Plasmid optimization for AAV production

Abstract

The disclosure features Rep / Helper plasmids comprising a cytomegalovirus (CMV) promoter, an E2A gene, an E4 gene, a VA gene and a rep gene, or a cytomegalovirus (CMV) promoter, an E2A gene, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), an E4 gene, a VA gene and a rep gene, or an E2A gene, an E4 gene, a VA gene, a Kozak consensus sequence and a rep gene, where the plasmid does not comprise a nucleic acid sequence encoding a cap gene.

Description

[0001] PLASMID OPTIMIZATION FOR AAV PRODUCTION

[0002] SUMMARY

[0003] In one aspect, the disclosure features a Rep / Helper plasmid comprising a cytomegalovirus (CMV) promoter, an E2A gene, an E4 gene, a VA gene and a rep gene, wherein the plasmid does not comprise a nucleic acid sequence encoding a cap gene. In some embodiments, the plasmid comprises the sequence of SEQ ID NO: 7.

[0004] In another aspect, the disclosure features a Rep / Helper plasmid comprising a cytomegalovirus (CMV) promoter, an E2A gene, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), an E4 gene, a VA gene and a rep gene, wherein the plasmid does not comprise a nucleic acid sequence encoding a cap gene. In some embodiments, the plasmid comprises the sequence of SEQ ID NO: 8.

[0005] In another aspect, the disclosure features a Rep / Helper plasmid comprising an E2A gene, an E4 gene, a VA gene, a Kozak consensus sequence and a rep gene, wherein the plasmid does not comprise a nucleic acid sequence encoding a cap gene. In some embodiments, the plasmid comprises the sequence of SEQ ID NO: 9.

[0006] In some embodiments, the E2A gene comprises the sequence of SEQ ID NO: 2.

[0007] In some embodiments, the E4 gene comprises the sequence of SEQ ID NO: 5.

[0008] In some embodiments, the VA gene comprises the sequence of SEQ ID NO: 6.

[0009] In some embodiments, the CMV promoter comprises the sequence of SEQ ID NO: 1 .

[0010] In some embodiments, the WPRE comprises the sequence of SEQ ID NO: 3.

[0011] In some embodiments, the Kozak consensus sequence comprises the sequence of SEQ ID NO: 4.

[0012] In another aspect, the disclosure features a composition comprising: the Rep / Helper plasmid of any one of the preceding aspects; and a Payload / Cap plasmid, wherein the Payload / Cap plasmid does not comprise a polynucleotide sequence encoding a rep gene.

[0013] In some embodiments, the Payload / Cap plasmid comprises: a polynucleotide sequence comprising a sequence encoding a cap gene; and a polynucleotide sequence encoding a gene of interest.

[0014] In some embodiments, the Payload / Cap plasmid comprises a Kozak consensus sequence.

[0015] In some embodiments, the Payload / Cap plasmid comprises the sequence of SEQ ID NO: 10 or SEQ ID NO: 1 1.

[0016] In another aspect, the disclosure features a universal backbone plasmid comprising the following elements arranged in order: a 5’ inverted terminal repeat (ITR) sequence, a gene of interest, a 3’ ITR sequence, a kanamycin resistance gene, a p40 promoter sequence and an LK03 AAV sequence.

[0017] In some embodiments, the 3’ ITR sequence is 145 base pairs in length.

[0018] In some embodiments, the plasmid ratio of the Rep / Helper plasmid to the Payload / Cap plasmid is greater than or equal to 1 .5:1 up to 10:1 . In some embodiments, the plasmid ratio of the Rep / Helper plasmid to the Payload / Cap plasmid is 1.5:1 to 1 :1.5.

[0019] In some embodiments, the composition of any one of the preceding aspects is for use in producing an AAV vector.

[0020] In another aspect, the disclosure features a method of manufacturing a packaged AAV vector, comprising delivering to a cell a composition of any one of the preceding aspects.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0023] FIG. 1 is a graph showing crude harvest ddPCR titer (vg / mL) for different ratios (Rep / Helper: Cap / Gene of Interest (GOI) molar ratio) of the Kozak-Rep / Helper plasmid tested for 2-plasmid (2P) production relative to the 2P control, pm-0555 is a control plasmid with intron.

[0024] FIG. 2A is a graph showing ddPCR result for whole lysate (vg / mL) for different ratios (Rep / Helper: Cap / GOI molar ratio) of the Kozak-Cap plasmid (a GOI / Cap plasmid) tested for 2-plasmid (2P) production relative to the 2P control.

[0025] FIG. 2B includes the construct design of pm-0538. pm-0538 is the Cap / GOI plasmid in 2P AAV production. It includes the LK03 capsid sequence, and a payload DNA sequence “Hm-hMUT” within two flanking ITR sequences. Together with pm-0555, it serves as a control in this study.

[0026] FIG. 3 is a graph showing crude harvest titer (vg / mL) for different ratios (Rep / Helper: Cap / GOI or GOI / Cap molar ratio) of the CMV-E2A-WPRE Rep / Helper plasmid tested for 2P production relative to the 2P control.

[0027] FIG. 4 is a graph showing crude harvest titer (vg / mL) for different ratios (Rep / Helper: Cap / GOI molar ratio) of several plasmids tested for 2P production relative to the 2P control. The plasmids tested include CMV-E2A-WPRE, Kozak-Rep, CMV-E2A and Kozak-Cap. CMV-E2A-WPRE, Kozak-Rep and CMV-E2A are Rep / Helper plasmids and Kozak-Cap is a GOI / Cap plasmid.

[0028] FIG. 5 is a graph showing ddPCR crude titer (vg / mL) for different ratios (Rep / Helper: Cap / GOI molar ratio) of several Rep / Helper plasmids tested for 2P production in combination with Kozak-Cap relative to the 2P control (red). The Rep / Helper plasmids tested include CMV-E2A-WPRE, Kozak-Rep and CMV-E2A.

[0029] FIG. 6 includes construct designs of the top performing Rep / Helper plasmids: CMV-E2A-WPRE, Kozak-Rep and CMV-E2A. It shows the core elements of the top performing Rep / Helper plasmids and orientation of the various core elements of the top performing Rep / Helper plasmids. The 2P production system includes 2 plasmids: the Rep / Helper plasmid and the Cap / GOI plasmid. It also shows the core elements and orientation of the Kozak-Cap plasmid (a GOI / Cap plasmid) and a Cap / GOI plasmid.

[0030] FIG. 7 includes construct designs of the 2 backbones: backbone 1 and backbone 2. It shows the core elements of the 2 backbones and orientation of the various core elements of the 2 backbones.

[0031] FIG. 8 includes construct designs of backbone 2 and four designs (Designs 1-4) obtained through different modifications of backbone 2. FIG. 9 is a graph showing ddPCR crude titer (vg / mL) for different modifications of backbone 2 (Designs 1-4 illustrated in FIG. 8) with backbone 2 as a control.

[0032] FIG. 10 is a graph showing ddPCR result for whole lysate (vg / mL) for backbone 1 + pm555 when compared with backbone 2 + pm555. pm555 is a control plasmid with intron.

[0033] FIG. 11 is a graph showing ddPCR result for whole lysate (vg / mL) for backbone 2 in combination with CMV-E2A when compared with backbone 2 in combination with pm555 and backbone 1 in combination with pm555. pm555 is a control plasmid with intron.

[0034] FIG. 12 is a graph showing crude harvest titer (vg / mL) for different ratios of several Rep / Helper plasmids tested for 2P production in combination with backbone 1 relative to the 2P control. The Rep / Helper plasmids tested include CMV-E2A-WPRE, Kozak-Rep and CMV-E2A.

[0035] FIG. 13 is a graph showing crude harvest ddPCR titer (vg / mL) for different ratios (Rep / Helper: Cap / GOI molar ratio) of the CMV-E2A-WPRE Rep / Helper plasmid when tested in combination with backbone 1 or backbone 2. The black dot on the graph shows a control plasmid.

[0036] FIG. 14 is a graph showing crude harvest titer (vg / mL) for different Rep / Helper plasmids tested for scalability during 2P production in ambr® 250 with condition 1 relative to the 2P control. The Rep / Helper plasmids tested include CMV-E2A-WPRE, Kozak-Rep and CMV-E2A. Condition 1 includes different % of non-empty capsids for the candidates tested (2P control, CMV-E2A-WPRE, Kozak-Rep and CMV-E2A) as described in Example 1 .

[0037] FIG. 15 is a graph showing crude harvest titer (vg / mL) for different Rep / Helper plasmids tested for scalability during 2P production in ambr® 250 with condition 2 relative to the 2P control. The Rep / Helper plasmids tested include CMV-E2A-WPRE, Kozak-Rep and CMV-E2A. Condition 2 includes different % of non-empty capsids for the candidates tested (2P control, CMV-E2A-WPRE, Kozak-Rep and CMV-E2A) as described in Example 1 .

[0038] FIG. 16 is a graph showing crude harvest titer (vg / mL) for different Rep / Helper plasmids tested for scalability during 2P production in 125 mL shake flasks relative to the 2P control. The Rep / Helper plasmids tested include CMV-E2A-WPRE, Kozak-Rep and CMV-E2A.

