Systems for amplification of aav cap protein

Abstract

Described herein are polynucleotides for increasing production of rAAV by increasing levels of AAV Capsid proteins, which is achieved by including a strong polyadenylation (polyA) signal sequence 3' to the sequence encoding the AAV capsid proteins and / or using an inducible promoter to drive expression of capsid proteins. Also provided are polynucleotide constructs, vectors, and system thereof, plasmids, and cells, such as, cell lines stably integrated with the polynucleotides, polynucleotide constructs, vectors, and system thereof, plasmids that enable increased production of recombinant AAV (rAAV) virions.

Description

SYSTEMS FOR AMPLIFICATION OF AAV CAP PROTEIN CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.63 / 542,480, filed on October 4, 2023; U.S. Provisional Application No.63 / 536,899, filed on September 6, 2023; U.S. Provisional Application No.63 / 522,304, filed on June 21, 2023; U.S. Provisional Application No.63 / 437,562, filed on January 6, 2023; and U.S. Provisional Application No.63 / 427,040, filed on November 21, 2022, the disclosures of which applications are herein incorporated by reference in their entirety. INTRODUCTION

[0002] Recombinant adeno-associated virus (rAAV) is the preferred vehicle for in vivo gene delivery. AAV has no known disease associations, infects dividing and non-dividing cells, rarely if ever integrates into the mammalian cell genome, and can persist essentially for the lifetime of infected cells as a transcriptionally active nuclear episome. The FDA has recently approved several rAAV gene therapy products and many other rAAV-based gene therapy and gene editing products are in development.

[0003] The most widely used method for producing rAAV virions is based on the helper-virus- free transient transfection of multiple plasmids, typically a triple transfection, into adherent cell lines. Although there is ongoing investment to increase production capacity, current AAV manufacturing processes are inefficient and expensive. In addition, they result in variable product quality, with low levels of encapsidation of a payload, such as a therapeutic payload. There is, therefore, a need for improved methods for producing rAAV. SUMMARY

[0004] The polynucleotides, polynucleotide constructs, vectors, and system thereof, plasmids, and cells, such as, cell lines stably integrated with the polynucleotides, polynucleotide constructs, vectors, and system thereof, plasmids provided herein enable increased production of recombinant AAV (rAAV) virions.

[0005] The increased production of rAAV is hypothesized to be achieved by providing an overall increase in the levels of AAV capsid proteins. In one embodiment, the increase in levels of AAV capsid proteins is achieved by including a strong polyadenylation (polyA) signal sequence 3’ to the sequence encoding the AAV capsid proteins. In another embodiment, the increase in levels of AAV capsid proteins is achieved by using an inducible promoter to driveexpression of capsid proteins which inducible promoter is stronger than the native promoter controlling expression of AAV capsid proteins. In a further embodiment, the increase in levels of AAV capsid proteins is achieved by including a strong polyadenylation (polyA) signal sequence 3’ to the sequence encoding the AAV capsid proteins and by using an inducible promoter to drive expression of capsid proteins which inducible promoter is stronger than the native promoter controlling expression of AAV capsid proteins. In another embodiment, the increase in levels of AAV capsid proteins is achieved by using two separate polynucleotide constructs each encoding AAV capsid proteins, one under the control of the native promoter and another under the control of an inducible promoter. One or more of these embodiments can also be combined to further increase capsid proteins expression and enable increased production of recombinant AAV (rAAV).

[0006] In certain embodiments, the polynucleotide constructs may be introduced into cells, e.g., stably integrated into the nuclear genome of the cells and used to express components for formation of rAAV in an inducible manner thereby avoiding the toxicity of AAV Rep proteins and AAV Cap proteins when constitutively expressed. Methods for generating such cells and cell lines useful for producing rAAV and methods for producing rAAV from the cells and the cell lines are also disclosed.

[0007] In some aspects, the present disclosure provides that polynucleotides comprising (i) a sequence encoding AAV Cap proteins operably linked to an inducible promoter; and (ii) a polyadenylation signal sequence. In some embodiments, the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence and is a 3' of the sequence encoding AAV Cap proteins.

[0008] In some aspects, the present disclosure provides polynucleotides comprising: (i) a first sequence encoding AAV Rep proteins operably linked to one or more promoters; and (ii) a second sequence encoding the polynucleotide comprising a sequence encoding AAV Cap proteins operably linked to an inducible promoter and a polyadenylation signal sequence. In some embodiments, the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence and is a 3’ of the sequence encoding AAV Cap proteins.

[0009] In some aspects, the present disclosure provides that systems of polynucleotides comprising: a) a first polynucleotide comprising a sequence encoding AAV Rep proteins operably linked to one or more promoters; and b) a second polynucleotide comprising (i) a sequence encoding AAV Cap proteins operably linked to an inducible promoter and(ii) a polyadenylation signal sequence, and one or more of c) a third polynucleotide comprising a sequence encoding one or more adenoviral helper proteins; and d) a fourth polynucleotide comprising a sequence encoding a payload. In some embodiments, the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence and is a 3’ of the sequence encoding AAV Cap proteins.

[0010] In other aspects, also disclosed herein are constructs that are capable of conditionally producing recombinant AAV (rAAV) virions within which are packaged an expressible payload, when introduced into a cell. In some embodiments, the constructs may or may not be integrated into the genome of the cell.

[0011] In still other aspects, further provided herein is a stable mammalian cell line and constructs, wherein the cells are capable of conditionally producing recombinant AAV (rAAV) virions within which are packaged an expressible payload; and production of virions is inducible upon addition of a triggering agent.

[0012] In some aspects, further provided herein is a stable mammalian cell line and constructs, wherein the cells are capable of conditionally producing recombinant AAV (rAAV) virions within which are packaged an expressible payload; and production of virions is not conditioned on the presence of a plasmid within the cell. In some aspects, a set of nucleic acids is provided, comprising: (i) a first recombinant nucleic acid sequence encoding AAV Rep proteins and AAV Cap proteins; (ii) a second recombinant nucleic acid sequence encoding the AAV Cap proteins; and (iii) a third recombinant nucleic acid sequence encoding one or more adenoviral helper proteins wherein when the one or more nucleic acids are integrated into the nuclear genome of a cell, e.g., a mammalian cell, the AAV Rep proteins, the AAV Cap proteins, and / or the one or more adenoviral helper proteins are conditionally expressible and thereby conditionally produce recombinant AAV (rAAV) virions.

[0013] In some aspects, a set of nucleic acids is provided, comprising: (i) a first recombinant nucleic acid sequence encoding AAV Rep proteins; (ii) a second recombinant nucleic acid sequence encoding the AAV Cap proteins; and (iii) a third recombinant nucleic acid sequence encoding one or more adenoviral helper proteins wherein when the one or more nucleic acids are integrated into the nuclear genome of a cell, e.g., a mammalian cell, the AAV Rep proteins, the AAV Cap proteins, and / or the one or more adenoviral helper proteins are conditionally expressible and thereby conditionally produce recombinant AAV (rAAV) virions. In some embodiments, a first polynucleotide comprises the first recombinant nucleic acid sequence and a second polynucleotide comprises the second recombinant nucleic acid sequence. In some embodiments, a first polynucleotide comprises the first recombinant nucleic acid sequence andthe second recombinant nucleic acid sequence. In some embodiments, the first recombinant nucleic acid sequence and the second recombinant nucleic acid sequence are separated by a transcriptional blocking element.

[0014] In some embodiments, the conditional expression of the AAV Rep proteins, the AAV Cap proteins, and / or the one or more adenoviral helper proteins is controlled by one or more excisable elements present in the first and third nucleic acids.

