Compositions and methods for the regulation of recombinant adeno-associated virus production

Abstract

The presently disclosed subject matter relates to compositions and methods for improving recombinant adeno-associated virus (rAAV) production. In particular, the presently disclosed subject matter relates to strategies for obtaining high titers of rAAV using novel constructs.

Description

[0001] COMPOSITIONS AND METHODS FOR THE REGULATION OF RECOMBINANT ADENO-ASSOCIATED VIRUS PRODUCTION

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 575,814, filed April 7, 2024, U.S. Provisional Application No. 63 / 636,820, filed on April 21, 2024, and U.S. Provisional Application No. 63 / 709,387, filed October 18, 2024, the contents of each of which are incorporated by reference in their entirety, and to each of which priority is claimed.

[0004] SEQUENCE LISTING

[0005] The present specification makes reference to a Sequence Listing (submitted electronically as an xml file named "089504_0401_SL.xml” on April 7, 2025). The 089504_0401_SL cml file was generated on April 3, 2025, and is 1,043,889 bytes in size. The entire contents of the Sequence Listing are hereby incorporated by reference.

[0006] FIELD

[0007] The presently disclosed subject matter relates to compositions and methods useful in the production of recombinant adeno-associated virus (rAAV). In particular, the presently disclosed subj ect matter relates to compositions and methods for regulation of rAAV titer using novel constructs.

[0008] BACKGROUND

[0009] AAV vectors offers many advantages in the gene therapy context over other viral vectors. The non-integrating, non-pathogenic properties of AAV vectors, coupled with ability to transduce both dividing and non-dividing cells and the potential for long-term stable transgene expression make them the vehicle of choice for clinical applications. In addition, AAV vectors can be engineered to alter their tissue tropism and to evade host immunity, thereby enabling more efficient transduction of the intended target tissue and successful establishment of transgene expression. There are, however, many challenges to balancing high titers of manufacturing-grade AAV and the desirability of minimizing the size and complexity of the sequences encoding helper virus functions to achieve enhanced rAAV titers. In view of the foregoing, there is a need in the art for new rAAV production strategies that enable efficient production of rAAV. SUMMARY

[0010] The presently disclosed subject matter is directed to compositions and methods for producing recombinant adeno-associated virus (rAAV) vector particles in cell culture. For example, but not by way of limitation, such methods can comprise culturing a population of cells comprising: AAV coding sequences comprising an AAV REP coding sequence; an AAV CAP coding sequence; or coding sequences providing helper virus function; followed by harvesting rAAV vector particles. In certain embodiments the coding sequences providing helper virus function comprise: a DBP coding sequence; an ORF6 and / or ORF6 / 7 coding sequence; and a VA coding sequence, where each of the DBP, ORF6 and / or ORF6 / 7, and VA coding sequences are operably linked to a constitutive promoter.

[0011] In certain embodiments, each of the DBP, ORF6 and / or ORF6 / 7, and VA coding sequences are independently (separately) operably linked to a constitutive promoter.

[0012] In certain embodiments, each of the DBP and either ORF6 and / or ORF6 / 7 coding sequences are operably linked to a common constitutive promoter and the VA coding sequence is operably linked to a separate constitutive promoter.

[0013] In certain embodiments, the constitutive promoter is selected from the group consisting of a Cytomegalovirus (CMV) promoter, a Simian Virus 40 (SV40) promoter, a bidirectional CMV (BICMV) promoter, a pl9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

[0014] In certain embodiments, the coding sequences providing helper virus function comprise a DBP coding sequence, an ORF6 and / or ORF6 / 7 coding sequence, and a VA coding sequence where each of the DBP, ORF6 and / or ORF6 / 7, and VA coding sequences are operably linked to a constitutive promoter, and further comprise an L4-22K coding sequence or an L4-22K_alt coding sequence operably linked to a constitutive promoter.

[0015] In certain embodiments, the L4-22K coding sequence is fused with the DBP coding sequence, the ORF6 coding sequence and / or the ORF6 / 7 coding sequence.

[0016] In certain embodiments, the L4-22K fusion comprises a 2A peptide coding sequence between the L4-22K coding sequence and the DBP coding sequence, the ORF6 coding sequence and / or the ORF6 / 7 coding sequence.

[0017] In certain embodiments, the 2A peptide is selected from the group consisting of T2A, P2A, E2A and F2A. In certain embodiments, the DBP sequence comprises the sequence set forth in any one of SEQ ID NOs: 1-8.

[0018] In certain embodiments, the ORF6 sequence comprises the sequence set forth in any one of SEQ ID NOs: 23, or 30-36.

[0019] In certain embodiments, the ORF6 / 7 sequence comprises the sequence set forth in any one of SEQ ID NOs: 37-41.

[0020] In certain embodiments, the L4-22K sequence comprises the sequence set forth in any one of SEQ ID NOs: 57-64.

[0021] In certain embodiments, the L4-22K_alt sequence comprises the sequence set forth in SEQ ID NO: 65.

[0022] In certain embodiments, the L4-22K coding sequence or the L4-22K_alt coding sequence comprises at least one mutation that enhances the production of rAAV particles. In certain embodiments, the L4-22K coding sequence comprises a Y20F mutation. In certain embodiments, the L4-22K coding sequence comprises a T79A mutation. In certain embodiments, the L4-22K coding sequence comprises a T99A mutation. In certain embodiments, the L4-22K coding sequence comprises a H169Q mutation. In certain embodiments, the L4-22K coding sequence comprises a Y171F mutation. In certain embodiments, the L4-22K coding sequence comprises a Y174F mutation. In certain embodiments, the L4-22K coding sequence comprises a T189A mutation.

[0023] In certain embodiments, the VA coding sequence comprises a VA_a coding sequence, a VA_b coding sequence, a VA_c coding sequence, a VA_d coding sequence, a VA_e coding sequence, or a VA_f coding sequence.

[0024] In certain embodiments, the VA sequence comprises the sequence set forth in SEQ ID NO: 42.

[0025] In certain embodiments, the VA_a sequence comprises the sequence set forth in SEQ ID NO: 43.

[0026] In certain embodiments, the VA_b sequence comprises the sequence set forth in any one of SEQ ID NOs: 44-48.

[0027] In certain embodiments, the VA_c sequence comprises the sequence set forth in SEQ ID NO: 49.

[0028] In certain embodiments, the VA_d sequence comprises the sequence set forth in SEQ ID NO: 50.

[0029] In certain embodiments, the VA_e sequence comprises the sequence set forth in SEQ

[0030] ID NO: 51. In certain embodiments, the VA_f sequence comprises the sequence set forth in any one of SEQ ID NOs: 52-55.

[0031] In certain embodiments, the AAV CAP coding sequence is contiguous with the AAV REP coding sequence.

[0032] In certain embodiments, the AAV REP coding sequence and the AAV CAP coding sequence are separated by a stuffer sequence. In certain embodiments, the stuffer sequence is about 0.5 kb to about 5.5 kb. In certain embodiments, the stuffer sequence is about 0.5 kb to about 3 kb. In certain embodiments, the stuffer sequence does not reduce production of rAAV vector particles.

[0033] In certain embodiments, the AAV CAP coding sequence encodes RHM4-1.

[0034] In certain embodiments, the AAV CAP coding sequence encodes LK03.

[0035] In certain embodiments, the AAV REP coding sequence and the AAV CAP coding sequence are operably linked to at least one promoter sequence selected from the group consisting of a full-length P5 promoter, a truncated P5 promoter, a T7 promoter, mouse mammary tumor virus (MMTV) promoter, a Cytomegalovirus (CMV), a Simian Virus 40 (SV40) promoter, a phosphoglycerate kinase promoter (PGK), ATP-citrate lyase promoter (ACLY), and actin gamma (ACTG) promoter, a bidirectional CMV (BICMV) promoter, a pl 9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

[0036] In certain embodiments, the promoter is at least the full-length P5 promoter sequence.

[0037] In certain embodiments, the full-length P5 promoter sequence does not decrease production of rAAV vector particles compared to an AAV coding sequence that lacks the full- length P5 promoter sequence.

[0038] In certain embodiments, the full-length P5 promoter sequence enhances production of rAAV vector particles compared to an AAV coding sequence that lacks the full-length P5 promoter sequence.

[0039] In certain embodiments, absence of the full-length P5 promoter sequence prevents formation of replication competent AAV (rcAAV) impurities.

[0040] In certain embodiments, absence of the full-length P5 promoter sequence results in reduced formation of replication competent AAV (rcAAV) impurities.

[0041] In certain embodiments, the AAV coding sequence has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 85-91 or 128-146. In certain embodiments, the AAV coding sequence comprises a sequence set forth in any one of SEQ ID NOs: 85-91, or 128-146. In certain embodiments, the coding sequences providing helper virus function has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 71-82.

[0042] In certain embodiments, reducing residual plasmid DNA (RPD) into the AAV capsid improves biosafety of the rAAV vector particles produced.

[0043] In certain embodiments, the presently disclosed subject matter is directed to a nucleic acid comprising a DBP coding sequence, an 0RF6 coding sequence and / or an ORF6 / 7 coding sequence, and a VA coding sequence, where each of the DBP coding sequence, the 0RF6 coding sequence and / or the ORF6 / 7 coding sequence, and the VA coding sequence is operably linked to a constitutive promoter.

[0044] In certain embodiments, each of the DBP, either 0RF6 and / or ORF6 / 7, and VA coding sequences are independently (separately) operably linked to a constitutive promoter.

[0045] In certain embodiments, each of the DBP and either 0RF6 and / or ORF6 / 7 coding sequences are operably linked to a common constitutive promoter and the VA coding sequence is operably linked to a separate constitutive promoter.

[0046] In certain embodiments, the constitutive promoter is selected from the group consisting of a Cytomegalovirus (CMV) promoter, a Simian Virus 40 (SV40) promoter, a bidirectional CMV (BICMV) promoter, a pl9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

[0047] In certain embodiments, the coding sequences providing helper virus function comprise a DBP coding sequence, an ORF6 and / or ORF6 / 7 coding sequence, and a VA coding sequence where each of the DBP, ORF6, ORF6 / 7 and VA coding sequences are operably linked to a constitutive promoter, and further comprise an L4-22K coding sequence or an L4-22K_alt coding sequence operably linked to a constitutive promoter.

[0048] In certain embodiments, the L4-22K coding sequence is fused with the DBP coding sequence, the ORF6 coding sequence and / or the ORF6 / 7 coding sequence.

[0049] In certain embodiments, the L4-22K fusion comprises a 2A peptide coding sequence between the L4-22K coding sequence and the DBP coding sequence, the ORF6 coding sequence and / or the ORF6 / 7 coding sequence.

[0050] In certain embodiments, the 2A peptide is selected from the group consisting of T2A, P2A, E2A and F2A.

[0051] In certain embodiments, the DBP sequence comprises the sequence set forth in any one of SEQ ID NOs: 1-8. In certain embodiments, the ORF6 sequence comprises the sequence set forth in any one of SEQ ID NOs: 23, or 30-36.

[0052] In certain embodiments, the ORF6 / 7 sequence comprises the sequence set forth in any one of SEQ ID NOs: 37-41.

[0053] In certain embodiments, the L4-22K sequence comprises the sequence set forth in any one of SEQ ID NOs: 57-64.

[0054] In certain embodiments, the L4-22K_alt sequence comprises the sequence set forth in SEQ ID NO: 65.

[0055] In certain embodiments, the L4-22K coding sequence or the L4-22K_alt coding sequence comprises at least one mutation that enhances the production of rAAV particles. In certain embodiments, the L4-22K coding sequ mce comprises a Y20F mutation. In certain embodiments, the L4-22K coding sequence comprises a T79A mutation. In certain embodiments, the L4-22K coding sequence comprises a T99A mutation. In certain embodiments, the L4-22K coding sequence comprises a H169Q mutation. In certain embodiments, the L4-22K coding sequence comprises a Y171F mutation. In certain embodiments, the L4-22K coding sequence comprises a Y174F mutation. In certain embodiments, the L4-22K coding sequence com prises a T189A mutation.

[0056] In certain embodiments, the VA coding s equence comprises a VA_a coding sequence, a VA_b coding sequence, a VA_c coding sequence, a VA_d coding sequence, a VA_e coding sequence, or a VA_f coding sequence.

[0057] In certain embodiments, the VA sequence comprises the sequence set forth in SEQ ID NO: 42.

[0058] In certain embodiments, the VA_a sequence comprises the sequence set forth in SEQ ID NO: 43.

[0059] In certain embodiments, the VA_b sequence comprises the sequence set forth in any one of SEQ ID NOs: 44-48.

[0060] In certain embodiments, the VA_c sequence comprises the sequence set forth in SEQ ID NO: 49.

[0061] In certain embodiments, the VA_d sequence comprises the sequence set forth in SEQ ID NO: 50.

[0062] In certain embodiments, the VA_e sequence comprises the sequence set forth in SEQ ID NO: 51.

[0063] In certain embodiments, the VA_f sequence comprises the sequence set forth in any one of SEQ ID NOs: 52-55. In certain embodiments, the presently disclosed subject matter is directed to a transfected cell comprising an AAV coding sequence comprising an AAV REP coding sequence, an AAV CAP coding sequence and coding sequences providing helper virus function. In certain embodiments, the coding sequences providing helper virus function comprise a DBP coding sequence, an ORF6 and / or ORF6 / 7 coding sequence, and a VA coding sequence, where each of the DBP, 0RF6, ORF6 / 7 and VA coding sequences are operably linked to a constitutive promoter, and a gene of interest (GOI) coding sequence.

[0064] In certain embodiments, each of the DBP, 0RF6 and / or ORF6 / 7, and VA coding sequences are independently (separately) operably linked to a constitutive promoter.

[0065] In certain embodiments, each of the DBP and either ORF6 and / or ORF6 / 7 coding sequences are operably linked to a common constitutive promoter and the VA coding sequence is operably linked to a separate constitutive promoter.

[0066] In certain embodiments, the constitutive promoter is selected from the group consisting of a Cytomegalovirus (CMV) promoter, a Simian Virus 40 (SV40) promoter, a bidirectional CMV (BICMV) promoter, a pl9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

[0067] In certain embodiments, the coding sequences providing helper virus function comprise a DBP coding sequence, an ORF6 and / or ORF6 / 7 coding sequence, and a VA coding sequence where each of the DBP, ORF6, ORF6 / 7 and VA coding sequences are operably linked to a constitutive promoter, and further comprise an L4-22K coding sequence or an L4-22K_alt coding sequence operably linked to a constitutive promoter.

[0068] In certain embodiments, the L4-22K coding sequence is fused with the DBP coding sequence, the ORF6 coding sequence, and / or the ORF6 / 7 coding sequence.

[0069] In certain embodiments, the L4-22K fusion comprises a 2A peptide coding sequence between the L4-22K coding sequence and the DBP coding sequence, the ORF6 coding sequence and / or the ORF6 / 7 coding sequence.

[0070] In certain embodiments, the 2A peptide is selected from the group consisting of T2A, P2A, E2A and F2A.

[0071] In certain embodiments, the DBP sequence comprises the sequence set forth in any one of SEQ ID NOs: 1-8.

[0072] In certain embodiments, the ORF6 sequence comprises the sequence set forth in any one of SEQ ID NOs: 23, or 30-36. In certain embodiments, the ORF6 / 7 sequence comprises the sequence set forth in any one of SEQ ID NOs: 37-41.

[0073] In certain embodiments, the L4-22K sequence comprises the sequence set forth in any one of SEQ ID NOs: 57-64.

[0074] In certain embodiments, the L4-22K_alt sequence comprises the sequence set forth in SEQ ID NO: 65.

[0075] In certain embodiments, the L4-22K coding sequence or the L4-22K_alt coding sequence comprises at least one mutation that enhances the production of rAAV particles. In certain embodiments, the L4-22K coding sequence comprises a Y20F mutation. In certain embodiments, the L4-22K coding sequence comprises a T79A mutation. In certain embodiments, the L4-22K coding sequence comprises a T99A mutation. In certain embodiments, the L4-22K coding sequence comprises a H169Q mutation. In certain embodiments, the L4-22K coding sequence comprises a Y171F mutation. In certain embodiments, the L4-22K coding sequence comprises a Y174F mutation. In certain embodiments, the L4-22K coding sequence comprises a T189A mutation.

[0076] In certain embodiments, the VA coding sequence comprises a VA_a coding sequence, a VA_b coding sequence, a VA_c coding sequence, a VA_d coding sequence, a VA_e coding sequence, or a VA_f coding sequence.

[0077] In certain embodiments, the VA sequence comprises the sequence set forth in SEQ ID NO: 42.

[0078] In certain embodiments, the VA_a sequence comprises the sequence set forth in SEQ ID NO: 43.

[0079] In certain embodiments, the VA_b sequence comprises the sequence set forth in any one of SEQ ID NOs: 44-48.

[0080] In certain embodiments, the VA_c sequence comprises the sequence set forth in SEQ ID NO: 49.

[0081] In certain embodiments, the VA_d sequence comprises the sequence set forth in SEQ ID NO: 50.

[0082] In certain embodiments, the VA_e sequence comprises the sequence set forth in SEQ ID NO: 51.

[0083] In certain embodiments, the VA_f sequence comprises the sequence set forth in any one of SEQ ID NOs: 52-55.

[0084] In certain embodiments, the AAV CAP coding sequence is contiguous with the AAV REP coding sequence. In certain embodiments, the AAV REP coding sequence and the AAV CAP coding sequence are separated by a stuffer sequence. In certain embodiments, the stuffer sequence is about 0.5 kb to about 5.5 kb. In certain embodiments, the stuffer sequence is about 0.5 kb to about 3 kb. In certain embodiments, the stuffer sequence does not reduce production of rAAV vector particles.

[0085] In certain embodiments, the AAV CAP coding sequence encodes RHM4-1.

[0086] In certain embodiments, the AAV CAP coding sequence encodes LK03.

[0087] In certain embodiments, the AAV REP coding sequence and the AAV CAP coding sequence are operably linked to at least one promoter sequence selected from the group consisting of a full-length P5 promoter, a truncated P5 promoter, a T7 promoter, mouse mammary tumor virus (MMTV) promoter, a Cytomegalovirus (CMV), a Simian Virus 40 (SV40) promoter, a phosphoglycerate kinase promoter (PGK), ATP-citrate lyase promoter (ACLY), and actin gamma (ACTG) promoter, a bidirectional CMV (BICMV) promoter, a pl9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

[0088] In certain embodiments, the promoter is at least the full-length P5 promoter sequence.

[0089] In certain embodiments, the full-length P5 promoter sequence does not decrease production of rAAV vector particles compared to an AAV coding sequence that lacks the full-length P5 promoter sequence.

[0090] In certain embodiments, the full-length P5 promoter sequence enhances production of rAAV vector particles compared to an AAV coding sequence that lacks the full-length P5 promoter sequence.

[0091] In certain embodiments, absence of the full-length P5 promoter sequence prevents formation of replication competent AAV (rcAAV) impurities.

[0092] In certain embodiments, absence of the full-length P5 promoter sequence results in reduced formation of replication competent AAV (rcAAV) impurities.

[0093] In certain embodiments, the AAV coding sequence has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 85-91 or 128-146. In certain embodiments, the coding sequence providing helper function has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 71-82. In certain embodiments, the AAV coding sequence comprises a sequence set forth in any one of SEQ ID NOs: 85-91, or 128-146. In certain embodiments, the coding sequence providing helper function comprises a sequence set forth in any one of SEQ ID NOs: 71-82. In certain embodiments, the GOI is Factor VIII, acid a-glucosidase (GAA), a- synuclein, or Apolipoprotein E (ApoE).

[0094] In certain embodiments, reducing residual plasmid DNA (RPD) into the AAV capsid improves biosafety of the rAAV vector particles produced.

[0095] In certain embodiments, the AAV coding sequence comprises a sequence set forth in any one of SEQ ID NOs: 85-91, 128-148, 150, or 156-158.

[0096] In certain embodiments the methods and compositions useful to produce rAAV particles comprise systems using less than three plasmids for producing rAAV vector particles. Such systems can, in certain embodiments, comprise a first nucleic acid construct and a second nucleic acid construct, where the first nucleic acid construct comprises one or more of an rAAV coding sequence, an AAV REP coding sequence comprising an AAV CAP coding sequence, and coding sequences providing helper virus function. For example, but not by way of limitation, the first nucleic acid construct can comprise one or more coding sequence providing helper virus function selected from a DBP coding sequence, an ORF6 and / or ORF6 / 7 coding sequence, and a VA coding sequence. In certain embodiments the second nucleic acid construct comprises a gene of interest (GOI) coding sequence and one or more of an rAAV coding sequence, an AAV REP coding sequence comprising an AAV CAP coding sequence, and coding sequences providing helper virus function. For example, but not by way of limitation, the second nucleic acid construct can comprise one or more coding sequence providing helper virus function selected from a DBP coding sequence, an ORF6 and / or ORF6 / 7 coding sequence, and a VA coding sequence.

[0097] In certain embodiments, each of the first a DBP coding sequence, the ORF6 and / or ORF 6 / 7 coding sequence, and the VA coding sequence (in the first nucleic acid construct and / or the second nucleic acid construct) can be operably linked to a constitutive promoter. In certain embodiments, the AAV REP coding sequence is operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity.

[0098] In certain embodiments, the AAV REP coding sequence is operably linked to at least one promoter selected from the group consisting of a P5 promoter, a truncated P5 promoter, a T7 promoter, mouse mammary tumor virus (MMTV) promoter, a Cytomegalovirus (CMV), a Simian Virus 40 (SV40) promoter, a phosphoglycerate kinase promoter (PGK), ATP-citrate lyase promoter (ACLY), and actin gamma (ACTG) promoter, a bidirectional CMV (BICMV) promoter, a pl9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter. In certain embodiments, the AAV REP coding sequence is operably linked to a T7 promoter and a truncated P5 promoter.

[0099] In certain embodiments, the constitutive promoter is selected from the group consisting of a P5 promoter, a truncated P5 promoter, a T7 promoter, a mouse mammary tumor virus (MMTV) promoter, a Cytomegalovirus (CMV), a Simian Virus 40 (SV40) promoter, a phosphoglycerate kinase promoter (PGK), ATP-citrate lyase promoter (ACLY), and actin gamma (ACTG) promoter, a bidirectional CMV (BICMV) promoter, a pl9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

[0100] In certain embodiments, the nucleic acid sequence with nonspecific transcription activity comprises from about 156 nucleotides to about 729 nucleotides.

[0101] In certain embodiments, the nucleic acid sequence with nonspecific transcription activity promulgates Rep protein expression compared to Rep protein expression when the nucleic acid sequence with nonspecific transcription activity is absent.

[0102] In certain embodiments, the nucleic acid sequence with nonspecific transcription activity enhances rAAV titer from about 2-fold to about 3-fold compared to rAAV titer when the nucleic acid sequence with nonspecific transcription activity is absent.

[0103] In certain embodiments, the coding sequences providing helper virus function comprise a DBP coding sequence, an ORF6 and / or ORF6 / 7 coding sequence, and a VA coding sequence where each of the DBP, ORF6, ORF6 / 7 and VA coding sequences are operably linked to a constitutive promoter, and further comprise an L4-22K coding sequence or an L4-22K_alt coding sequence operably linked to a constitutive promoter.

[0104] In certain embodiments, the L4-22K coding sequence is fused with the DBP coding sequence, the ORF6 coding sequence and / or the ORF6 / 7 coding sequence.

[0105] In certain embodiments, the L4-22K fusion comprises a 2A peptide coding sequence between the L4-22K coding sequence and the DBP coding sequence, the ORF6 coding sequence and / or the ORF6 / 7 coding sequence.

[0106] In certain embodiments, the 2A peptide is selected from the group consisting of T2A, P2A, E2A and F2A.

[0107] In certain embodiments, the DBP sequence comprises the sequence set forth in any one of SEQ ID NOs: 1-8.

[0108] In certain embodiments, the ORF6 sequence comprises the sequence set forth in any one of SEQ ID NOs: 23, or 30-36. In certain embodiments, the ORF6 / 7 sequence comprises the sequence set forth in any one of SEQ ID NOs: 37-41.

[0109] In certain embodiments, the L4-22K sequence comprises the sequence set forth in any one of SEQ ID NOs: 57-64.

[0110] In certain embodiments, the L4-22K_alt sequence comprises the sequence set forth in SEQ ID NO: 65.

[0111] In certain embodiments, the L4-22K coding sequence or the L4-22K_alt coding sequence comprises at least one mutation that enhances the production of rAAV particles. In certain embodiments, the L4-22K coding sequence comprises a Y20F mutation. In certain embodiments, the L4-22K coding sequence comprises a T79A mutation. In certain embodiments, the L4-22K coding sequence comprises a T99A mutation. In certain embodiments, the L4-22K coding sequence comprises a H169Q mutation. In certain embodiments, the L4-22K coding sequence comprises a Y171F mutation. In certain embodiments, the L4-22K coding sequence comprises a Y174F mutation. In certain embodiments, the L4-22K coding sequence comprises a T189A mutation.

[0112] In certain embodiments, the VA coding sequence comprises a VA_a coding sequence, a VA_b coding sequence, a VA_c coding sequence, a VA_d coding sequence, a VA_e coding sequence, or a VA_f coding sequence.

[0113] In certain embodiments, the VA sequence comprises the sequence set forth in SEQ ID NO: 42.

[0114] In certain embodiments, the VA_a sequence comprises the sequence set forth in SEQ ID NO: 43.

[0115] In certain embodiments, the VA_b sequence comprises the sequence set forth in any one of SEQ ID NOs: 44-48.

[0116] In certain embodiments, the VA_c sequence comprises the sequence set forth in SEQ ID NO: 49.

[0117] In certain embodiments, the VA_d sequence comprises the sequence set forth in SEQ ID NO: 50.

[0118] In certain embodiments, the VA_e sequence comprises the sequence set forth in SEQ ID NO: 51.

[0119] In certain embodiments, the VA_f sequence comprises the sequence set forth in any one of SEQ ID NOs: 52-55.

[0120] In certain embodiments, the AAV CAP coding sequence is contiguous with the AAV REP coding sequence. In certain embodiments, the AAV REP coding sequence and the AAV CAP coding sequence are separated by a stuffer sequence. In certain embodiments, the stuffer sequence is about 0.5 kb to about 5.5 kb. In certain embodiments, the stuffer sequence is about 0.5 kb to about 3 kb. In certain embodiments, the stuffer sequence does not reduce production of rAAV vector particles.

[0121] In certain embodiments, the AAV CAP coding sequence encodes RHM4-1.

[0122] In certain embodiments, the AAV CAP coding sequence encodes LK03.

[0123] In certain embodiments, the AAV REP coding sequence and the AAV CAP coding sequence are operably linked to at least one promoter sequence selected from the group consisting of a full-length P5 promoter, a truncated P5 promoter, a T7 promoter, mouse mammary tumor virus (MMTV) promoter, a Cytomegalovirus (CMV), a Simian Virus 40 (SV40) promoter, a phosphoglycerate kinase promoter (PGK), ATP-citrate lyase promoter (ACLY), and actin gamma (ACTG) promoter, a bidirectional CMV (BICMV) promoter, a pl9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

[0124] In certain embodiments, the promoter is at least the full-length P5 promoter sequence.

[0125] In certain embodiments, the full-length P5 promoter sequence does not decrease production of rAAV vector particles compared to a first nucleic acid construct that lacks the full-length P5 promoter sequence.

[0126] In certain embodiments, the full-length P5 promoter sequence enhances production of rAAV vector particles compared to a first nucleic acid construct that lacks the full-length P5 promoter sequence.

[0127] In certain embodiments, absence of the full-length P5 promoter sequence prevents formation of replication competent AAV (rcAAV) impurities.

[0128] In certain embodiments, absence of the full-length P5 promoter sequence results in reduced formation of replication competent AAV (rcAAV) impurities.

[0129] In certain embodiments, the first nucleic acid construct comprises an rAAV coding sequence that has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 85-91, or 128-146. In certain embodiments, the first nucleic acid construct comprises a coding sequence providing helper function has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 71-82. In certain embodiments, the first nucleic acid construct comprises a sequence set forth in any one of SEQ ID NOs: 152-154. In certain embodiments, the second nucleic acid construct comprises an rAAV coding sequence that has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 85-91 or 128-146. In certain embodiments, the second nucleic acid construct comprises a coding sequence providing helper function has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 71-82. In certain embodiments, the second nucleic acid construct comprises a sequence set forth in any one of SEQ ID NOs: 152-154.

[0130] In certain embodiments, the GOI is Factor VIII, acid a-glucosidase (GAA), a- synuclein, or Apolipoprotein E (ApoE).

[0131] In certain embodiments, reducing residual plasmid DNA (RPD) into the AAV capsid improves biosafety of the rAAV vector particles produced.

[0132] The presently disclosed subject matter is also directed, in certain embodiments, to systems for producing rAAV vector particles comprising a first nucleic acid construct and a second nucleic acid construct where the first nucleic acid construct comprises one or more of an AAV coding sequence comprising an AAV REP coding sequence, an AAV CAP coding sequence, and coding sequences providing helper virus function comprising a DBP coding sequence, an 0RF6 and / or ORF6 / 7 coding sequence, or a VA coding sequence and the second nucleic acid construct comprises a gene of interest (GOI) coding sequence. In certain embodiments, each of the first a DBP coding sequence, the 0RF6 and / or ORF6 / 7 coding sequence, or the VA coding sequence in the first nucleic acid construct are operably linked to a constitutive promoter. In certain embodiments, the AAV REP coding sequence is operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity disposed upstream from the at least one promoter. In certain embodiments, the AAV REP coding sequence is operably linked to at least one promoter selected from the group consisting of a P5 promoter, a truncated P5 promoter, a T7 promoter, mouse mammary tumor virus (MMTV) promoter, a Cytomegalovirus (CMV), a Simian Virus 40 (SV40) promoter, a phosphoglycerate kinase promoter (PGK), ATP-citrate lyase promoter (ACLY), and actin gamma (ACTG) promoter, a bidirectional CMV (BICMV) promoter, a pl9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

[0133] The presently disclosed subject matter is also directed, in certain embodiments, to systems for producing rAAV vector particles comprising a first nucleic acid construct and a second nucleic acid construct where the first nucleic acid construct comprises one or more of an rAAV coding sequence, an AAV REP coding sequence comprising an AAV CAP coding sequence, and coding sequences providing helper virus function comprising a DBP coding sequence, an ORF6 and / or ORF6 / 7 coding sequence, a VA coding sequence, or an L4-22K coding sequence or an L4-22K_alt coding sequence and the second nucleic acid construct comprises a gene of interest (GO I) coding sequence. In certain embodiments, each of the first a DBP coding sequence, the ORF6 and / or ORF 6 / 7 coding sequence, and the VA coding sequence in the first nucleic acid construct are operably linked to a constitutive promoter. In certain embodiments, the AAV REP coding sequence is operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity disposed upstream from the at least one promoter. In certain embodiments, the AAV REP coding sequence is operably linked to at least one promoter selected from the group consisting of a full-length P5 promoter, a truncated P5 promoter, a T7 promoter, mouse mammary tumor virus (MMTV) promoter, a Cytomegalovirus (CMV), a Simian Virus 40 (SV40) promoter, a phosphoglycerate kinase promoter (PGK), ATP-citrate lyase promoter (ACLY), and actin gamma (ACTG) promoter, a bidirectional CMV (BICMV) promoter, a pl9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter. In certain embodiments, the coding sequences providing helper virus function further comprise an L4-22K coding sequence or an L4-22K_alt coding sequence operably linked to a constitutive promoter.

[0134] In certain embodiments, the first nucleic acid construct and second nucleic acid construct comprise the sequences set forth any one of SEQ ID NOs: 85-113.

[0135] In certain embodiments, the AAV REP is one or more of Rep52, Rep40, Rep78, and Rep68.

[0136] In certain embodiments, the Rep78 sequence comprises the sequence set forth in any one of SEQ ID NOs: 115-125.

[0137] In certain embodiments, the AAV REP coding sequence is operably linked to a T7 promoter and a truncated P5 promoter.

[0138] In certain embodiments, the constitutive promoter is selected from the group consisting of a full-length P5 promoter, a truncated P5 promoter, a T7 promoter, a mouse mammary tumor virus (MMTV) promoter, a Cytomegalovirus (CMV), a Simian Virus 40 (SV40) promoter, a phosphoglycerate kinase promoter (PGK), ATP-citrate lyase promoter (ACLY), and actin gamma (ACTG) promoter, a bidirectional CMV (BICMV) promoter, a p!9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

[0139] In certain embodiments, the nucleic acid sequence with nonspecific transcription activity comprises from about 156 nucleotides to about 729 nucleotides.

[0140] In certain embodiments, the nucleic acid sequence with nonspecific transcription activity promulgates Rep protein expression compared to Rep protein expression when the nucleic acid sequence with nonspecific transcription activity is absent.

[0141] In certain embodiments, the nucleic acid sequence with nonspecific transcription activity enhances rAAV titer from about 2-fold to about 3-fold compared to rAAV titer when the nucleic acid sequence with nonspecific transcription activity is absent.

[0142] In certain embodiments, the coding sequences providing helper virus function comprise a DBP coding sequence, an 0RF6 and / or ORF6 / 7 coding sequence, and a VA coding sequence where each of the DBP, ORF6, ORF6 / 7 and VA coding sequences are operably linked to a constitutive promoter, and further comprise an L4-22K coding sequence or an L4-22K_alt coding sequence operably linked to a constitutive promoter.

[0143] In certain embodiments, the L4-22K coding sequence is fused with the DBP coding sequence, the 0RF6 coding sequence and / or the ORF6 / 7 coding sequence.

[0144] In certain embodiments, the L4-22K fusion comprises a 2A peptide coding sequence between the L4-22K coding sequence and the DBP coding sequence, the ORF6 coding sequence and / or the ORF6 / 7 coding sequence.

[0145] In certain embodiments, the 2A peptide is selected from the group consisting of T2A, P2A, E2A and F2A.

[0146] In certain embodiments, the DBP sequence comprises the sequence set forth in any one of SEQ ID NOs: 1-8.

[0147] In certain embodiments, the ORF6 sequence comprises the sequence set forth in any one of SEQ ID NOs: 23, or 30-36.

[0148] In certain embodiments, the ORF6 / 7 sequence comprises the sequence set forth in any one of SEQ ID NOs: 37-41.

[0149] In certain embodiments, the L4-22K sequence comprises the sequence set forth in any one of SEQ ID NOs: 57-64.

[0150] In certain embodiments, the L4-22K_alt sequence comprises the sequence set forth in SEQ ID NO: 65.

[0151] In certain embodiments, the L4-22K coding sequence or the L4-22K_alt coding sequence comprises at least one mutation that enhances the production of rAAV particles. In certain embodiments, the L4-22K coding sequence comprises a Y20F mutation. In certain embodiments, the L4-22K coding sequence comprises a T79A mutation. In certain embodiments, the L4-22K coding sequence comprises a T99A mutation. In certain embodiments, the L4-22K coding sequence comprises a H169Q mutation. In certain embodiments, the L4-22K coding sequence comprises a Y171F mutation. In certain embodiments, the L4-22K coding sequence comprises a Y174F mutation. In certain embodiments, the L4-22K coding sequence comprises a T189A mutation.