[0039] DEFINITIONS

[0040] As used herein, a “vector” includes reference to both polynucleotide vectors and viral vectors, each of which are capable of delivering a transgene contained within the vector into a host cell. Vectors can be episomal, i.e., do not integrate into the genome of a host cell, or can integrate into the host cell genome. The vectors may also be replication competent or replication deficient. Exemplary polynucleotide vectors include, but are not limited to, plasmids, cosmids and transposons. Exemplary viral vectors include, for example, AAV, lentiviral, retroviral, adenoviral, herpes viral and hepatitis viral vectors.

[0041] As used herein, “adeno-associated virus” or “AAV” refers to a non-pathogenic virus, which is a member of the human parvovirus family and depends on helper viruses for its replication. AAV is a parvovirus composed of an icosahedral protein capsid and a single-stranded DNA genome. The AAV viral capsid comprises three subunits or viral proteins (VPs): VP1 , VP2 and VP3 and two inverted terminal repeat (ITR) regions, which are at the ends of the genomic sequence. The ITRs serve as origins of replication and play a role in viral packaging. The viral genome also comprises rep and cap genes, which are associated with replication and capsid packaging, respectively. In most wild-type AAVs, the rep gene encodes four proteins required for viral replication, Rep78, Rep68, Rep52 and Rep40. The cap gene encodes the capsid subunits as well as the assembly activating protein (AAP), which promotes assembly of viral particles. AAVs are generally replication-deficient, requiring the presence of a helper virus or helper virus functions (e.g., herpes simplex virus (HSV) and / or adenovirus (AdV)) to replicate within an infected cell. For example, AAVs require adenoviral E1 A, E2A, E4 and VA RNA genes to replicate within a host cell.

[0042] As used herein, “adeno-associated viral vector” or “AAV vector” refers to a vector in which the capsid is derived from an adeno-associated virus, including without limitation, AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAV13, AAV from other clades or isolates, or AAV that comprises synthetic, bioengineered or modified AAV capsid proteins, including chimeric capsid proteins.

[0043] As used herein, an “ITR” is a palindromic nucleic acid, e.g., an inverted terminal repeat, that is about 120 nucleotides to about 250 nucleotides in length and capable of forming a hairpin. The term “ITR” includes the site of the viral genome replication that can be recognized and bound by a parvoviral protein (e.g., Rep78 / 68). An ITR may be from any AAV. The ssDNA AAV genome consists of two open reading frames, Rep and Cap, flanked by two 145 base ITRs. An ITR includes a replication protein binding element (RBE) and a terminal resolution sequence (TRS). The term “ITR” does not require a wild-type parvoviral ITR (e.g., a wild-type nucleic acid sequence may be altered by insertion, deletion, truncation, or missense mutations), as long as the ITR functions to mediate virus packaging, replication, integration, and / or provirus rescue, and the like.

[0044] As used herein, the term “5’ ITR sequence” or “5’ ITR” is intended to mean the parvoviral ITR located at the 5’ boundary of the nucleic acid molecule.

[0045] As used herein, the term “3’ ITR sequence” or “3’ ITR” is intended to mean the parvoviral ITR located at the 3’ boundary of the nucleic acid molecule. Packaging originates at the 3’ ITR.

[0046] As used herein, a “gene of interest” or “GOI” refers to a transgene, which comprises a gene encoding a functional nucleic acid or protein. A transgene or GOI can be a therapeutic agent, which comprises an agent that has a therapeutic effect upon a host cell or subject (including, e.g., a ribozyme, guide RNA (gRNA), antisense oligonucleotide (ASO), miRNA, siRNA and / or shRNA). For example, a therapeutic agent promotes a biological process to treat a medical condition, e.g., at least one symptom of a disease, disorder, or condition. A GOI may also comprise a polynucleotide sequence encoding a diagnostic and / or therapeutic agent, alone or in combination. A GOI or transgene can also be referred to as a payload, which comprises one or more heterologous nucleic acid sequences. A GOI can encode a reporter gene (e.g., a fluorescent or luminescent reporter) or comprise a polynucleotide sequence encoding a peptide or polypeptide.

[0047] As used herein, the term “plasmid” refers to an extrachromosomal circular double-stranded DNA molecule into which additional DNA segments may be ligated. A plasmid is a type of vector, a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Certain plasmids are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial plasmids having a bacterial origin of replication and episomal mammalian plasmids). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Certain plasmids are capable of directing the expression of genes to which they are operably linked.

[0048] As used herein, a “two-plasmid” or “2P” system comprises a plasmid comprising an AAV rep sequence and relevant sequences from helper viruses (“Rep / Helper Plasmid”) and a plasmid comprising an AAV cap sequence and a payload (“Payload / Cap Plasmid”). A Payload / Cap plasmid can also be referred to as a Cap / GOI plasmid.

[0049] As used herein, a “Rep / Helper plasmid” comprises a polynucleotide sequence encoding a rep gene and a polynucleotide sequence comprising one or more viral helper genes. The Rep / Helper plasmid does not comprise a polynucleotide sequence encoding a cap gene. Rep is translated to produce multiple distinct proteins such as Rep78, Rep68, Rep52 and Rep40, which are required for the AAV life cycle and viral replication. Rep78 / 68 play a role in AAV genome replication and genome integration. Rep52 / 40 play a role in AAV genome packaging. AAVs are generally replication-deficient, requiring the presence of a helper virus or helper virus functions (e.g., herpes simplex virus (HSV) and / or adenovirus (AdV)) to replicate within an infected cell. For example, AAVs require adenoviral E1 A, E2A, E4 and VA RNA genes to replicate within a host cell. In some embodiments, the Rep / Helper plasmids include CMV-E2A-WPRE, Kozak-Rep and CMV-E2A.

[0050] As used herein, “rep gene” encodes distinct proteins such as Rep78, Rep68, Rep52 and Rep40, which are required for the AAV life cycle and viral replication. Rep78 / 68 play a role in AAV genome replication and genome integration. Rep52 / 40 play a role in AAV genome packaging.

[0051] As used herein, a “Payload / Cap plasmid” comprises a polynucleotide sequence encoding a cap gene and a polynucleotide sequence encoding a payload. The Payload / Cap plasmid does not comprise a polynucleotide encoding a rep gene. Cap is translated to produce multiple distinct proteins such as VP1 , VP2 and VP3, which are capsid proteins. Cap VP1 , VP2 and VP3 form the capsid that packages the AAV genome. A Payload / Cap plasmid can also be referred to as a Cap / GOI plasmid. In some embodiments, the Cap / GOI plasmids include the Kozak-Cap plasmid (a GOI / Cap plasmid) and a Cap / GOI plasmid. A payload may comprise a transgene (also referred to herein as a GOI). A payload may comprise one or more inverted terminal repeat (ITR) sequences (e.g., one or more AAV ITRs). A payload may comprise one or more transgenes with flanking ITR sequences.

[0052] As used herein, “cap gene” encodes the capsid subunits as well as the assembly activating protein (AAP), which promotes assembly of viral particles. Cap is translated to produce multiple distinct proteins such as VP1 , VP2 and VP3, which are capsid proteins. Cap VP1 , VP2 and VP3 form the capsid that packages the AAV genome. As used herein, VP1 may possess a surface binding site that interacts with one or more molecules on the surface of a cell to initiate the process of cell entry (e.g., endocytic entry and receptor-mediated fusion). As used herein, VP2 and / or VP3 may facilitate capsid entry into a host cell, for example, by mediating associations with and exit from the endoplasmic reticulum of a host cell and by facilitating the entry of a nucleic acid molecule into a host cell nucleus.

[0053] As used herein, the term “promoter” refers to a recognition site on DNA that is bound by an RNA polymerase. The polymerase drives transcription of the transgene.

[0054] As used herein, the term “cytomegalovirus promoter” or “CMV promoter” refers to the cytomegalovirus immediate early enhancer and promoter. The CMV promoter can promote the expression of a transgene in target cells when the transgene is operably linked to the promoter. The CMV promoter is a constitutive and ubiquitous promoter.

[0055] As used herein, “E2A gene” refers to one of the adenoviral genes required for the replication of replication-deficient AAVs in a host cell. E2A encodes a single-stranded DNA binding protein (ssDBP) and is required for AAV promoter regulation, AAV genome replication, Rep splicing, and capsid protein production.

[0056] As used herein, “E4 gene” refers to one of the adenoviral genes required for the replication of replication-deficient AAVs in a host cell. E4 promotes AAV second-strand synthesis and inhibits the Mre11-Rad50-Nbs1 (MRN) complex. E4 also helps transport the AAV mRNA to the cytoplasm by forming a protein complex with E1 B.

[0057] As used herein, “VA gene” or “VA RNA” refers to one of the adenoviral genes required for the replication of replication-deficient AAVs in a host cell. VA RNA is a viral associated RNA and is non-coding in nature. VA RNA prevents E4orf6 / E1 B mediated degradation of AAV capsids and Rep52.

[0058] As used herein, the term “Woodchuck hepatitis virus (WHP) post-transcriptional regulatory element” or “WPRE” refers to a DNA sequence, which when transcribed, creates a tertiary structure enhancing expression. An AAV vector described herein may include a WPRE. The WPRE acts at the transcriptional level by promoting nuclear export of transcripts and / or by increasing the efficiency of polyadenylation of the nascent transcript, thus increasing the total amount of mRNA in the cells. The addition of the WPRE to the AAV vector results in a substantial improvement in the level of transgene expression from several different promoters, both in vitro and in vivo. WPRE is most effective when placed downstream of the transgene, proximal to the poly A.