[0015] In some embodiments, the one or more excisable elements comprise one or more introns and / or one or more exons. In some embodiments, the first recombinant nucleic acid sequence comprises: a) a first part of the AAV Rep proteins coding sequence; b) a second part of the AAV Rep proteins coding sequence; c) an excisable element between the first part of the AAV Rep protein coding sequence and the second part of the AAV Rep proteins coding sequence; and d) the AAV Cap proteins coding sequence. In some embodiments, the excisable element comprises a) a first spacer segment comprising a first intron, b) a second spacer segment comprising a coding sequence of a detectable marker; and c) a third spacer segment comprising a second intron, and wherein the first spacer segment and the third spacer segment are capable of being excised by endogenous cellular machinery of a mammalian cell.

[0016] In some embodiments, the first recombinant nucleic acid sequence comprises: a) a first part of the AAV Rep proteins coding sequence; b) a second part of the AAV Rep proteins coding sequence; and c) an excisable element between the first part of the AAV Rep protein coding sequence and the second part of the AAV Rep proteins coding sequence. In some embodiments, the excisable element comprises a) a first spacer segment comprising a first intron, b) a second spacer segment comprising a coding sequence of a detectable marker; and c) a third spacer segment comprising a second intron, and wherein the first spacer segment and the third spacer segment are capable of being excised by endogenous cellular machinery of a mammalian cell.

[0017] In some embodiments, the excisable element comprises from 5’ to 3’: a) a 5’ splice site; b) a first spacer segment comprising a first intron; c) a second spacer segment comprising: i) a first lox sequence; ii) a 3’ splice site; iii) an exon; iv) a stop signaling sequence; and v) a second lox sequence; and d) a third spacer segment comprising a second intron and another 3’ splice site.

[0018] In some embodiments, the detectable marker is a luminescent marker, a radiolabel or a fluorescent marker, optionally a fluorescent marker which is GFP, EGFP, RFP, CFP, BFP, YFP, or mCherry.

[0019] In some embodiments, a) the first spacer segment comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 1; and / or b) the second spacer segment comprises anucleic acid sequence having at least 80% identity to SEQ ID NO: 2; and / or c) the third spacer segment comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 3. In some embodiments, the second spacer segment is capable of being excised by a Cre polypeptide.

[0020] In some embodiments, the expression of the AAV Rep proteins and / or the AAV Cap proteins is driven by native promoters. In some embodiments, a) the native promoters P5 and / or P19 drive the expression of the AAV Rep proteins; and / or b) the native promoter P40 drives the expression of the AAV Cap proteins. In some embodiments, a) the native promoters P5 and / or P19 drive the expression of the AAV Rep proteins; and / or b) an inducible promoter drives the expression of the AAV Cap proteins.

[0021] In some embodiments, the second recombinant nucleic acid sequence encodes the AAV Cap proteins under the control of an inducible promoter and a selectable marker under the control of a constitutive promoter. In some embodiments, the selectable marker is an antibiotic resistance gene, such as, hygromycin. In some embodiments, the inducible promoter is a tetracycline inducible promoter and the constitutive promoter is a CMV or EF1alpha promoter.

[0022] In some embodiments, the third recombinant nucleic acid sequence encodes: a) one or more adenoviral helper proteins; b) a conditionally self-excising element; and c) an inducible promoter; wherein, once integrated into the nuclear genome of a mammalian cell, the expression of the one or more adenoviral helper protein coding sequences is under the control of the conditionally self-excising element and the inducible promoter.

[0023] In some embodiments, the one or more adenoviral helper proteins comprise E2A and E4. In some embodiments, the self-excising element comprises a sequence which encodes a polypeptide, e.g., a recombinase polypeptide, such as, a Cre polypeptide. In some embodiments, the polypeptide encoded by the self-excising element is conditionally expressible and is expressed only in the presence of a triggering agent. In some embodiments, the triggering agent is a hormone, e.g., tamoxifen. In some embodiments, the inducible promoter is a tetracycline- inducible promoter (“Tet inducible promoter”). In some embodiments, the third recombinant nucleic acid sequence further comprises a sequence that encodes a Tet responsive activator protein, e.g., Tet-on 3G. In some embodiments, the expression of Tet-on 3G activator protein is driven by an EF1alpha promoter. In some embodiments, the third recombinant nucleic acid sequence comprises a sequence with at least 80% homology, at least 90% homology, at least 95% homology, at least 99% homology, or a sequence identical to SEQ ID NO: 11 or SEQ ID NO: 12.

[0024] In some embodiments, the set of nucleic acids or any of the recombinant nucleic acids as disclosed herein further comprises a nucleic acid sequence encoding a viral associated RNA (“VA-RNA”) sequence. In some embodiments, the third recombinant nucleic acid sequencecomprises a nucleic acid sequence encoding a VA-RNA sequence. In some embodiments, the expression of VA-RNA is constitutive. In some embodiments, the expression of VA-RNA is inducible. In some embodiments, the VA-RNA sequence comprises one or more mutations in the VA-RNA internal promoter, preferably G16A and G60A. In some embodiments, the expression of VA-RNA is driven by a EF1alpha promoter, a U6 promoter, or a U7 promoter. In some embodiments, the expression of VA-RNA is driven by a U6 promoter or a U7 promoter. In some embodiments, the U6 promoter or the U7 promoter comprises a) a first part of a U6 or U7 promoter sequence, b) a stuffer sequence, and c) a second part of a U6 or U7 promoter sequence, and wherein the stuffer sequence is capable of being excised by a Cre polypeptide.

[0025] In some embodiments, a serotype of the AAV Cap proteins is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16 and AAVhu68. In some embodiments, the serotype is an AAV5 Cap protein which comprises one or more mutations or insertions.

[0026] In some embodiments, the set of nucleic acids or any of the recombinant nucleic acids as disclosed herein further encode a fourth recombinant nucleic acid sequence encoding a payload, optionally wherein the payload is: (a) a polynucleotide payload, such as a guide RNA for RNA editing, a guide RNA for Cas protein-directed DNA editing, a tRNA suppressor, or a gene for replacement gene therapy; or (b) a protein such as a therapeutic antibody or a vaccine immunogen.

[0027] In some embodiments, the set of nucleic acids or any of the recombinant nucleic acids as disclosed herein comprise one or more mammalian cell selection elements. In some embodiments, one or more of the mammalian cell selection elements encodes an antibiotic resistance gene, optionally a blasticidin resistance gene. In some embodiments, one or more of the mammalian cell selection elements is an auxotrophic selection element which encodes an active protein. In some embodiments the auxotrophic selection element is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, one or more of the mammalian cell selection elements is a first auxotrophic selection element which encodes an inactive protein that requires expression of a second inactive protein from a second auxotrophic selection coding sequence for activity. In some embodiments, the first auxotrophic selection coding sequence encodes forDHFR Z-Cter (SEQ ID NO: 5) activity, and / or wherein the second auxotrophic selection coding sequence encodes for DHFR Z-Nter (SEQ ID NO: 4).

[0028] In some embodiments, a) the first recombinant nucleic acid comprises a mammalian cell selection element which encodes an antibiotic resistance gene, e.g., a blasticidin resistance gene; b) the second recombinant nucleic acid comprises a mammalian cell selection element which encodes an antibiotic resistance gene, e.g., hygromycin; and c) the third recombinant nucleic acid comprises a mammalian cell selection element which encodes an antibiotic resistance gene, e.g., puromycin.

[0029] The elements of the previous embodiments are capable of being in one or more separate constructs (e.g., polynucleotide constructs), in any combination, wherein the one or more constructs are capable of conditionally producing recombinant AAV (rAAV) virions within which are packaged an expressible payload, when introduced into a cell.

[0030] In some aspects, disclosed herein is a mammalian cell wherein the nuclear genome of the cell comprises a plurality of integrated recombinant nucleic acid constructs which together encode for a recombinant adeno-associated virus (rAAV) virions, wherein the rAAV virions can be conditionally expressed from the cell.

[0031] In some embodiments, the plurality of integrated recombinant nucleic acid constructs comprises the one or more recombinant nucleic acids of any one of previous embodiments, wherein the AAV Rep proteins, the AAV Cap proteins and / or the adenoviral helper proteins can be conditionally expressed from the cell. In some embodiments, the cell is from a cell line that expresses adenoviral helper proteins E1A and E1B, e.g., from nucleic acids integrated into the nuclear genome of the cell.

