expression vector according to any one of claims 12 to 14, further comprising an expression cassette comprising an AAV rep gene and an AAV cap gene, with a target sequence for the first recombinase on one side and the first sequence on the other side.

16. An expression vector comprising: (a) a first sequence comprising ITRs and a palindromic sequence, wherein the ITRs flank the palindromic sequence on each side, wherein the palindromic sequence comprises an expression cassette comprising a nucleic acid sequence of interest and a complementary sequence to the expression cassette, and wherein the ITRs comprise an AAV replication sequence and an AAV packaging signal, (b) a target sequence for a first recombinase flanking each side of said first sequence, and (c) one or more additional target sequences for one or more additional recombinases, said one or more additional target sequences being integrated within a non-binding region of the target sequence of said first recombinase, The expression vector is used to produce a bacterial sequence-free vector with linear covalently closed ends. The expression vector according to claim 16 , wherein the complementary sequence is separated from the expression cassette containing the nucleic acid sequence of interest by a non-complementary spacer sequence. The expression vector according to claim 16 or 17, further comprising a spacer sequence between the target sequence of the first recombinase and the first sequence. The expression vector according to claim 18 , wherein the spacer sequence is 10 to 500 nucleotides.

20. The expression vector according to any one of claims 16 to 19, further comprising an expression cassette comprising an AAV rep gene and an AAV cap gene, flanked on one side by the target sequence of the first recombinase and on the other side by the first sequence.

21. An expression vector comprising: (a) a first sequence comprising a portion of an expression cassette containing a nucleic acid sequence of interest flanked on one side by a splicing sequence, (b) ITRs flanking each of the first sequence, wherein the ITRs comprise an AAV replication sequence and an AAV packaging signal, (c) the target sequence for the first recombinase flanking each ITR, and (d) one or more additional target sequences for one or more additional recombinases, said one or more additional target sequences being integrated within a non-binding region of the target sequence of said first recombinase, The expression vector is used to produce a bacterial sequence-free vector with linear covalently closed ends.

22. The expression vector according to claim 21, wherein the partial expression cassette comprises a 5' portion which, in combination with the rest of the expression cassette, provides the complete sequence of the expression cassette, and the splicing sequence is located at the 3' side of the 5' portion.

23. The expression vector according to claim 21, wherein the partial expression cassette comprises a 3' portion which, in combination with the rest of the expression cassette, provides the complete sequence of the expression cassette, and the splicing sequence is located on the 5' side of the 3' portion. 24 . The expression vector according to claim 21 , further comprising a spacer sequence between the target sequence of the first recombinase and the first sequence.

25. The expression vector of claim 24, wherein the spacer sequence is 10 to 500 nucleotides.

26. The expression vector of any one of claims 1 to 25, wherein the AAV replication sequence comprises an AAV ITR replication (Rep) protein binding element (RBE) and a terminal dissociation site (TRS).

27. The expression vector of any one of claims 1 to 26, wherein the AAV packaging signal comprises an AAV ITRD sequence.

28. An expression vector comprising: (a) an expression cassette comprising the AAV rep gene and the AAV cap gene, (b) target sequences for a first recombinase flanking each side of the expression cassette, and (c) one or more additional target sequences for one or more additional recombinases, said one or more additional target sequences being integrated within a non-binding region of the target sequence of said first recombinase, The expression vector is used to produce a bacterial sequence-free vector with linear covalently closed ends.

29. An expression vector comprising: (a) an expression cassette comprising one or more helper viral genes for producing AAV, (b) target sequences for a first recombinase flanking each side of the expression cassette, and (c) one or more additional target sequences for one or more additional recombinases, said one or more additional target sequences being integrated within a non-binding region of the target sequence of said first recombinase, The expression vector is used to produce a bacterial sequence-free vector with linear covalently closed ends.

30. The expression vector of claim 29, wherein the one or more helper virus genes are from an adenovirus, a herpes virus, a retrovirus, a pox virus, and / or a lentivirus.

31. The expression vector of claim 30, wherein the one or more helper virus genes comprise an adenovirus early 4 (E4) gene, an adenovirus early 2A (E2A) gene, and an adenovirus virus-associated (VA) gene.

32. The expression vector according to any one of claims 1 to 31, wherein the target sequence of the first recombinase and the one or more additional target sequences of the one or more additional recombinases are selected from the group consisting of the PY54 pal site, the N15 telRL site and the telRL site.

33. The expression vector of claim 32, wherein the expression vector comprises each of the target sequences.

34. The expression vector of claim 32, wherein the expression vector comprises a Tel recombinase pal site and a telRL recombinase target binding sequence integrated into the pal site.

35. The expression vector of any one of claims 1 to 31, wherein the target sequence for the first recombinase is the bacteriophage PY54 Tel 142 base pair target site.

36. A vector production system comprising a recombinant cell designed to encode at least a first recombinase under the control of an inducible promoter, wherein the cell comprises the expression vector of any one of claims 7 to 35.

37. The vector production system of claim 36, wherein the inducible promoter is heat-regulated, chemically-regulated, IPTG-regulated, glucose-regulated, arabinose-inducible, T7 polymerase-regulated, cold-shock-inducible, pH-inducible, or a combination thereof.

38. The vector production system according to claim 36 or 37, wherein the first recombinase is selected from TelN and Tel, and the expression vector incorporates at least a target sequence for the first recombinase.

39. The vector production system of any one of claims 36 to 38, wherein the recombinant cell has been further engineered to encode a nuclease genome editing system, and wherein the expression vector further comprises a backbone sequence containing a cleavage site for the nuclease genome editing system.

40. The vector production system of claim 39, wherein the nuclease genome editing system is a CRISPR nuclease system comprising a Cas nuclease and a gRNA, and the expression vector comprises a target sequence for the gRNA in a backbone sequence.