[0059] As used herein, “Kozak consensus sequence” refers to a translation initiation sequence in eukaryotes. A translation initiation sequence can be operably linked to an expression sequence. Kozak consensus sequence is a nucleic acid motif that functions as the protein translation initiation site for a majority of eukaryotic mRNA transcripts.

[0060] As used herein, “kanamycin resistance gene” refers to a polynucleotide sequence encoding an antibiotic resistance gene wherein the antibiotic is kanamycin. This gene confers resistance to kanamycin such that kanamycin is unable to destroy or kill a microorganism when provided.

[0061] As used herein, “p40 promoter sequence” refers to a native AAV serotype 2 (AAV2) promoter that is located at 40 map units. The p40 promoter is required for regulating the transcription of capsid proteins VP1 , VP2 and VP3. In addition, the p40 promoter can be transactivated by Rep78 / 68.

[0062] As used herein, “LK03 AAV sequence” refers to a novel capsid generated through capsid shuffling and library selection in a xenograft humanized mouse liver model resulting in the production of a chimeric AAV serotype, AAV-LK03 (Lisowski, L. et al., Nature, 506:382-6, 2014). LK03 is a capsid variant generated for the purposes of increasing transduction efficiency and transgene expression.

[0063] DETAILED DESCRIPTION

[0064] The disclosure is directed towards optimizing plasmids to increase AAV yield and improve the quality of AAVs produced. Plasmids are essential for AAV production, and the core elements of plasmids play a role in increasing the yield and improving the quality of AAV vectors. Following are some of the core elements of plasmids including their functions: E2A gene is required for AAV promoter regulation, AAV genome replication, Rep splicing, and capsid protein production.

[0065] E4 gene promotes AAV second-strand synthesis and inhibits the Mre11-Rad50-Nbs1 (MRN) complex.

[0066] VA RNA prevents E4orf6 / E1 B mediated degradation of AAV capsids and Rep52.

[0067] Rep78 / 68 play a role in AAV genome replication and genome integration.

[0068] Rep52 / 40 play a role in AAV genome packaging.

[0069] Cap proteins VP1 , VP2 and VP3 form the capsid that packages the AAV genome.

[0070] Kozak consensus sequence is a nucleic acid motif that functions as the protein translation initiation site.

[0071] CMV promoter is a human cytomegalovirus (CMV) immediate early enhancer and promoter.

[0072] Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) increases transgene expression. WPRE is most effective when placed downstream of the transgene, proximal to the poly A.

[0073] The 2-plasmid (2P) production system includes 2 plasmids- the Rep / Helper plasmid and the Cap / GOI plasmid. Aspects of a 2P production system are described in WO 2022 / 182986, which is hereby incorporated by reference in its entirety. In some embodiments, the 2P production system uses engineered human embryonic kidney (HEK)293 cells or cells derived from HEK293 cells.

[0074] The currently disclosed studies were performed to optimize plasmids to increase AAV yield and improve the quality of AAVs produced. Top performing Rep / Helper plasmids are as follows: CMV-E2A- WPRE, Kozak-Rep and CMV-E2A. The structure of the top performing Rep / Helper plasmids were identified as follows: E2A-E4-VA-Kozak-Rep, CMV-E2A-WPRE-E4-VA-Rep and CMV-E2A-E4-VA-Rep. The Cap / GOI plasmids include the Kozak-Cap plasmid (a GOI / Cap plasmid) and a Cap / GOI plasmid. The structure of the Kozak-Cap plasmid is as follows: ITR-GOI-ITR-Kozak-Cap. The structure of the Cap / GOI plasmid is as follows: Cap-ITR-GOI-ITR.

[0075] The sequences of the core elements are provided in Table 1 below.

[0076] Table 1 .

[0077] In some embodiments, the plasmid comprises a sequence that is at least 95%, 98%, 99%, or 00% identical to a sequence listed in the below table.

[0078] The sequences of the top performing Rep / Helper plasmids are provided in Table 2 below.

[0079] Table 2.

[0080] The sequences of the Payload / Cap plasmids are as follows: pm-0984: CAP-GOI plasmid (LK03-ITR-GOI-ITR), ITR=130bp (SEQ ID NO: 10)

[0081] SEQ ID NO: 10 is also the sequence of Backbone 1. SEQ ID NO: 10 is shown below.

[0082] TTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTT

[0083] TTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCC

[0084] TGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAAT

[0085] AAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGC

[0086] ATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAA

[0087] ACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTA

[0088] CAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAAT

[0089] CAGGATATTCTTCTAATACCTGGAATGCTGTTTTTCCGGGGATCGCAGTGGTGAGTAACCATGCATC

[0090] ATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTG

[0091] ACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATC

[0092] GGGCTTCCCATACAAGCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATAC

[0093] CCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGACGTTTCCCGTTGAATATGGCTCAT

[0094] ACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGA

[0095] ATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCT

[0096] AAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCG

[0097] CGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTG

[0098] TAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGG

[0099] GCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATTCGACGCTCTCCCTTAT

[0100] GCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGG

[0101] AATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCAC

[0102] GCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGAT

[0103] ATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGGGTCACCAAGCAGGAAGTCAAAGACTTTTTCC

[0104] GGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGA

[0105] AAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAG

[0106] CCATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCAGACAGGTACCAAAACAAATGTTCTCGTC

[0107] ACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGAATGAATCAGAATTCAAATAT

[0108] CTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCT

[0109] GTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGGAAAGGTGCCAGACGCTT

[0110] GCACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACTGCATCTTTGAACAATAAATGATTTAAATC

[0111] AGGTATGGCTGCTGACGGTTATCTTCCAGATTGGCTCGAGGACAACCTTTCTGAAGGCATTCGAGAG

[0112] TGGTGGGCGCTGCAACCTGGAGCCCCTAAACCCAAGGCAAATCAACAACATCAGGACAACGCTCGG

[0113] GGTCTTGTGCTTCCGGGTTACAAATACCTCGGACCCGGCAACGGACTCGACAAGGGGGAACCCGTC

[0114] AACGCAGCGGACGCGGCAGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGTGA CAACCCCTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTC TTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCT GGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGATCAGTCTCCTCAGGAACC GGACTCATCATCTGGTGTTGGCAAATCGGGCAAACAGCCTGCCAGAAAAAGACTAAATTTCGGTCAG ACTGGCGACTCAGAGTCAGTCCCAGACCCTCAACCTCTCGGAGAACCACCAGCAGCCCCCACAAGT TTGGGATCTAATACAATGGCTTCAGGCGGTGGCGCACCAATGGCAGACAATAACGAGGGTGCCGAT GGAGTGGGTAATTCCTCAGGAAATTGGCATTGCGATTCCCAATGGCTGGGCGACAGAGTCATCACC ACCAGCACCAGAACCTGGGCCCTGCCCACTTACAACAACCATCTCTACAAGCAAATCTCCAGCCAAT CAGGAGCTTCAAACGACAACCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTTAACAG ATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATTAACAACAACTGGGGATTCCGGCCC AAGAAACTCAGCTTCAAGCTCTTCAACATCCAAGTTAAAGAGGTCACGCAGAACGATGGCACGACGA CTATTGCCAATAACCTTACCAGCACGGTTCAAGTGTTTACGGACTCGGAGTATCAGCTCCCGTACGT GCTCGGGTCGGCGCACCAAGGCTGTCTCCCGCCGTTTCCAGCGGACGTCTTCATGGTCCCTCAGTA TGGATACCTCACCCTGAACAACGGAAGTCAAGCGGTGGGACGCTCATCCTTTTACTGCCTGGAGTAC TTCCCTTCGCAGATGCTAAGGACTGGAAATAACTTCCAATTCAGCTATACCTTCGAGGATGTACCTTT TCACAGCAGCTACGCTCACAGCCAGAGTTTGGATCGCTTGATGAATCCTCTTATTGATCAGTATCTGT ACTACCTGAACAGAACGCAAGGAACAACCTCTGGAACAACCAACCAATCACGGCTGCTTTTTAGCCA GGCTGGGCCTCAGTCTATGTCTTTGCAGGCCAGAAATTGGCTACCTGGGCCCTGCTACCGGCAACA GAGACTTTCAAAGACTGCTAACGACAACAACAACAGTAACTTTCCTTGGACAGCGGCCAGCAAATAT CATCTCAATG GCCG CG ACTCG CTG GTG AATCCAGG ACCAG CTATG GCCAGTCAC AAGG ACG ATG AA GAAAAATTTTTCCCTATGCACGGCAATCTAATATTTGGCAAAGAAGGGACAACGGCAAGTAACGCAG AATTAGATAATGTAATGATTACGGATGAAGAAGAGATTCGTACCACCAATCCTGTGGCAACAGAGCA GTATGGAACTGTGGCAAATAACTTGCAGAGCTCAAATACAGCTCCCACGACTAGAACTGTCAATGAT