[0032] In some embodiments, the plurality of integrated recombinant nucleic acid constructs comprises a first integrated polynucleotide construct comprising: a) a first part of an AAV Rep proteins coding sequence; b) a second part of the AAV Rep proteins coding sequence; c) an excisable element between the first part and the second part of the AAV Rep proteins coding sequence, wherein the excisable element comprises: i) a first spacer segment comprising a first intron; ii) a second spacer segment comprising a coding sequence of a detectable marker, wherein the second spacer segment is capable of being excised by a Cre polypeptide; and iii) a third spacer segment comprising a second intron; and d) an AAV Cap proteins coding sequence; wherein the AAV Rep proteins and the AAV Cap proteins expression is driven by the native promoters P5, P19, and P40.

[0033] In some embodiments, the plurality of integrated recombinant nucleic acid constructs comprises a first integrated polynucleotide construct comprising: a) a first part of an AAV Rep proteins coding sequence; b) a second part of the AAV Rep proteins coding sequence; c) anexcisable element between the first part and the second part of the AAV Rep proteins coding sequence, wherein the excisable element comprises: i) a first spacer segment comprising a first intron; ii) a second spacer segment comprising a coding sequence of a detectable marker, wherein the second spacer segment is capable of being excised by a Cre polypeptide; and iii) a third spacer segment comprising a second intron; and d) an AAV Cap proteins coding sequence; wherein the AAV Rep proteins expression is driven by native promoters P5 and P19, and the AAV Cap proteins coding sequence is driven by an inducible promoter.

[0034] In some embodiments, the plurality of integrated recombinant nucleic acid constructs further comprises a second integrated polynucleotide construct comprising a sequence encoding the same AAV Cap proteins as the first integrated polynucleotide construct. In some embodiments, the plurality of integrated recombinant nucleic acid constructs further comprises a second integrated polynucleotide construct comprising a sequence encoding the same AAV Cap proteins as the first integrated polynucleotide construct, wherein the AAV Cap proteins are operably linked to an inducible promoter.

[0035] In some embodiments, the plurality of integrated recombinant nucleic acid constructs further comprises a third integrated polynucleotide construct comprising a) a conditionally expressible VA-RNA coding sequence which comprises a mutation in the VA-RNA internal promoter, wherein the expression of VA-RNA is driven by a U6 or a U7 promoter, optionally wherein the VA-RNA sequence comprises G16A and G60A mutations; b) one or more adenoviral helper protein coding sequences, wherein the adenoviral helper proteins are E2A and E4; c) a conditionally self-excising element which encodes a Cre polypeptide which translocates to the nucleus and self-excises only in the presence of a triggering agent which is tamoxifen, and d) an inducible promoter which is a Tet inducible promoter, and wherein the expression of the one or more adenoviral helper protein coding sequences is under the control of the conditionally self-excising element and the inducible promoter.

[0036] In some embodiments, the plurality of integrated recombinant nucleic acid constructs further comprises a fourth integrated polynucleotide construct comprising encodes for the payload, wherein the payload is a polynucleotide payload.

[0037] In some aspects, a method of producing a population of rAAV virions comprises: (a) culturing the cell of any one of the embodiments disclosed herein in conditions which allow for the expression of the rAAV virions; and (b) isolating the rAAV virions from the cell culture.

[0038] In some aspects, a method of preparing the cell of any one of the previous embodiments comprises: i) providing a mammalian cell and the one or more nucleic acids of any one of the previous embodiments; and ii) selecting for integration of the one or more nucleic acids of any one of the previous embodiments into the nuclear genome of the mammalian cell.

[0039] In other aspects, also provided herein are cells comprising: a) a first polynucleotide construct comprising a first polynucleotide coding for AAV Rep proteins and AAV Cap proteins; b) a second polynucleotide construct coding the AAV Cap proteins; c) a third polynucleotide construct coding for one or more adenoviral helper proteins; wherein when the one or more nucleic acids are integrated into the nuclear genome of a mammalian cell, the AAV Rep proteins, the AAV Cap proteins, and / or the one or more adenoviral helper proteins are conditionally expressible and thereby conditionally produce recombinant AAV (rAAV) virions. In some embodiments, the first polynucleotide coding for AAV Rep proteins and AAV Cap proteins comprises a transcriptional blocking element separating the sequence coding for the AAV Rep proteins and the sequence coding for the AAV Cap proteins.

[0040] In some embodiments, the third polynucleotide construct comprises a sequence coding for: a) one or more helper proteins; b) a self-excising element upstream of the one or more helper proteins; and c) an inducible promoter upstream of the self-excising element. In some embodiments, the self-excising element is operably linked to the inducible promoter. In some embodiments, expression of the self-excising element is driven by the inducible promoter and expression of the AAV Cap proteins encoded by the second recombinant nucleic acid sequence is driven by the same inducible promoter. In some embodiments, expression of the self-excising element is driven by the inducible promoter and expression of the AAV Cap proteins encoded by the second polynucleotide construct is driven by the same inducible promoter. In some embodiments, expression of the self-excising element is driven by the inducible promoter and expression of the AAV Cap proteins encoded by the first polynucleotide construct is driven by the same inducible promoter.

[0041] In some embodiments, the inducible promoter is a tetracycline-responsive promoter element (TRE). In some embodiments, the TRE comprises Tet operator (tetO) sequence concatemers fused to a minimal promoter. In some embodiments, the minimal promoter is a human cytomegalovirus promoter. In some embodiments, the minimal promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 63-68. In some embodiments, the inducible promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 22, 46-48, or 50-62. In some embodiments, transcription is activated from the inducible promoter upon binding of an activator. In some embodiments, the activator binds to the inducible promoter in the presence of a first triggering agent. In some embodiments, the third polynucleotide construct further comprises a sequence coding for an activator. In some embodiments, the activator is operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter. In someembodiments, the EF1alpha promoter comprises at least one mutation. In some embodiments, the constitutive promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 20. In some embodiments, the activator is reverse tetracycline-controlled transactivator (rTA) comprising a Tet Repressor binding protein (TetR) fused to a VP16 transactivation domain. In some embodiments, the rTA comprises four mutations in the tetR DNA binding moiety. In some embodiments, the rTA comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 21, 40-45, or 69-85, or variants thereof.

[0042] In some embodiments, the inducible promoter is a cumate operator sequence. In some embodiments, the cumate operator sequence is downstream of a constitutive promoter. In some embodiments, the constitutive promoter is a human cytomegalovirus promoter. In some embodiments, the inducible promoter is bound by a cymR repressor in the absence of a first triggering agent. In some embodiments, the inducible promoter is activated in the presence of a first triggering agent. In some embodiments, the first triggering agent binds to the cymR repressor. In some embodiments, the third polynucleotide construct further comprises a cymR repressor. In some embodiments, the cymR repressor is operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is EF1alpha promoter. In some embodiments, the EF1alpha promoter comprises at least one mutation. In some embodiments, the constitutive promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 20. In some embodiments, the first triggering agent is a cumate.

[0043] In some embodiments, the sequence coding for the self-excising element comprises a poly A sequence. In some embodiments, the self-excising element is a recombinase. In some embodiments, the recombinase is fused to a ligand binding domain. In some embodiments, the recombinase is Cre polypeptide or flippase polypeptide. In some embodiments, the Cre polypeptide is fused to a ligand binding domain. In some embodiments, the ligand binding domain is a hormone receptor. In some embodiments, the recombinase is a Cre-ERT2 polypeptide. In some embodiments, the self-excising element translocates to the nucleus in the presence of a second triggering agent. In some embodiments, the second triggering agent is an estrogen receptor ligand. In some embodiments, the second triggering agent is a selective estrogen receptor modulator (SERM). In some embodiments, the second triggering agent is tamoxifen. In some embodiments, the recombinase is flanked by recombination sites. In some embodiments, the recombination sites are lox sites or flippase recognition target (FRT) sites. In some embodiments, the lox sites are loxP sites.