[0152] In certain embodiments, the VA coding sequence comprises a VA_a coding sequence, a VA_b coding sequence, a VA_c coding sequence, a VA_d coding sequence, a VA_e coding sequence, or a VA_f coding sequence.

[0153] In certain embodiments, the VA sequence comprises the sequence set forth in SEQ ID NO: 42.

[0154] In certain embodiments, the VA_a sequence comprises the sequence set forth in SEQ ID NO: 43.

[0155] In certain embodiments, the VA_b sequence comprises the sequence set forth in any one of SEQ ID NOs: 44-48.

[0156] In certain embodiments, the VA_c sequence comprises the sequence set forth in SEQ ID NO: 49.

[0157] In certain embodiments, the VA_d sequence comprises the sequence set forth in SEQ ID NO: 50.

[0158] In certain embodiments, the VA_e sequence comprises the sequence set forth in SEQ ID NO: 51.

[0159] In certain embodiments, the VA_f sequence comprises the sequence set forth in any one of SEQ ID NOs: 52-55.

[0160] In certain embodiments, the first nucleic acid construct and second nucleic acid construct comprise the sequences set forth any one of SEQ ID NOs: 85-113

[0161] In certain embodiments, the AAV REP is one or more of Rep52, Rep40, Rep78, and Rep68.

[0162] In certain embodiments, the Rep78 sequence comprises the sequence set forth in any one of SEQ ID NOs: 115-125.

[0163] In certain embodiments, the AAV CAP coding sequence is contiguous with the AAV REP coding sequence.

[0164] In certain embodiments, the AAV REP coding sequence and the AAV CAP coding sequence are separated by a stuffer sequence. In certain embodiments, the stuffer sequence is about 0.5 kb to about 5.5 kb. In certain embodiments, the stuffer sequence is about 0.5 kb to about 3 kb. In certain embodiments, the stuffer sequence does not reduce production of rAAV vector particles.

[0165] In certain embodiments, the AAV CAP coding sequence encodes RHM4-1.

[0166] In certain embodiments, the AAV CAP coding sequence encodes LK03.

[0167] In certain embodiments, the AAV REP coding sequence and the AAV CAP coding sequence are operably linked to at least one promoter sequence selected from the group consisting of a full-length P5 promoter, a truncated P5 promoter, a T7 promoter, mouse mammary tumor virus (MMTV) promoter, a Cytomegalovirus (CMV), a Simian Virus 40 (SV40) promoter, a phosphoglycerate kinase promoter (PGK), ATP-citrate lyase promoter (ACLY), and actin gamma (ACTG) promoter, a bidirectional CMV (BICMV) promoter, a pl9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

[0168] In certain embodiments, the promoter is at least the full-length P5 promoter sequence.

[0169] In certain embodiments, the full-length P5 promoter sequence does not decrease production of rAAV vector particles compared to a first nucleic acid construct that lacks the full-length P5 promoter sequence.

[0170] In certain embodiments, the full-length P5 promoter sequence enhances production of rAAV vector particles compared to a first nucleic acid construct that lacks the full-length P5 promoter sequence.

[0171] In certain embodiments, absence of the full-length P5 promoter sequence prevents formation of replication competent AAV (rcAAV) impurities.

[0172] In certain embodiments, absence of the full-length P5 promoter sequence results in reduced formation of replication competent AAV (rcAAV) impurities.

[0173] In certain embodiments, the first nucleic acid construct comprises an rAAV coding sequence that has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 85-91, or 128-146. In certain embodiments, the first nucleic acid construct comprises a coding sequence providing helper function has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 71-82. In certain embodiments, the first nucleic acid construct comprises a sequence set forth in any one of SEQ ID NOs: 152-154.

[0174] In certain embodiments, the second nucleic acid construct comprises an rAAV coding sequence that has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 85-91, or 128-146. In certain embodiments, the second nucleic acid construct comprises a coding sequence providing helper function has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 71-82. In certain embodiments, the second nucleic acid construct comprises a sequence set forth in any one of SEQ ID NOs: 152-154.

[0175] In certain embodiments, the GOI is Factor VIII, acid a-glucosidase (GAA), a- synuclein, or Apolipoprotein E (ApoE).

[0176] In certain embodiments, reducing residual plasmid DNA (RPD) into the AAV capsid improves biosafety of the rAAV vector particles produced.

[0177] The presently disclosed subject matter is further directed, in certain embodiments, to methods of producing rAAV vector particles. Such methods comprise, in certain embodiments, the culturing of a population of cells, wherein the cells comprise a first nucleic acid construct and a second nucleic acid construct and harvesting rAAV vector particles. In certain embodiments, the first nucleic acid construct comprises one or more of an AAV coding sequence comprising an AAV REP coding sequence, an AAV CAP coding sequence, and coding sequences providing helper virus function comprising a DBP coding sequence, an ORF6 and / or ORF6 / 7 coding sequence, or a VA coding sequence. In certain embodiments, the second nucleic acid construct comprises a gene of interest (GOI) coding sequence and one or more of an AAV coding sequence comprising an AAV REP coding sequence, an AAV CAP coding sequence, and coding sequences providing helper virus function comprising a DBP coding sequence, an ORF6 and / or ORF6 / 7 coding sequence, or a VA coding sequence. In certain embodiments, each of the first a DBP coding sequence, the ORF6 and / or ORF6 / 7 coding sequence, and the VA coding sequence in either or both of the first nucleic acid construct and the second nucleic acid construct are operably linked to a constitutive promoter. In certain embodiments, the AAV REP coding sequence is operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity disposed upstream from the at least one promoter.

[0178] The presently disclosed subject matter is further directed, in certain embodiments, to methods of producing rAAV vector particles. Such methods comprise, in certain embodiments, the culturing of a population of cells, wherein the cells comprise a first nucleic acid construct and a second nucleic acid construct and harvesting rAAV vector particles. In certain embodiments, the first nucleic acid construct comprises one or more of: an AAV coding sequence comprising an AAV REP coding sequence; an AAV CAP coding sequence; and one or more coding sequence providing helper virus function selected from a DBP coding sequence, an 0RF6 and / or ORF6 / 7 coding sequence, a VA coding sequence, and an L4-22K coding sequence or an L4-22K_alt coding sequence. In certain embodiments, the second nucleic acid construct comprises: a gene of interest (GOI) coding sequence; and one or more of an AAV coding sequence comprising an AAV REP coding sequence, an AAV CAP coding sequence; and one or more coding sequence providing helper virus function selected from a DBP coding sequence, an ORF6 and / or ORF6 / 7 coding sequence, a VA coding sequence, and an L4-22K coding sequence or an L4-22K_alt coding sequence. In certain embodiments, each of the DBP coding sequence, the ORF6 and / or ORF6 / 7 coding sequence, and the VA coding sequence (in either or both of the first nucleic acid construct and the second nucleic acid construct) are operably linked to a constitutive promoter. In certain embodiments, the AAV REP coding sequence is operably linked to: (1) at least one promoter; and (2) a nucleic acid sequence with nonspecific transcription activity disposed upstream from the at least one promoter.

[0179] In certain embodiments, the first nucleic acid construct and second nucleic acid construct comprise a sequence set forth in any one of SEQ ID NOs: 85-113.

[0180] In certain embodiments, the AAV REP coding sequence is operably linked to at least one promoter selected from the group consisting of a full-length P5 promoter, a truncated P5 promoter, a T7 promoter, mouse mammary tumor virus (MMTV) promoter, a Cytomegalovirus (CMV), a Simian Virus 40 (SV40) promoter, a phosphoglycerate kinase promoter (PGK), ATP-citrate lyase promoter (ACLY), and actin gamma (ACTG) promoter, a bidirectional CMV (BICMV) promoter, a pl9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

[0181] In certain embodiments, the AAV REP coding sequence is operably linked to a T7 promoter and a truncated P5 promoter.

[0182] In certain embodiments, the constitutive promoter is selected from the group consisting of a full-length P5 promoter, a truncated P5 promoter, a T7 promoter, a mouse mammary tumor virus (MMTV) promoter, a Cytomegalovirus (CMV), a Simian Virus 40 (SV40) promoter, a phosphoglycerate kinase promoter (PGK), ATP-citrate lyase promoter (ACLY), and actin gamma (ACTG) promoter, a bidirectional CMV (BICMV) promoter, a pl9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

[0183] In certain embodiments, the nucleic acid sequence with nonspecific transcription activity comprises from about 156 nucleotides to about 729 nucleotides. In certain embodiments, the nucleic acid sequence with nonspecific transcription activity promulgated Rep protein expression compared to Rep protein expression when the nucleic acid sequence is absent.

[0184] In certain embodiments, the nucleic acid sequence with nonspecific transcription activity enhanced rAAV titer from about 2-fold to about 3 -fold compared to rAAV titer when the nucleic acid sequence with nonspecific transcription activity is absent.

[0185] In certain embodiments, the coding sequences providing helper virus function comprise a DBP coding sequence, an 0RF6 and / or ORF6 / 7 coding sequence, and a VA coding sequence where each of the DBP, 0RF6, ORF6 / 7 and VA coding sequences are operably linked to a constitutive promoter, and further comprise an L4-22K coding sequence or an L4-22K_alt coding sequence operably linked to a constitutive promoter.

[0186] In certain embodiments, the L4-22K coding sequence is fused with the DBP coding sequence, the 0RF6 coding sequence and / or the ORF6 / 7 coding sequence.

[0187] In certain embodiments, the L4-22K fusion comprises a 2A peptide coding sequence between the L4-22K coding sequence and the DBP coding sequence, the ORF6 coding sequence and / or the ORF6 / 7 coding sequence.

[0188] In certain embodiments, the 2A peptide is selected from the group consisting of T2A, P2A, E2A and F2A.

[0189] In certain embodiments, the DBP sequence comprises the sequence set forth in any one of SEQ ID NOs: 1-8.

[0190] In certain embodiments, the ORF6 sequence comprises the sequence set forth in any one of SEQ ID NOs: 23, or 30-36.

[0191] In certain embodiments, the ORF6 / 7 sequence comprises the sequence set forth in any one of SEQ ID NOs: 37-41.

[0192] In certain embodiments, the L4-22K sequence comprises the sequence set forth in any one of SEQ ID NOs: 57-64.

[0193] In certain embodiments, the L4-22K_alt sequence comprises the sequence set forth in SEQ ID NO: 65.

[0194] In certain embodiments, the L4-22K coding sequence or the L4-22K_alt coding sequence comprises at least one mutation that enhances the production of rAAV particles. In certain embodiments, the L4-22K coding sequence comprises a Y20F mutation. In certain embodiments, the L4-22K coding sequence comprises a T79A mutation. In certain embodiments, the L4-22K coding sequence comprises a T99A mutation. In certain embodiments, the L4-22K coding sequence comprises a H169Q mutation. In certain embodiments, the L4-22K coding sequence comprises a Y171F mutation. In certain embodiments, the L4-22K coding sequence comprises a Y174F mutation. In certain embodiments, the L4-22K coding sequence comprises a T189A mutation.

[0195] In certain embodiments, the VA coding sequence comprises a VA_a coding sequence, a VA_b coding sequence, a VA_c coding sequence, a VA_d coding sequence, a VA_e coding sequence, or a VA_f coding sequence.

[0196] In certain embodiments, the VA sequence comprises the sequence set forth in SEQ ID NO: 42.

[0197] In certain embodiments, the VA_a sequence comprises the sequence set forth in SEQ ID NO: 43.

[0198] In certain embodiments, the VA_b sequence comprises the sequence set forth in any one of SEQ ID NOs: 44-48.

[0199] In certain embodiments, the VA_c sequence comprises the sequence set forth in SEQ ID NO: 49.

[0200] In certain embodiments, the VA_d sequence comprises the sequence set forth in SEQ ID NO: 50.

[0201] In certain embodiments, the VA_e sequence comprises the sequence set forth in SEQ ID NO: 51.

[0202] In certain embodiments, the VA_f sequence comprises the sequence set forth in any one of SEQ ID NOs: 52-55.

[0203] In certain embodiments, the first nucleic acid construct and second nucleic acid construct comprise the sequences set forth any one of SEQ ID NOs: 85-113

[0204] In certain embodiments, the AAV REP is one or more of Rep52, Rep40, Rep78, and Rep68.

[0205] In certain embodiments, the Rep78 sequence comprises the sequence set forth in any one of SEQ ID NOs: 115-125.

[0206] In certain embodiments, the AAV CAP coding sequence is contiguous with the AAV REP coding sequence.

[0207] In certain embodiments, the AAV REP coding sequence and the AAV CAP coding sequence are separated by a stuffer sequence. In certain embodiments, the stuffer sequence is about 0.5 kb to about 5.5 kb. In certain embodiments, the stuffer sequence is about 0.5 kb to about 3 kb. In certain embodiments, the stuffer sequence does not reduce production of rAAV vector particles.

[0208] In certain embodiments, the AAV CAP coding sequence encodes RHM4-1. In certain embodiments, the AAV CAP coding sequence encodes LK03.

[0209] In certain embodiments, the AAV REP coding sequence and the AAV CAP coding sequence are operably linked to at least one promoter sequence selected from the group consisting of a full-length P5 promoter, a truncated P5 promoter, a T7 promoter, mouse mammary tumor virus (MMTV) promoter, a Cytomegalovirus (CMV), a Simian Virus 40 (SV40) promoter, a phosphoglycerate kinase promoter (PGK), ATP-citrate lyase promoter (ACLY), and actin gamma (ACTG) promoter, a bidirectional CMV (BICMV) promoter, a pl9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

[0210] In certain embodiments, the promoter is at least the full-length P5 promoter sequence.

[0211] In certain embodiments, the full-length P5 promoter sequence does not decrease production of rAAV vector particles compared to a first nucleic acid construct that lacks the full-length P5 promoter sequence.

[0212] In certain embodiments, the full-length P5 promoter sequence enhances production of rAAV vector particles compared to a first nucleic acid construct that lacks the full-length P5 promoter sequence.

[0213] In certain embodiments, absence of the full-length P5 promoter sequence prevents formation of replication competent AAV (rcAAV) impurities.

[0214] In certain embodiments, absence of the full-length P5 promoter sequence results in reduced formation of replication competent AAV (rcAAV) impurities.

[0215] In certain embodiments, the first nucleic acid construct comprises an rAAV coding sequence that has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 85-91, or 128-146. In certain embodiments, the first nucleic acid construct comprises a coding sequence providing helper function has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 71-82. In certain embodiments, the first nucleic acid construct comprises a sequence set forth in any one of SEQ ID NOs: 152-154.

[0216] In certain embodiments, the second nucleic acid construct comprises an rAAV coding sequence that has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 85-91, or 128-146. In certain embodiments, the second nucleic acid construct comprises a coding sequence providing helper function has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 71-82. In certain embodiments, the second nucleic acid construct comprises a sequence set forth in any one of SEQ ID NOs: 152-154. In certain embodiments, the GOI is Factor VIII, acid a-glucosidase (GAA), a- synuclein, or Apolipoprotein E (ApoE).

[0217] In certain embodiments, reducing residual plasmid DNA (RPD) into the AAV capsid improves biosafety of the rAAV vector particles produced.

[0218] In certain embodiments, the presently disclosed subject matter is directed to transfected cells comprising a first nucleic acid construct and a second nucleic acid construct where the first nucleic acid construct comprises one or more of an AAV coding sequence comprising an AAV REP coding sequence, an AAV CAP coding sequence, and coding sequences providing helper virus function comprising a DBP coding sequence, an 0RF6 and / or ORF6 / 7 coding sequence, or a VA coding sequence and the second nucleic acid construct comprises a gene of interest (GOI) coding sequence and one or more of an AAV coding sequence comprising an AAV REP coding sequence, an AAV CAP coding sequence, and coding sequences providing helper virus function comprising a DBP coding sequence, an ORF6 and / or ORF6 / 7 coding sequence, or a VA coding sequence. In certain embodiments, each of the DBP coding sequence, the ORF6 and / or ORF6 / 7 coding sequence, and the VA coding sequence in the first nucleic acid construct and the second nucleic acid construct are operably linked to a constitutive promoter. In certain embodiments, the AAV REP coding sequence is operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity disposed upstream to the at least one promoter. In certain embodiments, the AAV REP coding sequence is operably linked to: (1) at least one promoter; and (2) a nucleic acid sequence with nonspecific transcription activity disposed downstream to the at least one promoter.

[0219] In certain embodiments, the presently disclosed subject matter is directed to transfected cells comprising a first nucleic acid construct and a second nucleic acid construct where the first nucleic acid construct comprises one or more of an rAAV coding sequence, an AAV REP coding sequence comprising an AAV CAP coding sequence, and coding sequences providing helper virus function comprising a DBP coding sequence, an 0RF6 and / or ORF6 / 7 coding sequence, a VA coding sequence, or an L4-22K coding sequence or an L4-22K_alt coding sequence, and the second nucleic acid construct comprises a gene of interest (GOI) coding sequence and one or more of an AAV coding sequence comprising an AAV REP coding sequence, an AAV CAP coding sequence, and coding sequences providing helper virus function comprising a DBP coding sequence, an 0RF6 and / or ORF6 / 7 coding sequence, or a VA coding sequence, or an L4-22K coding sequence or an L4-22K_alt coding sequence. In certain embodiments, each of the DBP coding sequence, the ORF6 and / or ORF 6 / 7 coding sequence, and the VA coding sequence in the first nucleic acid construct and the second nucleic acid construct are operably linked to a constitutive promoter. In certain embodiments, the AAV REP coding sequence is operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity disposed upstream to the at least one promoter. In certain embodiments, the AAV REP coding sequence is operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity disposed downstream to the at least one promoter.

[0220] In certain embodiments, the first nucleic acid construct and second nucleic acid construct comprise a sequence set forth in any one of SEQ ID NOs: 85-113.

[0221] In certain embodiments, the AAV REP coding sequence is operably linked to at least one promoter selected from the group consisting of a full-length P5 promoter, a truncated P5 promoter, a T7 promoter, mouse mammary tumor virus (MMTV) promoter, a Cytomegalovirus (CMV), a Simian Virus 40 (SV40) promoter, a phosphoglycerate kinase promoter (PGK), ATP-citrate lyase promoter (ACLY), and actin gamma (ACTG) promoter, a bidirectional CMV (BICMV) promoter, a pl9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

[0222] In certain embodiments, the AAV REP coding sequence is operably linked to a T7 promoter and a truncated P5 promoter.

[0223] In certain embodiments, the constitutive promoter is selected from the group consisting of a full-length P5 promoter, a truncated P5 promoter, a T7 promoter, a mouse mammary tumor virus (MMTV) promoter, a Cytomegalovirus (CMV), a Simian Virus 40 (SV40) promoter, a phosphoglycerate kinase promoter (PGK), ATP-citrate lyase promoter (ACLY), and actin gamma (ACTG) promoter, a bidirectional CMV (BICMV) promoter, a pl9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

[0224] In certain embodiments, the nucleic acid sequence with nonspecific transcription activity comprises from about 156 nucleotides to about 729 nucleotides.

[0225] In certain embodiments, the nucleic acid sequence with nonspecific transcription activity promulgated Rep protein expression compared to Rep protein expression when the nucleic acid sequence with nonspecific transcription activity is absent. In certain embodiments, the nucleic acid sequence with nonspecific transcription activity enhanced rAAV titer from about 2-fold to about 3 -fold compared to rAAV titer when the nucleic acid sequence with nonspecific transcription activity is absent.

[0226] In certain embodiments, the coding sequences providing helper virus function comprise a DBP coding sequence, an 0RF6 and / or ORF6 / 7 coding sequence, and a VA coding sequence where each of the DBP, 0RF6, ORF6 / 7 and VA coding sequences are operably linked to a constitutive promoter, and further comprise an L4-22K coding sequence or an L4-22K_alt coding sequence operably linked to a constitutive promoter.

[0227] In certain embodiments, the L4-22K coding sequence is fused with the DBP coding sequence, the 0RF6 coding sequence, and / or the ORF6 / 7 coding sequence.

[0228] In certain embodiments, the L4-22K fusion comprises a 2A peptide coding sequence between the L4-22K coding sequence and the DBP coding sequence, the ORF6 coding sequence and / or the ORF6 / 7 coding sequence.

[0229] In certain embodiments, the 2A peptide is selected from the group consisting of T2A, P2A, E2A and F2A.

[0230] In certain embodiments, the DBP sequence comprises the sequence set forth in any one of SEQ ID NOs: 1-8.

[0231] In certain embodiments, the ORF6 sequence comprises the sequence set forth in any one of SEQ ID NOs: 23, or 30-36.

[0232] In certain embodiments, the ORF6 / 7 sequence comprises the sequence set forth in any one of SEQ ID NOs: 37-41.

[0233] In certain embodiments, the L4-22K sequence comprises the sequence set forth in any one of SEQ ID NOs: 57-64.

[0234] In certain embodiments, the L4-22K_alt sequence comprises the sequence set forth in SEQ ID NO: 65.

[0235] In certain embodiments, the L4-22K coding sequence or the L4-22K_alt coding sequence comprises at least one mutation that enhances the production of rAAV particles. In certain embodiments, the L4-22K coding sequence comprises a Y20F mutation. In certain embodiments, the L4-22K coding sequence comprises a T79A mutation. In certain embodiments, the L4-22K coding sequence comprises a T99A mutation. In certain embodiments, the L4-22K coding sequence comprises a H169Q mutation. In certain embodiments, the L4-22K coding sequence comprises a Y171F mutation. In certain embodiments, the L4-22K coding sequence comprises a Y174F mutation. In certain embodiments, the L4-22K coding sequence comprises a T189A mutation. In certain embodiments, the VA coding sequence comprises a VA_a coding sequence, a VA_b coding sequence, a VA_c coding sequence, a VA_d coding sequence, a VA_e coding sequence, or a VA_f coding sequence.

[0236] In certain embodiments, the VA sequence comprises the sequence set forth in SEQ ID NO: 42.

[0237] In certain embodiments, the VA_a sequence comprises the sequence set forth in SEQ ID NO: 43.

[0238] In certain embodiments, the VA_b sequence comprises the sequence set forth in any one of SEQ ID NOs: 44-48.

[0239] In certain embodiments, the VA_c sequence comprises the sequence set forth in SEQ ID NO: 49.

[0240] In certain embodiments, the VA_d sequence comprises the sequence set forth in SEQ ID NO: 50.

[0241] In certain embodiments, the VA_e sequence comprises the sequence set forth in SEQ ID NO: 51.

[0242] In certain embodiments, the VA_f sequence comprises the sequence set forth in any one of SEQ ID NOs: 52-55.

[0243] In certain embodiments, the first nucleic acid construct and second nucleic acid construct comprise the sequences set forth any one of SEQ ID NOs: 85-113

[0244] In certain embodiments, the AAV REP is one or more of Rep52, Rep40, Rep78, and Rep68.

[0245] In certain embodiments, the Rep78 sequence comprises the sequence set forth in any one of SEQ ID NOs: 115-125.

[0246] In certain embodiments, the AAV CAP coding sequence is contiguous with the AAV REP coding sequence.

[0247] In certain embodiments, the AAV REP coding sequence and the AAV CAP coding sequence are separated by a stuffer sequence. In certain embodiments, the stuffer sequence is about 0.5 kb to about 5.5 kb. In certain embodiments, the stuffer sequence is about 0.5 kb to about 3 kb. In certain embodiments, the stuffer sequence does not reduce production of rAAV vector particles.

[0248] In certain embodiments, the AAV CAP coding sequence encodes RHM4-1.

[0249] In certain embodiments, the AAV CAP coding sequence encodes LK03.

[0250] In certain embodiments, the AAV REP coding sequence and the AAV CAP coding sequence are operably linked to at least one promoter sequence selected from the group consisting of a full-length P5 promoter, a truncated P5 promoter, a T7 promoter, mouse mammary tumor virus (MMTV) promoter, a Cytomegalovirus (CMV), a Simian Virus 40 (SV40) promoter, a phosphoglycerate kinase promoter (PGK), ATP-citrate lyase promoter (ACLY), and actin gamma (ACTG) promoter, a bidirectional CMV (BICMV) promoter, a pl9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

[0251] In certain embodiments, the promoter is at least the full-length P5 promoter sequence.

[0252] In certain embodiments, the full-length P5 promoter sequence does not decrease production of rAAV vector particles compared to first nucleic acid construct that lacks the full-length P5 promoter sequence.

[0253] In certain embodiments, the full-length P5 promoter sequence enhances production of rAAV vector particles compared to first nucleic acid construct that lacks the full-length P5 promoter sequence.

[0254] In certain embodiments, absence of the full-length P5 promoter sequence prevents formation of replication competent AAV (rcAAV) impurities.

[0255] In certain embodiments, absence of the full-length P5 promoter sequence results in reduced formation of replication competent AAV (rcAAV) impurities.

[0256] In certain embodiments, the first nucleic acid construct comprises an rAAV coding sequence that has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 85-91 or 128-146. In certain embodiments, the first nucleic acid construct comprises a coding sequence providing helper function has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 71-82. In certain embodiments, the first nucleic acid construct comprises a sequence set forth in any one of SEQ ID NOs: 152-154.

[0257] In certain embodiments, the second nucleic acid construct comprises an rAAV coding sequence that has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 85-91 or 128-146. In certain embodiments, the second nucleic acid construct comprises a coding sequence providing helper function has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 71-82. In certain embodiments, the second nucleic acid construct comprises a sequence set forth in any one of SEQ ID NOs: 152-154.

[0258] In certain embodiments, the GOI is Factor VIII, acid a-glucosidase (GAA), a- synuclein, or Apolipoprotein E (ApoE).

[0259] In certain embodiments, reducing residual plasmid DNA (RPD) into the AAV capsid improves biosafety of the rAAV vector particles produced. In certain embodiments, the presently disclosed subject matter is directed to transfected cells comprising a first nucleic acid construct and a second nucleic acid construct where the first nucleic acid construct comprises an AAV REP coding sequence, an AAV CAP coding sequence, and coding sequences providing helper virus function comprising a DBP coding sequence, an 0RF6 and / or ORF6 / 7 coding sequence, a VA coding sequence, or an L4-22K coding sequence or an L4-22K_alt coding sequence, and the second nucleic acid construct comprises a gene of interest (GO I) coding sequence. In certain embodiments, each of the DBP coding sequence, the 0RF6 and / or ORF6 / 7 coding sequence, and the VA coding sequence in the first nucleic acid construct are operably linked to a constitutive promoter. In certain embodiments, the AAV REP coding sequence is operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity disposed upstream to the at least one promoter. In certain embodiments, the AAV REP coding sequence is operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity disposed downstream to the at least one promoter. In certain embodiments, the first nucleic acid construct comprises a sequence set forth in any one of SEQ ID NOs: 85-91, 128-148, 150, or 156-158.

[0260] In certain embodiments, the presently disclosed subject matter is directed to transfected cells comprising a first nucleic acid construct and a second nucleic acid construct where the first nucleic acid construct comprises an AAV CAP coding sequence, and coding sequences providing helper virus function comprising a DBP coding sequence, an ORF6 and / or ORF6 / 7 coding sequence, a VA coding sequence, or an L4-22K coding sequence or an L4-22K_alt coding sequence, and the second nucleic acid construct comprises a gene of interest (GOI) coding sequence and an AAV REP coding sequence. In certain embodiments, each of the DBP coding sequence, the ORF6 and / or ORF6 / 7 coding sequence, and the VA coding sequence in the first nucleic acid construct are operably linked to a constitutive promoter. In certain embodiments, the AAV REP coding sequence is operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity disposed upstream to the at least one promoter. In certain embodiments, the AAV REP coding sequence is operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity disposed downstream to the at least one promoter. In certain embodiments, the first nucleic acid construct comprises a sequence set forth in SEQ ID NO: 108, or 150 and / or the second nucleic acid construct comprises a sequence set forth in SEQ ID NO: 109, or 152-154. In certain embodiments, the presently disclosed subject matter is directed to transfected cells comprising a first nucleic acid construct and a second nucleic acid construct where the first nucleic acid construct comprises an AAV REP coding sequence, and coding sequences providing helper virus function comprising a DBP coding sequence, an 0RF6 and / or ORF6 / 7 coding sequence, a VA coding sequence, or an L4-22K coding sequence or an L4-22K_alt coding sequence, and the second nucleic acid construct comprises a gene of interest (GOI) coding sequence and an AAV CAP coding sequence. In certain embodiments, each of the DBP coding sequence, the ORF6 and / or ORF6 / 7 coding sequence, and the VA coding sequence in the first nucleic acid construct are operably linked to a constitutive promoter. In certain embodiments, the AAV REP coding sequence is operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity disposed upstream to the at least one promoter. In certain embodiments, the AAV REP coding sequence is operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity disposed downstream to the at least one promoter. In certain embodiments, the first nucleic acid construct comprises a sequence set forth in any one of SEQ ID NOs: 98-107, or 110 and / or the second nucleic acid construct comprises a sequence set forth in any one of SEQ ID NOs: 111, 127, or 149.

[0261] In certain embodiments, the presently disclosed subject matter is directed to transfected cells comprising a first nucleic acid construct and a second nucleic acid construct where the first nucleic acid construct comprises an AAV REP52 coding sequence, an AAV CAP coding sequence, and coding sequences providing helper virus function comprising a DBP coding sequence, an ORF6 and / or ORF6 / 7 coding sequence, a VA coding sequence, or an L4-22K coding sequence or an L4-22K_alt coding sequence, and the second nucleic acid construct comprises a gene of interest (GOI) coding sequence and an AAV Rep78 coding sequence. In certain embodiments, each of the DBP coding sequence, the ORF6 and / or ORF 6 / 7 coding sequence, and the VA coding sequence in the first nucleic acid construct are operably linked to a constitutive promoter. In certain embodiments, the AAV REP52 and / or AAV REP78 coding sequences are operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity disposed upstream to the at least one promoter. In certain embodiments, the AAV REP52 and / or AAV REP78 coding sequences are operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity disposed downstream to the at least one promoter. In certain embodiments, the Rep78 sequence comprises the sequence set forth in any one of SEQ ID NOs: 115-125. In certain embodiments, the first nucleic acid construct comprises a sequence set forth in SEQ ID NO: 112 and / or the second nucleic acid construct comprises a sequence set forth in SEQ ID NO: 113.

[0262] In certain embodiments, the presently disclosed subject matter is directed to transfected cells comprising a first nucleic acid construct and a second nucleic acid construct where the first nucleic acid construct comprises an AAV large REP coding sequence, an AAV CAP coding sequence, and coding sequences providing helper virus function comprising a DBP coding sequence, an 0RF6 and / or ORF6 / 7 coding sequence, a VA coding sequence, or an L4-22K coding sequence or an L4-22K_alt coding sequence, and the second nucleic acid construct comprises a gene of interest (GOI) coding sequence and an AAV small REP coding sequence. In certain embodiments, each of the DBP coding sequence, the 0RF6 and / or ORF6 / 7 coding sequence, and the VA coding sequence in the first nucleic acid construct are operably linked to a constitutive promoter. In certain embodiments, the AAV large REP and / or AAV small REP coding sequences are operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity disposed upstream to the at least one promoter. In certain embodiments, the AAV large REP and / or AAV small REP coding sequences are operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity disposed downstream to the at least one promoter.

[0263] In certain embodiments, the presently disclosed subject matter is directed to transfected cells comprising a first nucleic acid construct and a second nucleic acid construct where the first nucleic acid construct comprises an AAV CAP coding sequence, and coding sequences providing helper virus function comprising one or more of a DBP coding sequence, an 0RF6 and / or ORF6 / 7 coding sequence, a VA coding sequence, or an L4-22K coding sequence or an L4-22K_alt coding sequence, and the second nucleic acid construct comprises a gene of interest (GOI) coding sequence, an AAV REP coding sequence and coding sequences providing helper virus function comprising one or more of a DBP coding sequence, an 0RF6 and / or ORF6 / 7 coding sequence, a VA coding sequence, or an L4-22K coding sequence or an L4-22K_alt coding sequence. In certain embodiments, each of the DBP coding sequence, the 0RF6 and / or ORF6 / 7 coding sequence, and the VA coding sequence in the first nucleic acid construct are operably linked to a constitutive promoter. In certain embodiments, the AAV REP coding sequences are operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity disposed upstream to the at least one promoter. In certain embodiments, the AAV REP coding sequences are operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity disposed downstream to the at least one promoter.

[0264] In certain embodiments, the presently disclosed subject matter is directed to transfected cells comprising a first nucleic acid construct and a second nucleic acid construct where the first nucleic acid construct comprises an AAV REP coding sequence, and coding sequences providing helper virus function comprising one or more of a DBP coding sequence, an 0RF6 and / or ORF6 / 7 coding sequence, a VA coding sequence, or an L4-22K coding sequence or an L4-22K_alt coding sequence, and the second nucleic acid construct comprises a gene of interest (GO I) coding sequence, an AAV CAP coding sequence and coding sequences providing helper virus function comprising one or more of a DBP coding sequence, an 0RF6 and / or ORF6 / 7 coding sequence, a VA coding sequence, or an L4-22K coding sequence or an L4-22K_alt coding sequence. In certain embodiments, each of the DBP coding sequence, the 0RF6 and / or ORF6 / 7 coding sequence, and the VA coding sequence in the first nucleic acid construct are operably linked to a constitutive promoter. In certain embodiments, the AAV REP coding sequences are operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity disposed upstream to the at least one promoter. In certain embodiments, the AAV REP coding sequences are operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity disposed downstream to the at least one promoter.

[0265] In certain embodiments, the presently disclosed subject matter is directed to transfected cells comprising a first nucleic acid construct and a second nucleic acid construct where the first nucleic acid construct comprises an AAV small REP coding sequence, an AAV CAP coding sequence, and coding sequences providing helper virus function comprising one or more of a DBP coding sequence, an 0RF6 and / or ORF6 / 7 coding sequence, a VA coding sequence, or an L4-22K coding sequence or an L4-22K_alt coding sequence, and the second nucleic acid construct comprises a gene of interest (GOI) coding sequence, an AAV large REP coding sequence and coding sequences providing helper virus function comprising one or more of a DBP coding sequence, an ORF6 and / or ORF6 / 7 coding sequence, a VA coding sequence, or an L4-22K coding sequence or an L4-22K_alt coding sequence. In certain embodiments, each of the DBP coding sequence, the ORF6 and / or ORF 6 / 7 coding sequence, and the VA coding sequence in the first nucleic acid construct are operably linked to a constitutive promoter. In certain embodiments, the AAV large REP and / or small REP coding sequences are operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity disposed upstream to the at least one promoter. In certain embodiments, the AAV large REP and / or small REP coding sequences are operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity disposed downstream to the at least one promoter.