[0115] CAGGGGGCCTTACCTGGCATGGTGTGGCAAGATCGTGACGTGTACCTTCAAGGACCTATCTGGGCA AAGATTCCTCACACGGATGGACACTTTCATCCTTCTCCTCTGATGGGAGGCTTTGGACTGAAACATC CGCCTCCTCAAATCATGATCAAAAATACTCCGGTACCGGCAAATCCTCCGACGACTTTCAGCCCGGC CAAGTTTGCTTCATTTATCACTCAGTACTCCACTGGACAGGTCAGCGTGGAAATTGAGTGGGAGCTA CAGAAAGAAAACAGCAAACGTTGGAATCCAGAGATTCAGTACACTTCCAACTACAACAAGTCTGTTAA TGTGGACTTTACTGTAGACACTAATGGTGTTTATAGTGAACCTCGCCCCATTGGCACCCGTTACCTTA CCCGTCCCCTGTAATTGCTTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTCTGC GTATTTCTTTCTTATCTAGTTTCCATATGCATGTAGATAAGTAGCATGGCGGGTTAATCATTAACTAAC CGGTACCTCTAGAACTATAGCTAGCGATGACCCTGCTGATTGGTTCGCTGACCATTTCCGGGTGCGG GACGGCGTTACCAGAAACTCAGAAGGTTCGTCCAACCAAACCGACTCTGACGGCAGTTTACGAGAG AGATGATAGGGTCTGCTTCAGTAAGCCAGATGCTACACAATTAGGCTTGTACATATTGTCGTTAGAAC G CGG CTACAATTAATACATAACCTTATGTATCATACAC ATACG ATTTAG GTG ACACTATAG AATACAC GGAATTAATTCCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCG ACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCAC TAGGGGTTCCTTACGTAACTCCATGAAAGTGGATTTTATTATCCTCATCATGCAGATGAGAATATTGA GACTTATAGCGGTATGCCTGAGCCCCAAAGTACTCAGAGTTGCCTGGCTCCAAGATTTATAATCTTAA ATGATGGGACTACCATCCTTACTCTCTCCATTTTTCTATACGTGAGTAATGTTTTTTCTGTTTTTTTTTT TTCTTTTTCCATTCAAACTCAGTGCACTTGTTGAGCTTGTGAAACACAAGCCCAAGGCAACAAAAGAG CAACTG AAAG CTGTTATG G ATG ATTTCG CAG CTTTTGTAG AG AAGTGCTGCAAGG CTG ACG ATAAG G AGACCTGCTTTGCCGAGGAGGTACTACAGTTCTCTTCATTTTAATATGTCCAGTATTCATTTTTGCATG TTTGGTTAGGCTAGGGCTTAGGGATTTATATATCAAAGGAGGCTTTGTACATGTGGGACAGGGATCT TATTTTACAAACAATTGTCTTACAAAATGAATAAAACAGCACTTTGTTTTTATCTCCTGCTCTATTGTGC CATACTGTTAAATGTTTATAATGCCTGTTCTGTTTCCAAATTTGTGATGCTTATGAATATTAATAGGAAT ATTTGTAAGGCCTGAAATATTTTGATCATGAAATCAAAACATTAATTTATTTAAACATTTACTTGAAATG TGGTGGTTTGTGATTTAGTTGATTTTATAGGCTAGTGGGAGAATTTACATTCAAATGTCTAAATCACTT AAAATTGCCCTTTATGGCCTGACAGTAACTTTTTTTTATTCATTTGGGGACAACTATGTCCGTGAGCTT CCGTCCAGAGATTATAGTAGTAAATTGTAATTAAAGGATATGATGCACGTGAAATCACTTTGCAATCA TCAATAGCTTCATAAATGTTAATTTTGTATCCTAATAGTAATGCTAATATTTTCCTAACATCTGTCATGT CTTTGTGTTCAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCCTTAGGCTTAGGCAGCGGCGCCA CCAACTTCAGCCTGCTGAAACAGGCCGGCGACGTGGAAGAGAACCCTGGCCCCCTGAGAGCCAAA AACCAGCTGTTCCTGCTGAGCCCCCACTATCTGAGACAGGTCAAAGAAAGTTCCGGGAGTAGACTG ATCCAGCAGAGACTGCTGCACCAGCAGCAGCCACTGCATCCTGAGTGGGCCGCTCTGGCCAAGAAA CAG CTG AAG GG CAAAAACCCAG AAG ACCTG ATCTGG CACACTCCAG AG GG G ATTTCAATCAAG CCC CTGTACAGCAAAAGGGACACTATGGATCTGCCAGAGGAACTGCCAGGAGTGAAGCCTTTCACCCGC GGACCTTACCCAACTATGTATACCTTTCGACCCTGGACAATTCGGCAGTACGCCGGCTTCAGTACTG TGGAGGAATCAAACAAGTTTTATAAGGACAACATCAAGGCTGGACAGCAGGGCCTGAGTGTGGCATT CGATCTGGCCACACATCGCGGCTATGACTCAGATAATCCCAGAGTCAGGGGGGACGTGGGAATGGC AGGAGTCGCTATCGACACAGTGGAAGATACTAAGATTCTGTTCGATGGAATCCCTCTGGAGAAAATG TCTGTGAGTATGACAATGAACGGCGCTGTCATTCCCGTGCTGGCAAACTTCATCGTCACTGGCGAGG AACAGGGGGTGCCTAAGGAAAAACTGACCGGCACAATTCAGAACGACATCCTGAAGGAGTTCATGG TGCGGAATACTTACATTTTTCCCCCTGAACCATCCATGAAAATCATTGCCGATATCTTCGAGTACACC GCTAAGCACATGCCCAAGTTCAACTCAATTAGCATCTCCGGGTATCATATGCAGGAAGCAGGAGCCG ACGCTATTCTGGAGCTGGCTTACACCCTGGCAGATGGCCTGGAATATTCTCGAACCGGACTGCAGG CAGGCCTGACAATCGACGAGTTCGCTCCTAGACTGAGTTTCTTTTGGGGAATTGGCATGAACTTTTA CATGGAGATCGCCAAGATGAGGGCTGGCCGGAGACTGTGGGCACACCTGATCGAGAAGATGTTCCA GCCTAAGAACTCTAAGAGTCTGCTGCTGCGGGCCCATTGCCAGACATCCGGCTGGTCTCTGACTGA ACAGGACCCATATAACAATATTGTCAGAACCGCAATCGAGGCAATGGCAGCCGTGTTCGGAGGAAC

[0116] CCAGAGCCTGCACACAAACTCCTTTGATGAGGCCCTGGGGCTGCCTACCGTGAAGTCTGCTAGGAT TGCACGCAATACACAGATCATTATCCAGGAGGAATCCGGAATCCCAAAGGTGGCCGATCCCTGGGG AGGCTCTTACATGATGGAGTGCCTGACAAACGACGTGTATGATGCTGCACTGAAGCTGATTAATGAA ATCGAGGAAATGGGGGGAATGGCAAAGGCCGTGGCTGAGGGCATTCCAAAACTGAGGATCGAGGA ATGTGCAGCTAGGCGCCAGGCACGAATTGACTCAGGAAGCGAAGTGATCGTCGGGGTGAATAAGTA CCAGCTGGAGAAAGAAGACGCAGTCGAAGTGCTGGCCATCGATAACACAAGCGTGCGCAATCGACA GATTGAGAAGCTGAAGAAAATCAAAAGCTCCCGCGATCAGGCACTGGCCGAACGATGCCTGGCAGC CCTGACTGAGTGTGCTGCAAGCGGGGACGGAAACATTCTGGCTCTGGCAGTCGATGCCTCCCGGG CTAG ATG CACTGTGG GG G AAATCACCG ACG CCCTG AAG AAAGTCTTCG G AG AGCACAAGG CCAATG ATCGGATGGTGAGCGGCGCTTATAGACAGGAGTTCGGGGAATCTAAAGAGATTACCAGTGCCATCA AGAGGGTGCACAAGTTCATGGAGAGAGAAGGGCGACGGCCCAGGCTGCTGGTGGCAAAGATGGGA CAGGACGGACATGATCGCGGAGCAAAAGTCATTGCCACCGGGTTCGCTGACCTGGGATTTGACGTG G ATATCG G CCCTCTGTTCCAG ACACCACG AG AG GTCGCACAG CAG GCAGTCG ACG CTG ATGTG CAC G CAGTCG G AGTGTCCACTCTGG CAG CTG GCCATAAG ACCCTGGTG CCTG AACTG ATCAAAG AG CTG AACTCTCTGGGCAGACCAGACATCCTGGTCATGTGCGGCGGCGTGATCCCACCCCAGGATTACGAA TTCCTGTTTGAGGTCGGGGTGAGCAACGTGTTCGGACCAGGAACCAGGATCCCTAAGGCCGCAGTG CAGGTCCTGGATGATATTGAAAAGTGTCTGGAAAAGAAACAGCAGTCAGTGTAACATCACATTTAAAA GCATCTCAGGTAACTATATTTTGAATTTTTTAAAAAAGTAACTATAATAGTTATTATTAAAATAGCAAAG ATTGACCATTTCCAAGAGCCATATAGACCAGCACCGACCACTATTCTAAACTATTTATGTATGTAAATA TTAGCTTTTAAAATTCTCAAAATAGTTGCTGAGTTGGGAACCACTATTATTTCTATTTTGTAGATGAGA AAATGAAGATAAACATCAAAGCATAGATTAAGTAATTTTCCAAAGGGTCAAAATTCAAAATTGAAACCA AAGTTTCAGTGTTGCCCATTGTCCTGTTCTGACTTATATGATGCGGTACACAGAGCCATCCAAGTAAG TGATGGCTCAGCAGTGGAATACTCTGGGAATTAGGCTGAACCACATGAAAGAGTGCTTTATAGGGCA AAAACAGTTGAATATCAGTGATTTCACATGGTTCAACCTAATAGTTCAACTCATCCTTTCCATTGGAGA ATATGATGGATCTACCTTCTGTGAACTTTATAGTGAAGAATCTGCTATTACATTTCCAATTTGTCAACA TG CTG AG CTTTAATAG G ACTTATCTTCTTATG ACAACATTTATTG GTGTGTCCCCTTGCCTAG CCCAA CAGAAGAATTCAGCAGCCGTAAGTCTAGGACAGGCTTAAATTGTTTTCACTGGTGTAAATTGCAGAAA GATGATCTAAGTAATTTGGCATTTATTTTAATAGGTTTGAAAAACACATGCCATTTTACAAATAAGACTT ATATTTGTCCTTTTGTTTTTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAAAAGCTTATT CATCTGTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATT TTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAATCTAATAGAGTGGTACAGCACTGTT ATTTTTCAAAGATGTGTTGTACGTAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCG CTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGC CTCAGTGAGCGAGCGAGCGCGCAGCTTATTCCACAGCTGCATTAATGAATCGGCCAACGCGCGGGG AGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTT CGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGAT AACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTG CTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGT GGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTC CTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTT CTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCA CGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGT AAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGG CGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATC TGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCA CCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGA AGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTG GTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCT AAAGTATATATGAGTAAACTTGGTCTGACAG pm-0903: CAP-GOI plasmid (LK03-ITR-GOI-ITR), ITR=145bp (SEQ ID NO: 11)