[0044] In some embodiments, the one or more adenoviral helper proteins comprise E2A and E4. In some embodiments, the E2A is FLAG-tagged E2A. In some embodiments, the sequence coding for E2A and the sequence coding for E4 are separated by an internal ribosome entry site (IRES) or by P2A.

[0045] In some embodiments, the third polynucleotide construct further comprises a sequence coding for a selectable marker. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is an auxotrophic protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the auxotrophic protein or split intein linked to a C-terminus of the auxotrophic protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the auxotrophic protein or leucine zipper linked to a C-terminus of the auxotrophic protein. In some embodiments, the auxotrophic protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal fragment of the split intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal fragment of the split intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.

[0046] In some embodiments, the third polynucleotide construct further comprises a sequence coding for VA-RNA. In some embodiments, the sequence coding for VA-RNA is a transcriptionally dead sequence. In some embodiments, the sequence coding for VA-RNA comprises at least two mutations in the internal promoter. In some embodiments, expression of VA-RNA is driven by a U6 or U7 promoter. In some embodiments, the third polynucleotide construct further comprises upstream of the sequence coding for VA-RNA gene sequence, from 5’ to 3’: a) a first part of a U6 or U7 promoter sequence; b) a first recombination site; c) a stuffersequence; d) a second recombination site; and e) a second part of a U6 or U7 promoter sequence. In some embodiments, the stuffer sequence is excisable by the recombinase. In some embodiments, the stuffer sequence comprises a sequence encoding a gene. In some embodiments, the stuffer sequence comprises a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a CMV promoter.

[0047] In some embodiments, the first polynucleotide construct comprises: a) a sequence of a first part of a Rep gene; b) a sequence of a second part of the Rep gene; c) a sequence of a Cap gene; and d) an excisable element positioned between the first part of the sequence of Rep gene and the second part of the sequence of the Rep gene.

[0048] In some embodiments, the excisable element comprises a stop signaling sequence. In some embodiments, the excisable element comprises a rabbit beta globin intron. In some embodiments, the excisable element comprises an exon. In some embodiments, the excisable element comprises an intron and an exon. In some embodiments, the excisable element comprises an intron.

[0049] In some embodiments, two splice sites are positioned between the sequence of the first part of the Rep gene and the sequence of the second part of the Rep gene. In some embodiments, the two splice sites are a 5’ splice site and a 3’ splice site. In some embodiments, the 5’ splice site is a rabbit beta globin 5’ splice site. In some embodiments, the 3’ splice site is a rabbit beta globin 3’ splice site. In some embodiments, three splice sites are positioned between the sequence of the first part of the Rep gene and the sequence of the second part of the Rep gene. In some embodiments, the three splice sites are a 5’ splice site, a first 3’ splice site, and a second 3’ splice site. In some embodiments, a first 3’ splice site is a duplicate of the second 3’ splice site. In some embodiments, the first 3’ splice site is a rabbit beta globin 3’ splice site. In some embodiments, the second 3’ splice site is a rabbit beta globin 3’ splice site.

[0050] In some embodiments, the excisable element comprises a recombination site. In some embodiments, the recombination site is a lox site or FRT site. In some embodiments, the lox site is a loxP site.

[0051] In some embodiments, the excisable element comprises from 5’ to 3’: a) the 5’ splice site; b) a first recombination site; c) the first 3’ splice site; d) a stop signaling sequence; e) a second recombination site; and f) the second 3’ splice site.

[0052] In some embodiments, the excisable element comprises from 5’ to 3’: a) the 5’ splice site; b) a first spacer segment; c) a second spacer segment comprising: i) a first recombination site; ii) the first 3’ splice site; iv) a stop signaling sequence; and v) a second recombination site; and d) a third spacer segment comprising the second 3’ splice site. In some embodiments, the first spacer sequence comprises an intron. In some embodiments, the first spacer segmentcomprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the second spacer segment comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the third spacer segment comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 3. In some embodiments, the third spacer segment comprises an intron. In some embodiments, the first spacer segment and the third spacer segment are capable of being excised by endogenous cellular machinery. In some embodiments, the second spacer segment comprises an exon. In some embodiments, the second spacer segment further comprises a polyA sequence. In some embodiments, the polyA sequence is 3’ of the exon. In some embodiments, the polyA sequence comprises a rabbit beta globin (RBG) polyA sequence.

[0053] In some embodiments, the second spacer segment comprises from 5’ to 3’: a) a first recombination site; b) the first 3’ splice site; c) an exon; d) a stop signaling sequence; and e) a second recombination site. In some embodiments, the first recombination site is a first lox sequence and the second recombination site is a second lox sequence. In some embodiments, the first lox sequence is a first loxP sequence and a second lox sequence is a second loxP sequence. In some embodiments, the first recombination site is a first FRT site and the second recombination site is a second FRT site. In some embodiments, the stop signaling sequence is a termination codon of the exon or a polyA sequence. In some embodiments, the polyA sequence comprises a rabbit beta globin (RBG) polyA sequence. In some embodiments, the exon encodes a detectable marker or a selectable marker. In some embodiments, the detectable marker comprises a luminescent marker or a fluorescent marker. In some embodiments, the fluorescent marker is GFP, EGFP, RFP, CFP, BFP, YFP, or mCherry.

[0054] In some embodiments, the second spacer segment is excisable by a recombinase. In some embodiments, the recombinase is a Cre polypeptide or a Flippase polypeptide. In some embodiments, the Cre polypeptide is fused to a ligand binding domain. In some embodiments, the ligand binding domain is a hormone receptor. In some embodiments, the recombinase is a Cre-ERT2 polypeptide.

[0055] In some embodiments, the Rep gene codes for Rep polypeptides. In some embodiments, the Cap gene codes for Cap polypeptides. In some embodiments, transcription of the Rep gene and the Cap gene are driven by native promoters. In some embodiments, the native promoters comprise P5, P19, and P40.

[0056] In some embodiments, the Rep proteins are wildtype Rep polypeptides. In some embodiments, the Rep polypeptides comprise Rep78, Rep68, Rep52, and Rep40. In some embodiments, a truncated replication associated protein comprising a polypeptide expressedfrom the sequence of first part of a Rep gene and the exon is capable of being expressed in the absence of the recombinase.

[0057] In some embodiments, the Cap polypeptides are wildtype Cap polypeptides. In some embodiments, the Cap polypeptides are AAV capsid proteins. In some embodiments, the AAV capsid proteins comprise VP1, VP2, and VP3. In some embodiments, a serotype of the AAV capsid proteins is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, and AAVhu68.

[0058] In some embodiments, the first polynucleotide construct further comprises a sequence coding for a selectable marker. In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is DHFR, GS, TYMS, or PAH. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the auxotrophic selection element or split intein linked to a C-terminus of the auxotrophic selection element. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the active protein or split intein linked to a C-terminus of the active protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the auxotrophic selection element or leucine zipper linked to a C-terminus of the auxotrophic selection element. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the active protein or leucine zipper linked to a C-terminus of the active protein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance.

[0059] In some embodiments, the first polynucleotide construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 1 – SEQ ID NO: 3, SEQ ID 6 – SEQ ID NO: 8, SEQ ID NO: 32, SEQ ID NO: 90 – SEQ ID NO: 99, SEQ ID NO: 101 – SEQ ID NO: 109, SEQ ID NO: 112 – SEQ ID NO: 131, or SEQ ID NO: 136– SEQ ID NO: 138. In some embodiments, the first polynucleotide construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 1 – SEQ ID NO: 3, SEQ ID 6 – SEQ ID NO: 8, SEQ ID NO: 32, SEQ ID NO: 90 – SEQ ID NO: 99, SEQ ID NO: 101 – SEQ ID NO: 109, SEQ ID NO: 112 – SEQ ID NO: 131, or SEQ ID NO: 136 – SEQ ID NO: 138, but wherein these sequences lack SEQ ID NO: 145 downstream of the sequence encoding the AAV Cap proteins.