[0266] In certain embodiments, the presently disclosed subject matter is directed to transfected cells comprising a first nucleic acid construct and a second nucleic acid construct where the first nucleic acid construct comprises an AAV large REP coding sequence, an AAV CAP coding sequence, and coding sequences providing helper virus function comprising one or more of a DBP coding sequence, an 0RF6 and / or ORF6 / 7 coding sequence, a VA coding sequence, or an L4-22K coding sequence or an L4-22K_alt coding sequence, and the second nucleic acid construct comprises a gene of interest (GOI) coding sequence, an AAV small REP coding sequence and coding sequences providing helper virus function comprising one or more of a DBP coding sequence, an ORF6 and / or ORF6 / 7 coding sequence, a VA coding sequence, or an L4-22K coding sequence or an L4-22K_alt coding sequence. In certain embodiments, each of the DBP coding sequence, the ORF6 and / or ORF 6 / 7 coding sequence, and the VA coding sequence in the first nucleic acid construct are operably linked to a constitutive promoter. In certain embodiments, the AAV large REP and / or small REP coding sequences are operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity disposed upstream to the at least one promoter. In certain embodiments, the AAV large REP and / or small REP coding sequences are operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity disposed downstream to the at least one promoter.

[0267] In certain embodiments, the presently disclosed subject matter is directed to transfected cells comprising a first nucleic acid construct and a second nucleic acid construct where the first nucleic acid construct comprises an AAV REP coding sequence, an AAV CAP coding sequence, and the second nucleic acid construct comprises a gene of interest (GOI) coding sequence, and coding sequences providing helper virus function comprising a DBP coding sequence, an ORF6 and / or ORF6 / 7 coding sequence, a VA coding sequence, or an L4-22K coding sequence or an L4-22K_alt coding sequence. In certain embodiments, each of the DBP coding sequence, the 0RF6 and / or ORF6 / 7 coding sequence, and the VA coding sequence in the first nucleic acid construct are operably linked to a constitutive promoter. In certain embodiments, the AAV REP coding sequences are operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity disposed upstream to the at least one promoter. In certain embodiments, the AAV REP coding sequences are operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity disposed downstream to the at least one promoter.

[0268] In certain embodiments, the presently disclosed subject matter is directed to transfected cells comprising a first nucleic acid construct and a second nucleic acid construct where the first nucleic acid construct comprises an AAV REP coding sequence, an AAV CAP coding sequence, and coding sequences providing helper virus function comprising one or more of a DBP coding sequence, an ORF6 and / or ORF6 / 7 coding sequence, a VA coding sequence, or an L4-22K coding sequence or an L4-22K_alt coding sequence, and the second nucleic acid construct comprises a gene of interest (GOI) coding sequence, and coding sequences providing helper virus function comprising one or more of a DBP coding sequence, an 0RF6 and / or ORF6 / 7 coding sequence, a VA coding sequence, or an L4-22K coding sequence or an L4-22K_alt coding sequence. In certain embodiments, each of the DBP coding sequence, the 0RF6 and / or ORF6 / 7 coding sequence, and the VA coding sequence in the first nucleic acid construct are operably linked to a constitutive promoter. In certain embodiments, the AAV REP coding sequences are operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity disposed upstream to the at least one promoter. In certain embodiments, the AAV REP coding sequences are operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity disposed downstream to the at least one promoter.

[0269] In certain embodiments, the L4-22K coding sequence or the L4-22K_alt coding sequence comprises at least one mutation that enhances the production of rAAV particles. In certain embodiments, the L4-22K coding sequence comprises a Y20F mutation. In certain embodiments, the L4-22K coding sequence comprises a T79A mutation. In certain embodiments, the L4-22K coding sequence comprises a T99A mutation. In certain embodiments, the L4-22K coding sequence comprises a H169Q mutation. In certain embodiments, the L4-22K coding sequence comprises a Y171F mutation. In certain embodiments, the L4-22K coding sequence comprises a Y174F mutation. In certain embodiments, the L4-22K coding sequence comprises a T189A mutation.

[0270] BRIEF DESCRIPTION OF THE DRAWINGS

[0271] Figures 1A-1L illustrate exemplary helper plasmids. Figure 1 A shows a helper plasmid comprising an E2A, DNA Binding Protein (DBP) coding sequence; an E4, Open Reading Frame 6 (ORF6) sequence; and a Viral Associated (VA) sequence. Figure IB shows a first exemplary helper plasmid comprising a DBP sequence and an ORF6 sequence each under independent Cytomegalovirus (CMV) promoters. Figure 1C shows a second exemplary helper plasmid that comprises the sequences shown in Figure IB and further comprises a Viral Associated_b sequence (VA_b) sequence under a Simian Virus 40 (SV40) promoter. Figure ID shows a third exemplary helper plasmid comprising DBP and ORF6 sequences linked by a T2A cleavage sequence (DBP-T2A-ORF6 configuration), in which transcription is under a CMV promoter. Figure IE shows a fourth exemplary helper plasmid similar to Figure ID but with an ORF-T2A-DBP configuration. Figure IF shows a fifth exemplary helper plasmid comprising a DBP, ORF6 and VA_b sequences under a CMV promoter, in which DBP and ORF6 are linked via a T2A sequence, whereas ORF6 and VA_b are linked via a P2A cleavage sequence. Figure 1G shows a sixth exemplary helper plasmid comprising the DBP, ORF6 and VA sequences as in Figure 1 A, in which sequences between the DBP start site and ORF6 start site were replaced with a bidirectional CMV (BICMV) promoter. Figure 1H shows a seventh exemplary helper plasmid as in Figure 1C in which, additionally, an L4-22K coding sequence linked via the T2A sequence upstream to the ORF6 sequence is operably linked to the CMV promoter. Figure II shows an eight exemplary helper plasmid as in Figure 1C in which, additionally, an L4-22K coding sequence linked via the T2A sequence upstream to the DBP sequence is operably linked to the CMV promoter. Figure 1 J shows a ninth exemplary helper plasmid as in Figure 1G in which, additionally, an L4-22K coding sequence linked via the T2A sequence upstream to the DBP sequence is operably linked to the BICMV promoter. Figure IK shows a tenth exemplary helper plasmid as in Figure 1G in which, additionally, an L4-22K coding sequence linked via the T2A sequence upstream to the ORF6 sequence is operably linked to the BICMV promoter. Figure IL shows an eleventh exemplary helper plasmid as in Figure 1G in which, additionally, an L4-22K coding sequence operably linked to a CMV promoter is fused to the DBP sequence.

[0272] Figure 2 depicts locations of possible Open Reading Frames in E2A, E4 and VA genes in the helper plasmid shown in Figure 1 A.

[0273] Figure 3 depicts the results of analyzing the minimal elements needed for rAAV production.

[0274] Figure 4 depicts the results of analyzing the effects of independent versus common promoters on rAAV production.

[0275] Figure 5 shows a comparison between the first and second exemplary helper plasmids shin in Figures IB and 1C respectively for rAAV production using the Ambr®250 system. Figure 6 depicts the results of co-transfecting host cells with the first exemplary helper plasmid shown in Figure IB with a plasmid comprising VA_a, VA_b, VA_c, VA_d, VA_e, or VA_f or VA sequences under CMV promoter, on rAAV production.

[0276] Figure 7 depicts the results of co-transfecting host cells with the second exemplary helper plasmid shown in Figure 1C with a plasmid comprising E2A, DBP, MastC, L4, L4- 22K, L4pVIII or L4 33K sequences under CMV promoter, on rAAV production.

[0277] Figure 8 depicts the results of co-transfecting host cells with the second exemplary helper plasmid shown in Figure 1C with a plasmid comprising E4, 0RF6, 16kDa, ORF1, ORF2, ORF3, ORF6 / 7, revORFl, revORF2, revORF3, revORF4 or revORF5 sequences under CMV promoter, on rAAV production.

[0278] Figures 9A-9B depicts the results of L4-22K co-transfection on rAAV production efficiency. Figure 9A depicts the results of co-transfecting host cells with the helper plasmid shown in Figure 1 A (positive control) or the sixth exemplary helper plasmid shown in Figure 1G, with a plasmid comprising the L4-22K sequence under CMV promoter, on RHM4-1 rAAV production. Figure 9A depicts the results of co-transfecting host cells with the helper plasmid shown in Figure 1 A (positive control) or the sixth exemplary helper plasmid shown in Figure 1G, with a plasmid comprising the L4-22K sequence under CMV promoter, on LK03 rAAV production.

[0279] Figure 10 depicts the results of co-transfecting host cells with the second exemplary helper plasmid shown in Figure 1C with, a plasmid comprising an L4-22K Alternative (L4- 22K_Alt) sequence under CMV promoter, or a plasmid comprising an L4-33K sequence under CMV promoter.

[0280] Figures 11A-11B depicts the effect of L4-22K incorporated helper plasmids on rAAV production. Figure 11 A depicts the results of L4-22K incorporated helper plasmids either co-transfected in a separate plasmid (“+”) or fused to a DBP or ORF6 sequence on RHM4-1 rAAV production. Figure 11 A depicts the results of L4-22K incorporated helper plasmids either co-transfected in a separate plasmid (“+”) or fused to a DBP or ORF6 sequence on LK03 rAAV production.

[0281] Figure 12 depicts the effect of L4-22K on RHM4-1 rAAV production using the Ambr®250 system.

[0282] Figures 13A-13C depicts a comparative analysis showing the effect of incorporating the L4-22K sequence on rAAV production using the Ambr®250 system. Figure 13 A depicts a comparison between the helper plasmids shown in Figures 1 A, IK and IL for RHM4-1 rAAV production for 2 versions of transgene 1 (vl and v2). Figure 13B depicts a comparison between the helper plasmids shown in Figures 1 A, IK and IL for RHM4-1 rAAV production for transgene 2 using qPCR. Figure 13C depicts a comparison between the helper plasmids shown in Figures 1 A, IK and IL for RHM4-1 rAAV production for transgene 3.

[0283] Figure 14 depicts the effect of L4-22K on Rep and Cap expression using transgene-2, transgene-3 and 2 versions of transgene-1.

[0284] Figure 15 depicts promoter dependence of the effect of L4-22K on Rep and Capsid expression levels. In plasmid PL0845, Rep78, Rep52 and capsid genes are discretely positioned, with the original promoters substituted with constitutive and ubiquitously active promoters. Specifically, the codon-optimized Rep78 is under the control of the CMV promoter, while Rep52 is expressed by the ACLY (ATP-citrate lyase) promoter. The LK03 expression is governed by ACTG (actin gamma) promoter.

[0285] Figures 16A-16E illustrate exemplary dual plasmid systems for rAAV production. Figure 16A shows a first dual plasmid system comprising replication (Rep), capsid (Cap), and helper elements carried in one plasmid, and the gene of interest (GOI) in a second plasmid. Figure 16B shows a second dual plasmid system comprising Cap and helper elements in one plasmid, and Rep and GOI in a second plasmid. Figure 16C shows a third dual plasmid system comprising Rep and helper elements in one plasmid, and Cap and GOI in a second plasmid. Figure 16D shows a fourth dual plasmid system comprising small Rep, Cap and helper elements in one plasmid, and Rep781arge rep and GOI in a second plasmid. Figure 16E shows a comparison of Rep78 protein expression between the dual plasmid systems shown in Figures 16A-16D (16A, 16B, 16C, 16D) and triple / quadruple plasmid systems described in Figures 1-15.

[0286] Figures 17A-17B illustrates two plasmid configurations that can be used for the presently disclosed subject matter. Figure 17A shows a configuration of the Rep / Cap plasmid where expression of Rep78, Rep52, and Cap proteins is driven using truncated P5, Pl 9, and P40 promoters, respectively. Figure 17B shows a configuration wherein the Rep, Cap, and helper elements were combined into one single plasmid.

[0287] Figures 18A-18D show an exemplary dual plasmid design with or without the backbone elements. Figure 18A is a schematic of an exemplary Rep / Cap / Helper dual plasmid design and plasmid backbone elements upstream of the Rep protein. Variants of this plasmid with different backbone elements (Dl-Il) were generated to investigate their impact on Rep expression. Figure 18B shows a comparison of Rep78 protein expression between the dual plasmid systems shown in Figure 18A and triple / quadruple plasmid systems. Figure 18C shows a comparison between triple and dual plasmid transfections for genome titers. Figure 18D shows a comparison between triple and dual plasmid transfections for capsid titers.

[0288] Figures 19A-19D illustrates the effect of replacing backbone elements and truncated P5 promoter with other promoters for driving Rep78 protein expression. Figure 19A is a schematic depicting various promoters for driving Rep78 expression. These include the complete P5 promoter, mouse mammary tumor virus (MMTV) promoter, CMV promoter, SV40 promoter, phosphoglycerate kinase promoter (PGK), ATP-citrate lyase promoter (ACLY), and actin gamma (ACTG) promoter. Figure 19B illustrates expression of Rep78 protein relative to Rep52 protein in cells transfected with the constructs shown in Figure 19A. Figure 19C shows a comparison between triple and dual plasmid transfections for genome titers. Figure 19D shows a comparison between triple and dual plasmid transfections for capsid titers.

[0289] Figure 20A-20B illustrates exemplary Rep / Cap / Helper plasmids encoding the LK03 or RHM4-1 capsid. Figure 20A is a schematic of exemplary Rep / Cap / Helper plasmids Ml, QI, R1 and SI, each comprising a sequence encoding the LK03 capsid. Figure 20B is a schematic of exemplary Rep / Cap / Helper plasmids Tl, Ul, VI, M2 and N2 each comprising a sequence encoding the RHM4-1 capsid.

[0290] Figures 21A-21B illustrate exemplary inducible Rep / Cap / Helper plasmids. Figure 21A is a schematic of an exemplary Rep / Cap / Helper plasmids Wl, XI, Yl, Zl, A2 and B2, each comprising a sequence encoding the LK03 capsid. Figure 2 IB is a schematic of exemplary Rep / Cap / Helper plasmids, C2, D2, E2, F2 and G2 each comprising a sequence encoding the RHM4-1 capsid.

[0291] Figures 22A-22B illustrate an exemplary dual plasmid system comprising an inducible Rep / Helper first plasmid and second plasmid comprising the Cap and GOI sequences. Figure 22A is a schematic of exemplary first plasmids H2, 12 and J2, wherein Rep is under control of a BiTet inducible promoter. Figure 22B is a schematic of exemplary second plasmids K2 and L2, each comprising a RHM4-1 capsid sequence and a GOI (transgene) sequence.

[0292] Figures 23A-2G illustrate additional exemplary dual plasmid systems for rAAV production. Figure 23 A shows a fifth dual plasmid system comprising large Rep, Cap and helper genes in one plasmid, and small Rep and GOI in a second plasmid. Figure 23B shows a sixth dual plasmid system comprising Cap and some of the helper genes in one plasmid, and Rep, some helper genes and GOI in a second plasmid. Figure 23C shows a seventh dual plasmid system comprising Rep and some helper genes in one plasmid, and Cap, some helper genes and GOI in a second plasmid. Figure 23D shows an eight dual plasmid system comprising small Rep, Cap and some helper elements in one plasmid, and large Rep, some helper genes and GOI in a second plasmid. Figure 23E shows a ninth dual plasmid system comprising large Rep, Cap, and some helper genes in one plasmid, and small Rep, some helper genes and GOI in a second plasmid. Figure 23F shows a tenth dual plasmid system comprising Rep and Cap genes in one plasmid, and helper genes and GOI in a second plasmid. Figure 23G shows an eleventh dual plasmid system comprising Rep, Cap and some helper genes in one plasmid, and some helper genes and GOI in a second plasmid.

[0293] Figures 24A-24C illustrate the impact of full-length P5 promoter removal on Rep78 expression and genome titer in Rep / Cap plasmids with RHM4-1 capsid.

[0294] Figures 25A-25C illustrate the impact of full-length P5 promoter removal on Rep78 expression and genome titer in Rep / Cap plasmids with LK03 capsid.

[0295] Figures 26A-26C illustrates backbone elements drive Rep78 expression non- specifically, while full-length P5 promoter may have additional roles with LK03 capsid.

[0296] Figures 27A-27D illustrate the removal of the P5 promoter eliminated the replication competent AAV (rcAAV) impurity.

[0297] Figures 28A-28F demonstrate how the SV40 promoter was used to drive Rep78 expression.

[0298] Figures 29A-29D demonstrates that Helper 11 boosts rAAV production in combination with Rep / Cap A4, Y2, Z2, and A3.

[0299] Figures 30A-30E demonstrates AAV production and RPD (residual plasmid DNA) levels using different configurations of dual plasmids selected from B4-H4.

[0300] Figures 31A-31F demonstrates AAV production and RPD (residual plasmid DNA) levels using different configurations of dual plasmids selected from 14-04.

[0301] Figures 32A-32C demonstrate that mutating certain amino acid residues in L4-22K protein results in changes to the vg titer.

[0302] DETAILED DESCRIPTION

[0303] Production of rAAV requires transfecting a host cell with a AAV REP coding sequence, AAV CAP coding sequence, and coding sequences providing helper virus function. Often, such transfections use “triple transfection” methods where three plasmids are employed - one comprising the coding sequences providing helper virus function, a second comprising the REP and CAP coding sequences, and a third comprising the gene of interest (GOI) sequences. The high per plasmid costs associated with production and storage makes triple transfection approaches less cost-effective. The presently disclosed subject matter relates to compositions and methods for boosting efficiency and reducing costs associated with rAAV production. In particular, the presently disclosed subject matter relates, in certain embodiments, to compositions comprising coding sequences providing helper virus function to enhance rAAV titers.

[0304] In one aspect, the subject matter of the present disclosure is directed to cell culture methods for producing rAAV vector particles comprising culturing a population of cells comprising an rAAV coding sequence; an AAV Rep coding sequence; an AAV CAP coding sequence; and coding sequences providing helper virus function comprising a DNA Binding Protein (DBP) coding sequence; an Open Reading Frame 6 (0RF6), or an Open Reading Frame 6 / 7 (ORF6 / 7) coding sequence; and a Viral Associated (VA) coding sequence where, each of the DBP, the 0RF6 (or the ORF 6 / 7) and VA coding sequences are operably linked to a constitutive promoter; and harvesting the rAAV vector particles.

[0305] In another aspect, the subject matter of the present disclosure is directed to a nucleic acid comprising a DBP coding sequence; an 0RF6 and / or the ORF6 / 7 coding sequence; and a VA coding sequence where, each of the DBP, the 0RF6 and / or the ORF 6 / 7 and the VA coding sequences are operably linked to a constitutive promoter.

[0306] In another aspect, the subject matter of the present disclosure is directed to a transfected cells comprising an AAV Rep coding sequence; an AAV CAP coding sequence; and coding sequences providing helper virus function comprising a DNA Binding Protein (DBP) coding sequence; an Open Reading Frame 6 (0RF6), or an Open Reading Frame 6 / 7 (ORF6 / 7) coding sequence; and a Viral Associated (VA) coding sequence where, each of the DBP, the 0RF6 (or the ORF 6 / 7) and the VA coding sequences are operably linked to a constitutive promoter.

[0307] For clarity, but not by way of limitation, the detailed description of the presently disclosed subject matter is divided into the following subsections:

[0308] 1. Definitions

[0309] 2. Nucleic acids providing helper virus function

[0310] 3. rAAV producing cells

[0311] 4. Method of producing rAAV vector particles in a cell culture

[0312] 5. Exemplary constructs providing helper virus function

[0313] 6. Examples 1. Definitions

[0314] The terms used in this specification generally have their ordinary meanings in the art, within the context of this disclosure and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the present disclosure and how to make and use them.

[0315] As used herein, the use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one” and “one or more than one.”

[0316] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s)” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms or words that do not preclude the possibility of additional acts or structures. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0317] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.

[0318] In certain embodiments, the cells of the present disclosure harbor a chromosomally integrated rep gene but require helper virus function in order to express Rep protein. “Helper virus” or “helper virus function” or “AAV Helper” as used herein refers to at least one of adenovirus (Ad) El (e.g., Ad Ela or Ad Elb), Ad E2A, Ad E4 and virus associated RNA (VA RNA), or to corresponding functions of other viruses, such as herpesviruses and poxviruses, which are able to impart helper function to support replication and packaging of AAV vector genomes. In particular embodiments, a hybrid virus made of adenovirus with an E1ZE3 deletion, but containing Ad E2A, Ad E4 and VA RNA which provide helper virus function, as well as AAV ITRs flanking a heterologous nucleic acid. In other embodiments, hybrid viruses comprise helper virus functions from herpesvirus or poxvirus, along with AAV ITRs flanking a heterologous nucleic acid.

[0319] As used herein, the term “helper virus function(s)” or “helper function(s)” refers to function(s) enabled by nucleic acid sequences that encode proteins and / or nucleic acid molecules (e.g., RNA) that are relevant for AAV production. Such helper functions are encoded in a helper virus genome, which allow rAAV vector genome replication and packaging (in conjunction with Rep and Cap). AAV generally cannot be propagated by itself and therefore, to establish a productive viral infection, AAV is coinfected with a helper virus. Adenovirus is believed to be the natural helper virus in the wild because clinical isolates of adenovirus are frequently contaminated with AAV, but herpesvirus and baculovirus also can supply complete helper activity in cell culture. Contemporary recombinant AAV (rAAV) virion production generally involves co-transfection of a host cell with an AAV vector plasmid and a construct which provides AAV helper functions to complement functions missing from the AAV vector plasmid. In this manner, the host cell is capable of AAV replication and packaging. The host cell is then infected with a helper virus to provide accessory functions. The helper virus is generally an infectious adenovirus (type 2 or 5), or herpesvirus. For adenovirus, helper functions have been identified as Ad Ela, E1B, E4, E2A, and viral associated RNA (VA RNA) (see Samulski et al., Annu. Rev. Virol. (2014), 1:427- 451 and WO 1997 / 017458 incorporated herein by reference in their entirety). DNA Binding Protein (DBP) is expressed from gene E2A, while the proteins ORF6 and a splice variant, ORF6 / 7, are expressed from gene E4. As disclosed herein, helper functions are provided by helper elements, DBP, ORF6, ORF6 / 7, and VA RNA.

[0320] As disclosed herein, “helper virus function” or “helper function” may be provided in a number of different ways. For example, helper virus function can be provided by a virus or, for example, provided by polynucleotide sequences encoding the requisite helper function(s) to a cell in trans. In another example, a plasmid or other expression vector comprising polynucleotide sequences encoding one or more viral (e.g., adenoviral) proteins provides helper function when after transfection into a cell line along with a rAAV vector genome allows rAAV vector genome replication and packaging into rAAV vector particles. In certain embodiments, a helper virus function is provided by a virus selected from adenovirus, herpesvirus, poxvirus, or a hybrid virus thereof. In certain embodiments, a helper virus function comprises one or more viruses, vectors or plasmids that provide the helper virus function. In certain embodiments, the helper virus function comprises at least one of Ad El protein (e.g., Ad Ela protein or Ad Elb protein), Ad E2A protein, Ad E4 protein and Ad VA RNA. In certain embodiments the helper virus function comprises a fusion protein comprising a helper protein e.g., DBP, and ORF6 where the amino acid sequence of the helper protein is fused, directly or via a linker, to an amino acid sequence of another helper protein, e.g., a L4-22K sequence.

[0321] The terms “plasmid” and “construct” are used interchangeably herein.

[0322] As used herein, the term “AAV protein” refers to any wild-type or modified protein that is derived from the AAV genome and useful for AAV production. “AAV protein” includes any form of the Rep, Cap, or Helper proteins, whether wild-type or modified. Modifications of the wild-type AAV genes does not necessarily result in a change in amino acid sequence of the expressed protein.

[0323] The term “vector” refers to small carrier nucleic acid molecule, e.g., a plasmid, a virus (e.g., AAV vector), or other vehicle, which can be manipulated by insertion or incorporation of a nucleic acid. Such vectors can be used for genetic manipulation (i.e., “cloning vectors”), to introduce / transfer polynucleotides into cells, and to transcribe or translate the inserted polynucleotide in cells. An “expression vector” is a specialized vector that contains a gene or nucleic acid sequence with the necessary regulatory regions needed for expression in a host cell.

[0324] A vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), intron, an inverted terminal repeat (ITR), selectable marker (e.g., antibiotic resistance), and polyadenylation signal.

[0325] A viral vector is derived from or based upon one or more nucleic acid elements that comprise a viral genome. A particular viral vector is an adeno-associated virus (AAV) vector.

[0326] The term “recombinant,” as a modifier of vector, such as recombinant AAV vector, as well as a modifier of sequences such as recombinant polynucleotides and polypeptides, means that the compositions have been manipulated (i.e., engineered) in a fashion that generally does not occur in nature. A particular example of a recombinant AAV vector would be where a nucleic acid sequence that is not normally present in the wild-type AAV genome (e.g., a heterologous nucleic acid sequence) is inserted within the AAV genome. Although the term “recombinant” is not always used herein in reference to AAV vectors, as well as sequences such as polynucleotides, recombinant forms including polynucleotides, are expressly included in spite of any such omission. A “recombinant AAV vector” or “rAAV” is derived from the wild type (wt or wild type) genome of AAV by using molecular methods to remove the wild type genome from the AAV genome, and replacing with a non-native nucleic acid sequence, referred to as a heterologous nucleic acid, i.e., engineering the vector. Typically, for AAV one or both inverted terminal repeat (ITR) sequences of AAV genome are retained in the AAV vector. rAAV is distinguished from an AAV genome, since all or a part of the AAV genome has been replaced with a non-native sequence with respect to the AAV genomic nucleic acid. Incorporation of a non-native sequence therefore defines the AAV vector as a “recombinant” vector, which can be referred to as a “rAAV vector.” The terms “AAV” and “rAAV” are used interchangeably herein. It is understood that an engineered AAV is an rAAV.

[0327] A rAAV sequence can be packaged - referred to herein as a “particle”- for subsequent infection (transduction) of a cell, ex vivo, in vitro or in vivo. Where a recombinant AAV vector sequence is encapsidated or packaged into an AAV particle, the particle can also be referred to as a “rAAV vector” or “rAAV particle.” Such rAAV particles include proteins that encapsidate or package the vector genome. In the case of AAV, they are referred to as capsid proteins.

[0328] A vector “genome” refers to the portion of the recombinant plasmid sequence that is ultimately packaged or encapsidated to form a viral (e.g., AAV) particle. In cases where recombinant plasmids are used to construct or manufacture recombinant vectors, the vector genome does not include the portion of the “plasmid” that does not correspond to the vector genome sequence of the recombinant plasmid. This non vector genome portion of the recombinant plasmid is referred to as the “plasmid backbone,” which is important for cloning and amplification of the plasmid, a process that is needed for propagation of the plasmid but is not itself packaged or encapsidated into virus (e.g., AAV) particles. Thus, a vector “genome” refers to the nucleic acid that is packaged or encapsidated by virus (e.g., AAV).

[0329] The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to refer to all forms of nucleic acid, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids include genomic DNA, cDNA and antisense DNA, and spliced or unspliced mRNA, rRNA tRNA and inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh)RNA, microRNA (miRNA), small or short interfering (si)RNA, trans-splicing RNA, or antisense RNA). Nucleic acids include naturally occurring, synthetic, and intentionally modified or altered polynucleotides (e.g., variant nucleic acid). The nucleic acids such as cDNA, genomic DNA, RNA, and fragments thereof which may be single- or double-stranded. Polynucleotides can be single, double, or triplex, linear, or circular, and can be of any length. In discussing polynucleotides, a sequence or structure of a particular polynucleotide may be described herein according to the convention of providing the sequence in the 5’ to 3’ direction.

[0330] A “transgene” is used herein to conveniently refer to a heterologous nucleic acid that is intended or has been introduced into a cell or organism. Transgenes include any heterologous nucleic acid, such as a gene that encodes a polypeptide or protein or encodes an inhibitory RNA. In a plasmid or AAV, the transgene, or heterologous nucleic acid sequence, is often referred to as the “gene of interest” or “GOI”. The heterologous nucleic acid sequence and associated DNA elements employed for transgene expression (e.g., promoters, introns, polyadenylation sequences), often flanked by the AAV inverted terminal repeat (ITR) sequences employed for viral packaging and replication is referred to as the ITR cassette. Since the ITR flanks both ends of the sequence, it is also referred to as the ITR-to- ITR cassette. The ITR cassette is in the plasmid containing the GOI and the genome packaged in the AAV. In certain embodiments, the ITR cassette is separated from the elements of the construct by flanking nucleotides upstream and downstream of the ITR-to- ITR cassette, e.g., inserting a stuffer sequence or cloning site outside (flanking) the ITR-to- ITR cassette. The stuffer sequence or cloning site can be of varied size (number of nucleotides) depending on the size of the ITR-to-ITR cassette and capacity of the plasmid or vector.

[0331] A heterologous nucleic acid can be introduced / transferred by way of vector, such as AAV, through “transduction” or without AAV vector through “transfection” into a cell. The term “transduce,” and grammatical variations thereof refer to introduction of a molecule such as an rAAV vector into a cell or host organism. The introduced heterologous nucleic acid may also exist in the recipient cell or host organism extrachromosomally, or transiently.

[0332] A “transduced cell” is a cell into which the transgene has been introduced. Accordingly, a “transduced” cell (e.g., in a mammal, such as a cell or tissue or organ cell), means a genetic change in a cell following incorporation of an exogenous molecule, for example, a nucleic acid (e.g., a transgene) into the cell. Thus, a “transduced” cell is a cell into which, or a progeny thereof in which an exogenous nucleic acid has been introduced. The cell(s) can be propagated, and the introduced protein expressed, or nucleic acid transcribed. For gene therapy uses and methods, a transduced cell can be in a subject.

[0333] A “transfected” cell is a cell into which at least one plasmid comprising a nucleic acid sequence has been introduced. For example, but not by way of limitation, for AAV production, two to three plasmids can be introduced into a cell and the cell’s transcription and translation machinery utilized to transcribe and translate the nucleic acid sequences encoded by the introduced plasmids. This process leads to the production of AAV components, including capsid proteins and a transgene. After transcription and translation, the AAV components can assemble to form functional AAV vector particles. These particles can contain the transgene within the AAV genome and are capable of transducing target cells. The AAV vector particles thus produced can exit the host cell and transduce neighboring cells.

[0334] A “promoter” is a type of regulatory element and refers to nucleic acid sequence(s) that influence expression of an operably linked nucleic acid. Vector sequences including AAV vectors can include one or more promoter elements. Typically, such promoter elements are included to drive proper heterologous polynucleotide transcription. Such elements typically act in cis, referred to as a “cis acting” element, but may also act in trans.

[0335] Promoters are typically juxtaposed near the 5’ end (z.e., “upstream”) of a transcribed nucleic acid. Promoters can also be located at the 3’ end (z.e., “downstream”) of the transcribed sequence or within the transcript (e.g., in an intron).

[0336] Functionally, expression of operably linked nucleic acid is at least in part controllable by regulatory elements such that the element modulates transcription of the nucleic acid. Regulatory elements typically increase an amount expressed from operably linked nucleic acid as compared to an amount expressed in their absence.

[0337] Promoters are typically located at a distance from the transcribed sequence. In particular embodiments, an expression control element, such as a promoter, is positioned at a specific distance upstream of the rep gene start codon. This distance can vary, ranging from at least about 25 nucleotides to about 5,000 nucleotides upstream of the start codon. In nonlimiting examples, the promoter may be situated about 25-5,000, 250-2,500, 500-2,000, 1,000-1,900, 1,500-1,900, 1,600-1,800, 1,700-1,800 nucleotides, or approximately 1,750 nucleotides upstream of the rep gene start codon.

[0338] Promoters include ubiquitous or promiscuous promoters, which are capable of driving expression of a polynucleotide in many different cell types. Such promoters include but are not limited to the cytomegalovirus (CMV) promoter, the Simian Virus 40 (SV40) promoter, the bidirectional CMV (BICMV) promoter, the Rous sarcoma virus (RSV) promoter and the other viral promoters active in a variety of mammalian cell types, or synthetic elements (see, e.g., Boshart et al., Cell, 41 :521-530 (1985)). Other promoters include, the dihydrofolate reductase promoter, the cytoplasmic P-actin promoter and the phosphoglycerol kinase (PGK) promoter, a pl9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

[0339] Expression control elements also include the native elements(s) for the heterologous polynucleotide. A native control element (e.g., promoter) may be used when it is desired that expression of the heterologous polynucleotide should mimic the native expression. Other native control elements, such as introns, polyadenylation sites or Kozak consensus sequences may also be used.

[0340] The term “operably linked” means that the regulatory sequences necessary for expression of a nucleic acid sequence are placed in the appropriate positions relative to the sequence so as to effect expression of the nucleic acid sequence. This same definition is sometimes applied to the arrangement of nucleic acid sequences and transcription control elements (e.g., promoters, enhancers, and termination elements) in an expression vector, e.g., rAAV vector.

[0341] In the example of an expression control element in operable linkage with a nucleic acid, the relationship is such that the control element modulates expression of the nucleic acid. More specifically, for example, two DNA sequences operably linked means that the two DNAs are arranged (cis or trans) in such a relationship that at least one of the DNA sequences is able to exert a physiological effect upon the other sequence.

[0342] As used herein, nucleic acid sequence with “nonspecific transcription activity” or “NTA” refers to a nucleotide sequence located upstream or downstream of the promoter, where the presence of the nucleic acid sequence with NTA promulgates transcription of a gene downstream to the promoter. For example, and not by way of limitation, the presence in a nucleic acid construct of a LacZ fragment upstream to a T7 promoter can promulgate transcription of a gene downstream to the T7 promoter, whereby expression of the encoded protein is elevated compared to a nucleic acid construct that has all off the above elements except for the nucleic acid sequence with NTA.

[0343] As disclosed herein, a nucleic acid spacer sequence positioned between an expression control element and an AAV rep gene can substantially reduce or eliminate expression of the rep gene thereby in turn reducing or eliminating expression of the Rep protein and allowing cells to survive even while the cells also express adenovirus El a protein. Addition of helper virus function to such cells, such as provided by a hybrid virus, adenovirus, poxvirus or herpesvirus, can overcome the attenuating effect of the spacer nucleic acid on rep gene expression and in turn drive expression of rep gene thereby providing Rep protein expression. As used herein, the term “small Rep” refers to a Rep52 or Rep40 unless otherwise stated. As used herein, the term “large Rep” refers to a Rep78 or Rep68 unless otherwise stated.

[0344] Additional elements for rAAV vectors include, without limitation, a transcription termination signal or stop codon, 5’ or 3’ untranslated regions (e.g., polyadenylation (poly A) sequences) which flank a sequence, such as one or more copies of an AAV ITR sequence, or an intron.

[0345] Further elements include, for example, filler or stuffer polynucleotide sequences, for example to improve packaging and reduce the presence of contaminating nucleic acid. AAV vectors typically accept inserts of DNA having a size range which is generally about 0.5kb to about 5.2 kb, or slightly more. Thus, for shorter sequences, inclusion of a stuffer or filler in order to adjust the length to near or at the normal size of the virus genomic sequence acceptable for AAV vector packaging into virus particle. In various embodiments, a filler / stuffer nucleic acid sequence is an untranslated (non-protein encoding) segment of nucleic acid. For an ITR cassette where the nucleic acid sequence is less than 4.7 kb, the filler or stuffer polynucleotide sequence has a length that when combined (e.g., inserted into a vector) with the sequence has a total length between about 3.0-5.5 kb, or between about 4.0- 5.0 kb, or between about 4.3-4.8 kb. The stuffer sequence is inert and does not cause a reduction in rAAV vector titers.

[0346] In certain embodiments, the stuffer sequence separates the ITR cassette from the elements of a transgene (e.g., GO I) by flanking nucleotides upstream and downstream of the ITR cassette.