[0117] SEQ ID NO: 1 1 is also the sequence of Backbone 2. SEQ ID NO: 11 is shown below. AGAGGGTGCACAAGTTCATGGAGAGAGAAGGGCGACGGCCCAGGCTGCTGGTGGCAAAGATGGGA CAGGACGGACATGATCGCGGAGCAAAAGTCATTGCCACCGGGTTCGCTGACCTGGGATTTGACGTG G ATATCG G CCCTCTGTTCCAG ACACCACG AG AG GTCGCACAG CAG GCAGTCG ACG CTG ATGTG CAC G CAGTCG G AGTGTCCACTCTGG CAG CTG GCCATAAG ACCCTGGTG CCTG AACTG ATCAAAG AG CTG AACTCTCTGGGCAGACCAGACATCCTGGTCATGTGCGGCGGCGTGATCCCACCCCAGGATTACGAA TTCCTGTTTGAGGTCGGGGTGAGCAACGTGTTCGGACCAGGAACCAGGATCCCTAAGGCCGCAGTG CAGGTCCTGGATGATATTGAAAAGTGTCTGGAAAAGAAACAGCAGTCAGTGTAACATCACATTTAAAA GCATCTCAGGTAACTATATTTTGAATTTTTTAAAAAAGTAACTATAATAGTTATTATTAAAATAGCAAAG ATTGACCATTTCCAAGAGCCATATAGACCAGCACCGACCACTATTCTAAACTATTTATGTATGTAAATA TTAGCTTTTAAAATTCTCAAAATAGTTGCTGAGTTGGGAACCACTATTATTTCTATTTTGTAGATGAGA AAATGAAGATAAACATCAAAGCATAGATTAAGTAATTTTCCAAAGGGTCAAAATTCAAAATTGAAACCA AAGTTTCAGTGTTGCCCATTGTCCTGTTCTGACTTATATGATGCGGTACACAGAGCCATCCAAGTAAG TGATGGCTCAGCAGTGGAATACTCTGGGAATTAGGCTGAACCACATGAAAGAGTGCTTTATAGGGCA AAAACAGTTGAATATCAGTGATTTCACATGGTTCAACCTAATAGTTCAACTCATCCTTTCCATTGGAGA ATATGATGGATCTACCTTCTGTGAACTTTATAGTGAAGAATCTGCTATTACATTTCCAATTTGTCAACA TG CTG AG CTTTAATAG G ACTTATCTTCTTATG ACAACATTTATTG GTGTGTCCCCTTGCCTAG CCCAA CAGAAGAATTCAGCAGCCGTAAGTCTAGGACAGGCTTAAATTGTTTTCACTGGTGTAAATTGCAGAAA GATGATCTAAGTAATTTGGCATTTATTTTAATAGGTTTGAAAAACACATGCCATTTTACAAATAAGACTT ATATTTGTCCTTTTGTTTTTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAAAAGCTTATT CATCTGTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATT TTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAATCTAATAGAGTGGTACAGCACTGTT ATTTTTCAAAGATGTGTTGTACGTAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCG CTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGC CTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTTATTCCACAGCTGCATTAATGAATC GGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTC GCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATC CACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACC GTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATC GACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAA GCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTC GGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTC CAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCG TCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAG CAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAG AAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCT TGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCA GAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAA CTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAA ATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTAGAAAAACTCATCGAG

[0118] CATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTG TAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATT CCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAA ATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTT CAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGAT TGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCA ACCG G CG CAG G AAC ACTG CCAG CG CATCAACAATATTTTCACCTG AATCAG G ATATTCTTCTAATACC TGGAATGCTGTTTTTCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAAT GCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATC ATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAAGCGA TAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCAT GTTGGAATTTAATCGCGGCCTCGACGTTTCCCGTTGAATATGGCTCATACTCTTCCTTTTTCAATATTA TTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACA AATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATG ACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTG AAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCA GACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCA TCAG AG CAG ATTGTACTG AG AGTG CACCATTCG ACGCTCTCCCTTATG CG ACTCCTGCATTAG G AAG CAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGAT GGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGA GCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCA CCTGTGGCGCCGGTGGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGT GGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGA CGCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGG AAGCTTCGATCAACTACGCAGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGAT GCTGTTTCCCTGCAGACAATGCGAGAGAATGAATCAGAATTCAAATATCTGCTTCACTCACGGACAG AAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCA G AAACTGTG CTACATTCATCATATCATGG G AAAGGTG CCAG ACG CTTG CACTGCCTG CG ATCTG GTC AATGTGGATTTGGATGACTGCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTGCTGACGGTT ATCTTCCAGATTGGCTCGAGGACAACCTTTCTGAAGGCATTCGAGAGTGGTGGGCGCTGCAACCTG GAGCCCCTAAACCCAAGGCAAATCAACAACATCAGGACAACGCTCGGGGTCTTGTGCTTCCGGGTT ACAAATACCTCGGACCCGGCAACGGACTCGACAAGGGGGAACCCGTCAACGCAGCGGACGCGGCA GCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGTGACAACCCCTACCTCAAGTAC AACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGG GCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAA GACGGCTCCTGGAAAGAAGAGGCCTGTAGATCAGTCTCCTCAGGAACCGGACTCATCATCTGGTGT TGGCAAATCGGGCAAACAGCCTGCCAGAAAAAGACTAAATTTCGGTCAGACTGGCGACTCAGAGTC AGTCCCAGACCCTCAACCTCTCGGAGAACCACCAGCAGCCCCCACAAGTTTGGGATCTAATACAATG GCTTCAGGCGGTGGCGCACCAATGGCAGACAATAACGAGGGTGCCGATGGAGTGGGTAATTCCTCA GGAAATTGGCATTGCGATTCCCAATGGCTGGGCGACAGAGTCATCACCACCAGCACCAGAACCTGG