[0060] In some embodiments, the second polynucleotide construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 149. In some embodiments, the second polynucleotide construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 148.

[0061] In some embodiments, the third polynucleotide construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 9 – SEQ ID NO: 19, SEQ ID 23 – SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 90 – SEQ ID NO: 99, SEQ ID NO: 101 – SEQ ID NO: 109, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the third polynucleotide construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 9 – SEQ ID NO: 19, SEQ ID 23 – SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 90 – SEQ ID NO: 99, SEQ ID NO: 101 – SEQ ID NO: 109, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138.In some embodiments, the first polynucleotide construct, the second polynucleotide construct, and the third polynucleotide construct are stably integrated in the cell’s genome.

[0062] In some embodiments, the cell further comprises a payload construct, wherein the payload construct is a polynucleotide coding for a payload. In some embodiments, the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 33. In some embodiments, the payload construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, the payload construct comprises a sequence of a payload flanked by ITR sequences. In some embodiments, the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 139. In some embodiments, expression of the sequence of the payload is driven by a constitutive promoter. In some embodiments, the constitutive promoter and sequence of the payload are flanked by ITR sequences. In some embodiments, the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 146.

[0063] In some embodiments, the sequence of the payload comprises a polynucleotide sequence coding for a gene. In some embodiments, the gene codes for a selectable marker or detectable marker. In some embodiments, the gene codes for a therapeutic polypeptide or transgene.

[0064] In some embodiments, the sequence of the payload comprises a polynucleotide sequence coding for a therapeutic polynucleotide. In some embodiments, the therapeutic polynucleotide is a tRNA suppressor or a guide RNA. In some embodiments, the guide RNA is a polyribonucleotide capable of binding to a protein. In some embodiments, the protein is nuclease. In some embodiments, the protein is a Cas protein, an ADAR protein, or an ADAT protein. In some embodiments, the Cas protein is catalytically inactive Cas protein. In some embodiments, the payload construct is stably integrated into the genome of the cell.

[0065] In some embodiments, a plurality of the payload construct is stably integrated into the genome of the cell. In some embodiments, the plurality of the payload constructs is separately stably integrated into the genome of the cell. In some embodiments, the payload construct further comprises a sequence coding for a selectable marker or detectable marker outside of the ITR sequences. In some embodiments, the payload construct is integrated into the genome of the cell. In some embodiments, the selectable marker is a first part of a split blasticidin and wherein the first polynucleotide sequence encodes the second part of the split blasticidin.

[0066] In some embodiments, a method for increasing production of rAAV virions from a cell, comprises amplifying expression of AAV Rep and capsid proteins, helper proteins, and / or payload in the cell, wherein the amplifying comprises: increasing copy number of a polynucleotide construct comprising a sequence encoding one or more AAV Rep proteins and a sequence encoding one or more AAV cap proteins, a polynucleotide construct comprising a sequence encoding the one or more AAV cap proteins, a polynucleotide construct comprising a sequence encoding one or more AAV helper proteins, and / or a polynucleotide construct comprising a sequence encoding the payload by introducing an agent to amplify expression of the Rep / Cap genes, helper genes, and / or payload.

[0067] In some embodiments, the increasing copy number of the polynucleotide construct(s) comprises culturing the cell under conditions that select for the presence of the selectable marker encoded by the polynucleotide construct(s) under the control of an attenuated promoter, thereby producing the cell comprising an increased copy number of the polynucleotide construct(s) compared to a cell comprising the polynucleotide construct comprising a selectable marker operably linked to a nonattenuated promoter. In some embodiments, the attenuated promoter is an attenuated EF1alpha promoter and the nonattenuated promoter is an EF1alpha promoter; optionally, wherein the attenuated EF1alpha promoter is SEQ ID NO: 132 and the EF1alpha promoter is SEQ ID NO: 133.

[0068] In some embodiments, the polynucleotide construct further comprises a mutated selectable marker having decreased enzymatic activity compared to an unmutated selectable marker. In some embodiments, the increasing copy number of the polynucleotide constructcomprises culturing the cell under conditions that select for the presence of the mutated selectable marker, thereby producing the cell comprising an increased copy number of the polynucleotide construct compared to the polynucleotide construct further comprising the unmutated selectable marker. In some embodiments, the mutated selectable marker is a mutated GS and the unmutated selectable marker is GS; optionally, wherein the mutated GS having a R324C, R324S, or R341C mutation as compared to SEQ ID NO: 112 and the GS is SEQ ID NO: 112; optionally, wherein the mutated GS is SEQ ID NO: 142, SEQ ID NO: 143, or SEQ ID NO: 144. In some embodiments, the polynucleotide construct further comprises a selectable marker. In some embodiments, the increasing copy number of the polynucleotide construct comprises culturing the cell under conditions that select for the presence of the selectable marker and in the presence of an inhibitor of the selectable marker, thereby producing the cell comprising an increased copy number of the polynucleotide construct compared to the polynucleotide construct further comprising the selectable marker cultured in the absence of the inhibitor of the selectable marker. In some embodiments, the polynucleotide construct is any polynucleotide construct as described herein.

[0069] Also provided herein are methods of producing a stable cell line comprising expanding a cell described above.

[0070] Also provided herein are methods of producing a plurality of rAAV virion comprising culturing a cell described above in the presence of a first triggering agent and a second triggering agent. In some embodiments, the first triggering agent is doxycycline and the second triggering agent is tamoxifen.

[0071] In some embodiments, the plurality of rAAV virion has an encapsidation ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99 prior to purification. In some embodiments, the plurality of rAAV virion has a F:E ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99 prior to purification. In some embodiments, the plurality of rAAV virion have a concentration of greater than 1 × 1011or no less than 5 × 1011, 1 × 1012, 5 × 1012, 1 × 1013or 1 × 1014viral genomes per milliliter prior to purification. In some embodiments, the plurality of rAAV virion have an infectivity of no less than 50%, 60%, 70%, 80%, 90%, 95%, or 99% at an MOI of 1 × 105vg / target cell or less. In some embodiments, the culturing is in a bioreactor.

[0072] Also provided herein are pharmaceutical compositions comprising the rAAV virion produced by the cell or the method described above and a pharmaceutically acceptable carrier. Also provided herein are methods of treating a condition or disorder, the method comprising administering a therapeutically effective amount of the pharmaceutical composition to a patient in need thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0073] FIG.1A depicts an exemplary system of polynucleotides for inducibly producing rAAV. In absence of first and second triggering agents, the system is in an off state. This system includes Construct 1, Construct 3, Construct 4, and Construct 2, and is referred to as v1.2 system. The v1.2 system is distinct from the v1.0 system in that the v1.2 system comprises a recombinant nucleic acid that expresses Cap protein driven by an inducible promoter (see, e.g., Construct 2). In this schematic, which depicts an exemplary v1.2 system, Construct 1 and Construct 2 are two separate polynucleotides which are configured for inducibly expressing AAV capsid proteins. Construct 1, in addition to inducibly expressing AAV Cap proteins also inducibly expresses full-length AAV Rep proteins, and both AAV Cap proteins and AAV Rep proteins are driven by their native promoters (p40, and p5 and p19, respectively).

[0074] FIG.1B depicts the post-triggered state (also referred to as the on state) of Constructs 1- 4 shown in FIG.1A following the addition of the first triggering agent, doxycycline, and the second triggering agent, tamoxifen. The Cre coding element is positioned between LoxP sites and is additionally fused to estrogen response elements (“ER2”), which allows for control over the localization of Cre in response to estrogen agonists, such as tamoxifen. Upon addition of a first triggering agent, e.g., doxycycline, Cre is expressed, and upon addition of the second triggering agent, e.g., tamoxifen, Cre translocates to the nucleus. Following translation and translocation of the Cre protein into the cell nucleus, the Cre protein effects excision of its own coding sequence, leaving the integrated constructs as shown in FIG.1B. Therefore, adenoviral E2A and E4 helper proteins are expressed. Cre also excises out the excisable element flanked by LoxP sites in Construct 1, allowing AAV rep and cap coding sequences expression under control of native promoters. rAAV virions encapsidating the payload, such as a GOI, are therefore produced.