[0347] In certain embodiments, the stuffer sequence is inserted inside the ITR cassette (ITR- to-ITR cassette). In certain embodiments, one or more stuffer sequences are used to facilitate an appropriate sizing of the ITR cassette within the AAV, and / or create separation between AAV genome elements. For example, in certain non-limiting embodiments, the stuffer sequence may be inserted inside the ITR cassette between one or more of rep, cap, or helper genes. In certain non-limiting embodiments, the stuffer sequence may be inserted inside the ITR cassette between rep and cap. The size of the stuffer sequence can, in certain embodiments, depend on the size of the ITR cassette and capacity of the plasmid or vector.

[0348] Where a wild type heterologous nucleic acid or transgene is too large to be packaged within an AAV vector particle, the heterologous nucleic acid may be provided in modified, fragmented or truncated form for packaging in and delivery by an AAV vector, such that a functional protein or nucleic acid product, such as a therapeutic protein or nucleic acid product, is ultimately provided.

[0349] In some embodiments, the heterologous nucleic acid that encodes a protein (e.g., therapeutic protein) is provided in modified or truncated forms or the heterologous nucleic acid is provided in multiple constructs, delivered by separate and multiple AAV vectors.

[0350] In certain aspects, the heterologous nucleic acid is provided as a truncated variant that maintains functionality of the encoded protein (e.g., therapeutic protein), including removal of portions unnecessary for function, such that the encoding heterologous polynucleotide is reduced in size for packaging in an AAV vector.

[0351] In certain aspects the heterologous nucleic acid is provided in split AAV vectors, each providing nucleic acid encoding different portions of a protein (e.g., therapeutic protein), thus delivering multiple portions of a protein (e.g., therapeutic protein) which assemble and function in the cell.

[0352] In other aspects, the heterologous nucleic acid is provided by dual AAV vectors using overlapping, trans-splicing or hybrid trans-splicing dual vector technology. In certain embodiments, two overlapping AAV vectors are used which combine in the cell to generate a full expression cassette, from which a full-length protein (e.g., therapeutic protein) is expressed.

[0353] A “hemostasis related disorder” refers to bleeding disorders such as hemophilia A, hemophilia A with inhibitory antibodies, hemophilia B, hemophilia B with inhibitory antibodies, a deficiency in any coagulation Factor: VII, VIII, IX, X, XI, V, XII, II, von Willebrand factor, combined FV / FVIII deficiency, thalassemia, vitamin K epoxide reductase Cl deficiency, or gamma-carboxylase deficiency; bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, or disseminated intravascular coagulation (DIC); over-anticoagulation associated with heparin, low molecular weight heparin, pentasaccharide, warfarin, or small molecule antithrombotics (i.e., Fxa inhibitors); and platelet disorders such as, Bernard Soulier syndrome, Glanzmann thrombasthenia, and storage pool deficiency.

[0354] The term “isolated,” when used as a modifier of a composition, means that the compositions are made by the hand of man or are separated, completely or at least in part, from their naturally occurring in vivo environment. Generally, isolated compositions are substantially free of one or more materials with which they normally associate with in nature, for example, one or more protein, nucleic acid, lipid, carbohydrate, cell membrane. The term “isolated” does not exclude combinations produced by the hand of man, for example, a rAAV sequence, or rAAV particle that packages or encapsidates an AAV vector genome and a pharmaceutical formulation. The term “isolated” also does not exclude alternative physical forms of the composition, such as hybrids / chimeras, multimers / oligomers, modifications (e.g., phosphorylation, glycosylation, lipidation) or derivatized forms, or forms expressed in host cells produced by the hand of man.

[0355] The term “substantially pure” refers to a preparation comprising at least 50-60% by weight of the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). The preparation can comprise at least 75% by weight, or at least 85% by weight, or about 90-99% by weight of the compound of interest. Purity is measured by methods appropriate for the compound of interest (e.g., chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).

[0356] The phrase “consisting essentially of’ when referring to a particular nucleotide sequence or amino acid sequence means a sequence having the properties of a given SEQ ID NO. For example, when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the basic and novel characteristics of the sequence.

[0357] The term “identity,” “homology” and grammatical variations thereof, mean that two or more referenced entities are the same, when they are “aligned” sequences. Thus, by way of example, when two protein sequences are identical, they have the same amino acid sequence, at least within the referenced region or portion. Where two nucleic acid sequences are identical, they have the same nucleic acid sequence, at least within the referenced region or portion. The identity can be over a defined area (region or domain) of the sequence.

[0358] An “area” or “region” of identity refers to a portion of two or more referenced entities that are the same. Thus, where two protein or nucleic acid sequences are identical over one or more sequence areas or regions, they share identity within that region. An “aligned” sequence refers to multiple protein (amino acid) or nucleic acid sequences, often containing corrections for missing or additional bases or amino acids (gaps) as compared to a reference sequence.

[0359] The identity can extend over the entire length or a portion of the sequence. In certain embodiments, the length of the sequence sharing the percent identity is 2, 3, 4, 5 or more contiguous amino acids or nucleic acids, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. contiguous nucleic acids or amino acids. In additional embodiments, the length of the sequence sharing identity is 21 or more contiguous amino acids or nucleic acids, e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, etc. contiguous amino acids or nucleic acids. In further embodiments, the length of the sequence sharing identity is 41 or more contiguous amino acids or nucleic acids, e.g., 42, 43, 44, 45, 45, 47, 48, 49, 50, etc., contiguous amino acids or nucleic acids. In yet further embodiments, the length of the sequence sharing identity is 50 or more contiguous amino acids or nucleic acids, e.g., 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 100-150, 150-200, 200-250, 250-300, 300-500, 500-1,000, etc. contiguous amino acids or nucleic acids.

[0360] The extent of identity (homology) or “percent identity” between two sequences can be ascertained using a computer program and / or mathematical algorithm. For purposes of this disclosure comparisons of nucleic acid sequences are performed using the GCG Wisconsin Package version 9.1, available from the Genetics Computer Group in Madison, Wisconsin. For convenience, the default parameters (gap creation penalty = 12, gap extension penalty = 4) specified by that program are intended for use herein to compare sequence identity. Alternately, the Blastn 2.0 program provided by the National Center for Biotechnology Information (found on the world wide web at ncbi.nlm.nih.gov / blast / ; Altschul et al., 1990, J Mol Biol 215:403-410) using a gapped alignment with default parameters, may be used to determine the level of identity and similarity between nucleic acid sequences and amino acid sequences. For polypeptide sequence comparisons, a BLASTP algorithm is typically used in combination with a scoring matrix, such as PAM100, PAM 250, BLOSUM 62 or BLOSUM 50. FASTA (e.g., FASTA2 and FASTA3) and SSEARCH sequence comparison programs are also used to quantitate extent of identity (Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444 (1988); Pearson, Methods Mol Biol. 132: 185 (2000); and Smith et al., J. Mol. Biol. 147: 195 (1981)). Programs for quantitating protein structural similarity using Delaunay -based topological mapping have also been developed (Bostick et al., Biochem Biophys Res Commun. 304:320 (2003)).

[0361] Nucleic acid molecules, expression vectors (e.g., AAV vector genomes), plasmids, including nucleic acid encoding modified / variant AAV capsids of the subject matter disclosed herein, and heterologous nucleic acids may be prepared by using recombinant DNA technology methods. The availability of nucleotide sequence information enables preparation of isolated nucleic acid molecules of the subject matter disclosed herein by a variety of means. For example, nucleic acid sequences can be made using various standard cloning, recombinant DNA technology, via cell expression or in vitro translation and chemical synthesis techniques. Purity of polynucleotides can be determined through sequencing, gel electrophoresis and the like. For example, nucleic acids can be isolated using hybridization or computer-based database screening techniques. Such techniques include, but are not limited to: (1) hybridization of genomic DNA or cDNA libraries with probes to detect homologous nucleotide sequences; (2) antibody screening to detect polypeptides having shared structural features, for example, using an expression library; (3) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers capable of annealing to a nucleic acid sequence of interest; (4) computer searches of sequence databases for related sequences; and (5) differential screening of a subtracted nucleic acid library.

[0362] 2. Nucleic acids providing helper virus function.

[0363] In one aspect, the subject matter of the present disclosure is directed to a nucleic acid comprising coding sequences providing helper virus function. In certain embodiments, the coding sequences providing a helper virus function are derived from a virus selected from adenovirus, herpesvirus, poxvirus, or a hybrid virus thereof. In certain embodiments, a helper virus function comprises vectors or plasmids that provide the helper virus function.

[0364] In certain embodiments, the nucleic acid providing helper virus function comprises a DNA Binding Protein (DBP) coding sequence; an Open Reading Frame 6 (0RF6), or an Open Reading Frame 6 / 7 (ORF 6 / 7) coding sequence; and a Viral Associated (VA) coding sequence, where each of the DBP, the 0RF6 (or the ORF 6 / 7) and the VA coding sequences are operably linked to a constitutive promoter. In certain embodiments at least one of the coding sequences providing a helper virus function are operably linked independently to a constitutive promoter. In certain embodiments two of the coding sequences providing a helper virus function are operably linked to a common constitutive promoter, and the third coding sequence providing helper virus function is operably linked to a different constitutive promoter.

[0365] In certain embodiments, the VA coding sequence comprises a VA_a coding sequence, a VA_b coding sequence, a VA_c coding sequence, a VA_d coding sequence, a VA_e coding sequence, or a VA_f coding sequence. In certain embodiments, the VA coding sequence comprises a VA_a coding sequence. In some embodiments, the VA coding sequence comprises a VA_b coding sequence. In some embodiments, the VA coding sequence comprises a VA_f coding sequence.

[0366] In certain embodiments, the nucleic acid providing helper virus function comprise DBP, L4-22K, ORF6 and VA_b.

[0367] In certain embodiments, the nucleic acid providing helper virus function comprise DBP, L4-22K, ORF6 and VA_f. Exemplary nucleic acid sequences for the DBP, 0RF6, ORF 6 / 7, and VA coding sequences are shown below in Table 1.

[0368] Table 1. Exemplary Coding Sequences

[0369]

[0370] In certain embodiments, the nucleic acid providing helper virus function further comprises an L4-22K coding sequence operably linked to a constitutive promoter. In certain embodiments, the L4-22K coding sequence is fused to the DBP coding sequence. In certain embodiments, the L4-22K coding sequence is fused to the 0RF6 coding sequence. In certain embodiments, the L4-22K coding sequence is fused to the ORF6 / 7 coding sequence. In any of the above embodiments, the L4-22K fusion construct comprises a 2A peptide coding sequence between the L4-22K coding sequence and the DBP coding sequence, the 0RF6 coding sequence and / or the ORF6 / 7 coding sequence. Non-limiting examples of 2A peptide coding sequence that find use in such embodiments include, T2A, P2A, E2A and F2A.

[0371] In certain embodiments, the nucleic acid providing helper virus function comprises an L4-22K_Alt coding sequence operably linked to a constitutive promoter.

[0372] In certain embodiments, the L4-22K_Alt coding sequence is fused to the DBP coding sequence. In certain embodiments, the L4-22K_Alt coding sequence is fused to the ORF6 coding sequence. In certain embodiments, the L4-22K_Alt coding sequence is fused to the ORF6 / 7 coding sequence. In any of the above embodiments, the L4-22K_Alt fusion comprises a 2A peptide coding sequence between the L4-22K_Alt coding sequence and the DBP coding sequence, the 0RF6 coding sequence and / or the ORF6 / 7 coding sequence. Nonlimiting examples of 2A peptide coding sequence that find use in such embodiments include T2A, P2A, E2A and F2A.

[0373] Exemplary nucleic acid sequences for the L4-22K and L4-24K_Alt coding sequences and their fusion constructs are shown in Table 1.

[0374] In certain embodiments, the L4-22K coding sequence or the L4-22K_alt coding sequence comprises at least one mutation. In identifying these mutations, the amino acid and its position are stated first, followed by the new amino acid replacing the initial residue at that position. For example, Y20F indicates that the tyrosine at position 20 is replaced with phenylalanine.

[0375] In certain embodiments, L4-22K mutation(s) enhance production of rAAV particles (vg titers). Non-limiting examples of L4-22K mutations that enhance viral titers include, Y20F, T79A, T99A, H169Q, Y171F, Y174F and T189A. In certain embodiments, one or more of the mutations identified as associated with enhanced viral titers in L4-22K (e.g., Y20F, T79A, T99A, H169Q, Y171F, Y174F and T189A) can be introduced into the equivalent position in the L4-22K_alt coding sequence.

[0376] In certain embodiments, L4-22K mutation(s) decrease production of rAAV particles (vg titers). Non-limiting examples of L4-22K mutations that decrease viral titers include, P13A / P14 / A, T15A, T55A, S64A, T72A, T100A, Y123F, R148A / L151A, R149A / L152A, H165Q, Y166F and Y167F. In certain embodiments, one or more of the mutations identified as associated with decreased viral titers in L4-22K (e.g., P13A / P14 / A, T15A, T55A, S64A, T72A, T100A, Y123F, R148A / L151A, R149A / L152A, H165Q, Y166F and Y167F) can be introduced into the equivalent position(s) in the L4-22K_alt coding sequence.

[0377] In certain embodiments, L4-22K mutation(s) do not change production of rAAV particles (vg titers). Non-limiting examples of L4-22K mutations that have no effect on viral titers include, P13A, P14A, S80A, T102A, S125A, S145A, H165Q / H169Q, S172A, C175S, T176A, S181A, and T194A. In certain embodiments, one or more of the mutations identified as associated with no effect on viral titers in L4-22K (e.g., P13A, P14A, S80A, T102A, S125A, S145A, H165Q / H169Q, S172A, C175S, T176A, S181A, and T194A) can be introduced into the equivalent position(s) in the L4-22K_alt coding sequence.

[0378] The methods of the present disclosure comprise the use of suitable regulatory elements, including promoters to drive the expression of Rep and Cap proteins. In certain embodiments, suitable promoters may be eukaryotic, prokaryotic, or viral promoters. Suitable promoters include non-inducible promoters and non-tissue specific promoters. In certain embodiments, the promoter is an AAV p5 promoter, which in its native state drives Rep protein expression from the rep gene. In certain embodiments, the promoter is the cytomegalovirus (CMV) immediate early promoter / enhancer. Additional nonlimiting examples of suitable promoters include ubiquitous or promiscuous promoters which are capable of driving expression of a polynucleotide in many different cell types. Such elements include but are not limited to the Rous sarcoma virus (RSV) promoter sequences and the other viral promoters active in a variety of mammalian cell types, or synthetic elements (see, e.g., Boshart et al., Cell, 41 :521-530 (1985)), the Simian Virus 40 (SV40) promoter, the bidirectional CMV (BICMV) promoter, the dihydrofolate reductase promoter, the cytoplasmic b-actin promoter and the phosphoglycerol kinase (PGK) promoter. In certain embodiments, the promoter is selected from the human elongation factor- la EFl alpha promoter, the CMV early enhancer / chicken P actin (CAG) promoter, the CBA promoter, the SFFV promoter, the pl9 promoter, the herpes simplex virus thymidine kinase (HSV-TK) promoter, the p40 promoter, the p41 promoter, the tetracycline (Tet) inducible promoter, and the Bidirectional Tet (BiTet) inducible promoter. In certain embodiments promoters may be located proximal or distal to the Rep, the Cap genes and the coding sequences providing helper virus function. In certain embodiments the promoters, may be in cis or trans to the Rep genes, Cap genes and genes providing helper virus function. Nucleic acid sequences for exemplary promoters are shown in Table 1. In certain embodiments, the methods of the present disclosure are directed to expression of a nucleic acid encoding an AAV Rep protein, wherein the AAV Rep protein is an AAV1 Rep protein, an AAV2 Rep protein, an AAV3 Rep protein, anAAV4 Rep protein, an AAV5 Rep protein, an AAV6 Rep protein, an AAV7 Rep protein, an AAV8 Rep protein, an AAV9 Rep protein, an AAV 10 Rep protein, or an AAV 11 Rep protein. Because the methods of the present disclosure are applicable to any cell type that produces AAV, the AAV can be, e.g., human, avian, bovine, canine, equine, primate, non-primate, ovine, or any derivation thereof.

[0379] In certain embodiments, the rAAV produced by the methods of the present disclosure include any viral strain or serotype. In certain, non-limiting, embodiments, an rAAV can be based upon any AAV genome, including, but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hul4), AAV10, AAV11, AAV12, AAV13, Rh8, RhlO, Rh74, AAV3B, AAV-2i8, LK03, RHM4-1, DJ, DJ8, NP59, Anc-80 and variants thereof, including the variants of AAV capsids set forth in Pulicherla et al., Mol. Ther., 19(6) 1070-1078 (2011) (describing AAV9 variants including AAV9.47 among others), U.S. Patent No. 7,906,111 (describing AAV9(hul4) among others), U.S. Patent No. 10,532,111 (describing NP59 among others), U.S. Patent No. US10738087 (describing Anc- 80 among others), W02012 / 145601, WO2013 / 158879, W02015 / 013313, WO2018 / 156654, US2013 / 0059732, U.S. Patent Nos. 9,169,299 (describing LK03), 9,840,719 (describing RHM4-1), 7,749,492, 7,588,772 (describing DJ and DJ8), and 9,587,282, all of which are incorporated herein by reference in their entireties. rAAV vectors therefore include gene / protein sequences identical to gene / protein sequences characteristic for a particular serotype, as well as mixed serotypes. In certain embodiments, the AAV serotypes can be matched to target cell types. For example, in certain non-limiting embodiments, AAV serotypes selected from the group consisting of AAV3, AAV5, AAV8, and AAV9 can be used to target liver cells.

[0380] In certain embodiments, the nucleic acid encoding AAV Rep and Cap proteins is arranged as in the native AAV genome. In certain embodiments, the nucleic acids encoding AAV Rep and Cap proteins are not arranged, directly or indirectly, in a tandem configuration. In certain embodiments, the nucleic acids encoding AAV Rep and Cap proteins are not covalently linked. Additional suitable arrangements of the AAV Rep and Cap coding sequences can be employed, e.g., the order of the AAV Rep and Cap coding sequences can be reversed relative to their native AAV genomic order, one or both of the AAV Rep and Cap coding sequences can be preceded or followed by a sequence comprising an IRES, a selfcleaving protein (e.g., a 2 A peptide) coding sequence, or stuff er region having no function.

[0381] In certain embodiments, AAV coding sequence or a nucleic acid construct comprising the AAV coding sequence comprises a Cap sequence that is contiguous with a Rep sequence. In certain embodiments, AAV coding sequence or a nucleic acid construct comprising the AAV coding sequence comprises stuffer sequence between the Cap sequence and the Rep sequence. In certain embodiments, the stuffer sequence has no effect on production of rAAV vector particles. In certain embodiments, the stuffer sequence is about 4 kb to about 5.2 kb. In certain embodiments, the stuffer sequence is about 3.0 kb to about 5.5 kb. In certain embodiments, the stuffer sequence is about 4.0 kb to about 5.0 kb. In certain embodiments, the stuffer sequence is about 4.3 kb to about 4.8 kb. In certain embodiments, the stuffer sequence is about 0.5 kb to about 1 kb. In certain embodiments, the stuffer sequence is about 1 kb to about 3 kb. In certain embodiments, the stuffer sequence is about 2 kb to about 2.5 kb. In certain embodiments, the stuffer sequence is about 2 kb to about 3 kb. For example, in some non-limiting embodiments the AAV coding sequence or a nucleic acid construct comprising the AAV coding sequence comprises a 1 kb stuffer sequence between the Rep gene and Cap gene. In some non-limiting embodiments, the AAV coding sequence or a nucleic acid construct comprising the AAV coding sequence comprises a 3 kb stuffer sequence between the Rep gene and Cap gene.

[0382] In certain embodiments, one or both of the AAV Rep and Cap coding sequences can be incorporated into a cell genome. In certain embodiments, one or both of the AAV Rep and Cap coding sequences can be combined with factors that increase DNA sequences that increase episomal plasmid maintenance.

[0383] In certain embodiments, a helper virus function is provided by a virus selected from adenovirus, herpesvirus, poxvirus, or a hybrid virus thereof. In certain embodiments, a helper virus function comprises one or more viruses, vectors or plasmids that provide the helper virus function. In certain embodiments, the helper virus function comprises at least one of adenovirus (Ad) El protein (e.g., Ad Ela or Ad Elb protein), Ad E2A protein, Ad E4 protein and Ad VA RNA.

[0384] The helper virus function can be provided in a number of different ways. In certain embodiments, the helper virus function can be provided by a virus or, for example, provided by polynucleotide sequences encoding the requisite helper function(s) to a cell in trans.

[0385] Exemplary nucleic acid sequences for plasmids providing helper virus are shown in

[0386] Table 1 In certain embodiments, the one or more nucleic acid sequences providing helper virus function in connection with the compositions and methods disclosed herein are located on two nucleotide constructs (dual nucleic acid constructs) that comprise a dual transfection system.

[0387] In certain embodiments of such dual transfection systems, there is provided a first nucleic acid construct comprising: one or more of an rAAV coding sequence; an AAV REP coding sequence; an AAV CAP coding sequence; and one or more coding sequence providing helper virus function selected from a DBP coding sequence, an ORF6 and / or ORF 6 / 7 coding sequence and a VA coding sequence. In certain embodiments, the AAV REP coding sequence is operably linked to: (1) at least one promoter; and (2) a nucleic acid sequence with nonspecific transcription activity disposed upstream or downstream to the at least one promoter. In certain embodiments of such dual transfection systems, there is provided a second nucleic acid construct comprising: a gene of interest (GOI) coding sequence; and one or more of an rAAV coding sequence; an AAV REP coding sequence; and an AAV CAP coding sequence; and one or more coding sequence providing helper virus function selected from, a DBP coding sequence, an ORF6 and / or ORF6 / 7 coding sequence, and a VA coding sequence. In certain embodiments, each of the DBP coding sequence, ORF6 and / or ORF6 / 7 coding sequence, and VA coding sequence is operably linked to a constitutive promoter.

[0388] In certain embodiments, of such dual transfection systems, there is provided a first nucleic acid construct comprising: one or more of an rAAV coding sequence; an AAV REP coding sequence; and an AAV CAP coding sequence; and one or more coding sequence providing helper virus function selected from a DBP coding sequence, an ORF6 and / or ORF 6 / 7 coding sequence, and a VA coding sequence. In certain embodiments of such dual transfection systems, the AAV REP coding sequence is operably linked to: (1) at least one promoter; and (2) a nucleic acid sequence with nonspecific transcription activity disposed upstream to the at least one promoter. In certain embodiments, of such dual transfection systems, an AAV REP coding sequence is operably linked to: (1) at least one promoter; and (2) a nucleic acid sequence with nonspecific transcription activity disposed downstream to the at least one promoter. In certain embodiments of such dual transfection systems, each of the DBP coding sequence, the ORF6 and / or ORF6 / 7 coding sequence, and the VA coding sequence is operably linked to a constitutive promoter. In certain embodiments of such dual transfection systems, there is provided a second nucleic acid construct comprising a gene of interest (GOI) coding sequence. Exemplary nucleic acid sequences for the dual nucleic acid constructs are shown in Table 2, below.

[0389] Table 2. Exemplary Construct Sequences

[0390] 3. AA V Producing Cells

[0391] In another aspect, the subject matter of the present disclosure is directed to rAAV producing cells. For example, but not limitation, the present disclosure is directed to rAAV producing cells comprising coding sequences that provide helper virus function.

[0392] The embodiments of the rAAV producing cells of the present disclosure include any cell type or system that can produce rAAV or AAV. Several examples of various systems have been previously described, e.g., Conlon and Mavilio, Mol. Therapy, 8: 181-182 (2018), which is incorporated herein by reference in its entirety. Embodiments of the present disclosure include making rAAV through transient transfection of plasmids in mammalian cells, production of rAAV in stable cell lines, rAAV through herpes simplex virus in mammalian cells, and rAAV production through baculovirus in Sf9 cells.

[0393] In certain embodiments, the rAAV producing cells of the present disclosure are eukaryotic cells. In certain embodiments, the rAAV producing cells of the present disclosure are animal cells. In certain embodiments, the rAAV producing cells of the present disclosure are insect cells. In certain embodiments, the rAAV producing cells of the present disclosure mammalian cells. In certain embodiments, the rAAV producing cells of the present disclosure are human cells. In certain embodiments, the rAAV producing cells of the present disclosure human embryonic kidney (HEK) cells. In certain embodiments, the rAAV producing cells of the present disclosure are HEK293 cells, HEK293F cells, or Expi293 cells.

[0394] In certain embodiments, the rAAV producing cell is a Ela expressing cell. In certain embodiments, the rAAV producing cell is a mammalian cell. In certain embodiments, the rAAV producing cell is a human cell. In certain embodiments, the rAAV producing cell is a transfected rAAV producing cell. In certain embodiments, the rAAV producing cells of the present disclosure are HEK293 cells. In certain embodiments, the cells are HEK293F cells.

[0395] In certain embodiments, the rAAV producing cells of the present disclosure are PERC6 cells.

[0396] In certain embodiments, the rAAV producing cells of the present disclosure are Chinese hamster ovary (CHO) cells. In certain embodiments, the rAAV producing cells of the present disclosure are insect (Sf9) cells.

[0397] In certain embodiments, the rAAV producing cells of the present disclosure do not express SV40 large T antigen.

[0398] In certain embodiments, the rAAV producing cells of the present disclosure are suspension cells. In certain embodiments, the rAAV producing cells of the present disclosure are adherent cells.

[0399] In certain embodiments, the rAAV producing cells of the present disclosure can be cultured at a cell density of at least about IxlO6, at least about 5xl06, at least about IxlO7or at least about 2xl07cells / mL. In certain embodiments, the rAAV producing cells of the present disclosure can be cultured at a cell density from about IxlO6- 5xl06, from about 5xl06-lxl07, or from about 1X107-2X107cells / mL.

[0400] In certain embodiments, the rAAV producing cells of the present disclosure are present in a culture or growth medium.

[0401] In certain embodiments, the rAAV producing cells of the present disclosure are in medium suitable for storage. In certain embodiments, the rAAV producing cells of the present disclosure are in a medium suitable for long-term storage at or below 0°, at or below - 30°, at or below -80° or at or below -160° C.

[0402] In certain embodiments, the present disclosure is directed to a rAAV producing cell, wherein the cell comprises nucleic acids comprising an rAAV coding sequence; an AAV Rep coding sequence; an AAV CAP coding sequence; and coding sequences providing helper virus function comprising a DNA Binding Protein (DBP) coding sequence; an Open Reading Frame 6 (ORF6), or an Open Reading Frame 6 / 7 (ORF6 / 7) coding sequence; and a Viral Associated (VA) coding sequence where each of the DBP, the ORF6 (or the ORF 6 / 7) and the VA coding sequences are operably linked to a constitutive promoter.

[0403] In certain embodiments, the rAAV producing cell of the present disclosure is a Ela- expressing cell. In certain embodiments, the E la-expressing cell is a HEK293 cell.

[0404] In certain embodiments, the rAAV producing cell of the present disclosure comprises a nucleic acid encoding AAV Rep and Cap proteins arranged as in the native AAV genome. In certain embodiments, the Rep protein is a Rep78, Rep68, Rep52, or Rep40 protein. In certain embodiments, the nucleic acids encoding AAV Rep and Cap proteins are not arranged, directly or indirectly, in a tandem configuration. In certain embodiments, the nucleic acids encoding AAV Rep and Cap proteins are not covalently linked. Additional suitable arrangements of the AAV Rep and Cap coding sequences can be employed, e.g., the order of the AAV Rep and Cap coding sequences can be reversed relative to their native AAV genomic order, one or both of the AAV Rep and Cap coding sequences can be preceded or followed by a sequence comprising an IRES, a self-cleaving protein (e.g., a 2A peptide) coding sequence, or stuffer region having no function. In certain embodiments, one or both of the AAV Rep and Cap coding sequences can be incorporated into a cell genome. In certain embodiments, one or both of the AAV Rep and Cap coding sequences can be combined with factors that increase DNA sequences that increase episomal plasmid maintenance.

[0405] In certain embodiments, AAV coding sequence or a nucleic acid construct comprising the AAV coding sequence comprises a Cap sequence that is contiguous with a Rep sequence. In certain embodiments, AAV coding sequence or a nucleic acid construct comprising the AAV coding sequence comprises stuffer sequence between the Cap sequence and the Rep sequence. In certain embodiments, the stuffer sequence has no effect on production of rAAV vector particles. In certain embodiments, the stuffer sequence is about 4 kb to about 5.2 kb. In certain embodiments, the stuffer sequence is about 3.0 kb to about 5.5 kb. In certain embodiments, the stuffer sequence is about 4.0 kb to about 5.0 kb. In certain embodiments, the stuffer sequence is about 4.3 kb to about 4.8 kb. In certain embodiments, the stuffer sequence is about 0.5 kb to about 1 kb. In certain embodiments, the stuffer sequence is about 1 kb to about 3 kb. In certain embodiments, the stuffer sequence is about 2 kb to about 2.5 kb. In certain embodiments, the stuffer sequence is about 2 kb to about 3 kb. For example, in some non-limiting embodiments the AAV coding sequence or a nucleic acid construct comprising the AAV coding sequence comprises a 1 kb stuffer sequence between the Rep gene and Cap gene. In some non-limiting embodiments, the AAV coding sequence or a nucleic acid construct comprising the AAV coding sequence comprises a 3 kb stuffer sequence between the Rep gene and Cap gene. As noted above, a stuffer sequence can be of varied size (i.e., number of nucleotides) depending on the size of the ITR-to-ITR cassette, the desired spacing between genetic elements within the ITR-to-ITR cassette or outside of the ITR-to-ITR cassette, as well as depending on the capacity of the plasmid or vector.

[0406] In certain embodiments, the stuffer sequence separates the ITR cassette from the elements of a transgene (e.g., GO I) by flanking nucleotides upstream and downstream of the ITR cassette.

[0407] In certain embodiments, the stuffer sequence is inserted inside the ITR cassette (ITR- to-ITR cassette). In some embodiments, stuffer sequences are used to make the ITR cassette fit the AAV, and / or create separation between AAV genome elements. For example, in certain non-limiting embodiments, the stuffer sequence may be inserted inside the ITR cassette between one or more of rep, cap, or helper genes. In certain non-limiting embodiments, the stuffer sequence may be inserted inside the ITR cassette between rep and cap. The size of the stuffer sequence depends on the size of the ITR cassette and capacity of the plasmid or vector.

[0408] Exemplary codon optimized sequences for Rep78 are shown in Table 1.

[0409] In certain embodiments, the rAAV producing cell of the present disclosure comprise coding sequences capable of providing helper virus function. In certain embodiments, the coding sequences are derived from a virus selected from adenovirus, herpesvirus, poxvirus, or a hybrid virus thereof. In certain embodiments, the rAAV producing cell of the present disclosure comprises one or more vectors or plasmids that provide the helper virus function. In certain embodiments, the helper virus function comprises DBP, the ORF6 (or the ORF 6 / 7) and the VA coding sequences, where these sequences are operably linked to a constitutive promoter.

[0410] In some embodiments, the VA coding sequence in the rAAV producing cell of the present disclosure comprises a VA_a coding sequence, a VA_b coding sequence, a VA_c coding sequence, a VA_d coding sequence, a VA_e coding sequence, or a VA_f coding sequence. In some embodiments, the VA coding sequence comprises a VA_a coding sequence. In some embodiments, the VA coding sequence comprises a VA_b coding sequence. In some embodiments, the VA coding sequence comprises a VA_f coding sequence.

[0411] Exemplary nucleic acid sequences for the DBP, ORF6, ORF6 / 7, and VA coding sequences are shown in Table 1.

[0412] In certain embodiments, the rAAV producing cell of the present disclosure further comprises an L4-22K coding sequence for providing helper virus function, wherein the L4- 22K sequence is operably linked to a constitutive promoter. In certain embodiments, the rAAV producing cell of the present disclosure further comprises an L4-22K_Alt coding sequence for providing helper virus function, wherein the L4-22K_Alt sequence is operably linked to a constitutive promoter. In either embodiment, the L4-22K coding sequence or the L4-22K_Alt coding sequence is fused to one or more of the DBP, the ORF6, and the ORF6 / 7 coding sequences to form a fusion construct.

[0413] In some embodiments, the L4-22K fusion construct comprises a 2A peptide coding sequence between the L4-22K coding sequence and the DBP coding sequence, the ORF6 coding sequence and / or the ORF6 / 7 coding sequence. Non-limiting examples of 2A peptide coding sequence include, T2A, P2A, E2A and F2A.

[0414] Exemplary nucleic acid sequences for the L4-22K and L4-22K_Alt coding sequences and their fusion constructs are shown in Table 2.

[0415] In some embodiments, the rAAV producing cell comprises a nucleic acid sequence comprising DBP, L4-22K, ORF6 and VA_b coding sequences for providing helper virus function.

[0416] In some embodiments, the rAAV producing cell comprises a nucleic acid sequence comprising DBP, L4-22K, ORF6 and VA_f coding sequences for providing helper virus function.

[0417] In certain embodiments, the rAAV producing cell of the present disclosure comprises amino acid sequence that is not normally present in the wild-type AAV genome, e.g., a heterologous nucleic acid sequence, also referred to herein as a gene of interest or (GOI). In certain non-limiting embodiments, the GOI comprises a nucleic acid sequence encoding a therapeutic protein or an inhibitory nucleic acid sequence. In certain embodiments, the GOI can be introduced / transferred by way of vector, such as AAV transduction or transfection into a cell. In certain embodiments, the introduced GOI can also exist in the recipient cell or host organism extrachromosomally, or only transiently.

[0418] In certain embodiments, the GOI encodes a protein (e.g., therapeutic protein) that is provided in modified or truncated forms or the GOI is provided in multiple constructs, delivered by separate and multiple AAV vectors.

[0419] In certain embodiments, the GOI is provided as a truncated variant that maintains functionality of the encoded protein (e.g., therapeutic protein), including removal of portions unnecessary for function, such that the GOI is reduced in size for packaging in an AAV vector.

[0420] In certain embodiments, the GOI is provided in split AAV vectors, each providing nucleic acid encoding different portions of a protein (e.g., therapeutic protein), thus delivering multiple portions of a protein (e.g., therapeutic protein) which assemble and function in the cell.

[0421] In certain embodiments, the GOI is provided by dual AAV vectors using overlapping, trans- splicing or hybrid trans- splicing dual vector technology. In certain embodiments, two overlapping AAV vectors are used which combine in the rAAV producing cell of the present disclosure to generate a full expression cassette, from which a full-length protein (e.g., therapeutic protein) is expressed. Non-limiting examples of heterologous nucleic acids encoding gene products (e.g., therapeutic proteins) which are useful in accordance with the invention include those that may be used in the treatment of a disease or disorder including, but not limited to, "hemostasis" or blood clotting disorders such as hemophilia A, hemophilia A patients with inhibitory antibodies, hemophilia B, deficiencies in coagulation Factors, VII, VIII, IX and X, XI, V, XII, II, von Willebrand factor, combined FV / FVIII deficiency, thalassemia, vitamin K epoxide reductase CI deficiency, gamma-carboxylase deficiency; anemia, bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC); over-anticoagulation associated with heparin, low molecular weight heparin, pentasaccharide, warfarin, small molecule antithrombotics (i.e. FXa inhibitors); and platelet disorders such as, Bernard Soulier syndrome, Glanzman thrombasthenia, and storage pool deficiency.