[0119] G CCCTG CCCACTTACAACAACCATCTCTACAAGC AAATCTCCAG CCAATCAGG AG CTTCAAACG ACA ACCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTTAACAGATTCCACTGCCACTTCTCA CCACGTGACTGGCAGCGACTCATTAACAACAACTGGGGATTCCGGCCCAAGAAACTCAGCTTCAAG CTCTTCAACATCCAAGTTAAAGAGGTCACGCAGAACGATGGCACGACGACTATTGCCAATAACCTTA CCAGCACGGTTCAAGTGTTTACGGACTCGGAGTATCAGCTCCCGTACGTGCTCGGGTCGGCGCACC AAGGCTGTCTCCCGCCGTTTCCAGCGGACGTCTTCATGGTCCCTCAGTATGGATACCTCACCCTGAA CAACGGAAGTCAAGCGGTGGGACGCTCATCCTTTTACTGCCTGGAGTACTTCCCTTCGCAGATGCTA AGGACTGGAAATAACTTCCAATTCAGCTATACCTTCGAGGATGTACCTTTTCACAGCAGCTACGCTCA CAG CCAG AGTTTG G ATCG CTTG ATG AATCCTCTTATTG ATCAGTATCTGTACTACCTG AACAG AACGC AAGGAACAACCTCTGGAACAACCAACCAATCACGGCTGCTTTTTAGCCAGGCTGGGCCTCAGTCTAT GTCTTTGCAGGCCAGAAATTGGCTACCTGGGCCCTGCTACCGGCAACAGAGACTTTCAAAGACTGCT AACGACAACAACAACAGTAACTTTCCTTGGACAGCGGCCAGCAAATATCATCTCAATGGCCGCGACT CGCTGGTGAATCCAGGACCAGCTATGGCCAGTCACAAGGACGATGAAGAAAAATTTTTCCCTATGCA CGGCAATCTAATATTTGGCAAAGAAGGGACAACGGCAAGTAACGCAGAATTAGATAATGTAATGATTA CGGATGAAGAAGAGATTCGTACCACCAATCCTGTGGCAACAGAGCAGTATGGAACTGTGGCAAATAA CTTGCAGAGCTCAAATACAGCTCCCACGACTAGAACTGTCAATGATCAGGGGGCCTTACCTGGCATG GTGTGGCAAGATCGTGACGTGTACCTTCAAGGACCTATCTGGGCAAAGATTCCTCACACGGATGGA CACTTTCATCCTTCTCCTCTGATGGGAGGCTTTGGACTGAAACATCCGCCTCCTCAAATCATGATCAA AAATACTCCGGTACCGGCAAATCCTCCGACGACTTTCAGCCCGGCCAAGTTTGCTTCATTTATCACT CAGTACTCCACTGGACAGGTCAGCGTGGAAATTGAGTGGGAGCTACAGAAAGAAAACAGCAAACGT TGGAATCCAGAGATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTAGACAC TAATGGTGTTTATAGTGAACCTCGCCCCATTGGCACCCGTTACCTTACCCGTCCCCTGTAATTGCTTG TTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTCTGCGTATTTCTTTCTTATCTAGTTT CCATATGCATGTAGATAAGTAGCATGGCGGGTTAATCATTAACTAACCGGTACCTCTAGAACTATAGC TAGCGATGACCCTGCTGATTGGTTCGCTGACCATTTCCGGGTGCGGGACGGCGTTACCAGAAACTC AGAAGGTTCGTCCAACCAAACCGACTCTGACGGCAGTTTACGAGAGAGATGATAGGGTCTGCTTCA GTAAGCCAGATGCTACACAATTAGGCTTGTACATATTGTCGTTAGAACGCGGCTACAATTAATACATA ACCTTATGTATCATACACATACGATTTAGGTGACACTATAGAATACACGGAATTAATTCTTGGCCACTC CCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTT GGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGG GTTCCTTACGTAACTCCATGAAAGTGGATTTTATTATCCTCATCATGCAGATGAGAATATTGAGACTTA TAGCGGTATGCCTGAGCCCCAAAGTACTCAGAGTTGCCTGGCTCCAAGATTTATAATCTTAAATGATG GGACTACCATCCTTACTCTCTCCATTTTTCTATACGTGAGTAATGTTTTTTCTGTTTTTTTTTTTTCTTTT TCCATTCAAACTCAGTGCACTTGTTGAGCTTGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACTG AAAGCTGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGCTGACGATAAGGAGACCT GCTTTGCCGAGGAGGTACTACAGTTCTCTTCATTTTAATATGTCCAGTATTCATTTTTGCATGTTTGGT TAGGCTAGGGCTTAGGGATTTATATATCAAAGGAGGCTTTGTACATGTGGGACAGGGATCTTATTTTA CAAACAATTGTCTTACAAAATG AATAAAACAG CACTTTGTTTTTATCTCCTG CTCTATTGTG CCATACT GTTAAATGTTTATAATGCCTGTTCTGTTTCCAAATTTGTGATGCTTATGAATATTAATAGGAATATTTGT AAGGCCTGAAATATTTTGATCATGAAATCAAAACATTAATTTATTTAAACATTTACTTGAAATGTGGTG

[0120] GTTTGTGATTTAGTTGATTTTATAGGCTAGTGGGAGAATTTACATTCAAATGTCTAAATCACTTAAAAT TGCCCTTTATGGCCTGACAGTAACTTTTTTTTATTCATTTGGGGACAACTATGTCCGTGAGCTTCCGT CCAGAGATTATAGTAGTAAATTGTAATTAAAGGATATGATGCACGTGAAATCACTTTGCAATCATCAAT AGCTTCATAAATGTTAATTTTGTATCCTAATAGTAATGCTAATATTTTCCTAACATCTGTCATGTCTTTG TGTTCAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCCTTAGGCTTAGGCAGCGGCGCCACCAAC TTCAGCCTGCTGAAACAGGCCGGCGACGTGGAAGAGAACCCTGGCCCCCTGAGAGCCAAAAACCA GCTGTTCCTGCTGAGCCCCCACTATCTGAGACAGGTCAAAGAAAGTTCCGGGAGTAGACTGATCCA GCAGAGACTGCTGCACCAGCAGCAGCCACTGCATCCTGAGTGGGCCGCTCTGGCCAAGAAACAGC TGAAGGGCAAAAACCCAGAAGACCTGATCTGGCACACTCCAGAGGGGATTTCAATCAAGCCCCTGT ACAGC AAAAGG G ACACTATG G ATCTG CCAG AG G AACTG CCAG G AGTG AAGCCTTTCACCCGCG G AC CTTACCCAACTATGTATACCTTTCGACCCTGGACAATTCGGCAGTACGCCGGCTTCAGTACTGTGGA GGAATCAAACAAGTTTTATAAGGACAACATCAAGGCTGGACAGCAGGGCCTGAGTGTGGCATTCGAT CTGGCCACACATCGCGGCTATGACTCAGATAATCCCAGAGTCAGGGGGGACGTGGGAATGGCAGG AGTCGCTATCGACACAGTGGAAGATACTAAGATTCTGTTCGATGGAATCCCTCTGGAGAAAATGTCT GTGAGTATGACAATGAACGGCGCTGTCATTCCCGTGCTGGCAAACTTCATCGTCACTGGCGAGGAA CAG GG G GTG CCTAAGG AAAAACTG ACCG GCACAATTCAG AACG AC ATCCTG AAG G AGTTCATGGTG CGGAATACTTACATTTTTCCCCCTGAACCATCCATGAAAATCATTGCCGATATCTTCGAGTACACCGC TAAG CACATG CCCAAGTTCAACTCAATTAG CATCTCCG GGTATCATATG CAG G AAG CAGG AG CCG AC GCTATTCTGGAGCTGGCTTACACCCTGGCAGATGGCCTGGAATATTCTCGAACCGGACTGCAGGCA GGCCTGACAATCGACGAGTTCGCTCCTAGACTGAGTTTCTTTTGGGGAATTGGCATGAACTTTTACAT GGAGATCGCCAAGATGAGGGCTGGCCGGAGACTGTGGGCACACCTGATCGAGAAGATGTTCCAGC CTAAGAACTCTAAGAGTCTGCTGCTGCGGGCCCATTGCCAGACATCCGGCTGGTCTCTGACTGAAC AGGACCCATATAACAATATTGTCAGAACCGCAATCGAGGCAATGGCAGCCGTGTTCGGAGGAACCC AGAGCCTGCACACAAACTCCTTTGATGAGGCCCTGGGGCTGCCTACCGTGAAGTCTGCTAGGATTG CACGCAATACACAGATCATTATCCAGGAGGAATCCGGAATCCCAAAGGTGGCCGATCCCTGGGGAG GCTCTTACATGATGGAGTGCCTGACAAACGACGTGTATGATGCTGCACTGAAGCTGATTAATGAAAT CGAGGAAATGGGGGGAATGGCAAAGGCCGTGGCTGAGGGCATTCCAAAACTGAGGATCGAGGAAT GTGCAGCTAGGCGCCAGGCACGAATTGACTCAGGAAGCGAAGTGATCGTCGGGGTGAATAAGTACC AGCTGGAGAAAGAAGACGCAGTCGAAGTGCTGGCCATCGATAACACAAGCGTGCGCAATCGACAGA TTGAGAAGCTGAAGAAAATCAAAAGCTCCCGCGATCAGGCACTGGCCGAACGATGCCTGGCAGCCC

[0121] TGACTGAGTGTGCTGCAAGCGGGGACGGAAACATTCTGGCTCTGGCAGTCGATGCCTCCCGGGCTA GATGCACTGTGGGGGAAATCACCGACGCCCTGAAGAAAGTCTTCGGAGAGCACAAGGCCAATGATC GGATGGTGAGCGGCGCTTATAGACAGGAGTTCGGGGAATCTAAAGAGATTACCAGTGCCATCA

[0122] EXAMPLES

[0123] The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used and evaluated and are intended to be purely exemplary and are not intended to limit the scope of the disclosure.

[0124] Example 1. Plasmid optimization and its contributions for adeno-associated virus (AAV) production.

[0125] The aim of this study was to optimize plasmids to increase AAV yield and improve the quality of AAVs produced. Identification of the top performing Rep / Helper plasmids

[0126] FIG. 1 is a graph showing crude harvest ddPCR titer (vg / mL) for different ratios (Rep / Helper: Cap / GOI molar ratio) of the Kozak-Rep / Helper plasmid tested for 2-plasmid (2P) production relative to the 2P control, pm-0555 is the helper plasmid. It was observed that the 1 :3 Rep / Helper: Cap / GOI molar ratio was the best performing ratio and increased the titer by 3.08-fold.

[0127] FIG. 2A is a graph showing ddPCR result for whole lysate (vg / mL) for different ratios (Rep / Helper: Cap / GOI molar ratio) of the Kozak-Cap plasmid (a GOI / Cap plasmid) tested for 2P production relative to the 2P control. It was observed that the 1 :6 Rep / Helper: Cap / GOI molar ratio was the best performing ratio and increased the titer by more than 2-fold (approximately 2.27-fold).