[0075] FIG.2A depicts an exemplary system of polynucleotides for inducibly producing rAAV, which is also an exemplary v1.2 system. In absence of a first triggering agent, e.g., doxycycline, and a second triggering agent, e.g., tamoxifen, the system is in an off state. In Construct 1 of this schematic, the sequence encoding the AAV Cap proteins is operably linked to an inducible promoter (for example, Tet-On promoter). This is different from the v1.0 system in which the AAV Cap proteins are expressed under the control of a native AAV promoter. In this v1.2 system, the coding sequences for the Rep and Cap proteins are oriented in opposite directions such that the internal p40 promoter in Rep coding sequence which controls expression of the Cap proteins is spatially separated from the Cap coding sequence and does not control Cap proteins expression. The inducible promoter (for example, Tet-On promoter) controlling theexpression of the Cap proteins is stronger than the native p40 promoter, resulting in increased Cap proteins expression as compared to the v1.0 system. The coding sequences and promoters for the Rep proteins are separated from the coding sequence and the inducible promoter for the Cap proteins by a transcription blocking element (TBE). This construct may also be referred to as a Rep-Cap construct.

[0076] FIG.2B depicts the on state of the system of polynucleotides for inducibly producing rAAV depicted in FIG.2A, after induction by a first triggering agent, e.g., doxycycline, and a second triggering agent, e.g., tamoxifen. The Cre coding element is positioned between LoxP sites and is additionally fused to estrogen response elements (“ER2”), which allows for control over the localization of Cre in response to estrogen agonists, such as tamoxifen. Upon addition of a first triggering agent, e.g., doxycycline, Cre is expressed and Cap proteins are expressed, and upon addition of the second triggering agent, e.g., tamoxifen, Cre translocates to the nucleus. Following translation and translocation of the Cre protein into the cell nucleus, the Cre protein effects excision of its own coding sequence, leaving the integrated constructs as shown in FIG.2B. Therefore, adenoviral E2A and E4 helper proteins are expressed. Cre also excises out the excisable element flanked by LoxP sites in Construct 1, allowing AAV rep coding sequences expression under control of native promoters. rAAV virions encapsidating the payload, such as a GOI, are therefore produced.

[0077] FIG.3A shows an exemplary system of polynucleotides for inducibly producing rAAV, which is also an exemplary v1.2 system. In absence of a first triggering agent and a second triggering agent, the system is in an off state. In Construct 1 of this schematic, the sequence encoding the AAV Cap proteins is operably linked to an inducible promoter (for example, Tet- On promoter). The coding sequences and promoters for the Rep proteins are separated from the coding sequence and inducible promoter for the Cap proteins by a transcription blocking element (TBE). This system includes an additional polynucleotide, depicted as Construct 4, for expressing AAV Cap proteins driven by an inducible promoter. Construct 4 may be referred to as a Cap construct and is the same as Construct 2 shown in FIGs.1A and 1B.

[0078] FIG.3B depicts the on state of the system of polynucleotides for inducibly producing rAAV depicted in FIG.3A, after induction by the agents, e.g., doxycycline, and a second triggering agent, e.g., tamoxifen. The Cre coding element is positioned between LoxP sites and is additionally fused to estrogen response elements (“ER2”), which allows for control over the localization of Cre in response to estrogen agonists, such as tamoxifen. Upon addition of a first triggering agent, e.g., doxycycline, Cre is expressed and Cap proteins are expressed, and upon addition of the second triggering agent, e.g., tamoxifen, Cre translocates to the nucleus. Following translation and translocation of the Cre protein into the cell nucleus, the Cre proteineffects excision of its own coding sequence, leaving the integrated constructs as shown in FIG. 2B. Therefore, adenoviral E2A and E4 helper proteins are expressed. Cre also excises out the excisable element flanked by LoxP sites in Construct 1, allowing AAV rep and cap coding sequences expression under control of native promoters. rAAV virions encapsidating the payload, such as a GOI, are therefore produced.

[0079] FIG.4 shows an exemplary method for generating a pool of P2 cell clone that includes Construct 3 configured to express Adenovirus helper proteins, Construct 1 configured to express AAV Rep and Cap proteins and Construct 4 to express a payload (GOI, e.g., GFP or progranulin). Constructs 1, 3, and 4 are those depicted in FIG.1A.

[0080] FIG.5 shows an exemplary method for generating a P3 cell pool from the P2 cell pool by introducing a polynucleotide (e.g., Construct 2, as depicted in FIG.1A) that encodes for AAV Cap proteins “T231 (P3)” into the P2 polyclonal cell pool of FIG.4. A polynucleotide that does not encode for AAV Cap proteins is used as a control to generate “T232 (P3)”.

[0081] FIG.6 provides data for the titer of AAV Capsid proteins (Capsid Titer) and encapsidated payload, progranulin (PGRN), (PGRN titer) produced in T231 (P3) and T232 (P3) cells. Also shown is a table depicting percent packaging of the gene encoding PGRN for T231 (P3) cells and T232 (P3) cells.

[0082] FIG.7 depicts an exemplary v1.2 system of polynucleotide Constructs 1, 2, 3, and 4 that includes a first polynucleotide for encoding AAV Cap proteins under the control of the native promoter, p40, (see Construct 1) and a second polynucleotide for encoding AAV Cap proteins (Construct 2) under the control of an inducible promoter to achieve an overall increase in AAV Cap proteins expression as compared to a system of constructs that includes only the first polynucleotide for encoding AAV Cap proteins under the control of the native promoter, p40 (e.g., only Construct 1 and not including Construct 2).

[0083] FIG.8 provides a schematic summarizing induction of cells that include the v1.2 system of polynucleotides “v1.2 cells” and characterization of the Capsid levels, excision kinetics for production of AAV Rep and Cap, GOI levels, and levels of encapsidated GOI.

[0084] FIG.9 provides a timeline from the point of induction of cells with addition of tamoxifen and doxycycline (T=0) through 96 hours post-induction (T=96 hours). Expression of a protein marker, blue fluorescent protein, encoded by Construct 1 prior to the induction in v1.2 cells (T231) is measured and compared to a single cell clone generated after introduction and selection for integration of Construct 3 into viral producing cells and subsequent selection for that clone (T177CL9). Presence of the additional polynucleotide Construct 2 does not impact excision of the gene encoding BFP such that AAV Rep and Cap proteins can be expressed.

[0085] FIG.10 provides data for characterization of rAAV produced by v1.2 cells and v1.0 cells. The ratio of “encapsidated GOI / total GOI” in v1.2 cells was measured and compared to the ratio of “encapsidated GOI / total GOI” in v1.0 cells. Fig.10 shows that in v1.0 cells, the production of genome of interest (GOI) is higher than Capsid production, indicating that capsid production could be a rate limiting step for producing encapsidated GOI (e.g., a payload). Increased capsid production in the V1.2 cells resulted in increased generation of rAAV.

[0086] FIG.11 shows a time course of production of viral particles over a 7-day period after induction of v1.2 (pool) and v1.0 (clone) cells with tamoxifen and doxycycline. v1.2 cells produce viral particles that encapsidate a gene for expressing PRGN while v1.0 cells produce viral particles that encapsidate a gene for expressing green fluorescent protein (GFP). In FIG.11, the expression of capsids was measured (vp / ml).

[0087] FIG.12 shows a time course of production of viral particles over a 7-day period after induction of v1.2 (pool) and v1.0 (clone) cells with tamoxifen and doxycycline. v1.2 cells produce viral particles that encapsidate a gene for expressing PGRN while v1.0 cells produce viral particles that encapsidate a gene for expressing green fluorescent protein (GFP). In FIG. 12, encapsidated GOI (either the gene for expressing PGRN or the gene for expressing GFP) was measured (vg / ml).