[0422] In certain embodiments, the disease or disorder affects or originates in the central nervous system (CNS). In certain embodiments, the disease is a neurodegenerative disease. In certain embodiments, the CNS or neurodegenerative disease is Alzheimer’s disease, Huntington's disease, ALS, hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, Kennedy’s disease, a polyglutamine repeat disease, or Parkinson's disease. In certain embodiments, the CNS or neurodegenerative disease is a polyglutamine repeat disease. In certain embodiments, the polyglutamine repeat disease is a spinocerebellar ataxia (SCA1, SCA2, SCA3, SCA6, SCA7, or SCA17).

[0423] In certain embodiments, the AAV particles comprise a heterologous nucleic acid encoding a gene product selected from the group consisting of insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietins, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor a (TGFa), platelet-derived growth factor (PDGF), insulin growth factors I and II (IGF-I and IGF-II), TGFp, activins, inhibins, bone morphogenic protein (BMP), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell line derived neurotrophic factor (GDNF), neurturin, agrin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrins, noggin, sonic hedgehog and tyrosine hydroxylase, ApoE, a- synuclein, acid and alpha-glucosidase (GAA). In certain embodiments, the AAV particles comprise a heterologous nucleic acid encoding a gene product selected from the group consisting of thrombopoietin (TPO), interleukins (IL1 through IL- 17), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factors a and P, interferons a, P, and y, stem cell factor, flk-2 / flt3 ligand, IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cell receptors, class I and class II MHC molecules.

[0424] In certain embodiments, the AAV particles comprise a heterologous nucleic acid, i.e., a gene of interest (GOI), encoding a gene product selected from the group consisting of carbamoyl synthetase I, ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase, fumarylacetacetate hydrolase, phenylalanine hydroxylase, alpha- 1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, factor V, factor VIII, factor IX, cystathione beta-synthase, branched chain ketoacid decarboxylase, albumin, isovaleryl-coA dehydrogenase, propionyl CoA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, RPE65, H-protein, T-protein, a cystic fibrosis transmembrane regulator (CFTR) sequence, ApoE, a-synuclein, acid alphaglucosidase (GAA), and a dystrophin cDNA sequence

[0425] In certain embodiments, the AAV particles comprise a heterologous nucleic acid encoding a polypeptide, a nucleic acid that encodes a protein or is transcribed into a transcript of interest, or nucleic acid, selected from the group consisting of a siRNA, an antisense molecule, miRNA a ribozyme and a shRNA.

[0426] In certain embodiments, the AAV particles comprise a heterologous nucleic acid that encodes a protein selected from the group consisting of: GAA (acid alpha-glucosidase) for treatment of Pompe disease; a-synuclein for the treatment of one or more synucleinopathies, such as, but not limited to, Parkinson's disease, dementia with Lewy bodies (DLB), and multiple system atrophy (MSA); ApoE for the treatment of one or more neurological diseases (e.g., Alzheimer’s disease) or cardiovascular diseases; CFTR for the treatment cystic fibrosis; ATP7B (copper transporting ATPase2) for treatment of Wilson’s disease; alpha galactosidase for treatment of Fabry’s disease; ASS1 (arginosuccinate synthase) for treatment of citrullinemia Type 1; beta-glucocerebrosidase for treatment of Gaucher disease Type 1; betahexosaminidase A for treatment of Tay Sachs disease; SERPING1 (Cl protease inhibitor or Cl esterase inhibitor) for treatment of hereditary angioedema (HAE), also known as Cl inhibitor deficiency type I and type II); and glucose-6-phosphatase for treatment of glycogen storage disease type I (GSDI).

[0427] In certain embodiments, a heterologous nucleic acid encodes CFTR (cystic fibrosis transmembrane regulator protein), a blood coagulation (clotting) factor (Factor XIII, Factor IX, Factor VIII, Factor X, Factor VII, Factor Vila, protein C, etc.) a gain of function blood coagulation factor, an antibody, retinal pigment epithelium-specific 65 kDa protein (RPE65), erythropoietin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, P-globin, a- globin, spectrin, a-antitrypsin, adenosine deaminase (ADA), a metal transporter (ATP7A or ATP7), sulfamidase, an enzyme involved in lysosomal storage disease (ARSA), hypoxanthine guanine phosphoribosyl transferase, P-25 glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, branched-chain keto acid dehydrogenase, a hormone, a growth factor, insulin-like growth factor 1 or 2, platelet derived growth factor, epidermal growth factor, nerve growth factor, neurotrophic factor -3 and -4, brain-derived neurotrophic factor, glial derived growth factor, transforming growth factor a and P, a cytokine, a-interferon, P-interferon, interferon-y, interleukin-2, interleukin-4, interleukin 12, granulocyte-macrophage colony stimulating factor, lymphotoxin, a suicide gene product, herpes simplex virus thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, tumor necrosis factor, a drug resistance protein, a tumor suppressor protein (e.g., p53, Rb, Wt-1, NF1, Von Hippel-Lindau (VHL), adenomatous polyposis coli (APC)), a peptide with immunomodulatory properties, a tolerogenic or immunogenic peptide or protein Tregitope or hCDRl, insulin, glucokinase, guanylate cyclase 2D (LCA-GUCY2D), Rab escort protein 1 (choroideremia), LCA 5 (LCA-Lebercilin), ornithine ketoacid aminotransferase (gyrate atrophy), retinoschisin 1 (X-linked retinoschisis), USH1C (Usher’s Syndrome 1C), X-linked retinitis pigmentosa GTPase (XLRP), MERTK (AR forms of RP: retinitis pigmentosa), DFNB1 (connexin 26 deafness), ACHM 2, 3 and 4 (achromatopsia), PKD-1 or PKD-2 (polycystic kidney disease), TPP1, CLN2, a sulfatase, N- acetylglucosamine-1 -phosphate transferase, cathepsin A, GM2-AP, NPC1, VPC2, a sphingolipid activator protein, one or more zinc finger nucleases for genome editing, or one or more donor sequences used as repair templates for genome editing.

[0428] Nucleic acid molecules, vectors such as cloning, expression vectors (e.g., vector genomes) and plasmids, may be prepared using recombinant DNA technology methods. The availability of nucleotide sequence information enables preparation of nucleic acid molecules by a variety of means. For example, a heterologous nucleic acid encoding Factor IX (FIX) comprising a vector or plasmid can be made using various standard cloning, recombinant DNA technology, via cell expression or in vitro translation and chemical synthesis techniques. Purity of polynucleotides can be determined through sequencing, gel electrophoresis and the like. For example, nucleic acids can be isolated using hybridization or computer-based database screening techniques. Such techniques include, but are not limited to: (1) hybridization of genomic DNA or cDNA libraries with probes to detect homologous nucleotide sequences; (2) antibody screening to detect polypeptides having shared structural features, for example, using an expression library; (3) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers capable of annealing to a nucleic acid sequence of interest; (4) computer searches of sequence databases for related sequences; and (5) differential screening of a subtracted nucleic acid library.

[0429] 4. Method of producing rAA V vector particles in a cell culture

[0430] In another aspect, the subject matter of the present disclosure is directed to methods for producing rAAV vector particles comprising culturing a population of cells comprising an rAAV coding sequence, e.g., an AAV Rep coding sequence and / or an AAV CAP coding sequence; and one or more coding sequences providing helper virus function, e.g., a DNA Binding Protein (DBP) coding sequence; an Open Reading Frame 6 (0RF6), or an Open Reading Frame 6 / 7 (ORF6 / 7) coding sequence, and / or a Viral Associated (VA) coding sequence; where each of the DBP, the 0RF6 (or the ORF 6 / 7) and VA coding sequences are operably linked to a constitutive promoter; and harvesting the rAAV vector particles. In certain embodiments, the methods disclosed herein comprise culturing a population of cells comprising a first nucleic acid sequence comprising the rAAV coding sequence, a second nucleic acid comprising a coding sequence providing helper virus function, and a third nucleic acid comprising a GOI. In certain embodiments, the the methods disclosed herein comprise culturing a population of cells comprising a first nucleic acid sequence comprising a GOI and one or both of a rAAV coding sequence and a coding sequence providing helper virus function, and a second nucleic acid comprising one or both of a rAAV coding sequence and a coding sequence providing helper virus function.

[0431] In certain embodiments at least one of the coding sequences providing a helper virus function is operably linked independently to a constitutive promoter. In certain embodiments two of the coding sequences providing a helper virus function are operably linked to a common constitutive promoter, and the third coding sequence providing helper virus function is operably linked to a different constitutive promoter. In certain embodiments, the VA coding sequence in the cell comprises a VA_a coding sequence, a VA_b coding sequence, a VA_c coding sequence, a VA_d coding sequence, a VA_e coding sequence, or a VA_f coding sequence. In certain embodiments, the VA coding sequence comprises a VA_a coding sequence. In certain embodiments, the VA coding sequence comprises a VA_b coding sequence. In certain embodiments, the VA coding sequence comprises a VA_f coding sequence.

[0432] Exemplary nucleic acid sequences for the DBP, ORF6, ORF6 / 7, and VA coding sequences are shown in Table 1.

[0433] In certain embodiments, the cell further comprises an L4-22K coding sequence for providing helper virus function, wherein the L4-22K sequence is operably linked to a constitutive promoter. In certain embodiments, the rAAV producing cell of the present disclosure further comprises an L4-22K_Alt coding sequence for providing helper virus function, wherein the L4-22K_Alt sequence is operably linked to a constitutive promoter. In either embodiment, the L4-22K coding sequence or the L4-22K_Alt coding sequence is fused to one or more of the DBP, the ORF6, and the ORF6 / 7 coding sequences to form a fusion construct.

[0434] In certain embodiments, the L4-22K fusion construct comprises a 2A peptide coding sequence between the L4-22K coding sequence and the DBP coding sequence, the 0RF6 coding sequence and / or the ORF6 / 7 coding sequence. Non-limiting examples of 2A peptide coding sequence include, T2A, P2A, E2A and F2A.

[0435] Exemplary nucleic acid sequences for the L4-22K and L4-22K_Alt coding sequences and their fusion constructs are shown in Table 1.

[0436] In certain embodiments, the rAAV producing cell comprises a nucleic acid sequence comprising DBP, L4-22K, ORF6 and VA_b coding sequences for providing helper virus function.

[0437] In certain embodiments, the rAAV producing cell comprises a nucleic acid sequence comprising DBP, L4-22K, ORF6 and VA_f coding sequences for providing helper virus function.

[0438] In certain embodiments, the rAAV coding sequence, AAV REP coding sequence, AAV CAP coding sequence, the coding sequences providing helper virus function, and the GOI coding sequence are provided to the cell in two, three or four nucleic acid constructs.

[0439] In certain embodiments, the rAAV coding sequence, AAV REP coding sequence, AAV CAP coding sequence, the coding sequences providing helper virus function, and the GOI coding sequence are provided to the cell in two constructs, e.g., a dual vector system comprising a first nucleic acid construct and a second nucleic acid construct.

[0440] In certain embodiments, the first nucleic acid construct comprises one or more of an rAAV coding sequence, an AAV REP coding sequence, an AAV CAP coding sequence, and coding sequences providing helper virus function comprising a DBP coding sequence, an ORF6 and / or ORF6 / 7 coding sequence, and a VA coding sequence.

[0441] In certain embodiments, the second nucleic acid construct comprises a gene of interest (GOI) coding sequence.

[0442] In certain embodiments, the second nucleic acid construct comprises a GOI coding sequence and one or more of an rAAV coding sequence, an AAV REP coding sequence, an AAV CAP coding sequence, and coding sequences providing helper virus function comprising a DBP coding sequence, an ORF6 and / or ORF6 / 7 coding sequence, and a VA coding sequence.

[0443] In certain embodiments, each of the first a DBP coding sequence, the ORF6 and / or ORF 6 / 7 coding sequence, and the VA coding sequence in the first nucleic acid construct and the second nucleic acid construct is operably linked to a constitutive promoter.

[0444] In certain embodiments, the AAV REP coding sequence is operably linked to at least one promoter.

[0445] In certain embodiments, the AAV REP coding sequence and the AAV CAP coding sequence are operably linked to at least one promoter. In certain embodiments, the promoter sequence is selected from the group consisting of a full-length P5 promoter, a truncated P5 promoter, a T7 promoter, mouse mammary tumor virus (MMTV) promoter, a Cytomegalovirus (CMV), a Simian Virus 40 (SV40) promoter, a phosphoglycerate kinase promoter (PGK), ATP-citrate lyase promoter (ACLY), and actin gamma (ACTG) promoter, a bidirectional CMV (BICMV) promoter, a pl9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter. In one non-limiting embodiment, the AAV REP coding sequence and the AAV CAP coding sequence are operably linked to at least the full-length P5 promoter sequence.

[0446] In certain embodiments, the full-length P5 promoter sequence does not decrease production of rAAV vector particles compared to a AAV coding sequence, or a nucleic acid construct comprising a AAV coding sequence that lacks the full-length P5 promoter sequence. In certain embodiments, the full-length P5 promoter sequence enhances production of rAAV vector particles compared to a AAV coding sequence, or a nucleic acid construct comprising a AAV coding sequence that lacks the full-length P5 promoter sequence.

[0447] In certain embodiments, absence of the full-length P5 promoter sequence prevents formation of replication competent AAV (rcAAV) impurities.

[0448] In certain embodiments, absence of the full-length P5 promoter sequence results in reduced formation of replication competent AAV (rcAAV) impurities.

[0449] In certain embodiments, a nucleic acid sequence with nonspecific transcription activity is disposed upstream to the AAV REP coding sequence.

[0450] In certain embodiments, a nucleic acid sequence with nonspecific transcription activity is disposed downstream to the AAV REP coding sequence.

[0451] In certain embodiments, the nucleic acid sequence with nonspecific transcription activity comprises from about 156 nucleotides to about 729 nucleotides.

[0452] In certain embodiments, the Rep protein expression is promulgated to levels higher than in the absence of the nucleic acid sequence with nonspecific transcription activity.

[0453] In certain embodiments, the nucleic acid sequence with nonspecific transcription activity can enhance rAAV titer from about 2-fold to about 3 -fold compared to rAAV titer when the nucleic acid sequence with nonspecific transcription activity is absent.

[0454] In certain embodiments, the methods, transfected cells, and systems yield rAAV vector particles exhibiting increased biosafety (e.g., less immunogenicity, less inflammation and / or less genotoxicity) as the amount of residual plasmid DNA (RPD) in the AAV capsid is reduced relative to vector particles produced using alternative strategies.

[0455] 5. Exemplary constructs providing helper virus function.

[0456] The subject matter of the present disclosure is directed to a method of producing rAAV vector particles, a nucleic acid, and a transfected rAAV producing cell, wherein the cells and the nucleic acids comprise coding sequences that provide helper virus function. In certain embodiments, the coding sequences providing a helper virus function are derived from a virus selected from adenovirus, herpesvirus, poxvirus, or a hybrid virus thereof. In certain embodiments, coding sequences providing the helper virus function are comprised in one or more vectors or plasmids.

[0457] Figure 1 A shows a helper plasmid (Helperl) comprising an E2A, DNA Binding Protein (DBP) coding sequence; an E4, Open Reading Frame 6 (ORF6) sequence; and a Viral Associated (VA) sequence. Figure 2 depicts locations of possible Open Reading Frames in E2A, E4 and VA genes in Helperl . As seen from Figure 2, the Helperl plasmid has many open reading frames in E2A, E4 and VA. Black outlined ORFs, i.e., DBP, L4-22K, E4- ORF6, VA_b, and VA f, were found, as outlined in the Examples disclosed herein, to be non-limiting example of ORFs effective in providing helper function during rAAV production.

[0458] Exemplary helper plasmids comprising the coding sequences providing a helper virus function are shown in Figures IB- IL. The exemplary constructs described in this section are merely illustrative of the presently disclosed subject matter and should not be considered as limitations in any way.

[0459] Figure IB shows a first exemplary helper plasmid comprising a DBP sequence and an 0RF6 sequence each under independent Cytomegalovirus (CMV) promoters.

[0460] Figure 1C shows a second exemplary helper plasmid that comprises the sequences shown in Figure IB and further comprises a Viral Associated b sequence (VA_b) sequence under a Simian Virus 40 (SV40) promoter.

[0461] Figure ID shows a third exemplary helper plasmid comprising DBP and ORF6 sequences linked by a T2A cleavage sequence (DBP-T2A-ORF6 configuration), in which transcription is under a CMV promoter.

[0462] Figure IE shows a fourth exemplary helper plasmid similar to Figure ID but with an ORF-T2A-DBP configuration.

[0463] Figure IF shows a fifth exemplary helper plasmid comprising a DBP, ORF6 and VA_b sequences under a CMV promoter, in which DBP and ORF6 are linked via a T2A sequence, whereas ORF6 and VA_b are linked via a P2A cleavage sequence.

[0464] Figure 1G shows a sixth exemplary helper plasmid comprising the DBP, ORF6 and VA sequences as in Figure 1 A, in which sequences between the DBP start site and ORF6 start site were replaced with a bidirectional CMV (BICMV) promoter.

[0465] Figure 1H shows a seventh exemplary helper plasmid as in Figure 1C in which, additionally, an L4-22K coding sequence linked via the T2A sequence upstream to the ORF6 sequence is operably linked to the CMV promoter.

[0466] Figure II shows an eight exemplary helper plasmid as in Figure 1C in which, additionally, an L4-22K coding sequence linked via the T2A sequence upstream to the DBP sequence is operably linked to the CMV promoter.

[0467] Figure 1 J shows a ninth exemplary helper plasmid as in Figure 1G in which, additionally, an L4-22K coding sequence linked via the T2A sequence upstream to the DBP sequence is operably linked to the BICMV promoter. Figure IK shows a tenth exemplary helper plasmid as in Figure 1G in which, additionally, an L4-22K coding sequence linked via the T2A sequence upstream to the 0RF6 sequence is operably linked to the BICMV promoter.

[0468] Figure IL shows an eleventh exemplary helper plasmid as in Figure 1G in which, additionally, an L4-22K coding sequence operably linked to a CMV promoter is inserted upstream to the DBP sequence.

[0469] Figure 16A shows an exemplary first dual plasmid system, wherein the replication (Rep), capsid (Cap), and Helper coding sequences are located on one plasmid, and a gene of interest (GOI) coding sequence is located on a second plasmid.

[0470] Figure 16B shows a second dual plasmid system, wherein the Cap and Helper coding sequences are located on one plasmid, and the Rep and GOI coding sequences are located on a second plasmid.

[0471] Figure 16C shows a third dual plasmid system, wherein the Rep and helper coding sequences are located on one plasmid, and the Cap and GOI coding sequences are located on a second plasmid.

[0472] Figure 16D shows a fourth dual plasmid system, wherein the Rep52, Cap and Helper coding sequences are located on one plasmid, and the Rep78 and GOI coding sequences are located on a second plasmid.

[0473] Figure 17A shows a configuration of a Rep / Cap plasmid, wherein expression of large Rep (here, Rep78), small Ref (here, Rep52), and Cap proteins is driven using truncated P5, P19, and s, respectively. In this configuration, the Cap protein can be either RHM4-1 (plasmid Al) or LK03 (plasmid Bl). Additional elements included in this plasmid are the pUC19 Ori useful for plasmid replication in bacteria, and the Kanamycin resistance transposon for selection. Upstream of the truncated P5 promoter is a T7 promoter, two LacZ fragments, and the Fl origin of replication. Downstream of the Cap gene, a complete P5 promoter is located, followed by elements of the Lac operon.

[0474] Figure 17B shows a configuration of a Rep / Cap / Helper plasmid, wherein the Rep, Cap, and Helper coding sequences are combined into one single plasmid. Here, the helper elements include DNA binding protein (DBP), open reading frame 6 (E4 ORF6), viral associated (VA), and L4-22K coding sequences. DBP and E4 ORF6 are each driven by separate CMV promoters and were cloned from Helper 3 plasmid, while VA and L4-22K are driven by SV40 and EFl -a (elongation factor 1 alpha) promoters respectively. In this design, Rep and LK03 Cap are separated, with each under the control of truncated P5 and P40 promoters, respectively. Figure 18A depicts an exemplary Rep / Cap / Helper dual plasmid design with or without plasmid backbone elements upstream of the Rep protein. In this example, a bidirectional CMV promoter (Bi-CMV) is used to drive expression of DNA binding protein (DBP), E4 0RF6 and ORF6 / 7 in opposite directions. L4-22K expression is facilitated by an internal ribosome entry site (IRES) sequence whereas, LK03 cap expression is regulated by the P40 promoter. Further, to investigate the impact of different plasmid backbone elements on Rep expression, several plasmid variants (Dl-Il) were generated. Plasmid DI comprises a truncated P5 promoter and a fragment of the multicloning site (MCS); plasmid El comprises the MCS and T7 promoter; plasmid Fl comprises a truncated P5 promoter, MCS, and T7 promoter; plasmid G1 comprises a truncated P5 promoter, MCS, T7 promoter, and a LacZ fragment; plasmid Hl comprises a truncated P5 promoter, MCS, T7 promoter, LacZ fragment, and Fl origin; and plasmid II comprises a truncated P5 promoter, MCS, T7 promoter, two LacZ fragments, and Fl origin of replication.

[0475] Figure 19A depicts yet another exemplary Rep / Cap / Helper dual plasmid design, wherein a full-length P5 promoter, a mouse mammary tumor virus promoter (MMTV), a CMV promoter, a SV40 promoter, a phosphoglycerate kinase promoter (PGK), an ATP- citrate lyase promoter (ACLY), and a actin gamma promoter (ACTG) are used to drive Rep expression.

[0476] Figure 20A depicts four (4) exemplary plasmid configurations each comprising the Rep, Cap, and Helper coding sequences wherein the capsid is LK03. In this configuration, L4-22K expression is controlled by IRES (plasmid Ml), self-cleaving P2A peptide (plasmid QI), Bi-CMV promoter (plasmid Rl), or the ACTG promoter (plasmid SI). In plasmid Rl, expression of E4 ORF6 and E4 ORF6 / 7 is regulated by self-cleaving P2A peptide.

[0477] Figure 20B depicts three (3) exemplary plasmid configurations each comprising the Rep, Cap, and Helper coding sequences wherein the capsid is RHM4-1. In this configuration, L4-22K expression is controlled by IRES (plasmid Tl), self-cleaving P2A peptide (plasmid Ul), or Bi-CMV promoter (plasmid VI). In plasmid VI, expression of E4 ORF6 and E4 ORF6 / 7 is regulated by self-cleaving P2A peptide.

[0478] Figure 21A depicts six (6) exemplary inducible plasmid configurations each comprising the Rep, Cap, and Helper coding sequences wherein the capsid is LK03. In plasmids W1 and XI, expression of L4-22K is regulated by an IRES sequence. However, there is a difference in the PolyA signal sequence downstream of Rep52 between W1 and XI designs. In design Wl, both PolyA signals downstream of Rep78 and Rep52 are BGH- PolyA sequence while in design XI, the PolyA downstream of Rep78 is BGH and the PolyA downstream of Rep52 is HSV-TK-PolyA signal. Plasmid Y1 replaces the P40 promoter in W1 with a P41 promoter to drive LK03 expression. In plasmid Z1 and A2, expression of L4- 22K is controlled by a SV40 promoter. In plasmid A2, expression of LK03 is driven by a P41 promoter. Plasmid B2 features a LK03 lacking the intron region and driven by a P41 promoter.

[0479] Figure 2 IB depicts five (5) exemplary inducible plasmid configurations each comprising the Rep, Cap, and Helper coding sequences wherein the capsid is RHM4-1. Plasmids C2 and D2 are identical except for a (plasmid C2) and a P41 promoter (plasmid D2) to drive RHM4-1 capsid. Expression of L4-22K in plasmids E2, F2, and G2 is under a SV40 promoter. In plasmid F2, expression of RHM4-1 capsid is driven by a P41 promoter. Plasmid G2 features a RHM4-1 lacking the intron region and driven by a P41 promoter.

[0480] Figure 22A depicts three (3) exemplary inducible first plasmid configurations comprising the Rep, and Helper coding sequences, wherein expression of codon optimized Rep78 and Rep52 is driven by a BiTet inducible promoter. Further, plasmid 12 lacks the L4- 22K coding sequence while in plasmid J2 expression of L4-22K is driven by a ACTG promoter.

[0481] Figure 22B depicts two (2) exemplary second plasmid configurations each comprising a RHM4-1 capsid sequence and a GOI (transgene) sequence. Expression of the RHM4-1 capsid is driven by a or a P41 promoter in plasmids K2 or L2 respectively.

[0482] Figure 23 A shows an exemplary fifth dual plasmid system comprising large Rep, Cap and helper genes in one plasmid, and small Rep and GOI in a second plasmid.

[0483] Figure 23B shows an exemplary sixth dual plasmid system comprising Cap and some of the helper genes in one plasmid, and Rep, some helper genes and GOI in a second plasmid.

[0484] Figure 23C shows an exemplary seventh dual plasmid system comprising Rep and some helper genes in one plasmid, and Cap, some helper genes and GOI in a second plasmid.

[0485] Figure 23D shows an exemplary eight dual plasmid system comprising small Rep, Cap and some helper elements in one plasmid, and large Rep, some helper genes and GOI in a second plasmid.

[0486] Figure 23E shows an exemplary ninth dual plasmid system comprising large Rep, Cap and some helper genes in one plasmid, and small Rep, some helper genes and GOI in a second plasmid.

[0487] Figure 23F shows an exemplary tenth dual plasmid system comprising Rep and Cap genes in one plasmid, and helper genes and GOI in a second plasmid. Figure 23G shows an exemplary eleventh dual plasmid system comprising Rep, Cap and some helper genes in one plasmid, and some helper genes and GOI in a second plasmid.

[0488] For any of Figures 23A-23G, Rep genes shown in the figures can be separated as large Rep and small Rep within the same plasmid. Further, Cap genes can be separated from Rep Gene with a stuffer sequence within the same plasmid.

[0489] Figure 24A shows two exemplary constructs A4 and M3. Each construct comprises Rep and Cap genes with RHM4-1 as an example Cap gene. Upstream of the Rep gene is a truncated P5 promoter, alongside a multi-cloning site fragment (MCS), a T7 promoter, two LacZ fragments, and a segment of the Fl origin sequence. The M3 construct is distinguished from the A4 construct by the absence of a full-length P5 promoter.

[0490] Figure 25 A shows two exemplary constructs B3 and N3. Each construct comprises Rep and Cap genes, with LK03 as an example Cap gene. Upstream of the Rep gene is a truncated P5 promoter, alongside a multi-cloning site fragment (MCS), a T7 promoter, two LacZ fragments, and a segment of the Fl origin sequence. The N3 construct is distinguished from the B3 construct by the absence of a full-length P5 promoter.

[0491] Figure 26A shows eight exemplary promoters including the B3 construct described above. Construct 03 is characterized by the separation of the Rep and Cap genes by a 1-kb stuffer sequence, a MCS fragment and one LacZ fragment positioned upstream of the Rep sequence, and absence of both the full-length P5 promoter and truncated P5 promoter. Construct P2 is characterized by the separation of the Rep and Cap genes by a 1-kb stuffer sequence, a MCS fragment and one LacZ fragment positioned upstream of the Rep sequence, and absence of both the full-length P5 promoter and truncated P5 promoter, and further comprises an L4-22K element under the control of an SV40 promoter, positioned downstream of the LK03 gene. Construct Q2 is characterized by the separation of the Rep and Cap genes by a 1-kb stuffer sequence, a MCS fragment and one Fl origin positioned upstream of the Rep sequence, and absence of both the full-length P5 promoter and truncated P5 promoter. Construct R2 is characterized by the separation of the Rep and Cap genes by a 1-kb stuffer sequence, a MCS fragment and one Fl origin positioned upstream of the Rep sequence, absence of both the full-length P5 promoter and truncated P5 promoter, and further comprises an L4-22K element under the control of an SV40 promoter, positioned downstream of the LK03 gene. Construct S2 is characterized by absence of both the full- length P5 promoter, truncated P5 promoter and all backbone elements, including the MCS fragment, T7 promoter, Fl origin, and LacZ fragments, and presence of a SV40 promoter upstream of the Rep sequence that drives expression of Rep (e.g., Rep78). Construct T2 is characterized by absence of both the full-length P5 promoter, truncated P5 promoter and all backbone elements, including the MCS fragment, T7 promoter, Fl origin, and LacZ fragments, and presence of a SV40 promoter upstream of the Rep sequence that drives expression of Rep (e.g., Rep78), and a polyadenylation (poly A) sequence downstream of the LK03 sequence. Construct U2 is characterized by absence of both the full-length P5 promoter, truncated P5 promoter and all backbone elements, including the MCS fragment, T7 promoter, Fl origin, and LacZ fragments, and presence of a SV40 promoter upstream of the Rep sequence that drives expression of Rep (e.g., Rep78), a P41 promoter upstream of the LK03 gene, a poly A sequence downstream of the LK03 sequence, and a 1-kb stuff er sequence between the Rep and Cap genes.

[0492] Figure 27A shows three exemplary constructs including the B3 construct described above. Constructs V2 and W2 have a structure as for B3 and additionally comprises a 3-kb stuffer sequence between the Rep and Cap genes. Construct W2 differs from B3 and V2 by the absence of the full-length P5 promoter.

[0493] Figure 28A shows an exemplary X2 construct and the B3 construct described above. Construct X2 is characterized by absence of full-length and truncated P5 promoters, absence of the backbone elements upstream of Rep, and presence of a SV40 promoter upstream to Rep to drive Rep (e.g., Rep78) expression, and a 3-kb stuffer sequence between the Rep and Cap genes.

[0494] Figure 29A shows fours exemplary constructs including the A4 construct described above. Construct Y2 is characterized by absence of the truncated and full-length P5 promoters, the backbone elements upstream of the Rep gene, have been removed and presence of a SV40 promoter upstream to Rep to drive Rep (e.g., Rep78) expression. Construct Z2 is characterized by absence of the truncated and full-length P5 promoters, the backbone elements upstream of the Rep gene, have been removed, presence of a SV40 promoter upstream to Rep to drive Rep (e.g., Rep78) expression, and a 1-kb stuffer sequence between the Rep and Cap (RHM4-1) genes. Construct A3 is characterized by absence of the truncated and full-length P5 promoters, the backbone elements upstream of the Rep gene, have been removed, presence of a SV40 promoter upstream to Rep to drive Rep (e.g., Rep78) expression, and a 3-kb stuffer sequence between the Rep and Cap (RHM4-1) genes.

[0495] Figure 30A shows three exemplary constructs, B4, C4 and D4 comprising the Rep, helper and Cap (RHM4-1) coding sequences, wherein a SV40 promoter drives Rep expression, a Bi-CMV promoter drives expression of helper genes and a P40 promoter drives expression of Cap. Figure 30B shows two exemplary constructs, E4 and F4. Construct E4 comprises the Rep and helper coding sequences, wherein a SV40 promoter drives Rep expression and a Bi- CMV promoter drives expression of helper genes. Construct F4 comprises the Cap (RHM4- 1) coding sequences under control of a P40 promoter, and an ITR cassette comprising the GOI.

[0496] Figure 30C shows two exemplary constructs, G4 and H4. Construct G4 comprises helper and Cap (RHM4-1) coding sequences, wherein a Bi-CMV promoter drives expression of helper genes and P40 promoter drives expression of Cap. Construct H4 comprises the Rep coding sequence under control of a SV40 promoter, and an ITR cassette comprising the GOI.

[0497] Figure 31A shows two exemplary constructs, 14 and J4. Construct 14 comprises a Rep52 coding sequence under control of a pl9 promoter and an ITR cassette comprising the GOI. Construct J4 comprises a Rep52 and Cap (RHM4-1) coding sequences under control of a pl9 promoter and an ITR cassette comprising the GOI.

[0498] Figure 3 IB shows two exemplary constructs, K4 and L4. Construct K4 comprises a codon optimized Rep52 coding sequence under control of a pl9 promoter and an ITR cassette comprising the GOI. Construct L4 comprises a codon optimized Rep52 coding sequence and a Cap (RHM4-1) coding sequence under control of a pl9 promoter and an ITR cassette comprising the GOI.

[0499] Figure 31C shows two exemplary constructs, M4 and N4. Construct M4 comprises the Rep and helper coding sequences, wherein a S V40 promoter drives Rep expression, and a Bi-CMV promoter drives expression of helper genes. Construct M4 comprises the Rep68- Rep40 and helper coding sequences, wherein a SV40 promoter drives Rep expression, and a Bi-CMV promoter drives expression of helper genes.

[0500] Figure 3 ID shows an exemplary construct, 04, comprising the Rep68-Rep40 and helper coding sequences, wherein a SV40 promoter drives Rep expression, and a Bi-CMV promoter drives expression of helper genes.

[0501] For any of Figures 24A, 25A, 26A, 27A, 28A, 29 A, and 30A-30C and 31C (Construct M4), the Rep genes shown in the figures can be large Rep and / or small Rep within the same plasmid.

[0502] In certain embodiments, the AAV coding sequence has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 85-112, 126, 128-148, or 156-158. In certain embodiments, the coding sequences providing helper virus function has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 71-82.

[0503] In certain embodiments, the AAV coding sequence comprises a sequence set forth in any one of SEQ ID NOs: 85-112, 126, 128-148, or 156-158. In certain embodiments, the coding sequences providing helper virus function comprises a sequence set forth in any one of SEQ ID NOs: 71-82.

[0504] In certain embodiments, the first nucleic acid construct comprises an AAV coding sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 85-112, 126, 128-148, or 156-158.

[0505] In certain embodiments, the second nucleic acid construct comprises an AAV coding sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 109, 111, 113, 127, 149, or 151-155.

[0506] In certain embodiments, the first nucleic acid construct comprises coding sequences providing helper virus function comprising a sequence set forth in any one of SEQ ID NOs: 71-82.

[0507] In certain embodiments, the second nucleic acid construct comprises a sequence set forth in any one of SEQ ID NOs: 109, 111, 113, 127, 149, or 151-155.

[0508] 6. EXAMPLES

[0509] The following examples are merely illustrative of the presently disclosed subject matter and should not be considered as limitations in any way.

[0510] Materials and Methods

[0511] Transfection

[0512] Expi293 cells (Thermo Fisher Scientific) were seeded at 1.6E6 viable cells / mL in

[0513] Expi293 media the day before transfection. Transfection was performed when the viable cells were between 2.5E6-4E6 cells / mL. Cells were transfected with the DNA (0.6 pg DNA / million cells) complexed with polyethylenimine (PEI) transfection reagent (PELDNA, 2.5). Triple, quadruple, or dual plasmid transfections were performed with Rep / Cap, helper, and transgene plasmids or additional helper elements or dual plasmid constructs. 5 mM VA and free PEI were added after the addition of DNA-PEI complexes. Cells were harvested at 72 hr post-transfection, centrifuged and washed with cold PBS. Cell pellets were stored at - 80°C until use.