[0128] FIG. 2B includes the construct design of pm-0538. pm-0538 is the Cap / GOI plasmid in 2P AAV production. It includes the LK03 capsid sequence, and a payload DNA sequence “Hm-hMUT” within two flanking ITR sequences. Together with pm-0555, it serves as a control in this study.

[0129] FIG. 3 is a graph showing crude harvest titer (vg / mL) for different ratios (Rep / Helper: Cap / GOI or GOI / Cap molar ratio) of the CMV-E2A-WPRE Rep / Helper plasmid tested for 2P production relative to the 2P control. It was observed that CMV-E2A-WPRE enhanced the titer by more than 3-fold (approximately 3.3-fold).

[0130] FIG. 4 is a graph showing crude harvest titer (vg / mL) for different ratios (Rep / Helper: Cap / GOI molar ratio) of several plasmids tested for 2P production relative to the 2P control. The plasmids tested include CMV-E2A-WPRE, Kozak-Rep, CMV-E2A and Kozak-Cap. In this experiment, the top performing plasmids were tested and compared. The plasmids were tested in 250 mL shake flasks. It was observed that Kozak-Rep at the 1 :3 molar ratio was the best performing candidate.

[0131] FIG. 5 is a graph showing ddPCR crude titer (vg / mL) for different ratios (Rep / Helper: Cap / GOI molar ratio) of several Rep / Helper plasmids tested for 2P production in combination with Kozak-Cap relative to the 2P control (red). The Rep / Helper plasmids tested include CMV-E2A-WPRE, Kozak-Rep and CMV-E2A. In this experiment, the Rep / Helper plasmids were tested in combination with Kozak-Cap to determine if an increase in crude harvest titer could be achieved by combining the top performing Rep / Helper plasmids with Kozak-Cap (a GOI / Cap plasmid). It was observed that the 1 :3 molar ratio was the best performing ratio for all the candidates tested. At the 1 :3 molar ratio, Kozak-Rep, CMV-E2A- WPRE and CMV-E2A increased the titer by 2.5-fold, 2.3-fold and 2.6-fold, respectively.

[0132] FIG. 6 includes construct designs of the top performing Rep / Helper plasmids: CMV-E2A-WPRE, Kozak-Rep and CMV-E2A. It shows the core elements of the top performing Rep / Helper plasmids and orientation of the various core elements of the top performing Rep / Helper plasmids. The 2P production system includes 2 plasmids: the Rep / Helper plasmid and the Cap / GOI plasmid. The structure of the top performing Rep / Helper plasmids are as follows: E2A-E4-VA-Kozak-Rep, CMV-E2A-WPRE-E4-VA-Rep and CMV-E2A-E4-VA-Rep. It also shows the core elements and orientation of the Kozak-Cap plasmid (a GOI / Cap plasmid) and a Cap / GOI plasmid. The structure of the Kozak-Cap plasmid is as follows: ITR-GOI-ITR-Kozak-Cap. The structure of the Cap / GOI plasmid is as follows: Cap-ITR-GOI-ITR.

[0133] Evaluation of backbone sequences and their effects on AAV productivity FIG. 7 includes construct designs of the 2 backbones: backbone 1 and backbone 2. It shows the core elements of the 2 backbones and orientation of the various core elements of the 2 backbones. Backbone 1 has the following features: antibiotic - kanamycin resistance gene, 130 / 130 bp ITR, 10906 bp, and origin of replication (ORI) and kanamycin is downstream of cap. Backbone 2 has the following features: antibiotic - ampicillin resistance gene, 145 / 123 (base pair) bp ITR, 10563 bp, ORI and ampicillin is upstream of cap, and SV40 promoter is upstream of ORI.

[0134] The following table (Table 3) shows the impact of the backbone sequence on AAV productivity.

[0135] Table 3.

[0136] FIG. 8 includes construct designs of backbone 2 and four designs (Designs 1-4) obtained through different modifications of backbone 2. This experiment was performed to generate a universal backbone for AAV production so that the backbone could be matched with the top performing Rep / Helper plasmids for producing higher AAV titers and better quality AAV vectors. A universal backbone would also be beneficial for large scale AAV production. In Design 1 , the SV40 promoter was removed from backbone 2. Originally in backbone 2, the SV40 promoter was placed upstream of ORI. Human embryonic kidney (HEK)293T cells express the SV40 large T antigen, which increases the replication of transfected DNA plasmids carrying the SV40 ORI. SV40 large T antigen is known to be capable of transforming human and rodent cells in vitro and in vivo, which raises safety concerns. In Design 2, the ampicillin resistance gene was replaced with a kanamycin resistance gene. Kanamycin and neomycin are preferred antibiotics currently because they are rarely used in clinics and are known to have a low incidence of ototoxicity and nephrotoxicity. In Design 3, the SV40 promoter was removed from backbone 2 and the ampicillin resistance gene was replaced with a kanamycin resistance gene. In Design 4, the modifications of Design 3 were replicated and a 22 bp addition was made in the 3’ ITR. The 3’ ITR was increased from 123 bp to 145 bp.

[0137] FIG. 9 is a graph showing ddPCR crude titer (vg / mL) for different modifications of backbone 2 (Designs 1-4 (D1-4) described in FIG. 8) with backbone 2 as a control. This experiment was performed to compare various modifications of backbone 2. The plasmids were tested in 250 mL shake flasks. No significant difference in titer was observed after modifying backbone 2. Removing SV40 (D1) did not decrease the yield and changing ampicillin to kanamycin increased the titer slightly. Based on the results, D4 (No SV40 + KAN + fixed 3’ITR) was selected as the best candidate for the universal backbone.

[0138] FIG. 10 is a graph showing ddPCR result for whole lysate (vg / mL) for backbone 1 + pm555 when compared with backbone 2 + pm555. pm555 is a control plasmid with intron. It was observed that backbone 2 increased the titer by more than 2-fold (approximately 2.23-fold). FIG. 11 is a graph showing ddPCR result for whole lysate (vg / mL) for backbone 2 in combination with CMV-E2A when compared with backbone 2 in combination with pm555 and backbone 1 in combination with pm555. pm555 is a control plasmid with intron. It was observed that combining CMV- E2A with backbone 2 resulted in an approximately 30% increase in yield. In comparison to pm555 + backbone 1 , CMV-E2A + backbone 2 increased the yield by 2.85-fold. In comparison to pm555 + backbone 1 , pm555 + backbone 2 increased the yield by 2.23-fold. This comparison was made between backbone 1 and original backbone 2 (non-modified backbone 2).

[0139] FIG. 12 is a graph showing crude harvest titer (vg / mL) for different ratios of several Rep / Helper plasmids tested for 2P production in combination with backbone 1 relative to the 2P control. The Rep / Helper plasmids tested include CMV-E2A-WPRE, Kozak-Rep and CMV-E2A. The plasmids were tested in 250 mL shake flasks. It was observed that Kozak-Rep at the 1 :1 .5 molar ratio was the best performing candidate when in combination with backbone 1 .

[0140] FIG. 13 is a graph showing crude harvest ddPCR titer (vg / mL) for different ratios (Rep / Helper: Cap / GOI molar ratio) of the CMV-E2A-WPRE Rep / Helper plasmid when tested in combination with backbone 1 or backbone 2. The black dot on the graph shows a control plasmid. It was observed that CMV-E2A-WPRE in combination with backbone 2 provided the best yield, which was approximately 6 times higher than the control plasmid. Thus, combining CMV-E2A-WPRE with backbone 2 resulted in a crude harvest titer of 1 .05e12 vg / mL. CMV-E2A-WPRE in combination with backbone 1 resulted in an approximately 3.3 times higher crude harvest titer than the control plasmid. This comparison was made between backbone 1 and original backbone 2 (non-modified backbone 2).

[0141] Scalability of optimized plasmids

[0142] FIG. 14 is a graph showing crude harvest titer (vg / mL) for the top performing Rep / Helper plasmids tested for scalability during 2P production in ambr® 250 with condition 1 relative to the 2P control. The Rep / Helper plasmids tested include CMV-E2A-WPRE, Kozak-Rep, and CMV-E2A. Condition 1 includes different % of non-empty capsids for the candidates tested (2P control, CMV-E2A-WPRE, Kozak-Rep, and CMV-E2A) as described in Table 4 below. It was observed that the top performing Rep / Helper plasmids are scalable in ambr® 250 with condition 1 .

[0143] Table 4.

[0144] FIG. 15 is a graph showing crude harvest titer (vg / mL) for the top performing Rep / Helper plasmids tested for scalability during 2P production in ambr® 250 with condition 2 relative to the 2P control. The Rep / Helper plasmids tested include CMV-E2A-WPRE, Kozak-Rep and CMV-E2A. Condition 2 includes different % of non-empty capsids for the candidates tested (2P control, CMV-E2A-WPRE, Kozak-Rep and CMV-E2A) as described in Table 5 below. It was observed that the top performing Rep / Helper plasmids are scalable in ambr® 250 with condition 2 and an increase in titer relative to condition 1 was also observed for all candidates tested (2P control, CMV-E2A-WPRE, Kozak-Rep and CMV-E2A). Relative to control, the titer increased by 1 .59-fold for CMV-E2A-WPRE and by 1 .54-fold for CMV-E2A. The titer for Kozak-Rep was similar to the control.

[0145] Table 5.

[0146] The descriptions of conditions 1 and 2 are provided in Table 6 below.

[0147] Table 6.