[0088] FIG.13 shows high titer produced after induction of v1.2 cells from the T231 stable cell pool wherein the payload is progranulin (“v1.2 Pool (PGRN)”). The graph shows the capsid titer (vp / ml) from cell lysate produced after induction of v1.2 Pool (PGRN) cells compared to after induction of a v1.0 stable cell line pool wherein the payload is progranulin (“v1.0 Pool (PGRN)”), compared to after induction of a v1.0 stable cell line pool wherein the payload is eGFP (“v1.2 Pool (eGFP)”), and compared to after transient transfection of cells wherein the payload is eGFP (“Transient Transfection (eGFP)”). The v1.2 Pool (PGRN) cells show a greater than 10-fold improvement in viral titer over Transient Transfection (eGFP) titer and improvement in viral titer over both v1.0 Pool (PGRN) titer and v1.0 Pool (eGFP) titer.

[0089] FIG.14A shows high capsid titer produced after induction of v1.2 pool cells wherein the payload is progranulin (v1.2 Pool (PGRN)) and after induction of v1.2 single cell clones wherein the payload is progranulin (v1.2 Clones (PGRN)). The v1.2 Pool (PGRN) capsid titer and v1.2 Clones (PGRN) capsid titer are higher than the v1.0 stable pool cells wherein the payload is eGFP (v1.0 eGFP Pool) capsid titer, v1.0 single stable cell clones wherein the payload iseGFP (v1.0 eGFP clones) capsid titer, v1.0 stable pool cells wherein the payload is progranulin (v1.0 PGRN Pool) capsid titer, and v1.0 single stable cell clones wherein the payload is progranulin (v1.0 PGRN Clones) capsid titer, indicating the v1.2 stable cells have successfully increased capsid titer >2 log.

[0090] FIG.14B shows high vector genome production after induction of v1.2 pool cells wherein the payload is progranulin (v1.2 Pool (PGRN)) and after induction of v1.2 single cell clones wherein the payload is progranulin (v1.2 Clones (PGRN)). The v1.2 Pool (PGRN) vector genome titer and v1.2 Clones (PGRN) vector genome titer are higher than the v1.0 stable pool cells wherein the payload is eGFP (v1.0 eGFP Pool) vector genome titer, v1.0 single stable cell clones wherein the payload iseGFP (v1.0 eGFP clones) vector genome titer, v1.0 stable pool cells wherein the payload is progranulin (v1.0 PGRN Pool) vector genome titer, and v1.0 single stable cell clones wherein the payload is progranulin (v1.0 PGRN Clones) vector genome titer, indicating the v1.2 stable cells have successfully increased vector genome titer.

[0091] FIG.15 shows the parent cell line is T205 CL23 (Top clone from stable cells v1.0 pool, in which the payload is progranulin). T318 was generated from T205 CL23 clone that has integrated the sequence for Tet inducible capsid (Construct 2 of FIG.1A) and selected based on hygromycin-resistance to make a v1.2 pool. Selecting a top clone with v1.0 system integrated in the genome and introducing Construct 2 into the top clone provides for increased vector genome titer and capsid titer as compared to introducing Construct 2 into a pool of clones with v1.0 system integrated in the genome.

[0092] FIG.16 depicts an exemplary v1.0 system in the pre-triggered state. Cap proteins are expressed under the control of a native AAV p40 promoter. The brackets in construct 3 indicate the position of the flanking ITRs. V1.0 system is described in U.S. Patent Application Publication No.2022 / 0145328 which is incorporated herein. V1.0 cells are a pool of cells or a cell line that includes the constructs of the V1.0 system. DETAILED DESCRIPTION

[0093] To solve the problems associated with low rAAV titers, disclosed herein are polynucleotide constructs and cells, such as, cell lines stably integrated with the polynucleotide constructs that enable increased production of recombinant AAV (rAAV) virions. The increased production is hypothesized to be achieved by providing an overall increase in the levels of capsid proteins which in turn increases production of rAAV. In one embodiment, the increase in levels of AAV capsid proteins is achieved by including a strong polyadenylation (polyA) signal sequence 3’ to the sequence encoding the AAV capsid proteins In one embodiment, the increase in levels of capsid proteins is achieved by using an inducible promoter to drive expression of capsid proteins. In a further embodiment, the increase in levels of AAV capsid proteins is achieved by including a strong polyadenylation (polyA) signal sequence 3’ to the sequence encoding the AAV capsid proteins and by using an inducible promoter to drive expression ofcapsid proteins which inducible promoter is stronger than the native promoter controlling expression of AAV capsid proteins. In another embodiment, the increase in levels of capsid proteins is achieved by using two separate polynucleotide constructs each encoding AAV capsid proteins, one under the control of a native promoter and another under the control of an inducible promoter. One or more of these embodiments can also be combined to increase capsid proteins expression and enable increased production of recombinant AAV (rAAV).

[0094] In certain embodiments, the polynucleotide constructs may be stably integrated into the nuclear genome of the cells and may express components for formation of rAAV in an inducible manner thereby avoiding the toxicity of AAV Rep protein when constitutively expressed. 1.1. Definitions

[0095] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which the invention pertains.

[0096] The term "about", particularly in reference to a given quantity, is meant to encompass deviations of up to plus or minus five percent.

[0097] "AAV" is an abbreviation for adeno-associated virus, and may be used to refer to the virus itself or derivatives thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise. The components of the AAV DNA genome consist of two open reading frames, Rep and Cap, flanked by two 145 base inverted terminal repeats (ITRs). Rep gene encodes multiple distinct proteins including Rep78, Rep68, Rep52, and Rep40. These proteins are also referred to herein as Rep proteins or Rep and may encompass one or more of Rep78, Rep68, Rep52, and Rep40 and functional variants thereof and homologs thereof. Rep78 and Rep68 and functional variants thereof and homologs thereof are referred to herein as large Rep. Rep52, and Rep40 and functional variants thereof and homologs thereof are referred to herein as small Rep. Rep proteins from an AAV of a particular serotype may also be referred to as Rep1, Rep2, etc. where the Rep protein is derived from an AAV1 or an AAV2 serotype, respectively. Cap gene encodes capsid proteins VP1, VP2, and VP3 required for production of rAAV capsids. These proteins are also referred to herein as Cap proteins or Cap and may encompass one or more of VP1, VP2, and VP3 and functional variants thereof and homologs thereof. Cap proteins from an AAV of a particular serotype may also be referred to as Cap1, Cap2, Cap4, etc. where the Rep protein is derived from an AA1, an AAV2, or an AAV5 serotype, respectively. In addition to Rep and Cap, AAV requires a helper plasmid containing genes from a helper virus such as adenovirus, including E1a, E1b, E4, E2a, and VA genes for AAV replication.

[0098] "Recombinant", as applied to an AAV virion, means that the rAAV virion (synonymously, rAAV virus particle) is the product of one or more procedures that result in an AAV particle Construct that is distinct from an AAV virion in nature. The procedure may be genetic alteration, e.g., by the addition or insertion of a heterologous nucleic acid Construct into the virus.

[0099] "Recombinant virus" is meant to describe a virus that has been genetically altered, e.g., by the addition or insertion of a heterologous nucleic acid construct into the virus.