[0514] Western Blot

[0515] Cell pellets were lysed on ice for 30 minutes with Pierce™ RIP A Buffer (Thermo Fischer Scientific) containing protease, phosphatase inhibitors, and EDTA. Lysates were centrifuged at max speed for 15 minutes and supernatants analyzed by western blotting after SDS-PAGE (30 pg protein per lane). For western blotting, nitrocellulose membranes were blocked with blocking buffer (Li-Cor) for 30 minutes at room temperature. Membranes were incubated overnight (4°C) with primary antibody and corresponding loading control antibody (LiCor). Next day membranes were washed thrice with TBST buffer (Invitrogen) and incubated or 45 minutes at room temperature with goat anti-mouse Alexa Fluor 680 and goat anti-rabbit Alexa Fluor 800 secondary antibodies. The membranes were next washed 6 times with TBST buffer before scanning in an Odyssey® imagers (Li-Cor). Anti -AAV Rep clone 303.9 (ARP, cat# 03-61069) purified mouse monoclonal antibody (1 :500), Anti-AAV VP1 / VP2 / VP3 clone Bl (ARP, cat# 03-65158) mouse antibody (1 :500), DBP (Cusabio, cat# CSB-PA365892ZA01HIL) rabbit polyclonal antibody (1 :20000), L4-22K custom rabbit polyclonal antibody (1 : 1000), AAP custom rabbit polyclonal antibody (1 :1000) and ORF6 custom rabbit polyclonal antibody (1 :500) were used. For mouse primary antibodies, anti- GAPDH rabbit monoclonal antibody (Cell Signaling, cat# 2118) was used (1 :5000). For rabbit primary antibodies, anti-GAPDH mouse monoclonal antibody (Cell Signaling, cat# 97166) was used (1 :2000). qPCR

[0516] Viral genome titer was detected using either quantitative PCR (qPCR) or digital PCR (dPCR) with primers specific to each rAAV transgene. In general, samples were pretreated with endonuclease to digest DNA and RNA free in solution, followed by Proteinase-K treatment for endonuclease and capsid protein digestion, and then an incubation at 95° C for 15 minutes to inactivate the protease. Viral DNA concentrations were then measured with qPCR or dPCR (QuantStudio 7 Flex PCR system or QIAcuity 8 dPCR system, respectively) using appropriate primer and probes. All preparation steps were automated on a Tecan liquid handler. Cloning

[0517] Individual ORFs were subcloned to separate plasmids under CMV promoter. Helper2 was constructed by inserting CMV-ORF6 transcription cassette into CMV-DBP plasmid. Helper3 was created by switching Neomycin gene in helper2 by VA_b sequence. Helper4 was modified from CMV-DBP plasmid by adding T2A and ORF6 to the end of DBP sequence. Helper5 was modified from CMV-ORF6 plasmid by adding T2A and DBP to the end of ORF6 sequence. Helper6 was modified from CMV-DBP by adding T2A, ORF6, P2A and VA_b to the end of DBP sequence in this order. Helper? was created by switching the region in between DBP and ORF6 start sites with BICMV promoter sequence from Clontech. Helper8 was modified from helper3 by inserting L4-22K-T2A gene fragment to the beginning of ORF6. Helper9 was modified from helper3 by inserting L4-22K-T2A gene fragment to the beginning of DBP. HelperlO was modified from helper? by inserting L4- 22K-T2A gene fragment to the beginning of DBP. Helperl 1 was modified from helper? by inserting L4-22K-T2A gene fragment to the beginning of ORF6. Helperl2 was created by inserting CMV-L4-22K transcription cassette into helper? plasmid.

[0518] Example 1: DBP And ORF6 Are Useful For rAA V Production

[0519] This examples tests sequence elements useful for providing helper virus function using spin tube scale production. An AAV vector encoding GAA (AAV-GAA) as described in US20210222141 was used. Expi293 cells were co-transfected with Helper2 in the presence of separate plasmids comprising the VA or L4-22K sequences under the CMV promoter. Following transfection (72 hr), titers were quantitated based on testing the RHM4-1 capsid.

[0520] Results. As shown in Figure 3, DBP and ORF6 are useful for rAAV production. VA and L4-22K enhance rAAV titers but cannot produce AAV without DBP and ORF6. Best titers were achieved with the combination of DBP, ORF6, VA and L4-22K.

[0521] Example 2: Coding Sequences Under the Control of Separate Promoters Yield Higher AA V Titers Over Common Promoters

[0522] This example tests the importance of promoter sequence placement on rAAV production efficiency using spin tube scale production. An AAV vector encoding GAA (AAV-GAA) as described in US20210222141 was used. Expi293 cells were co-transfected with one of helper plasmids Helper2, Helper4 or Helper5 in the presence of one of plasmids comprising the VA, VA_a, VA_b or ORF6 / 7 sequences under the CMV promoter. The Rep / Cap plasmid used for this experiment had an inducible Rep. Helperl was used as positive control. Post-transfection (72 hr), titers were quantitated by testing the RHM4-1 capsid.

[0523] Results. As shown in Figure 4, transfection with plasmids comprising DBP and 0RF6 combined with a T2A linker (Helper4, Helper 5) under the control of a common CMV promoter led to AAV production. However, the AAV titers was relatively higher when DBP and ORF6 were each controlled by a separate CMV promoter. Co-transfection with plasmids comprising the VA or VA_b sequence significantly enhanced the AAV titer for helper2 and helper (p<0.05).

[0524] Example 3: Presence of The VA_b Coding Sequence Enhance rAA V Production

[0525] This example tests the importance of the VA_b sequence on rAAV production efficiency using Ambr®250 scale production. An AAV vector encoding GAA (AAV-GAA) as described in US20210222141 was used. Expi293 cells were co-transfected with one of helper plasmid Helper2 or Helper3. Helperl was used as positive control. Post-transfection (72 hr), titers were quantitated by testing the RHM4-1 capsid.

[0526] Results. As evident from Figure 5, transfecting with Helper3 yielded a higher rAAV titer compared to the Helper2 plasmid underscoring the benefit of including a VA_b coding sequence.

[0527] Example 4: VA bAnd VA _f Coding Sequence Enhance rAA V Production

[0528] This example tests the importance of the VA_b and VA_f coding sequences on rAAV production efficiency using spin tube scale production. An AAV vector encoding GAA (AAV-GAA) as described in US20210222141 was used. Expi293 cells were co-transfected with helper plasmid Helper2 and plasmids comprising the VA_a, VA_b, VA_c, VA_d, VA_e, VA_f or VA sequences under the CMV promoter. Helperl was used as positive control. Post-transfection (72 hr), titers were quantitated based on testing the RHM4-1 capsid.

[0529] Results. Figure 6 shows that co-transfection with a plasmid comprising the VA_b or VA_f coding sequence enhanced rAAV titers.

[0530] Example 5: Enhancement of AAV Titers By Co-Transfection With The L4-22K Coding Sequence

[0531] This example tests the contribution of the E2A derived coding sequences on rAAV production efficiency using spin tube scale production. An AAV vector encoding GAA (AAV-GAA) as described in US20210222141 was used. Expi293 cells were co-transfected with helper plasmid Helper3 and plasmids comprising the E2A, DBP, MastC, L4, L4-22K, L4pVIII or L4 33K sequences under the CMV promoter. Helperl was used as positive control. Titers were quantitated 72 hr post transfection based on testing the RHM4-1 capsid.

[0532] Results. Figure 7 shows that L4-22K boosted the AAV production when supplied from a separate plasmid. These data show the benefit of L4-22K expression.

[0533] Example 6: E4 Derived Sequences Do Not Provide Significant Enhancement of rAA V Production

[0534] This example tests the contribution of the E4 derived coding sequences on rAAV production efficiency using spin tube scale production. An AAV vector encoding GAA (AAV-GAA) as described in US20210222141 was used. Expi293 cells were co-transfected with helper plasmid Helper3 and plasmids comprising the E4, ORF6, 16kDa, ORF1, 0RF2, ORF3, ORF6 / 7, revORFl, revORF2, revORF3, revORF4 or revORF5 sequences under the CMV promoter. Helperl was used as positive control. Titers were quantitated 72 hr post transfection based on testing the RHM4-1 capsid.

[0535] Results. Figure 8 shows no significant benefits obtained by the inclusion of these E4 derived coding sequences.

[0536] Example 7: Enhancement of rAA V Production With L4-22K Coding Sequence

[0537] This example tests the contribution of the L4-22K coding sequence on rAAV production efficiency using spin tube scale production. An AAV vector encoding GAA (AAV-GAA) as described in US20210222141 was used. Expi293 cells were co-transfected with helper plasmid Helperl or Helper7 and plasmids comprising the L4-22K sequence under the CMV promoter. Helperl was used as positive control. Titers were quantitated 72 hr post transfection based on testing the RHM4-1 or LK03 capsid.

[0538] Spin tube scale production.

[0539] Results. The results shown in Figure 9A shows significant enhancement of rAAV production with the DBP, ORF6, VA and L4-22K coding sequences for the RHM4-1 capsid. With the LK03 capsid, significant enhancement in rAAV titers is observed in the presence of L4-22K for both Helperl and Helper 7 (Figure 9B). Example 8: Enhancement of rAA V Production With L4-22K Alternative Coding Sequences

[0540] This example tests the contribution of the L4-22K Alternative coding sequence on rAAV production efficiency using spin tube scale production. An AAV vector encoding GAA (AAV-GAA) as described in US20210222141 was used. Expi293 cells were cotransfected with helper plasmid Helper3 and plasmids comprising the L4-22K, L4-22K Alternative, or L4-33K coding sequences under the CMV promoter. Helperl was used as positive control. Titers were quantitated 72 hr post transfection based on testing the RHM4-1 capsid.

[0541] Results. The results shown in Figure 10 shows similar enhancement of rAAV titers when using the L4-22K and L4-22K Alternative coding sequences but not with the L4-33K sequence.

[0542] Example 9: Effect of an Incorporated L4-22K Coding Sequence on rAA V Production

[0543] This example tests the effect of an incorporated L4-22K sequences on rAAV production efficiency using spin tube scale production. An AAV vector encoding GAA (AAV-GAA) as described in US20210222141 was used. Expi293 cells were transfected with a L4-22K incorporated helper plasmid, or co-transfected with a helper plasmid and a plasmid comprising the L4-22K coding sequences under the CMV promoter. Helperl was used as positive control. Titers were quantitated 72 hr post transfection based on testing the RHM4-1 or LK03 capsid.

[0544] Results. The results shown in Figure 11 A and 1 IB show enhancement of rAAV titers for all samples where L4-22K was expressed.

[0545] Example 10: Effect of L4-22K on RHM4-1 rAA V production using the Ambr®250 system

[0546] This example tests the effect of L4-22K on RHM4-1 rAAV production for different transgenes using the Ambr®250 scale production system. An AAV vector encoding transgene- 1 or transgene-2 or transgene-3 was used. Expi293 cells were transfected with a L4-22K incorporated helper plasmid. Helperl was used as positive control. Titers were quantitated 72 hr post transfection based on testing the RHM4-1 capsid.

[0547] Results. The results in Figure 12 show that Helperl 1 and Helperl2 increased the titer 2-3-fold in Ambr®250 scale. Example 11: Comparative Analysis of Incorporating The L4-22K Sequence On rAAV Production Using The Ambr®250 System

[0548] This example compares the effect of incorporating L4-22K sequences on rAAV production efficiency using the Ambr®250 scale production system. An AAV vector encoding transgene- 1 or transgene-2 or transgene-3 was used. Expi293 cells were transfected with a L4-22K incorporated helper plasmid. Helper 1 was used as positive control. Titers were quantitated 72 hr post transfection based on testing the RHM4-1 capsid.

[0549] Results. The results shown in Figure 13A shows that Helperl 1 and Helperl2 increased the titer 2-3-fold for the 2 versions of the transgene-1 in Ambr®250 scale. Figure 13B shows that Helperl 1 and Helperl2 moderately increased the titer for transgene 2. Figure 13C shows a comparison between the helper plasmids shown for RHM4-1 rAAV production for transgene 3.

[0550] Example 12: Effect of L4-22K On Rep And Cap Expression

[0551] This example depicts the effect of L4-22K on Rep and Cap expression using transgene-2, transgene-3 and 2 versions of transgene- 1 using the Ambr®250 scale production system. An AAV vector encoding transgene- 1 or transgene-2 or transgene-3 was used. Expi293 cells were transfected with a L4-22K incorporated helper plasmid. Helperl was used as positive control. Protein expressions were based on testing the Expi293 cell lysates at 72 hr using western blotting.

[0552] Results. Western blotting (Figure 14) reveals that L4-22K increases Rep78, Rep52, VP1, VP2 and VP3 expressions. To test the promoter dependence of the effect of L4-22K on Rep and capsid expression levels a ubiquitously expressed plasmid PL0845 was compared with LK03 Rep Cap plasmid. L4-22K increased the Rep and capsid expression with LK03 Rep Cap plasmid but not with PL0845. The results in Figure 15 show that L4-22K activates viral promoters such as p5, pl9 and p40.

[0553] Example 13: Rep78 Expression in Dual Plasmid Systems

[0554] 13.1 Rep78 Expression in Cells transfected with Dual Plasmids

[0555] This example compares Rep78 expression in cells transfected with the dual and triple / quadruple plasmid systems of the disclosed subject matter. HEK293 cells were transfected with dual plasmid systems depicted in Figures 16A-16D. The triple and quadruple systems were used as controls. Triple plasmid systems comprising Helper 1, Bl Rep / Cap, and a transgene plasmid and quadruple plasmid system has an additional L4-22K plasmid. Following transfection (72 hr), cells were lysed, proteins resolved by SDS-PAGE and expression assessed by western blotting.

[0556] Results. Western blotting analysis revealed absence of Rep78 expression for all dual plasmid configurations (Figure 16E). In all these dual plasmid designs, backbone elements upstream of the truncated P5 promoter were removed which caused the loss of Rep78 expression. The dual plasmid system that had the upstream backbone elements was able to express Rep78 (Lane 3).

[0557] 13.2 Rep78 Expression in Cells transfected with Dual Plasmids With or Without Backbone Elements

[0558] To understand the contribution of backbone elements in the plasmid to Rep78 expression, HEK293 cells were co-transfected in Ambr®250 bioreactors with either of the plasmids Cl -Il (Figures 17B and 18 A). Protein expression was assessed 72 hr post transfection as described above to visualize viral proteins Rep78, Rep52, Vpl, Vp2, Vp3, DBP, and loading control GAPDH. Lysates from cells transfected with the GAA GOI plasmid, helperl, and plasmid Bl (ratio =1 : 1 : 1; lane 2); the GAA GOI plasmid, helperl, CMV-L4-22K, and plasmid Bl (ratio =1 : 1 :0.5 : 1 ; lane 3); or the GAA GOI plasmid, helper7, CMV-L4-22K, and plasmid Bl (ratio=l:l:0.5:l; lane 4) were used as positive control. Plasmid Bl is depicted in Figure 17A. Lanes 5-11 correspond to lysates from dual plasmid transfected cells that used Rep / Cap / helper plasmid variants Cl -Il and GAA GOI plasmid with a molar ratio of 1 : 1.

[0559] Results. As shown in Figure 18B, enhanced Rep78 expression was observed with the addition of the LacZ fragment (plasmids Gl, Hl, II). In the absence of the LacZ fragment, neither the truncated P5 promoter (plasmid DI) nor the T7 promoter (plasmid El) alone could drive Rep78 expression (lanes 6 and 7). However, the combination of the T7 and truncated P5 promoters induced marginal Rep78 expression (lane 8, plasmid FL Addition of LacZ (plasmid Gl) to this combination enhanced Rep78 expression to that of the positive controls (lanes 2 and 9). Similarly, transfection with plasmid Hl, which carried the Fl origin of replication upstream to a LacZ fragment (lane 10) or plasmid 11, which carried two LacZ fragments (lane 11) also resulted in Rep78 expression. Analysis of genome titer (qPCR, Figure 18C) revealed comparable yields for plasmid Gl to the positive control plasmids (triple or quadruple transfections), with higher capsid titers (Gyros Protein Technologies, Figure 18D) than the positive control plasmids. 13.3 Rep78 Expression in Cells transfected with Dual Plasmids With Various Promoters

[0560] To test the consequence of replacing backbone elements and truncated P5 promoter with other promoters on Rep78 expression, HEK293 cells were co-transfected with Rep / Cap / Helper plasmid (Cl, Figure 17B) carrying alternative promoters (plasmids JI -Pl, Figure 19 A) and the GAA GOI plasmid at a 1 : 1 molar ratio. The positive control plasmids were identical to that described above in section 13.2 of this example. Protein expression was assessed 72 hr post transfection as described above to visualize viral proteins Rep78, Rep52, VP 1-3, DBP, 0RF6, ORF6 / 7, and loading control GAPDH.

[0561] Results. As shown in Figure 19B, the SV40 promoter in plasmid Ml demonstrated efficient expression of the Rep78 protein relative to Rep52. Analysis of genome and capsid titer revealed that Rep78 expression under the SV40 promoter (Ml) yielded genome (Figure 19C) and capsid (Figure 19D) titers comparable to dual plasmid Gl.

[0562] Example 14: AA V production through triple transfection does not require P5 promoter.

[0563] As shown in Figure 24A, Rep / Cap plasmids with a RHM4-1 Cap gene were constructed with a truncated P5 promoter upstream of the Rep gene, alongside a multicloning site fragment (MCS), a T7 promoter, two LacZ fragments, and a segment of the Fl origin sequence. Importantly, the M3 construct is distinguished by the lack of the full-length P5 promoter.

[0564] Figure 24B shows Western blot analysis of Rep expression for the constructs in Figure 24 A. Expi293 cells were transfected with a plasmid encoding the GAA transgene as described in US20210222141, which is incorporated herein by reference in its entirety. Triple transfection with plasmids containing a GAA transgene, helper, and Rep / Cap at a molar ratio of 1 : 1 : 1 was performed in an Ambr®250 bioreactor. Samples were harvested after 72 hours, and Western blot analysis was performed. The results indicated no difference in the expression levels of Rep78 and Rep52, regardless of the presence or absence of the full- length P5 promoter. Quantification of the genome titers from the transfected cells using qPCR assay is shown in Figure 24C. Samples were harvested at 24, 48, and 72 hours, and genome titer was measured using a qPCR assay. The data indicated that the genome titer remained unchanged with the removal of the full-length P5 promoter.

[0565] Figure 25 shows constructs B3 and N3 which are similar to the A4 and M3 constructs in Figure 24A, respectively, but with the Cap gene as LK03 instead of RHM4-1. Situated upstream of the Rep gene is a truncated P5 promoter accompanied by a multi-cloning site (MCS), a T7 promoter, two LacZ fragments, and a portion of the Fl origin sequence. As with M3 in Figure 24A, the distinguishing feature of the N3 construct compared to B3 is the absence of the complete P5 promoter.

[0566] Figure 25B shows Western blot analysis of Rep and VP proteins for the constructs in Figure 25A. Expi293 cells were transfected with three plasmids: a plasmid encoding a b- domain deleted (BDD) factor eight (FVIII) transgene (BDD-FVIII) (as prepared in PCT / US2015 / 045142, incorporated herein by reference in its entirety), a Rep / Cap plasmid, and a Helper plasmid at a molar ratio of 2.6:1 :2.6 in an Ambr®250 bioreactor. After 72 hours, samples were harvested for Western blot analysis. The results demonstrated a decrease in the expression levels of both Rep and VP proteins when the full-length P5 promoter was absent. Quantification of the genome titer of the transfected cells using qPCR assay is shown in Figure 25C. Samples were collected at 48- and 72-hours post-transfection, and genome titer was quantified via qPCR assay. The data revealed a significant reduction in genome titer at both 48 and 72h, associated with the omission of the full-length P5 promoter.

[0567] Having determined that full length P5 is not required for AAV production, backbone elements that can drive Rep78 expression were examined. To investigate the impact of different plasmid backbone elements on Rep expression, several plasmid variants of B3 (described above) were generated. Figure 26A shows schematic representation of Rep / Cap plasmid variants with different backbone elements (03, and P2-U2).

[0568] Construct 03: is characterized by the separation of the Rep and Cap genes through a 1-kb stuffer sequence. In this construct, both the full-length and truncated P5 promoters have been removed. Despite the absence of these promoters, a fragment of the MCS and one LacZ fragment are positioned upstream of the Rep sequence.

[0569] Construct P2: closely resembles construct 03, with the primary difference being the addition of the L4-22K element under the control of an SV40 promoter, positioned downstream of the LK03 gene.

[0570] Construct Q2: is very similar to construct 03, with the difference being the absence of the LacZ fragment. Instead, this construct features the Fl origin positioned upstream of the Rep sequence.

[0571] Construct R2: closely mirrors construct Q2, with the addition of the L4-22K element placed downstream of the LK03 gene under the control of an SV40 promoter.

[0572] Constructs S2, T2, and U2: these constructs are characterized by the removal of both the full-length and truncated P5 promoters, as well as all backbone elements, including the MCS fragment, T7 promoter, Fl origin, and LacZ fragments. In these constructs, the SV40 promoter drives the expression of Rep78. The distinction between S2, T2, and U2 lies in the different polyadenylation (poly A) signals added downstream of the LK03 sequence in T2 and U2. Additionally, in construct U2, the Rep and Cap genes are separated by a 1-kb stuffer sequence, similar to constructs 03 and P2-R2. Furthermore, the LK03 gene in T2 is driven by a P41 promoter.

[0573] Figure 26B shows Western blot analysis of Rep, VP, and L4-22K proteins for the above constructs after triple transfection in Expi293 cells. Triple transfection was performed with the following plasmids: the BDD-FVIII transgene plasmid, a helper plasmid, and a Rep / Cap plasmid variant (03 and P2-U2) in an Ambrl 5 bioreactor at molar ratio of 1 : 1 : 1. Samples were collected 72 hours post-transfection for analysis. Notably, samples S2-U2, where Rep78 is regulated by the SV40 promoter, exhibited comparable or higher levels of Rep78 expression relative to the Rep / Cap B3 plasmid. However, there was a decrease in the expression of Rep52 and VP proteins. The U2 construct specifically showed a significant alteration in the ratio of VP proteins. Intriguingly, constructs 03 and P2-R2, which lack a P5 promoter, still expressed Rep78 through non-specific transcriptional activity associated with the LacZ and Fl origin fragments in the backbone elements. The presence of the L4-22K element in the P2 and R2 plasmids further increased Rep78 expression levels. In these constructs, VP protein expression either remained consistent with control levels (03 and P2- Q2) or was enhanced (R2).

[0574] Figure 26C shows post transfection quantification of genome titer using qPCR assay. The removal of the full-length P5 promoter and replacement of the backbone elements and the truncated P5 promoter with the SV40 promoter resulted in up to a 53% decrease in genome titer in constructs S2 and T2. Constructs containing the backbone elements (03, P2- R2) produced similar levels of genome titer, although the 03 construct exhibited a significantly lower genome titer compared to the others.

[0575] Example 15: Removal of the P5 promoter eliminates replication competent AAV (rcAAV) impurity.

[0576] Figures 27A-27D show that removal of the P5 promoter eliminated the replication competent AAV (rcAAV) impurity at El 1 genome titer. Figure 27A shows the schematic representation of the Rep / Cap plasmid design based on B3 above with LK03 as the capsid. Constructs V2 and W2 are the same as construct B3, with the difference that Rep and Cap genes are separated using a 3 -kb stuffer sequence. In addition, in the W2 construct the full- length P5 promoter downstream of Cap is removed. Figure 27B shows the Western blot analysis of Rep and VP proteins from these constructs after triple transfection in Expi293 cells. The Expi293 cells were transfected with the following plasmids: BDD-FVIII transgene, Rep / Cap (B3, V2, or W2), and a helper in an Ambr®250 bioreactor at molar ratios of 1 : 1 : 1 and 2.6: 1 :2.6. Samples were collected 72 hours post-transfection for Western blot analysis. At a molar ratio of 2.6: 1 :2.6 (transgene: Rep / Cap: helper), both V2 and W2 plasmids exhibited the same level of Rep expression as the B3 plasmid. However, the VP protein levels were decreased in the V2 and W2 samples. When using a 1 : 1 : 1 molar ratio, Rep78 expression increased in the V2 and W2 samples, and the VP protein levels were comparable to those observed with the B3 plasmid.

[0577] Figure 27C shows the quantification of genome titer using qPCR assay. Samples of the transfected cells were collected at 24, 48, and 72 hours post-transfection for genome titer analysis. At 48 hours, both V2 and W2 samples exhibited the same level of genome titer as the B3 construct at a 1 : 1 : 1 molar ratio (transgene: Rep / Cap: helper). However, the W2 construct showed a significant reduction in genome titer at the 2.6: 1 :2.6 molar ratio at both 48 and 72 hours. At 72 hours, the V2 and W2 constructs demonstrated similar levels of genome titer at the 1 : 1 : 1 ratio, showing around a 45% reduction in genome titer compared to the B3 construct.

[0578] AAV samples were obtained 72h post-transfection as described above. The AAV samples from the V2 and W2 constructs were assessed for the presence of rcAAV at different genome titer levels. The results indicated that the W2 sample did not exhibit rcAAV impurity at a genome titer of 1.00+E11, while the V2 sample was positive for rcAAV impurity at the same genome titer. As shown in Figure 27A, the difference between W2 and V2 is the absence of full length P5 promoter in V2. Thus, removal of the P5 promoter removes rcAAV impurity.

[0579] Example 16: SV40 promoter and a st ffer between Rep and Cap can produce AA V.

[0580] Figure 28A shows a schematic representation of construct X2. Construct X2 features a Rep / Cap plasmid where both the truncated and full-length P5 promoters, as well as the backbone elements upstream of Rep, have been removed. SV40 is utilized to drive Rep78 expression. In this construct, the Rep and Cap genes are separated by a 3 -kb stuff er sequence.

[0581] Figure 28B shows the Western blot analysis of Rep, VP, and DBP expression level from Expi293 cells that were subjected to triple transfection with either a BDD-FVIII or a modified BDD-FVIII transgene plasmid (modBDD-FVIII), a Rep / Cap plasmid (B3 or X2), and a helper plasmid (Helperl or Helperl 1) in an Ambr®250 bioreactor at molar ratios of 1 : 1 : 1 and 2.6: 1 :2.6 (transgene: Rep / Cap: helper). Samples were collected 72 hours post- transfection for Western blot analysis. The combination of Helperl 1 and B3 construct increased the expression of Rep52 in both the BDD-FVIII and modBDD-FVIII transgenes (lanes 4 and 8), however, the levels of Rep78 and VP proteins did not increase significantly. In the case of the X2 Rep / Cap plasmid, the combination of Helperl 1 and X2 Rep / Cap increased both Rep78 and Rep52 levels, and the VP protein level appeared to increase slightly at a molar ratio of 1 : 1 : 1 (lane 7 vs 9).

[0582] Quantification of the genome titer using qPCR assay for the BDD-FVIII transgene is shown in Figure 28C. Expi293 cells were subjected to triple transfection with plasmids including the BDD-FVIII transgene, Rep / Cap (B3 or X2), and a helper plasmid (Helperl or Helperl 1) in an Ambr®250 bioreactor at a molar ratio of 1 : 1 : 1 (transgene: Rep / Cap: helper). Samples were collected at 48 and 72 hours post-transfection for genome titer analysis. The combination of B3 Rep / Cap and Helperl 1 resulted in a 68% increase in genome titer compared to the combination of B3 Rep / Cap and Helperl at the 72-hour data point. In contrast, the combination of X2 Rep / Cap and Helperl 1 did not increase the genome titer, resulting in a genome titer level comparable to that of B3 Rep / Cap and Helperl at 72 hours.

[0583] Quantification of the genome titer using qPCR assay for the modBDD-FVIII transgene is shown in Figures 28D-E. Expi293 cells were transfected with three plasmids: the modBDD-FVIII transgene, Rep / Cap (B3 or X2), and a helper plasmid (Helperl or Helperl 1) in an Ambr®250 bioreactor at a molar ratio of 1 : 1 : 1 (Figure 28D) or 2.6: 1 :2.6 (Figure 28E) (transgene:Rep / Cap:helper). Samples were collected at 48 and 72 hours post-transfection for genome titer analysis. Figure 28D shows the combination of B3 Rep / Cap and Helperl 1 resulted in a 35% increase in genome titer compared to B3 Rep / Cap and Helperl at the 72- hour data point. In contrast, the combination of X2 Rep / Cap and Helperl 1 did not increase genome titer compared to X2 Rep / Cap and Helperl, and instead resulted in an approximate 50% reduction in genome titer compared to the combination of B3 Rep / Cap and Helperl at the same 72-hour time point. Figure 28E shows that at 72h, the combination of B3 Rep / Cap and Helperl 1 resulted in similar genome titer compared to B3 Rep / Cap and Helperl. In contrast, the combination of X2 Rep / Cap and Helperl 1 led to 30% decrease in genome titer compared to B3 Rep / Cap and Helperl 1.

[0584] Figure 28F shows the quantification of the host cell DNA (hcDNA) impurity when Expi293 cells were transfected with three plasmids: the transgene (either modBDD-FVIII or BDD-FVIII), Rep / Cap (B3 or X2), and a helper plasmid (Helperl or Helperl 1). Transfections were performed in an Ambr®250 bioreactor at molar ratios of 1 : 1 : 1 and 2.6: 1 :2.6 (transgene:Rep / Cap:helper). Host cell DNA (hcDNA) impurities were quantified using a qPCR assay following the purification process. The data indicated that the combination of Helper 11 with Rep / Cap B3 significantly decreased hcDNA impurities, achieving up to an 84% reduction. However, the combination of Helperl 1 with Rep / Cap X2 did not result in a significant decrease in hcDNA impurities.

[0585] Example 17: Helper 11 boosts rAA V production in combination with a variety of Rep / Cap constructs.

[0586] Figure 29A shows a schematic representation of several Rep / Cap constructs. Construct A4 as described above for Figure 24A, is a Rep / Cap plasmid with a RHM4-1 Cap gene, a truncated P5 promoter upstream of the Rep gene, alongside a multi-cloning site fragment (MCS), a T7 promoter, two LacZ fragments, and a segment of the Fl origin sequence. Construct Y2 features a Rep / Cap plasmid, with a RHM4-1 Cap gene, where both the truncated and full-length P5 promoters, as well as the backbone elements upstream of Rep, have all been removed. SV40 is utilized to drive Rep78 expression. Constructs Z2 and A3 are similar to Construct Y2, have the same Cap gene, but the difference being the inclusion of stuffer sequences. Construct Z2 incorporates a 1-kb stuffer, while Construct A3 includes a 3-kb stuffer, both of which serve to separate the Rep and Cap genes.

[0587] Figure 29B shows the Western blot analysis of viral proteins. VP2.0 cells (Thermo Fisher Scientific) were transfected with three plasmids: the a-Synuclein transgene (prepared as described in PCT / US2024 / 051907, which is incorporated herein by reference in its entirety), Rep / Cap (A4, Y2, Z2, or A3), and a helper plasmid (Helperl or Helperl 1). Transfections were performed in an Ambr®250 bioreactor at molar ratios of 1 : 1 : 1. Samples were harvested after 72 hours for Western blot analysis to evaluate protein expression. The combination of Helperl 1 with Rep / Cap A4 resulted in increased expression levels of both Rep78 and Rep52 proteins, with a more pronounced increase observed for Rep52. When Helperl 1 was used in combination with Rep / Cap Y2, Z2, and A3, there was also an increase in the expression of Rep78 and Rep52. However, the increase in Rep78 expression was more significant with these constructs than with Rep / Cap (construct A4), while the increase in Rep52 expression was not as substantial as with Rep / Cap A4. These results suggest that L4- 22K expressed from Helperl 1 may affect Rep78 levels through interactions with the SV40 promoter, translation, or splicing pathways, leading to differing ratios of Rep78 to Rep52 between the A4 construct and the Y2, Z2, and A3 constructs. Additionally, Helperl 1 increased the VP (viral protein) levels across all constructs. Figure 29C shows the quantification of the genome titer of the transfected cells using qPCR assay. Samples were harvested 72 hours post-transfection, lysed and the lysate purified for genome titer analysis using a qPCR assay. The addition of Helperl 1 to all Rep / Cap constructs resulted in a significant increase in genome titer, yielding approximately 1E+12 vg / ml.

[0588] Host cell DNA (hcDNA) impurity was also quantified as shown in Figure 29D using a qPCR assay following lysis and purification. The data indicated that the combination of Helperl 1 with all Rep / Cap plasmids significantly reduced hcDNA impurities, achieving up to an 82% reduction.

[0589] Example 18: AA V Production from a dual plasmid system with varying configurations.

[0590] To test the effect of different configurations and ratio of viral elements in a dual plasmid system on AAV production, HEK293 cells were co-transfected in Ambr®250 bioreactors with different dual plasmid systems (Figures 30A-C and 31A-D) at different molar ratios. The positive control includes triple plasmid transfection of Helper 11, either constructs of A3 or A4 Rep / Cap plasmids (from Figure 29A) with a RHM4-1 Cap gene, and a plasmid containing either the a-Synuclein transgene (prepared as described in PCT / US2024 / 051907, which is incorporated herein by reference in its entirety) or ApoE transgene (prepared as described in PCT / US2023 / 076406, which is incorporated herein by reference in its entirety). Residual plasmid DNA (RPD) and genome titer levels were measured for these constructs. Residual plasmid DNA incorporated into the AAV capsid may result in the unintended expression of plasmid sequences, including antibiotic resistance genes. This can potentially trigger immune responses and undermine the safety of gene therapy products. Thus, reducing RPD is preferred in AAV production.

[0591] Results. As illustrated in Figure 30D, constructs B4 and C4, each with the a- Synuclein transgene plasmid at a 1 : 1 molar ratio, produced genome titers (measured by qPCR) comparable to the triple plasmid transfection control (A4 / Helperl l / GOI (a- Synuclein)). Conversely, the combination of the E4 plasmid (which includes helper elements DBP, 0RF6, ORF6 / 7, VA, and Rep) with the F4 plasmid (featuring ITR-GOI and RHM4-1 as the Cap) resulted in a lower genome titer compared to both the triple plasmid transfection controls and the dual plasmid constructs B4 and C4. Furthermore, Figure 30E shows that the level of residual plasmid DNA for the dual plasmids B4 and C4 was similar to that observed in the controls (A3 / Helperl 1 and A4 / Helperl 1) with the GOI plasmid for a-Synuclein transgene. Notably, the combination of the E4 and F4 dual plasmids resulted in more than a threefold reduction in residual plasmid DNA.