[0148] FIG. 16 is a graph showing crude harvest titer (vg / mL) for the top performing Rep / Helper plasmids tested for scalability during 2P production in 125 mL shake flasks relative to the 2P control. The Rep / Helper plasmids tested include CMV-E2A-WPRE, Kozak-Rep and CMV-E2A. It was observed that the top performing Rep / Helper plasmids are scalable in shake flasks. Relative to control, the titer increased by 2.91 -fold for CMV-E2A-WPRE, by 2.8-fold for CMV-E2A and by 2.58-fold for Kozak-Rep.

[0149] The transfection mix used for the transfection in the shake flasks was the same as the transfection mix used with ambr® 250 with condition 2. When compared with the ambr® 250 with condition 2, the 2P control had a lower titer, while the top candidates had a slightly higher titer.

[0150] Summary

[0151] The above findings demonstrate that CMV-E2A-WPRE, Kozak-Rep and CMV-E2A are the top performing Rep / Helper plasmids, and these Rep / Helper plasmids are scalable in ambr® 250, with condition 2 resulting in a higher titer compared to condition 1.

[0152] The above findings also demonstrate that backbone 2 outperformed backbone 1 and combining the top performing Rep / Helper plasmids with backbone 2 resulted in a crude harvest titer of 1 .05e12 vg / mL, which was approximately 6 times higher than the control plasmid.

[0153] Numbered embodiments 1 . A Rep / Helper plasmid comprising a cytomegalovirus (CMV) promoter, an E2A gene, an E4 gene, a VA gene and a rep gene, wherein the plasmid does not comprise a nucleic acid sequence encoding a cap gene.

[0154] 2. The Rep / Helper plasmid of embodiment 1 , wherein the plasmid comprises the sequence of SEQ ID NO: 7.

[0155] 3. A Rep / Helper plasmid comprising a cytomegalovirus (CMV) promoter, an E2A gene, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), an E4 gene, a VA gene and a rep gene, wherein the plasmid does not comprise a nucleic acid sequence encoding a cap gene.

[0156] 4. The Rep / Helper plasmid of embodiment 3, wherein the plasmid comprises the sequence of SEQ ID NO: 8.

[0157] 5. A Rep / Helper plasmid comprising an E2A gene, an E4 gene, a VA gene, a Kozak consensus sequence and a rep gene, wherein the plasmid does not comprise a nucleic acid sequence encoding a cap gene.

[0158] 6. The Rep / Helper plasmid of embodiment 5, wherein the plasmid comprises the sequence of SEQ ID NO: 9.

[0159] 7. The Rep / Helper plasmid of embodiment 1 , 3, or 5, wherein the E2A gene comprises the sequence of SEQ ID NO: 2.

[0160] 8. The Rep / Helper plasmid of embodiment 1 , 3, or 5, wherein the E4 gene comprises the sequence of SEQ ID NO: 5.

[0161] 9. The Rep / Helper plasmid of embodiment 1 , 3, or 5, wherein the VA gene comprises the sequence of SEQ ID NO: 6.

[0162] 10. The Rep / Helper plasmid of embodiment 1 or 3, wherein the CMV promoter comprises the sequence of SEQ ID NO: 1 .

[0163] 11 . The Rep / Helper plasmid of embodiment 3, wherein the WPRE comprises the sequence of SEQ ID NO: 3.

[0164] 12. The Rep / Helper plasmid of embodiment 5, wherein the Kozak consensus sequence comprises the sequence of SEQ ID NO: 4.

[0165] 13. A composition comprising: the Rep / Helper plasmid of any one of embodiments 1 -12; and a Payload / Cap plasmid, wherein the Payload / Cap plasmid does not comprise a polynucleotide sequence encoding a rep gene.

[0166] 14. The composition of embodiment 13, wherein the Payload / Cap plasmid comprises: a polynucleotide sequence comprising a sequence encoding a cap gene; and a polynucleotide sequence encoding a gene of interest.

[0167] 15. The composition of embodiment 14, wherein the Payload / Cap plasmid comprises a Kozak consensus sequence.

[0168] 16. The composition of embodiment 13, wherein the Payload / Cap plasmid comprises the sequence of SEQ ID NO: 10 or SEQ ID NO: 11 . 17. A universal backbone plasmid comprising the following elements arranged in order: a 5’ inverted terminal repeat (ITR) sequence, a gene of interest, a 3’ ITR sequence, a kanamycin resistance gene, a p40 promoter sequence and an LK03 AAV sequence.

[0169] 18. The universal backbone plasmid of embodiment 17, wherein the 3’ ITR sequence is 145 base pairs in length.

[0170] 19. The composition of any one of embodiments 13-15, wherein the plasmid ratio of the Rep / Helper plasmid to the Payload / Cap plasmid is greater than or equal to 1 .5:1 up to 10:1 .

[0171] 20. The composition of embodiment 19, wherein the plasmid ratio of the Rep / Helper plasmid to the Payload / Cap plasmid is 1.5:1 to 1 :1.5.

[0172] 21. The composition of any one of embodiments 13-15, 19 and 20, for use in producing an AAV vector.

[0173] 22. A method of manufacturing a packaged AAV vector, comprising delivering to a cell a composition of any one of embodiments 13-15, 19 and 20.

[0174] Additional embodiments

[0175] All references cited in this specification, including, database-accessioned information (e.g., in GENBANK, UNIPROT, PUBMED), are herein incorporated by reference as though each reference was specifically and individually indicated to be incorporated by reference. The citation of any reference is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such reference by virtue of prior invention.

[0176] It will be understood that each of the elements described above, or two or more together may also find a useful application in other types of methods differing from the type described above. Without further analysis, the foregoing will so fully reveal the gist of the present disclosure that others can, by applying current knowledge, readily adapt it for various applications without omitting features that, from the standpoint of prior art, fairly constitute essential characteristics of the generic or specific aspects of this disclosure set forth in the appended claims. The foregoing embodiments are presented by way of example only.

Claims

CLAIMSWhat is claimed is:1 . A Rep / Helper plasmid comprising a cytomegalovirus (CMV) promoter, an E2A gene, an E4 gene, a VA gene and a rep gene, wherein the plasmid does not comprise a nucleic acid sequence encoding a cap gene.

2. The Rep / Helper plasmid of claim 1 , wherein the plasmid comprises the sequence of SEQ ID NO: 7.

3. A Rep / Helper plasmid comprising a cytomegalovirus (CMV) promoter, an E2A gene, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), an E4 gene, a VA gene and a rep gene, wherein the plasmid does not comprise a nucleic acid sequence encoding a cap gene.

4. The Rep / Helper plasmid of claim 3, wherein the plasmid comprises the sequence of SEQ ID NO: 8.

5. A Rep / Helper plasmid comprising an E2A gene, an E4 gene, a VA gene, a Kozak consensus sequence and a rep gene, wherein the plasmid does not comprise a nucleic acid sequence encoding a cap gene.

6. The Rep / Helper plasmid of claim 5, wherein the plasmid comprises the sequence of SEQ ID NO: 9.

7. The Rep / Helper plasmid of claim 1 , 3, or 5, wherein the E2A gene comprises the sequence of SEQ ID NO: 2.

8. The Rep / Helper plasmid of claim 1 , 3, or 5, wherein the E4 gene comprises the sequence of SEQ ID NO: 5.

9. The Rep / Helper plasmid of claim 1 , 3, or 5, wherein the VA gene comprises the sequence of SEQ ID NO: 6.

10. The Rep / Helper plasmid of claim 1 or 3, wherein the CMV promoter comprises the sequence of SEQ ID NO: 1.11 . The Rep / Helper plasmid of claim 3, wherein the WPRE comprises the sequence of SEQ ID NO: 3.

12. The Rep / Helper plasmid of claim 5, wherein the Kozak consensus sequence comprises the sequence of SEQ ID NO: 4.

13. A composition comprising: the Rep / Helper plasmid of claim 1 , 3, or 5; and a Payload / Cap plasmid,wherein the Payload / Cap plasmid does not comprise a polynucleotide sequence encoding a rep gene.

14. The composition of Claim 13, wherein the Payload / Cap plasmid comprises: a polynucleotide sequence comprising a sequence encoding a cap gene; and a polynucleotide sequence encoding a gene of interest.

15. The composition of Claim 14, wherein the Payload / Cap plasmid comprises a Kozak consensus sequence.

16. The composition of claim 13, wherein the Payload / Cap plasmid comprises the sequence of SEQ ID NO: 10 or SEQ ID NO: 11.

17. A universal backbone plasmid comprising the following elements arranged in order: a 5’ inverted terminal repeat (ITR) sequence, a gene of interest, a 3’ ITR sequence, a kanamycin resistance gene, a p40 promoter sequence and an LK03 AAV sequence.

18. The universal backbone plasmid of Claim 17, wherein the 3’ ITR sequence is 145 base pairs in length.

19. The composition of Claim 13, wherein the plasmid ratio of the Rep / Helper plasmid to the Payload / Cap plasmid is greater than or equal to 1 .5:1 up to 10:1 .

20. The composition of Claim 19, wherein the plasmid ratio of the Rep / Helper plasmid to the Payload / Cap plasmid is 1 .5:1 to 1 :1 .5.

21. The composition of claim 13, for use in producing an AAV vector.

22. A method of manufacturing a packaged AAV vector, comprising delivering to a cell a composition of Claim 13.

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