41. A method of producing a bacterial sequence-free vector having linear covalently closed ends, comprising incubating the vector production system according to any one of claims 36 to 38 under conditions suitable for the expression of a first recombinase.

42. A method for producing a bacterial sequence-free vector having linear covalently closed ends, comprising incubating the vector production system of claim 39 or 40 under conditions suitable for expression of a first recombinase and a nuclease genome editing system.

43. The method of claim 41 or 42, further comprising harvesting the vector free of bacterial sequences.

44. A bacterial sequence-free vector produced by the method of any one of claims 41 to 43.

45. The bacterial sequence-free vector of claim 44, wherein the bacterial sequence-free vector is generated from the expression vector of any one of claims 8 to 10.

46. The bacterial sequence-free vector of claim 44, wherein the bacterial sequence-free vector is generated from the expression vector of claim 11.

47. The bacterial sequence-free vector of claim 44, wherein the bacterial sequence-free vector is generated from the expression vector of any one of claims 12 to 14.

48. The bacterial sequence-free vector of claim 44, wherein the bacterial sequence-free vector is generated from the expression vector of claim 15.

49. The bacterial sequence-free vector of claim 44, wherein the bacterial sequence-free vector is generated from the expression vector of any one of claims 16 to 19.

50. The bacterial sequence-free vector of claim 44, wherein the bacterial sequence-free vector is generated from the expression vector of claim 20.

51. The bacterial sequence-free vector of claim 44, wherein the bacterial sequence-free vector is generated from an expression vector according to any one of claims 21 to 25.

52. The bacterial sequence-free vector of claim 42, wherein the bacterial sequence-free vector is generated from the expression vector of claim 28.

53. The bacterial sequence-free vector of claim 40, wherein the bacterial sequence-free vector is generated from an expression vector according to any one of claims 29 to 31.

54. A method for producing a single-chain AAV, wherein the method comprises: (a) Transfect AAV-producing cells with: i. The bacterial sequence-free vector according to claim 47, ii. The bacterial sequence-free vector according to claim 52, or an expression vector comprising an expression cassette comprising an AAV rep gene and an AAV cap gene, and iii. The bacterial sequence-free vector according to claim 53, or an expression vector comprising an expression cassette containing one or more helper virus genes for producing AAV; and (b) incubating the cells under conditions suitable for AAV production.

55. A method for producing a single-chain AAV, wherein the method comprises: (a) Transfect AAV-producing cells with: i. The bacterial sequence-free vector according to claim 48, ii. The bacterial sequence-free vector of claim 53, or an expression vector comprising an expression cassette containing one or more helper viral genes for producing AAV; and (b) incubating the cells under conditions suitable for AAV production.

56. A method for producing a single-chain AAV, wherein the method comprises: (a) transfecting a cell capable of producing AAV with the bacterial sequence-free vector of claim 47, wherein an AAV rep gene, an AAV cap gene, and one or more helper virus genes for producing AAV are each encoded by the cell or by the vector; (b) incubating the cells under conditions suitable for expressing the rep gene, cap gene and one or more helper viral genes and suitable for producing AAV.

57. A method of producing a self-complementary AAV, wherein the method comprises: (a) Transfect AAV-producing cells with: i. The bacterial sequence-free vector according to claim 45, ii. The bacterial sequence-free vector according to claim 52, or an expression vector comprising an expression cassette comprising an AAV rep gene and an AAV cap gene; and iii. a bacterial sequence-free vector according to claim 53, or an expression vector comprising an expression cassette containing one or more helper viral genes for producing AAV, and (b) incubating the cells under conditions suitable for AAV production.

58. A method of producing a self-complementary AAV, wherein the method comprises: (a) Transfect AAV-producing cells with: iv. a bacterial sequence-free vector according to claim 46, v. a bacterial sequence-free vector according to claim 53, or an expression vector comprising an expression cassette containing one or more helper viral genes for producing AAV, and (b) incubating the cells under conditions suitable for AAV production.

59. A method of producing a self-complementary AAV, wherein the method comprises: (a) Transfect AAV-producing cells with: i. The bacterial sequence-free vector according to claim 49, ii. The bacterial sequence-free vector according to claim 52, or an expression vector comprising an expression cassette comprising an AAV rep gene and an AAV cap gene, and iii. a bacterial sequence-free vector according to claim 53, or an expression vector comprising an expression cassette containing one or more helper viral genes for producing AAV, and (b) incubating the cells under conditions suitable for AAV production.

60. A method of producing a self-complementary AAV, wherein the method comprises: (a) Transfect AAV-producing cells with: i. The bacterial sequence-free vector according to claim 50, ii. a bacterial sequence-free vector according to claim 53, or an expression vector comprising an expression cassette containing one or more helper viral genes for producing AAV, and (b) incubating the cells under conditions suitable for AAV production.

61. A method of producing a self-complementary AAV, wherein the method comprises: (a) transfecting a cell capable of producing AAV with the bacterial sequence-free vector of claim 45 or 49, wherein an AAV rep gene, an AAV cap gene, and one or more helper virus genes for producing AAV are each encoded by the cell or by the vector, and (b) incubating the cells under conditions suitable for expressing the rep gene, cap gene and one or more helper viral genes and suitable for producing AAV.

62. The method of any one of claims 54 to 61, wherein the cells are HEK293T cells.

63. The method of any one of claims 54 to 62, further comprising harvesting the AAV.

64. AAV produced by the method of any one of claims 54 to 63.

65. A pharmaceutical composition comprising the AAV according to claim 64.

66. A method of treating a disease or condition in a subject in need thereof, comprising administering to the subject the AAV of claim 64 or the pharmaceutical composition of claim 63.

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