[0592] As depicted in Figure 3 IE, the construct D4 (containing Cap, Helper elements DBP, ORF6, ORF 6 / 7, VA RNA, and Rep without P40 promoter activity), combined with the plasmid containing ApoE transgene at a 1 :4 molar ratio (transgene plasmid: Rep / Cap / Helper plasmid), generated genome titers (measured by dPCR) comparable to the triple plasmid transfection control (A4 / Helperl 1 / GOI (ApoE)). Similarly, but not to the same extent as D4, construct B4 combined with the ApoE transgene at a 1 :4 molar ratio (transgene plasmid: Rep / Cap / Helper plasmid), generated genome titers as shown in Figure 3 IE. This difference in titer may be attributable to the absence of P40 promoter in the Rep gene of D4. Conversely, the combination of plasmid D4 with plasmid 14 (containing ITR-GOI (ApoE) and Rep52 without P40 promoter function) produced similar or lower genome titers, depending on the ratio between the two plasmids 14 and D4. For instance, the I4-D4 combination at ratios of 1 :3 and 1 :4 yielded higher genome titers compared to the 1.5: 1 ratio. This variation can be attributed to the lower levels of helper, Rep, and Cap elements at 1.5: 1 ratio. Similarly, the combination of plasmid 14 and plasmid B4 (containing Rep, Cap, and Helper elements) at a 1 :4 plasmid ratio also resulted in lower titers compared to the B4 and transgene combination. This outcome might be related to the disproportionate levels of viral elements collectively. Also, the combination of plasmid J4 (containing ITR-GOI (ApoE), Rep52, and RHM4-1 as the cap) with plasmid M4 (containing Helper elements DBP, ORF6, ORF 6 / 7, VA, and Rep without P40 promoter activity) produced genome titers comparable to those of the 14 and D4 combination, depending on the ratio between the two plasmids. For example, increasing the proportion of plasmid J4 relative to plasmid M4 (4: 1 ratio) resulted in higher genome titers compared to the 1.5: 1 ratio of J4 to M4 plasmids. The genome titer levels of the J4 plasmid in combination with the E4 plasmid (containing Helper elements DBP, ORF6, ORF6 / 7, VA, and Rep) showed a similar trend to that observed with the J4 and M4 plasmid combination, where increasing the proportion of J4 plasmid resulted in higher genome titers. In construct N4, Rep68 / 40 was used as a Rep sequence along with helper elements DBP, ORF6, ORF6 / 7, and VA. The J4 and N4 plasmids were used in a 4: 1 ratio, resulting in a lower genome titer compared to the triple plasmid transfection controls and other dual plasmid combinations. Constructs N4 and 04 are similar, with the primary difference being that Rep68 / 40 in 04 includes the P40 promoter sequence. As demonstrated with constructs B4 and D4, where the absence of the P40 promoter contributes to higher genome titers, a similar outcome is expected for the 04 and J4 combination compared to the N4 and J4 combination (data is not shown). Figure 30C illustrates the combination of plasmids G4 and H4, where plasmid G4 contains RHM4-1 as the cap along with helper elements DBP, ORF6, ORF6 / 7, and VA, and plasmid H4 contains ITR-GOI and Rep. This combination produced a lower level of genome titer compared to the triple plasmid transfection utilizing helper 11, Rep / Cap A4, and ApoE as the transgene plasmid (data not shown). Such effect can be attributed to the disproportional expression of viral elements in the plasmid combination G4 and H4. Figure 3 IF demonstrates that dual plasmid combinations produced similar or lower levels of residual plasmid DNA compared to triple plasmid transfection utilizing helper 11, Rep / Cap A4, and the ApoE transgene. Examples of dual plasmid constructs exhibiting an equal or more than 25% reduction in residual plasmid DNA include combinations of the D4 plasmid with the ApoE transgene plasmid, J4 and M4 plasmids, and J4 and N4 plasmids.

[0593] Example 19: Helper 11 Site-Directed Mutagenesis in the L4-22K Gene Changes vg Titers

[0594] This example compares the effect of mutations in L4-22K sequences in Helper 11 on rAAV production efficiency. The L4-22K gene in the Helper 11 plasmid codes for 195 amino acids. To determine which of these amino acids encoded by the L4-22K gene are relevant for Helper 11 function, site-directed mutagenesis analysis was performed. Expi293 cells were transfected with either a mutant L4-22K Helper 11 plasmid, or Helperl 1 (positive control). rAAV production efficiency was assayed using the 24-Deep-Well scale production system. Titers were quantitated 72 hr post transfection based on testing the RHM4-1 capsid.

[0595] Results. Site-directed mutagenesis identified several amino acid residues that were relevant for the function of L4-22K (Figures 32A-32C). Helper 11 plasmid with the following L4-22K mutations - P13A / P14A, T15A, T55A, S64A, T72A, T100A, Y123F, R148A / L151A, R149A / L152A, H165Q, Y166F and Y167F, yielded a relatively lower rAAV production (vg titer) compared to the positive control Helper 11 plasmid (Figure 32A, Table 3, column A). Among these, R148A / L151A, R149A / L152A, Y166F or Y167F mutations in L4-22K resulted in approximately 75% reduction in viral titers; S64A, T72A, T100A, or Y123F, mutations in L4-22K resulted in approximately 50% reduction in viral titers; whereas P13A / P14 / A, T15A, T55A or H165Q mutations in L4-22K resulted in approximately 25% reduction in viral titers.

[0596] In contrast to the above mutations, Y20F, T79A, T99A, H169Q, Y171F, Y174F and T189A mutations in L4-22K yielded a relative increase in rAAV production (vg titer) compared to the positive control Helper 11 plasmid (Figure 32B, Table 3, column B). By comparison, P13A, P14A, S80A, T102A, S125A, S145A, H165Q / H169Q, S172A, C175S, T176A, S181A, and T194A mutations in L4-22K did not change rAAV titers when compared with the Helper 11 plasmid (Figure 32C, Table 3, column C).

[0597] Table 3. Exemplary L4-22K Mutations in Helper 11 plasmid

[0598] All publications, patents and other references cited herein are incorporated by reference in their entirety into the present disclosure.

Claims

WHAT IS CLAIMED IS:

1. A method of producing recombinant adeno-associated virus (rAAV) vector particles in a cell culture comprising:(A) culturing a population of cells, wherein the cells comprise:(a) an AAV coding sequence comprising an AAV REP coding sequence and an AAV CAP coding sequence; and(b) coding sequences providing helper virus function comprising:(i) a DBP coding sequence;(ii) an ORF6 and / or ORF6 / 7 coding sequence; and(iii) a VA coding sequence where each of (i)-(iii) are operably linked to a constitutive promoter; and(B) harvesting rAAV vector particles.

2. The method of claim 1, wherein each of a) and b) are separately operably linked to a constitutive promoter.

3. The method of claim 1, wherein a) and b) are operably linked a common constitutive promoter and c) is operably linked to a separate constitutive promoter.

4. The method of any one of claims 1-3, wherein the constitutive promoter is selected from the group consisting of a Cytomegalovirus (CMV) promoter, a Simian Virus 40 (SV40) promoter, a bidirectional CMV (BICMV) promoter, a pl9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

5. The method of claim 1, wherein coding sequences providing helper virus function further comprise an L4-22K coding sequence or an L4-22K_alt coding sequence operably linked to a constitutive promoter.

6. The method of claim 5, wherein the L4-22K coding sequence is fused to the DBP coding sequence, the ORF6 coding sequence and / or the ORF6 / 7 coding sequence.

7. The method of claim 6, wherein L4-22K fusion comprises a 2A peptide coding sequence between the L4-22K coding sequence and the DBP coding sequence, the 0RF6 coding sequence and / or the ORF6 / 7 coding sequence.

8. The method of claim 7, wherein the 2A peptide is selected from the group consisting of T2A, P2A, E2A and F2A.

9. The method of any one of claims 1-8, wherein the DBP sequence comprises the sequence set forth in any one of SEQ ID NOs: 1-8.

10. The method of any one of claims 1-9, wherein the ORF6 sequence comprises the sequence set forth in any one of SEQ ID NOs: 23, or 30-36.

11. The method of any one of claims 1-10, wherein the ORF6 / 7 sequence comprises the sequence set forth in any one of SEQ ID NOs: 37-41.

12. The method of and one of claims 5-11, wherein the L4-22K sequence comprises the sequence set forth in any one of SEQ ID NOs: 57-64.

13. The method of and one of claims 5-11, wherein the L4-22K_alt sequence comprises the sequence set forth in SEQ ID NO: 65.

14. The method of claim 5, wherein the L4-22K coding sequence or the L4-22K_alt coding sequence comprises at least one mutation that enhances the production of rAAV particles.

15. The method of claim 14 wherein the L4-22K coding sequence comprises a Y20F mutation.

16. The method of claim 14 wherein the L4-22K coding sequence comprises a T79A mutation.

17. The method of claim 14 wherein the L4-22K coding sequence comprises a T99A mutation.

18. The method of claim 14 wherein the L4-22K coding sequence comprises a H169Q mutation.

19. The method of claim 14 wherein the L4-22K coding sequence comprises a Y171F mutation.

20. The method of claim 14 wherein the L4-22K coding sequence comprises a Y174F mutation.

21. The method of claim 14 wherein the L4-22K coding sequence comprises a T189A mutation.

22. The method of any one of claims 1-21, wherein the VA coding sequence comprises a VA_a coding sequence, a VA_b coding sequence, a VA_c coding sequence, a VA_d coding sequence, a VA_e coding sequence, or a VA_f coding sequence.

23. The method of any one of claims 1-22, wherein the VA sequence comprises the sequence set forth in SEQ ID NO: 42.

24. The method of claim 22, wherein the VA_a sequence comprises the sequence set forth in SEQ ID NO: 43.

25. The method of claim 22, wherein the VA_b sequence comprises the sequence set forth in any one of SEQ ID NOs: 44-48.

26. The method of claim 22, wherein the VA_c sequence comprises the sequence set forth in SEQ ID NO: 49.

27. The method of claim 22, wherein the VA_d sequence comprises the sequence set forth in SEQ ID NO: 50.

28. The method of claim 22, wherein the VA_e sequence comprises the sequence set forth in SEQ ID NO: 51.

29. The method of claim 22, wherein the VA_f sequence comprises the sequence set forth in any one of SEQ ID NOs: 52-55.

30. The method of any one of claims 1-29, wherein the AAV CAP coding sequence is contiguous with the AAV REP coding sequence.

31. The method of any one of claims 1-29, wherein the AAV REP coding sequence and the AAV CAP coding sequence are separated by a stuffer sequence.

32. The method of claim 31, wherein the stuffer sequence does not reduce production of rAAV vector particles.

33. The method of claim 31 or claim 32, wherein the stuffer sequence is about 0.5 kb to about 5.5 kb.

34. The method of claim 31 or claim 32, wherein the stuffer sequence is about 0.5 kb to about 3 kb.

35. The method of any one of claims 1 -34, wherein the AAV CAP coding sequence encodesRHM4-1.

36. The method of any one of claims 1-34, wherein the AAV CAP coding sequence encodes LK03.

37. The method of any one of claims 1-36, wherein the AAV REP coding sequence and the AAV CAP coding sequence are operably linked to at least one promoter sequence selected from the group consisting of a full-length P5 promoter, a truncated P5 promoter, a T7 promoter, mouse mammary tumor virus (MMTV) promoter, a Cytomegalovirus (CMV), a Simian Virus 40 (SV40) promoter, a phosphoglycerate kinase promoter (PGK), ATP-citrate lyase promoter (ACLY), and actin gamma (ACTG) promoter, a bidirectional CMV (BICMV) promoter, a pl 9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

38. The method of 37, wherein the promoter is at least the full-length P5 promoter sequence.

39. The method of claim 38, wherein:(a) the full-length P5 promoter sequence does not decrease production of rAAV vector particles compared to an AAV coding sequence that lacks the full-length P5 promoter sequence; or(b) the full-length P5 promoter sequence enhances production of rAAV vector particles compared to an AAV coding sequence that lacks the full-length P5 promoter sequence.

40. The method of claim 38 or claim 39, wherein absence of the full-length P5 promoter sequence prevents formation of replication competent AAV (rcAAV) impurities.

41. The method of claim 38 or claim 39, wherein absence of the full-length P5 promoter sequence results in reduced formation of replication competent AAV (rcAAV) impurities.

42. The method of any one of claims 1-41, wherein the AAV coding sequence has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 85-91 or 128-146, and / or wherein the coding sequences providing helper virus function sequence has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 71-82.

43. The method of any one of claims 1-41, wherein the AAV coding sequence comprises a sequence set forth in any one of SEQ ID NOs: 85-91 or 128-146, and / or wherein the coding sequences providing helper virus function sequence comprises a sequence set forth in any one of SEQ ID NOs: 71-82 .

44. The method of any one of claims 1-43, wherein reducing residual plasmid DNA (RPD) into the AAV capsid improves biosafety of the rAAV vector particles produced.

45. A nucleic acid comprising:A. a DBP coding sequence;B. an ORF6 coding sequence and / or an ORF6 / 7 coding sequence; andC. a VA coding sequence where each of A-C are operably linked to a constitutive promoter46. The nucleic acid of claim 45, wherein each of A - C are separately operably linked to a constitutive promoter.

47. The nucleic acid of claim 45, wherein A and B are operably linked a common constitutive promoter and C is operably linked to a separate constitutive promoter.

48. The nucleic acid of any one of claims 45-47, wherein the constitutive promoter is selected from the group consisting of a Cytomegalovirus (CMV) promoter, a Simian Virus 40 (SV40) promoter, a bidirectional CMV (BICMV) promoter, a pl9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

49. The nucleic acid of claim 45, wherein the nucleic acid further comprises an L4-22K coding sequence or an L4-22K_alt coding sequence operably linked to a constitutive promoter.

50. The nucleic acid of claim 49, wherein the L4-22K coding sequence is fused to the DBP coding sequence, the ORF6 coding sequence and / or the ORF6 / 7 coding sequence.51 The nucleic acid of claim 50, wherein L4-22K fusion comprises a 2A peptide coding sequence between the L4-22K coding sequence and the DBP coding sequence, the ORF6 coding sequence and / or the ORF6 / 7 coding sequence.

52. The nucleic acid of claim 51, wherein the 2A peptide is selected from the group consisting of T2A, P2A, E2A and F2A.

53. The nucleic acid of any one of claims 45-52, wherein the DBP sequence comprises the sequence set forth in any one of SEQ ID NOs: 1-8.

54. The nucleic acid of any one of claims 45-53, wherein the ORF6 sequence comprises the sequence set forth in any one of SEQ ID NOs: 23, or 30-36.

55. The nucleic acid of any one of claims 45-54, wherein the ORF6 / 7 sequence comprises the sequence set forth in any one of SEQ ID NOs: 37-41.

56. The nucleic acid of and one of claims 49-55, wherein the L4-22K sequence comprises the sequence set forth in any one of SEQ ID NOs: 57-64.

57. The nucleic acid of and one of claims 49-55, wherein the L4-22K_alt sequence comprises the sequence set forth in SEQ ID NO: 65.

58. The nucleic acid of claim 49, wherein the L4-22K coding sequence or the L4-22K_alt coding sequence comprises at least one mutation that enhances the production of rAAV particles.

59. The nucleic acid of claim 58, wherein the L4-22K coding sequence comprises a Y20F mutation.

60. The nucleic acid of claim 58, wherein the L4-22K coding sequence comprises a T79A mutation.

61. The nucleic acid of claim 58, wherein the L4-22K coding sequence comprises a T99A mutation.

62. The nucleic acid of claim 58, wherein the L4-22K coding sequence comprises a H169Q mutation.

63. The nucleic acid of claim 58, wherein the L4-22K coding sequence comprises a Y171F mutation.

64. The nucleic acid of claim 58, wherein the L4-22K coding sequence comprises a Y174F mutation.

65. The nucleic acid of claim 58, wherein the L4-22K coding sequence comprises a T189A mutation.

66. The nucleic acid of any one of claims 45-65, wherein the VA coding sequence comprises a VA_a coding sequence, a VA_b coding sequence, a VA_c coding sequence, a VA_d coding sequence, a VA_e coding sequence, or a VA_f coding sequence.

67. The nucleic acid of any one of claims 45-66, wherein the VA sequence comprises the sequence set forth in SEQ ID NO: 42.

68. The nucleic acid of claim 66, wherein the VA_a sequence comprises the sequence set forth in SEQ ID NO: 43.

69. The nucleic acid of claim 66, wherein the VA_b sequence comprises the sequence set forth in any one of SEQ ID NOs: 44-48.

70. The nucleic acid of claim 66, wherein the VA_c sequence comprises the sequence set forth in SEQ ID NO: 49.

71. The nucleic acid of claim 66, wherein the VA_d sequence comprises the sequence set forth in SEQ ID NO: 50.

72. The nucleic acid of claim 66, wherein the VA_e sequence comprises the sequence set forth in SEQ ID NO: 51.

73. The nucleic acid of claim 66, wherein the VA_f sequence comprises the sequence set forth in any one of SEQ ID NOs: 52-55.

74. A transfected cell comprising:(A) an AAV coding sequence comprising an AAV REP coding sequence and an AAV CAP coding sequence; and(B) coding sequences providing helper virus function comprising:(i) a DBP coding sequence;(ii) an ORF6 and / or ORF6 / 7 coding sequence; and(iii) a VA coding sequence; where each of (i)-(iii) are operably linked to a constitutive promoter; and(C) a gene of interest (GOI) coding sequence.

75. The transfected cell of claim 74, wherein each of A)-C) are separately operably linked to a constitutive promoter.

76. The transfected cell of claim 74, wherein A) and B) are operably linked a common constitutive promoter and C) is operably linked to a separate constitutive promoter.

77. The transfected cell of any one of claims 74-76, wherein the constitutive promoter is selected from the group consisting of a Cytomegalovirus (CMV) promoter, a Simian Virus 40 (SV40) promoter, a bidirectional CMV (BICMV) promoter, a pl9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

78. The transfected cell of any one of claims 7477, wherein coding sequences providing helper virus function further comprise an L4-22K coding sequence or an L4-22K_alt coding sequence operably linked to a constitutive promoter.

79. The transfected cell of claim 78, wherein the L4-22K coding sequence is fused to the DBP coding sequence, the ORF6 coding sequence and / or the ORF6 / 7 coding sequence.

80. The transfected cell of claim 79, wherein L4-22K fusion comprises a 2A peptide coding sequence between the L4-22K coding sequence and the DBP coding sequence, the ORF6 coding sequence and / or the ORF6 / 7 coding sequence.

81. The transfected cell of claim 80, wherein the 2A peptide is selected from the group consisting of T2A, P2A, E2A and F2A.

82. The transfected cell of any one of claims 74-81, wherein the DBP sequence comprises the sequence set forth in any one of SEQ ID NOs: 1-8.

83. The transfected cell of any one of claims 74-82, wherein the ORF6 sequence comprises the sequence set forth in any one of SEQ ID NOs: 23, or 30-36.

84. The transfected cell of any one of claims 74-83, wherein the ORF6 / 7 sequence comprises the sequence set forth in any one of SEQ ID NOs: 37-41.

85. The transfected cell of and one of claims 78-84, wherein the L4-22K sequence comprises the sequence set forth in any one of SEQ ID NOs: 57-64.

86. The transfected cell of and one of claims 78-84, wherein the L4-22K_alt sequence comprises the sequence set forth in SEQ ID NO: 65.

87. The transfected cell of claim 78, wherein the L4-22K coding sequence or the L4- 22K_alt coding sequence comprises at least one mutation that enhances the production of rAAV particles.

88. The transfected cell of claim 87, wherein the L4-22K coding sequence comprises a Y20F mutation.

89. The transfected cell of claim 87, wherein the L4-22K coding sequence comprises a T79A mutation.

90. The transfected cell of claim 87, wherein the L4-22K coding sequence comprises a T99A mutation.

91. The transfected cell of claim 87, wherein the L4-22K coding sequence comprises a H169Q mutation.

92. The transfected cell of claim 87, wherein the L4-22K coding sequence comprises a Y171F mutation.

93. The transfected cell of claim 87, wherein the L4-22K coding sequence comprises a Y174F mutation.

94. The transfected cell of claim 87, wherein the L4-22K coding sequence comprises a T189A mutation.

95. The transfected cell of any one of claims 74-94, wherein the VA coding sequence comprises a VA_a coding sequence, a VA_b coding sequence, a VA_c coding sequence, a VA_d coding sequence, a VA_e coding sequence, or a VA_f coding sequence.

96. The transfected cell of any one of claims 74-95, wherein the VA sequence comprises the sequence set forth in SEQ ID NO: 42.

97. The transfected cell of claim 95, wherein the VA_a sequence comprises the sequence set forth in SEQ ID NO: 43.

98. The transfected cell of claim 95, wherein the VA_b sequence comprises the sequence set forth in any one of SEQ ID NOs: 44-48.

99. The transfected cell of claim 95, wherein the VA_c sequence comprises the sequence set forth in SEQ ID NO: 49.

100. The transfected cell of claim 95, wherein the VA_d sequence comprises the sequence set forth in SEQ ID NO: 50.

101. The transfected cell of claim 95, wherein the VA_e sequence comprises the sequence set forth in SEQ ID NO: 51.

102. The transfected cell of claim 95, wherein the VA_f sequence comprises the sequence set forth in any one of SEQ ID NOs: 52-55.

103. The transfected cell of any one of claims 74-102, wherein the AAV CAP coding sequence is contiguous with the AAV REP coding sequence.

104. The transfected cell of any one of claims 74-102, wherein the AAV REP coding sequence and the AAV CAP coding sequence are separated by a stuffer sequence.

105. The transfected cell of claim 104, wherein the stuffer sequence does not reduce production of rAAV vector particles.

106. The transfected cell of claim 104 or claim 105, wherein the stuffer sequence is about 0.5 kb to about 5.5 kb.

107. The transfected cell of claim 104 or claim 105, wherein the stuffer sequence is about 0.5 kb to about 3 kb.

108. The transfected cell of any one of claims 74-107, wherein the AAV CAP coding sequence encodes RHM4-1.

109. The transfected cell of any one of claims 74-107, wherein the AAV CAP coding sequence encodes LK03.

110. The transfected cell of any one of claims 74-109, wherein the AAV REP coding sequence and the AAV CAP coding sequence are operably linked to at least one promoter sequence selected from the group consisting of a full-length P5 promoter, a truncated P5 promoter, a T7 promoter, mouse mammary tumor virus (MMTV) promoter, a Cytomegalovirus (CMV), a Simian Virus 40 (SV40) promoter, a phosphoglycerate kinase promoter (PGK), ATP-citrate lyase promoter (ACLY), and actin gamma (ACTG) promoter, a bidirectional CMV (BICMV) promoter, a pl9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

111. The transfected cell of 110, wherein the promoter is at least the full-length P5 promoter sequence.

112. The transfected cell of claim 110, wherein:(a) the full-length P5 promoter sequence does not decrease production of rAAV vector particles compared to an AAV coding sequence that lacks the full-length P5 promoter sequence; or(b) the full-length P5 promoter sequence enhances production of rAAV vector particles compared to an AAV coding sequence that lacks the full-length P5 promoter sequence.

113. The transfected cell of claim 111 or claim 112, wherein absence of the full-length P5 promoter sequence prevents formation of replication competent AAV (rcAAV) impurities.

114. The transfected cell of claim 111 or claim 112, wherein absence of the full-length P5 promoter sequence results in reduced formation of replication competent AAV (rcAAV) impurities.

115. The transfected cell of any one of claims 74-114, wherein the AAV coding sequence has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 85-91 or 128-146, and / or wherein the coding sequences providing helper virus function sequence has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 71-82116. The transfected cell of any one of claims 74-114, wherein the AAV coding sequence comprises a sequence set forth in any one of SEQ ID NOs: 85-91 or 128-146, and / or wherein the coding sequences providing helper virus function sequence comprises a sequence set forth in any one of SEQ ID NOs: 71-82.

117. The transfected cell of any one of claims 74-116, wherein the GOI is Factor VIII, acid a-glucosidase (GAA), a-synuclein, or Apolipoprotein E (ApoE).

118. The transfected cell of any one of claims 74-117, wherein reducing residual plasmid DNA (RPD) into the AAV capsid improves biosafety of the rAAV vector particles produced by the transfected cell.

119. A system for producing recombinant adeno-associated virus (rAAV) vector particles comprising:(A) a first nucleic acid construct comprising one or more of:(a) an AAV coding sequence comprising an AAV REP coding sequence and an AAV CAP coding sequence; and(b) coding sequences providing helper virus function comprising:(i) a DBP coding sequence;(ii) an ORF6 and / or ORF6 / 7 coding sequence; and / or(iii) a VA coding sequence; and(B) a second nucleic acid construct comprising a gene of interest (GOI) coding sequence and one or more of:(c) an AAV coding sequence comprising an AAV REP coding sequence and an AAV CAP coding sequence; or(d) coding sequences providing helper virus function comprising:(i) a DBP coding sequence;(ii) an ORF6 and / or ORF6 / 7 coding sequence; and / or(iii) a VA coding sequence; wherein each of (i)-(iii) in the first nucleic acid construct and the second nucleic acid construct is operably linked to a constitutive promoter; and wherein the AAV REP coding sequence is operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity.

120. A system for producing recombinant adeno-associated virus (rAAV) vector particles comprising:(A) a first nucleic acid construct comprising:(a) an AAV coding sequence comprising an AAV REP coding sequence and an AAV CAP coding sequence; and(b) coding sequences providing helper virus function comprising:(i) a DBP coding sequence;(ii) an ORF6 and / or ORF6 / 7 coding sequence; and / or(iii) a VA coding sequence; and(B) a second nucleic acid construct comprising a gene of interest (GOI) coding sequence; wherein each of (i)-(iii) in the first nucleic acid construct is operably linked to a constitutive promoter; and wherein the AAV REP coding sequence is operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity.

121. The system of claim 119 or claim 1206, wherein the AAV REP coding sequence is operably linked to at least one promoter selected from the group consisting of a P5 promoter, a truncated P5 promoter, a T7 promoter, mouse mammary tumor virus (MMTV) promoter, a Cytomegalovirus (CMV), a Simian Virus 40 (SV40) promoter, a phosphoglycerate kinase promoter (PGK), ATP-citrate lyase promoter (ACLY), and actin gamma (ACTG) promoter, a bidirectional CMV (BICMV) promoter, a pl9 promoter, a p40 promoter, a p41 promoter, aCMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

122. The system of any one of claims 119-121, wherein the promoter comprises a T7 promoter and a truncated P5 promoter.

123. The system of any one of claims 119-122, wherein the constitutive promoter is selected from the group consisting of a P5 promoter, a truncated P5 promoter, a T7 promoter, a mouse mammary tumor virus (MMTV) promoter, a Cytomegalovirus (CMV), a Simian Virus 40 (SV40) promoter, a phosphoglycerate kinase promoter (PGK), ATP-citrate lyase promoter (ACLY), and actin gamma (ACTG) promoter, a bidirectional CMV (BICMV) promoter, a pl 9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

124. The system of any one of claims 119-123, wherein the nucleic acid sequence with nonspecific transcription activity comprises from about 156 nucleotides to about 729 nucleotides.

125. The system of any one of claims 119-124, wherein the nucleic acid sequence with nonspecific transcription activity promulgates Rep protein expression compared to Rep protein expression when the nucleic acid sequence with nonspecific transcription activity is absent.

126. The system of any one of claims 119-125, wherein the nucleic acid sequence with nonspecific transcription activity enhances rAAV titer from about 2-fold to about 3 -fold compared to rAAV titer when the nucleic acid sequence with nonspecific transcription activity is absent.

127. The system of any one of claims 119-126, wherein the coding sequences providing helper virus function further comprise an L4-22K coding sequence or an L4-22K_alt coding sequence operably linked to a constitutive promoter.

128. The system of claim 127, wherein the L4-22K coding sequence is fused to the DBP coding sequence, the ORF6 coding sequence, and / or the ORF6 / 7 coding sequence.

129. The system of claim 128, wherein L4-22K fusion construct comprises a 2 A peptide coding sequence between the L4-22K coding sequence and the DBP coding sequence, the 0RF6 coding sequence and / or the ORF6 / 7 coding sequence.

130. The system of claim 129, wherein the 2A peptide is selected from the group consisting of T2A, P2A, E2A and F2A.

131. The system of any one of claims 119-130, wherein the DBP sequence comprises the sequence set forth in any one of SEQ ID NOs: 1-8.

132. The system of any one of claims 119-131, wherein the ORF6 sequence comprises the sequence set forth in any one of SEQ ID NO: 23, or 30-36.

133. The system of any one of claims 119-132, wherein the ORF6 / 7 sequence comprises the sequence set forth in any one of SEQ ID NOs: 37-41.

134. The system of any one of claims 127-133, wherein the L4-22K sequence comprises the sequence set forth in any one of SEQ ID NOs: 57-64.

135. The system of any one of claims 127-133, wherein the L4-22K_alt sequence comprises the sequence set forth in SEQ ID NO: 65.

136. The system of claim 127, wherein the L4-22K coding sequence or the L4-22K_alt coding sequence comprises at least one mutation that enhances the production of rAAV particles.

137. The system of claim 136, wherein the L4-22K coding sequence comprises a Y20F mutation.

138. The system of claim 136, wherein the L4-22K coding sequence comprises a T79A mutation.

139. The system of claim 136, wherein the L4-22K coding sequence comprises a T99A mutation.

140. The system of claim 136, wherein the L4-22K coding sequence comprises a H169Q mutation.

141. The system of claim 136, wherein the L4-22K coding sequence comprises a Y171F mutation.1428. The system of claim 136, wherein the L4-22K coding sequence comprises a Y174F mutation.

143. The system of claim 136, wherein the L4-22K coding sequence comprises a T189A mutation.

144. The system of any one of claims 119-143, wherein the VA coding sequence comprises a VA_a coding sequence, a VA_b coding sequence, a VA_c coding sequence, a VA_d coding sequence, a VA_e coding sequence, or a VA_f coding sequence.

145. The system of claim 144, wherein the VA sequence comprises the sequence set forth in SEQ ID NO: 42.

146. The system of claim 144, wherein the VA_a sequence comprises the sequence set forth in SEQ ID NO: 43.

147. The system of claim 144, wherein the VA_b sequence comprises the sequence set forth in any one of SEQ ID NO: 44-48.

148. The system of claim 144, wherein the VA_c sequence comprises the sequence set forth in SEQ ID NO: 49.

149. The system of claim 144, wherein the VA_d sequence comprises the sequence set forth in SEQ ID NO: 50.

150. The system of claim 144, wherein the VA_e sequence comprises the sequence set forth in SEQ ID NO: 51.

151. The system of claim 144, wherein the VA_f sequence comprises the sequence set forth in any one of SEQ ID NOs: 52-55.

152. The system of any one of claims 119-151, wherein the AAV REP is one or more of Rep52, Rep40, Rep78, and Rep68.

153. The system of claim 152, wherein the Rep78 sequence comprises the sequence set forth in any one of SEQ ID NOs: 115-125.

154. The system of any one of claims 119-153, wherein the AAV CAP coding sequence is contiguous with the AAV REP coding sequence.

155. The system of any one of claims 119-153, wherein the AAV REP coding sequence and the AAV CAP coding sequence are separated by a stuffer sequence.

156. The system of claim 155, wherein the stuffer sequence does not reduce production of rAAV vector particles.

157. The system of claim 155 or claim 156, wherein the stuffer sequence is about 0.5 kb to about 5.5 kb.

158. The system of claim 155 or claim 156, wherein the stuffer sequence is about 0.5 kb to about 3 kb.

159. The system of any one of claims 119-158, wherein the AAV CAP coding sequence encodes RHM4- 1.

160. The system of any one of claims 119-158, wherein the AAV CAP coding sequence encodes LK03.

161. The system of any one of claims 119-160, wherein the AAV REP coding sequence and the AAV CAP coding sequence are operably linked to at least one promoter sequence selected from the group consisting of a full-length P5 promoter, a truncated P5 promoter, a T7 promoter,mouse mammary tumor virus (MMTV) promoter, a Cytomegalovirus (CMV), a Simian Virus 40 (SV40) promoter, a phosphoglycerate kinase promoter (PGK), ATP-citrate lyase promoter (ACLY), and actin gamma (ACTG) promoter, a bidirectional CMV (BICMV) promoter, a pl 9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

162. The system of 161, wherein the promoter is at least the full-length P5 promoter sequence.

163. The system of claim 162, wherein:(a) the full-length P5 promoter sequence does not decrease production of rAAV vector particles compared to a first nucleic acid construct that lacks the full-length P5 promoter sequence; or(b) the full-length P5 promoter sequence enhances production of rAAV vector particles compared to a first nucleic acid construct that lacks the full-length P5 promoter sequence.

164. The system of claim 162 or claim 163, wherein absence of the full-length P5 promoter sequence prevents formation of replication competent AAV (rcAAV) impurities.

165. The system of claim 162 or claim 163, wherein absence of the full-length P5 promoter sequence results in reduced formation of replication competent AAV (rcAAV) impurities.

166. The system of any one of claims 119-165, wherein:(a) the first nucleic acid construct has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 71-82, 85-91, or 128-146, and / or(b) the second nucleic acid construct has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 152-154.

167. The system of any one of claims 119-165, wherein:(a) the first nucleic acid construct comprises a sequence set forth in any one of SEQ ID NOs: 71-82, 85-91, or 128-146, and / or(b) the second nucleic acid construct comprises a sequence set forth in any one of SEQ ID NOs: 152-154.168 The system of any one of claims 119-167, wherein the GOI is Factor VIII, acid a- glucosidase (GAA), a-synuclein, or Apolipoprotein E (ApoE).

169. The system of any one of claims 119-168, wherein reducing residual plasmid DNA (RPD) into the AAV capsid improves biosafety of the rAAV vector particles produced.

170. A method of producing recombinant adeno-associated virus (rAAV) vector particles comprising:(A) culturing a population of cells, wherein the cells comprise:(I) a first nucleic acid construct comprising one or more of:(a) an AAV coding sequence comprising an AAV REP coding sequence and an AAV CAP coding sequence; and(b) coding sequences providing helper virus function comprising:(i) a DBP coding sequence;(ii) an ORF6 and / or ORF6 / 7 coding sequence; or(iii) a VA coding sequence; and(II) a second nucleic acid construct comprising a gene of interest (GOI) coding sequence and one or more of:(c) an AAV coding sequence comprising an AAV REP coding sequence and an AAV CAP coding sequence; and(d) coding sequences providing helper virus function comprising:(i) a DBP coding sequence;(ii) an ORF6 and / or ORF6 / 7 coding sequence; or(iii) a VA coding sequence; wherein each of (i)-(iii) in the first nucleic acid construct and the second nucleic acid construct is operably linked to a constitutive promoter; and wherein the AAV REP coding sequence is operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity; and(B) harvesting rAAV vector particles.

171. The method of claim 170, wherein the AAV REP coding sequence is operably linked to at least one promoter selected from the group consisting of a P5 promoter, a truncated P5 promoter, a T7 promoter, mouse mammary tumor virus (MMTV) promoter, a Cytomegalovirus (CMV), a Simian Virus 40 (SV40) promoter, a phosphoglycerate kinase promoter (PGK), ATP-citrate lyase promoter (ACLY), and actin gamma (ACTG) promoter, a bidirectional CMV (BICMV) promoter, a pl9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

172. The method of claim 171, wherein the promoter is a T7 promoter and a truncated P5 promoter.

173. The method of any one of claims 170-172, wherein the constitutive promoter is selected from the group consisting of a P5 promoter, a truncated P5 promoter, a T7 promoter, a mouse mammary tumor virus (MMTV) promoter, a Cytomegalovirus (CMV), a Simian Virus 40 (SV40) promoter, a phosphoglycerate kinase promoter (PGK), ATP-citrate lyase promoter (ACLY), and actin gamma (ACTG) promoter, a bidirectional CMV (BICMV) promoter, a pl 9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

174. The method of any one of claims 170-173, wherein the nucleic acid sequence with nonspecific transcription activity comprises from about 156 nucleotides to about 729 nucleotides.

175. The method of any one of claims 170-174, wherein the nucleic acid sequence with nonspecific transcription activity promulgates Rep protein expression compared to Rep protein expression when the nucleic acid sequence with nonspecific transcription activity is absent.

176. The method of any one of claims 170-175, wherein the nucleic acid sequence with nonspecific transcription activity enhances rAAV titer from about 2-fold to about 3 -fold compared to rAAV titer when the nucleic acid sequence with nonspecific transcription activity is absent.

177. The method of any one of claims 170-1762, wherein the coding sequences providing helper virus function further comprise an L4-22K coding sequence or an L4-22K_alt coding sequence operably linked to a constitutive promoter.

178. The method of claim 177, wherein the L4-22K coding sequence is fused to the DBP coding sequence, the ORF6 coding sequence, and / or the ORF6 / 7 coding sequence.

179. The method of claim 178, wherein L4-22K fusion construct comprises a 2A peptide coding sequence between the L4-22K coding sequence and the DBP coding sequence, the ORF6 coding sequence and / or the ORF6 / 7 coding sequence.

180. The method of claim 179, wherein the 2A peptide is selected from the group consisting of T2A, P2A, E2A and F2A.

181. The method of any one of claims 170-180, wherein the DBP sequence comprises the sequence set forth in any one of SEQ ID NOs: 1-8.

182. The method of any one of claims 170-181, wherein the ORF6 sequence comprises the sequence set forth in any one of SEQ ID NO: 23, or 30-36.

183. The method of any one of claims 170-182, wherein the ORF6 / 7 sequence comprises the sequence set forth in any one of SEQ ID NOs: 37-41.

184. The method of any one of claims 177-183, wherein the L4-22K sequence comprises the sequence set forth in any one of SEQ ID NOs: 57-64.

185. The method of any one of claims 177-183, wherein the L4-22K_alt sequence comprises the sequence set forth in SEQ ID NO: 65.

186. The method of claim 177, wherein the L4-22K coding sequence or the L4-22K_alt coding sequence comprises at least one mutation that enhances the production of rAAV particles.

187. The method of claim 186, wherein the L4-22K coding sequence comprises a Y20F mutation.

188. The method of claim 186, wherein the L4-22K coding sequence comprises a T79A mutation.

189. The method of claim 186, wherein the L4-22K coding sequence comprises a T99A mutation.

190. The method of claim 186, wherein the L4-22K coding sequence comprises a H169Q mutation.

191. The method of claim 186, wherein the L4-22K coding sequence comprises a Y171F mutation.

192. The method of claim 186, wherein the L4-22K coding sequence comprises a Y174F mutation.

193. The method of claim 186, wherein the L4-22K coding sequence comprises a T189A mutation.

194. The method of any one of claims 170-193, wherein the VA coding sequence comprises a VA_a coding sequence, a VA_b coding sequence, a VA_c coding sequence, a VA_d coding sequence, a VA_e coding sequence, or a VA_f coding sequence.

195. The method of any one of claims 170-194, wherein the VA sequence comprises the sequence set forth in SEQ ID NO: 42.

196. The method of claim 194 wherein the VA_a sequence comprises the sequence set forth in SEQ ID NO: 43.

197. The method of claim 194, wherein the VA_b sequence comprises the sequence set forth in any one of SEQ ID NO: 44-48.

198. The method of claim 194, wherein the VA_c sequence comprises the sequence set forth in SEQ ID NO: 49.

199. The method of claim 194, wherein the VA_d sequence comprises the sequence set forth in SEQ ID NO: 50.

200. The method of claim 194, wherein the VA_e sequence comprises the sequence set forth in SEQ ID NO: 51.

201. The method of claim 194, wherein the VA_f sequence comprises the sequence set forth in any one of SEQ ID NOs: 52-55.

202. The method of any one of claims 170-201, wherein the AAV REP is one or more of Rep52, Rep40, Rep78, and Rep68.

203. The method of claim 202, wherein the Rep78 sequence comprises the sequence set forth in any one of SEQ ID NOs: 115-125.

204. The method of any one of claims 170-203, wherein the AAV CAP coding sequence is contiguous with the AAV REP coding sequence.

205. The method of any one of claims 170-204, wherein the AAV REP coding sequence and the AAV CAP coding sequence are separated by a stuffer sequence.

206. The method of claim 205, wherein the stuffer sequence does not reduce production of rAAV vector particles.

207. The method of claim 205 or claim 206, wherein the stuffer sequence is about 0.5 kb to about 5.5 kb.

208. The method of claim 205 or claim 206, wherein the stuffer sequence is about 0.5 kb to about 3 kb.

209. The method of any one of claims 170-208, wherein the AAV CAP coding sequence encodes RHM4- 1.

210. The method of any one of claims 170-208, wherein the AAV CAP coding sequence encodes LK03.

211. The method of any one of claims 170-210, wherein the AAV REP coding sequence and the AAV CAP coding sequence are operably linked to at least one promoter sequence selected from the group consisting of a full-length P5 promoter, a truncated P5 promoter, a T7 promoter, mouse mammary tumor virus (MMTV) promoter, a Cytomegalovirus (CMV), a Simian Virus 40 (SV40) promoter, a phosphoglycerate kinase promoter (PGK), ATP-citrate lyase promoter (ACLY), and actin gamma (ACTG) promoter, a bidirectional CMV (BICMV) promoter, a pl 9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

212. The method of 211, wherein the promoter is at least the full-length P5 promoter sequence.

213. The method of claim 212, wherein:(a) the full-length P5 promoter sequence does not decrease production of rAAV vector particles compared to a first nucleic acid construct that lacks the full-length P5 promoter sequence; or(b) the full-length P5 promoter sequence enhances production of rAAV vector particles compared to a first nucleic acid construct that lacks the full-length P5 promoter sequence.

214. The method of claim 212 or claim 213, wherein absence of the full-length P5 promoter sequence prevents formation of replication competent AAV (rcAAV) impurities.

215. The method of a claim 212 or claim 213, wherein absence of the full-length P5 promoter sequence results in reduced formation of replication competent AAV (rcAAV) impurities.

216. The method of any one of claims 170-215, wherein:(a) the first nucleic acid construct has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 71-82, 85-91, or 128-146, and / or(b) the second nucleic acid construct has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 152-154.

217. The method of any one of claims 170-215, wherein:(a) the first nucleic acid construct comprises a sequence set forth in any one of SEQ ID NOs: 71-82, 85-91, or 128-146, and / or(b) the second nucleic acid construct comprises a sequence set forth in any one of SEQ ID NOs: 152-154.

218. The method of any one of claims 170-217, wherein the GOI is Factor VIII, acid a- glucosidase (GAA), a-synuclein, or Apolipoprotein E (ApoE).

219. The method of any one of claims 170-218, wherein reducing residual plasmid DNA (RPD) into the AAV capsid improves biosafety of the rAAV vector particles.

220. A transfected cell comprising:(A) a first nucleic acid construct comprising one or more of:(a) an AAV coding sequence comprising an AAV REP coding sequence and an AAV CAP coding sequence; and(b) coding sequences providing helper virus function comprising:(i) a DBP coding sequence;(ii) an ORF6 and / or ORF6 / 7 coding sequence; or(iii) a VA coding sequence; wherein each of (i)-(iii)in the first nucleic acid construct and the second nucleic acid construct is operably linked to a constitutive promoter; and(B) a second nucleic acid construct comprising a gene of interest (GOI) coding sequence and one or more of:(c) an AAV coding sequence comprising an AAV REP coding sequence and an AAV CAP coding sequence; and(d) coding sequences providing helper virus function comprising:(i) a DBP coding sequence;(ii) an ORF6 and / or ORF6 / 7 coding sequence; or(iii) a VA coding sequence; wherein each of (i)-(iii) in the first nucleic acid construct and the second nucleic acid construct is operably linked to a constitutive promoter; and wherein the AAV REP coding sequence is operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity.

221. The transfected cell of claim 220, wherein the AAV REP coding sequence is operably linked to at least one promoter selected from the group consisting of a P5 promoter, a truncated P5 promoter, a T7 promoter, mouse mammary tumor virus (MMTV) promoter, a Cytomegalovirus (CMV), a Simian Virus 40 (SV40) promoter, a phosphoglycerate kinase promoter (PGK), ATP-citrate lyase promoter (ACLY), and actin gamma (ACTG) promoter, a bidirectional CMV (BICMV) promoter, a pl9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

222. The transfected cell of claim 221, wherein the promoter comprises a T7 promoter and a truncated P5 promoter.

223. The transfected cell of any one of claims 220-222, wherein the constitutive promoter is selected from the group consisting of a P5 promoter, a truncated P5 promoter, a T7 promoter, a mouse mammary tumor virus (MMTV) promoter, a Cytomegalovirus (CMV), a Simian Virus 40 (SV40) promoter, a phosphoglycerate kinase promoter (PGK), ATP-citrate lyase promoter (ACLY), and actin gamma (ACTG) promoter, a bidirectional CMV (BICMV) promoter, a pl 9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

224. The transfected cell of any one of claims 220-223, wherein the nucleic acid sequence with nonspecific transcription activity comprises from about 156 nucleotides to about 729 nucleotides.

225. The transfected cell of any one of claims 220-224, wherein the nucleic acid sequence with nonspecific transcription activity promulgates Rep protein expression compared to Rep protein expression when the nucleic acid sequence with nonspecific transcription activity is absent.

226. The transfected cell of any one of claims 220-225, wherein the nucleic acid sequence with nonspecific transcription activity enhances rAAV titer from about 2-fold to about 3 -fold compared to rAAV titer when the nucleic acid sequence with nonspecific transcription activity is absent.

227. The transfected cell of any one of claims 220-226, wherein the coding sequences providing helper virus function further comprise an L4-22K coding sequence or an L4-22K_alt coding sequence operably linked to a constitutive promoter.

228. The transfected cell of claim 227, wherein the L4-22K coding sequence is fused to the DBP coding sequence, the ORF6 coding sequence, and / or the ORF6 / 7 coding sequence.

229. The transfected cell of claim 228, wherein L4-22K fusion construct comprises a 2A peptide coding sequence between the L4-22K coding sequence and the DBP coding sequence, the ORF6 coding sequence and / or the ORF6 / 7 coding sequence.

230. The transfected cell of claim 229, wherein the 2A peptide is selected from the group consisting of T2A, P2A, E2A and F2A.

231. The transfected cell of any one of claims 220-230, wherein the DBP sequence comprises the sequence set forth in any one of SEQ ID NOs: 1-8.

232. The transfected cell of any one of claims 220-231, wherein the ORF6 sequence comprises the sequence set forth in any one of SEQ ID NO: 23, or 30-36.

233. The transfected cell of any one of claims 220-232, wherein the ORF6 / 7 sequence comprises the sequence set forth in any one of SEQ ID NOs: 37-41.

234. The transfected cell of any one of claims 227-233, wherein the L4-22K sequence comprises the sequence set forth in any one of SEQ ID NOs: 57-64.

235. The transfected cell of any one of claims 227-233, wherein the L4-22K_alt sequence comprises the sequence set forth in SEQ ID NO: 65.

236. The transfected cell of claim 227, wherein the L4-22K coding sequence or the L4- 22K_alt coding sequence comprises at least one mutation that enhances the production of rAAV particles.

237. The transfected cell of claim 236, wherein the L4-22K coding sequence comprises a Y20F mutation.

238. The transfected cell of claim 236, wherein the L4-22K coding sequence comprises a T79A mutation.

239. The transfected cell of claim 236, wherein the L4-22K coding sequence comprises a T99A mutation.

240. The transfected cell of claim 236, wherein the L4-22K coding sequence comprises a H169Q mutation.

241. The transfected cell of claim 236, wherein the L4-22K coding sequence comprises a Y171F mutation.

242. The transfected cell of claim 236, wherein the L4-22K coding sequence comprises a Y174F mutation.

243. The transfected cell of claim 236, wherein the L4-22K coding sequence comprises a T189A mutation.

244. The transfected cell of any one of claims 220-243, wherein the VA coding sequence comprises a VA_a coding sequence, a VA_b coding sequence, a VA_c coding sequence, a VA_d coding sequence, a VA_e coding sequence, or a VA_f coding sequence.

245. The transfected cell of claim 244, wherein the VA sequence comprises the sequence set forth in SEQ ID NO: 42.

246. The transfected cell of claim 244, wherein the VA_a sequence comprises the sequence set forth in SEQ ID NO: 43.

247. The transfected cell of claim 244, wherein the VA_b sequence comprises the sequence set forth in any one of SEQ ID NO: 44-48.

248. The transfected cell of claim 244, wherein the VA_c sequence comprises the sequence set forth in SEQ ID NO: 49.

249. The transfected cell of claim 244, wherein the VA_d sequence comprises the sequence set forth in SEQ ID NO: 50.

250. The transfected cell of claim 244, wherein the VA_e sequence comprises the sequence set forth in SEQ ID NO: 51.

251. The transfected cell of claim 244, wherein the VA_f sequence comprises the sequence set forth in any one of SEQ ID NOs: 52-55.

252. The transfected cell of any one of claims 230-251, wherein the AAV REP is one or more of Rep52, Rep40, Rep78, and Rep68.

253. The transfected cell of claim 252, wherein the Rep78 sequence comprises the sequence set forth in any one of SEQ ID NOs: 115-125.

254. The transfected cell of any one of claims 220-253, wherein the AAV CAP coding sequence is contiguous with the AAV REP coding sequence.

255. The transfected cell of any one of claims 220-254, wherein the AAV REP coding sequence and the AAV CAP coding sequence are separated by a stuffer sequence.

256. The transfected cell of claim 255, wherein the stuffer sequence does not reduce production of rAAV vector particles.

257. The transfected cell of claim 255 or claim 256, wherein the stuffer sequence is about 0.5 kb to about 5.5 kb.

258. The transfected cell of claim 255 or claim 256, wherein the stuffer sequence is about 0.5 kb to about 3 kb.

259. The transfected cell of any one of claims 220-258, wherein the AAV CAP coding sequence encodes RHM4-1.

260. The transfected cell of any one of claims 220-258, wherein the AAV CAP coding sequence encodes LK03.

261. The transfected cell of any one of claims 220-260, wherein the AAV REP coding sequence and the AAV CAP coding sequence are operably linked to at least one promoter sequence selected from the group consisting of a full-length P5 promoter, a truncated P5 promoter, a T7 promoter, mouse mammary tumor virus (MMTV) promoter, a Cytomegalovirus (CMV), a Simian Virus 40 (SV40) promoter, a phosphoglycerate kinase promoter (PGK), ATP-citrate lyase promoter (ACLY), and actin gamma (ACTG) promoter, a bidirectional CMV (BICMV) promoter, a pl9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

262. The transfected cell of 261, wherein the promoter is at least the full-length P5 promoter sequence.

263. The transfected cell of claim 262, wherein:(a) the full-length P5 promoter sequence does not decrease production of rAAV vector particles compared to a first nucleic acid construct that lacks the full-length P5 promoter sequence; or(b) the full-length P5 promoter sequence enhances production of rAAV vector particles compared to a first nucleic acid construct that lacks the full-length P5 promoter sequence.

264. The transfected cell of claim 262 or claim 263, wherein absence of the full-length P5 promoter sequence prevents formation of replication competent AAV (rcAAV) impurities.

265. The transfected cell of claim 262 or claim 263, wherein absence of the full-length P5 promoter sequence results in reduced formation of replication competent AAV (rcAAV) impurities.

266. The transfected cell of any one of claims 220-265, wherein:(a) the first nucleic acid construct has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 71-82, 85-91, or 128-146, and / or(b) the second nucleic acid construct has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 152-154.

267. The transfected cell of any one of claims 220-265, wherein:(a) the first nucleic acid construct comprises a sequence set forth in any one of SEQ ID NOs: 71-82, 85-91, or 128-146, and / or(b) the second nucleic acid construct comprises a sequence set forth in any one of SEQ ID NOs: 152-154.

268. The transfected cell of any one of claims 220-267, wherein the GOI is Factor VIII, acid a-glucosidase (GAA), a-synuclein, or Apolipoprotein E (ApoE).

269. The transfected cell of any one of claims 220-268, wherein reducing residual plasmid DNA (RPD) into the AAV capsid improves biosafety of the rAAV vector particles produced by the transfected cell.

270. A system for producing recombinant adeno-associated virus (rAAV) vector particles comprising:(A) a first nucleic acid construct comprising(a) an AAV REP coding sequence;(b) an AAV CAP coding sequence; and(c) coding sequences providing helper virus function comprising:(i) a DBP coding sequence;(ii) an ORF6 and / or ORF6 / 7 coding sequence; or(iii) a VA coding sequence; and(B) a second nucleic acid construct comprising a gene of interest (GOI) coding sequence;wherein (i)-(iii) in the first nucleic acid construct is operably linked to a constitutive promoter; and wherein the AAV REP coding sequence is operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity.

271. The system of claim 270, wherein the first nucleic acid construct comprises a sequence set forth in any one of SEQ ID NOs: 71-82, 85-91, or 128-146.

272. A system for producing recombinant adeno-associated virus (rAAV) vector particles comprising:(A) a first nucleic acid construct comprising(a)an AAV CAP coding sequence; and(b) coding sequences providing helper virus function comprising:(i) a DBP coding sequence;(ii) an ORF6 and / or ORF6 / 7 coding sequence; or(iii) a VA coding sequence; and(B) a second nucleic acid construct comprising:(c) a gene of interest (GO I) coding sequence; and(d) an AAV REP, coding sequence wherein (i)-(iii) in the first nucleic acid construct is operably linked to a constitutive promoter; and wherein the AAV REP coding sequence is operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity.273 The system of claim 272, wherein the first nucleic acid construct comprises a sequence set forth in SEQ ID NOs: 108, or 150 and / or the second nucleic acid construct comprises a sequence set forth in SEQ ID NO: 109, or 152-154.

274. A system for producing recombinant adeno-associated virus (rAAV) vector particles comprising:(A) a first nucleic acid construct comprising(a)an AAV REP coding sequence; and(b) coding sequences providing helper virus function comprising:(i) a DBP coding sequence;(ii) an 0RF6 and / or ORF6 / 7 coding sequence; or(iii) a VA coding sequence; and(B) a second nucleic acid construct comprising:(c) a gene of interest (GO I) coding sequence; and(d) an AAV CAP, coding sequence wherein (i)-(iii) in the first nucleic acid construct is operably linked to a constitutive promoter; and wherein the AAV REP coding sequence is operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity.

275. The system of claim 274, wherein the first nucleic acid construct comprises a sequence set forth in any one of SEQ ID NOs: 98-107, or 110 and / or the second nucleic acid construct comprises a sequence set forth in any one of SEQ ID NOs: 111, 127, or 149.

276. A system for producing recombinant adeno-associated virus (rAAV) vector particles comprising:(A) a first nucleic acid construct comprising(a) an AAV REP52 coding sequence;(b) an AAV CAP coding sequence; and(c) coding sequences providing helper virus function comprising:(i) a DBP coding sequence;(ii) an ORF6 and / or ORF6 / 7 coding sequence; or(iii) a VA coding sequence; and(B) a second nucleic acid construct comprising(c) a gene of interest (GO I) coding sequence; and(d) an AAV Rep78, coding sequence; wherein (i)-(iii) in the first nucleic acid construct is operably linked to a constitutive promoter; and wherein the AAV REP52 and / or REP78 coding sequences are operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity.

277. The system of claim 276, wherein the REP78 sequence comprises the sequence set forth in any one of SEQ ID NOs: 115-125.

278. The system of claim 277, wherein the first nucleic acid construct comprises a sequence set forth in SEQ ID NO: 112 and / or the second nucleic acid construct comprises a sequence set forth in SEQ ID NO: 113.

279. A system for producing recombinant adeno-associated virus (rAAV) vector particles comprising:(A) a first nucleic acid construct comprising(a) an AAV large REP coding sequence;(b) an AAV CAP coding sequence; and(c) coding sequences providing helper virus function comprising:(i) a DBP coding sequence;(ii) an ORF6 and / or ORF6 / 7 coding sequence; or(iii) a VA coding sequence; and(B) a second nucleic acid construct comprising(c) a gene of interest (GO I) coding sequence; and(d) an AAV small REP, coding sequence; wherein (i)-(iii) in the first nucleic acid construct is operably linked to a constitutive promoter; and wherein the AAV large REP and / or small REP coding sequences are operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity.

280. A system for producing recombinant adeno-associated virus (rAAV) vector particles comprising:(A) a first nucleic acid construct comprising(a) an AAV CAP coding sequence; and(b) coding sequences providing helper virus function comprising one or more of:(i) a DBP coding sequence;(ii) an ORF6 and / or ORF6 / 7 coding sequence; and(iii) a VA coding sequence; and(B) a second nucleic acid construct comprising(c) a gene of interest (GO I) coding sequence;(d) an AAV REP coding sequence; and(e) coding sequences providing helper virus function comprising one or more of:(i) a DBP coding sequence;(ii) an ORF6 and / or ORF6 / 7 coding sequence; and(iii) a VA coding sequence; wherein (i)-(iii) in the first nucleic acid construct and the second nucleic acid construct are operably linked to a constitutive promoter; and wherein the AAV REP coding sequence is operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity.

281. A system for producing recombinant adeno-associated virus (rAAV) vector particles comprising:(A) a first nucleic acid construct comprising(a) an AAV REP coding sequence; and(b) coding sequences providing helper virus function comprising one or more of:(i) a DBP coding sequence;(ii) an ORF6 and / or ORF6 / 7 coding sequence; and(iii) a VA coding sequence; and(B) a second nucleic acid construct comprising(c) a gene of interest (GO I) coding sequence;(d) an AAV CAP coding sequence; and(e) coding sequences providing helper virus function comprising one or more of:(i) a DBP coding sequence;(ii) an ORF6 and / or ORF6 / 7 coding sequence; and(iii) a VA coding sequence; wherein (i)-(iii) in the first nucleic acid construct and the second nucleic acid construct are operably linked to a constitutive promoter; and wherein the AAV REP coding sequences are operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity.

282. A system for producing recombinant adeno-associated virus (rAAV) vector particles comprising:(A) a first nucleic acid construct comprising(a) an AAV small REP coding sequence;(b) an AAV CAP coding sequence; and(c) coding sequences providing helper virus function comprising one or more of(i) a DBP coding sequence;(ii) an ORF6 and / or ORF6 / 7 coding sequence; and(iii) a VA coding sequence; and(B) a second nucleic acid construct comprising(c) a gene of interest (GO I) coding sequence;(d) an AAV large REP coding sequence; and(e) coding sequences providing helper virus function comprising one or more of(i) a DBP coding sequence;(ii) an ORF6 and / or ORF6 / 7 coding sequence; and(iii) a VA coding sequence; wherein (i)-(iii) in the first nucleic acid construct and the second nucleic acid construct are operably linked to a constitutive promoter; and wherein the AAV large REP and / or small REP coding sequences are operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity.

283. A system for producing recombinant adeno-associated virus (rAAV) vector particles comprising:(A) a first nucleic acid construct comprising(a) an AAV large REP coding sequence;(b) an AAV CAP coding sequence; and(c) coding sequences providing helper virus function comprising one or more of(i) a DBP coding sequence;(ii) an ORF6 and / or ORF6 / 7 coding sequence; and(iii) a VA coding sequence; and(B) a second nucleic acid construct comprising(c) a gene of interest (GO I) coding sequence;(d) an AAV small Rep coding sequence; and(e) coding sequences providing helper virus function comprising one or more of:(i) a DBP coding sequence;(ii) an ORF6 and / or ORF6 / 7 coding sequence; and(iii) a VA coding sequence; wherein (i)-(iii) in the first nucleic acid construct and the second nucleic acid construct are operably linked to a constitutive promoter; and wherein the AAV large REP and / or small Rep coding sequences are operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity.

284. A system for producing recombinant adeno-associated virus (rAAV) vector particles comprising:(A) a first nucleic acid construct comprising(a) an AAV REP coding sequence; and(b) an AAV CAP coding sequence; and(B) a second nucleic acid construct comprising(c) a gene of interest (GO I) coding sequence;(d) coding sequences providing helper virus function comprising:(i) a DBP coding sequence;(ii) an ORF6 and / or ORF6 / 7 coding sequence; or(iii) a VA coding sequence; wherein (i)-(iii) in the second nucleic acid construct is operably linked to a constitutive promoter; and wherein the AAV REP coding sequences are operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity.

285. A system for producing recombinant adeno-associated virus (rAAV) vector particles comprising:(A) a first nucleic acid construct comprising(a) an AAV REP coding sequence;(b) an AAV CAP coding sequence; and(c) coding sequences providing helper virus function comprising one or more of:(i) a DBP coding sequence;(ii) an 0RF6 and / or ORF6 / 7 coding sequence; and(iii) a VA coding sequence;(B) a second nucleic acid construct comprising(c) a gene of interest (GO I) coding sequence;(d) coding sequences providing helper virus function comprising one or more of:(i) a DBP coding sequence;(ii) an ORF6 and / or ORF6 / 7 coding sequence; and(iii) a VA coding sequence; wherein (i)-(iii) in the first nucleic acid construct and the second nucleic acid construct are operably linked to a constitutive promoter; and wherein the AAV REP coding sequences are operably linked to at least one promoter and a nucleic acid sequence with nonspecific transcription activity.

286. The system of any one of claims 270-285, wherein the coding sequences providing helper virus function further comprise an L4-22K coding sequence or an L4-22K_alt coding sequence operably linked to a constitutive promoter.

287. The system of claim 286, wherein the L4-22K coding sequence is fused to the DBP coding sequence, the ORF6 coding sequence, and / or the ORF6 / 7 coding sequence.

288. The system of claim 287, wherein L4-22K fusion construct comprises a 2A peptide coding sequence between the L4-22K coding sequence and the DBP coding sequence, the ORF6 coding sequence and / or the ORF6 / 7 coding sequence.

289. The system of claim 288, wherein the 2A peptide is selected from the group consisting of T2A, P2A, E2A and F2A.

290. The system of any one of claims 270-289, wherein the DBP sequence comprises the sequence set forth in any one of SEQ ID NOs: 1-8.

291. The system of any one of claims 270-290, wherein the ORF6 sequence comprises the sequence set forth in any one of SEQ ID NO: 23, or 30-36.

292. The system of any one of claims 270-291, wherein the ORF 6 / 7 sequence comprises the sequence set forth in any one of SEQ ID NOs: 37-41.

293. The system of any one of claims 286-292, wherein the L4-22K sequence comprises the sequence set forth in any one of SEQ ID NOs: 57-64.

294. The system of any one of claims 286-292, wherein the L4-22K_alt sequence comprises the sequence set forth in SEQ ID NO: 65.

295. The system of claim 286, wherein the L4-22K coding sequence or the L4-22K_alt coding sequence comprises at least one mutation that enhances the production of rAAV particles.

296. The system of claim 295, wherein the L4-22K coding sequence comprises a Y20F mutation.

297. The system of claim 295, wherein the L4-22K coding sequence comprises a T79A mutation.

298. The system of claim 295, wherein the L4-22K coding sequence comprises a T99A mutation.

299. The system of claim 295, wherein the L4-22K coding sequence comprises a H169Q mutation.

300. The system of claim 295, wherein the L4-22K coding sequence comprises a Y171F mutation.

301. The system of claim 295, wherein the L4-22K coding sequence comprises a Y174F mutation.

302. The system of claim 295, wherein the L4-22K coding sequence comprises a T189A mutation.

303. The system of any one of claims 270-302, wherein the VA coding sequence comprises a VA_a coding sequence, a VA_b coding sequence, a VA_c coding sequence, a VA_d coding sequence, a VA_e coding sequence, or a VA_f coding sequence.

304. The system of any one of claims 270-303, wherein the VA sequence comprises the sequence set forth in SEQ ID NO: 42.

305. The system of claim 303, wherein the VA_a sequence comprises the sequence set forth in SEQ ID NO: 43.

306. The system of claim 303, wherein the VA_b sequence comprises the sequence set forth in any one of SEQ ID NO: 44-48.

307. The system of claim 303, wherein the VA_c sequence comprises the sequence set forth in SEQ ID NO: 49.

308. The system of claim 303, wherein the VA_d sequence comprises the sequence set forth in SEQ ID NO: 50.

309. The system of claim 303, wherein the VA_e sequence comprises the sequence set forth in SEQ ID NO: 51.

310. The system of claim 303, wherein the VA_f sequence comprises the sequence set forth in any one of SEQ ID NOs: 52-55.

311. The system of any one of claims 270-310, wherein the AAV CAP coding sequence is contiguous with the AAV REP coding sequence.

312. The system of any one of claims 270-311, wherein the AAV REP coding sequence and the AAV CAP coding sequence are separated by a stuffer sequence.

313. The system of claim 312, wherein the stuffer sequence does not reduce production of rAAV vector particles.

314. The system of claim 312 or claim 313, wherein the stuffer sequence is about 0.5 kb to about 5.5 kb.

315. The system of claim 312 or claim 313, wherein the stuffer sequence is about 0.5 kb to about 3 kb.

316. The system of any one of claims 270-315, wherein the AAV CAP coding sequence encodes RHM4- 1.

317. The system of any one of claims 270-315, wherein the AAV CAP coding sequence encodes LK03.

318. The system of any one of claims 270-317, wherein the AAV REP coding sequence and the AAV CAP coding sequence are operably linked to at least one promoter sequence selected from the group consisting of a full-length P5 promoter, a truncated P5 promoter, a T7 promoter, mouse mammary tumor virus (MMTV) promoter, a Cytomegalovirus (CMV), a Simian Virus 40 (SV40) promoter, a phosphoglycerate kinase promoter (PGK), ATP-citrate lyase promoter (ACLY), and actin gamma (ACTG) promoter, a bidirectional CMV (BICMV) promoter, a pl 9 promoter, a p40 promoter, a p41 promoter, a CMV early enhancer / chicken P actin (CAG) promoter, a Rous sarcoma virus (RSV) promoter, a tetracycline (Tet) inducible promoter, and a Bidirectional Tet (BiTet) inducible promoter.

319. The system of 318, wherein the promoter is at least the full-length P5 promoter sequence.

320. The system of claim 319, wherein:(a) the full-length P5 promoter sequence does not decrease production of rAAV vector particles compared to a first nucleic acid construct that lacks the full-length P5 promoter sequence; or(b) the full-length P5 promoter sequence enhances production of rAAV vector particles compared to a first nucleic acid construct that lacks the full-length P5 promoter sequence.

321. The system of claim 319 or claim 320, wherein absence of the full-length P5 promoter sequence prevents formation of replication competent AAV (rcAAV) impurities.

322. The system of claim 319 or claim 320, wherein absence of the full-length P5 promoter sequence results in reduced formation of replication competent AAV (rcAAV) impurities.

323. The system of any one of claims 270-322, wherein:(a) the first nucleic acid construct has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 71-82, 85-91, or 128-146, and / or(b) the second nucleic acid construct has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 152-154.

324. The system of any one of claims 270-322, wherein:(a) the first nucleic acid construct comprises a sequence set forth in any one of SEQ ID NOs: 71-82, 85-91, or 128-146, and / or(b) the second nucleic acid construct comprises a sequence set forth in any one of SEQ ID NOs: 152-154.

325. The system of any one of claims 270-324, wherein the GOI is Factor VIII, acid a- glucosidase (GAA), a-synuclein, or Apolipoprotein E (ApoE).

326. The system of any one of claims 270-325, wherein reducing residual plasmid DNA (RPD) into the AAV capsid improves biosafety of the rAAV vector particles produced.

327. A system for producing recombinant adeno-associated virus (rAAV) vector particles comprising:(A) a first nucleic acid construct comprising:(a) an AAV coding sequence comprising an AAV REP coding sequence and an AAV CAP coding sequence;(B) a second nucleic acid construct comprising: coding sequences providing helper virus function comprising:(i) a DBP coding sequence;(ii) an ORF6 and / or ORF6 / 7 coding sequence; or(iii) a VA coding sequence;wherein each of (i)-(iii) in the second nucleic acid construct is operably linked to a constitutive promoter; and(C) a third nucleic acid construct comprising a gene of interest (GOI) coding sequence.

328. The system of claim 327, wherein the coding sequences providing helper virus function further comprise an L4-22K coding sequence or an L4-22K_alt coding sequence operably linked to a constitutive promoter.

329. The system of claim 328, wherein the L4-22K coding sequence is fused to the DBP coding sequence, the 0RF6 coding sequence, and / or the ORF6 / 7 coding sequence.

330. The system of claim 329, wherein L4-22K fusion construct comprises a 2A peptide coding sequence between the L4-22K coding sequence and the DBP coding sequence, the ORF6 coding sequence and / or the ORF6 / 7 coding sequence.

331. The system of any one of claims 327-330, wherein:(a) the first nucleic acid construct has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 85-91 or 128-146;(b) the second nucleic acid construct has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 71-82; and / or(c) the third nucleic acid construct has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 152-154.

332. The system of any one of claims 327-330, wherein:(a) the first nucleic acid construct comprises a sequence set forth in any one of SEQ ID NOs: 71-82, 85-91, or 128-146, and / or(b) the second nucleic acid construct comprises a sequence set forth in any one of SEQ ID NOs: 71-82; and / or(c) the third nucleic acid construct comprises a sequence set forth in any one of SEQ ID NOs: 152-154.

333. A method for producing recombinant adeno-associated virus (rAAV) vector particles comprising culturing a population of cells, wherein the cells comprise:(A) a first nucleic acid construct comprising:(a) an AAV coding sequence comprising an AAV REP coding sequence and an AAV CAP coding sequence;(B) a second nucleic acid construct comprising: coding sequences providing helper virus function comprising:(i) a DBP coding sequence;(ii) an ORF6 and / or ORF6 / 7 coding sequence; or(iii) a VA coding sequence; wherein each of (i)-(iii) in the second nucleic acid construct is operably linked to a constitutive promoter; and(C) a third nucleic acid construct comprising a gene of interest (GO I) coding sequence; and harvesting the rAAV vector particles.

334. The method of claim 333, wherein the coding sequences providing helper virus function further comprise an L4-22K coding sequence or an L4-22K_alt coding sequence operably linked to a constitutive promoter.

335. The method of claim 334, wherein the L4-22K coding sequence is fused to the DBP coding sequence, the 0RF6 coding sequence, and / or the ORF6 / 7 coding sequence.

336. The method of claim 335, wherein L4-22K fusion construct comprises a 2A peptide coding sequence between the L4-22K coding sequence and the DBP coding sequence, the ORF6 coding sequence and / or the ORF6 / 7 coding sequence.

337. The method of any one of claims 333-336, wherein:(a) the first nucleic acid construct has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 85-91 or 128-146;(b) the second nucleic acid construct has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 71-82; and / or(c) the third nucleic acid construct has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 152-154.

338. The method of any one of claims 333-336, wherein:(a) the first nucleic acid construct comprises a sequence set forth in any one of SEQ ID NOs: 71-82, 85-91, or 128-146, and / or (b) the second nucleic acid construct comprises a sequence set forth in any one of SEQID NOs: 71-82; and / or(c) the third nucleic acid construct comprises a sequence set forth in any one of SEQ ID NOs: 152-154.