[0100] The abbreviation "rAAV" refers to recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or "rAAV vector"). The term “AAV” includes any AAV serotype as well as AAV vectors based on the combination of different serotypes (also referred to as "hybrid AAV vectors" or "pseudotype AAV vectors"). AAV serotype may be AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV type 9 (AAV-9), AAV type 10 (AAV-10), AAV type 11 (AAV-11), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, ovine AAV, AAV-7m8, AAV-6.2, AAV- Dj, AAV-DJ / 8, AAV2-retro, AAV2-QuadYF and AAV2.7m8, AAV-PHP.B, AAV-PHP.B2, AAV-PHP.B3, AAV-PHP.A, AAV-PHP.eB, AAV-PHP.eS, evolved capsids that are less immunogenic to mice and humans, and variants thereof and combinations thereof. “Primate AAV” refers to AAV isolated from a primate, “non-primate AAV” refers to AAV isolated from a non-primate mammal, “bovine AAV” refers to AAV isolated from a bovine mammal (e.g., a cow), etc. An "rAAV vector" comprises a polynucleotide sequence not of AAV origin (i.e., a polynucleotide heterologous to AAV), typically a polynucleotide sequence of interest for introducing into a target cell. In general, the heterologous polynucleotide is flanked by at least one, and usually by two AAV inverted terminal repeat sequences (ITRs). The heterologous polynucleotide can also be referred to as a polynucleotide payload. The term rAAV vector encompasses both rAAV virions and rAAV vector plasmids.

[0101] An "AAV virus" or "AAV viral particle" or "rAAV vector particle" refers to a viral particle composed of at least one AAV capsid protein (typically by all of the capsid proteins of a wild-type AAV) and an encapsidated polynucleotide rAAV vector. If the particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as an "rAAV vector particle" or simply an "rAAV vector". Thus, production of a rAAV particle necessarily includes production of a rAAV vector, as such a vector contained within an rAAV particle.

[0102] "Packaging" refers to a series of intracellular events that result in the assembly, encapsidation, and production of an AAV particle.

[0103] AAV "rep" and "cap" genes refer to polynucleotide sequences encoding replication and capsid proteins of adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes."

[0104] By "AAV Rep coding region" or “sequence encoding one or more Rep proteins” or “Rep encoding sequence” and grammatical equivalents thereof is meant the art-recognized region of the AAV genome which encodes the replication proteins of the virus which are required to replicate the viral genome and / or a payload flanked by ITRs. The rep coding region, as used herein, may be derived from any viral serotype, such as those described above. The region need not include all of the wild-type genes but may be altered, e.g., by the insertion, deletion or substitution of nucleotides, so long as the rep genes provide for expression Rep proteins. Rep coding sequences are further described below. The terms “AAV Rep coding sequence” and “AAV Rep proteins coding sequence” are used interchangeably herein. The terms “AAV Rep proteins”, “Rep proteins”, “AAV Rep polypeptide”, and “Rep polypeptide” are interchangeably used herein.

[0105] By "AAV cap coding region" or “sequence encoding one or more cap proteins,” or “Cap encoding sequence” and grammatical equivalents thereof it is meant the art-recognized region of the AAV genome which encodes the coat proteins of the virus which are required for the capsid that viral genome or a payload is packaged into by the Rep proteins. For a further description of the cap coding region, see, e.g., Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158, 97-129; Kotin, R. M. (1994) Human Gene Therapy 5, 793-801. The AAV cap coding region, as used herein, may be derived from any AAV serotype, as described above. The region need not include all of the wild-type cap genes but may be altered, e.g., by the insertion, deletion or substitution of nucleotides, so long as the genes provide for sufficient packaging functions. Cap coding sequences are further described below. The term “AAV Cap coding sequence” and “AAV Cap proteins coding sequence” are interchangeably used herein. The terms “AAV Cap proteins”, “Cap proteins”, “AAV Cap polypeptide”, and “Cap polypeptide” are interchangeably used herein. The term “Capsid” and “Cap” are interchangeably used herein.

[0106] By "adeno-associated virus inverted terminal repeats" or "AAV ITRs" is meant the art-recognized regions found at each end of the AAV genome which function together in cis as origins of DNA replication and as packaging signals for the viral genome. The nucleotide sequences of AAV ITR regions are known. See, e.g., Kotin, R. M. (1994) Human Gene Therapy 5, 793-801; Berns, K. I. "Parvoviridae and their Replication" in Fundamental Virology, 2d ed., (B. N. Fields and D. M. Knipe, eds.) for the AAV-2 ITRs sequence. As used herein, an "AAV ITR" need not have a wild-type nucleotide sequence, but may be altered, e.g., by the insertion,deletion or substitution of nucleotides. The AAV ITR may be derived from any of several AAV serotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-7, etc. Furthermore, 5' and 3' ITRs which flank a selected nucleotide sequence in an AAV vector need not necessarily be identical or derived from the same AAV serotype or isolate. The ITRs may be single stranded (ssITRs) or self-complementary (scITRs).

[0107] A "helper virus" for AAV refers to a virus that allows AAV (e.g., wild-type AAV) to be replicated and packaged by a mammalian cell. A variety of such helper viruses for AAV are known in the art, including adenoviruses, herpesviruses and poxviruses such as vaccinia. The adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C is most commonly used. Numerous adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as the ATCC. Viruses of the herpes family include, for example, herpes simplex viruses (HSV) and Epstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV); which are also available from depositories such as ATCC.

[0108] The term “adenoviral helper proteins” and “AAV helper proteins” are interchangeably used herein.

[0109] "Helper virus function(s)" refers to function(s) encoded in a helper virus genome which allows AAV replication and packaging (in conjunction with other requirements for replication and packaging described herein). As described herein, "helper virus function" may be provided in a number of ways, including by providing helper virus or providing, for example, polynucleotide sequences encoding the requisite function(s) to a producer cell in trans.

[0110] An "infectious" virus or viral particle is one that comprises a polynucleotide component which the particle is capable of delivering into a cell for which the viral species is tropic. The term does not necessarily imply any replication capacity of the virus. As used herein, an “infectious” virus or viral particle is one that may access a target cell, may infect a target cell, and may express a heterologous nucleic acid in a target cell. Thus, “infectivity” refers to the ability of a viral particle to access a target cell, infect a target cell, and express a heterologous nucleic acid in a target cell. Infectivity may refer to in vitro infectivity or in vivo infectivity. Assays for counting infectious viral particles are described elsewhere in this disclosure and in the art. Viral infectivity may be expressed as the ratio of infectious viral particles to total viral particles. Total viral particles may be expressed as the number of viral genome (vg) copies. The ability of a viral particle to express a heterologous nucleic acid in a cell may be referred to as “transduction.” The ability of a viral particle to express a heterologous nucleic acid in a cell may be assayed using a number of techniques, including assessment of a marker gene, such as a green fluorescent protein (GFP) assay (e.g., where the virus comprises a nucleotide sequenceencoding GFP), where GFP is produced in a cell infected with the viral particle and is detected and / or measured; or the measurement of a produced protein, for example by an enzyme-linked immunosorbent assay (ELISA). Viral infectivity may be expressed as the ratio of infectious viral particles to total viral particles. Methods of determining the ratio of infectious viral particle to total viral particle are known in the art. See, e.g., Grainger et al. (2005) Mol. Ther.11:S337 (describing a TCID50 infectious titer assay); and Zolotukhin et al. (1999) Gene Ther.6:973.

[0111] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. The terms “polynucleotide” and “nucleic acid” are interchangeably used herein. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0112] As used herein, the term “polynucleotide construct” refers to a DNA segment of any size that includes one or more sequences encoding an RNA or protein and at least one promoter for driving expression from the one or more sequences. The term “polynucleotide construct” and “nucleic acid construct” are interchangeably used herein. A polynucleotide construct may be a circular DNA or a linear DNA. A polynucleotide construct may be single stranded or double stranded. As used herein, the term "vector" includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which may transfer gene sequences into and between cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors. The use of the term "vector" throughout this specification encompasses plasmid or viral vectors, which permit the desired components to be transferred to the host cell via transfection or infection. For example, an adeno-associated viral (AAV) vector is a plasmid comprising a recombinant AAV genome. In some embodiments, useful vectors are contemplated to be those vectors in which the nucleic acid segment to be transcribed is positioned under the transcriptional control of a promoter. A vector may be linear or circular, single stranded or double stranded, DNA or RNA. In certain aspects, the vector may be circular, double stranded DNA.

[0113] As used herein, the term “vector system” refers to two or more vectors that are used together, e.g., by simultaneous or sequential introduction into a cell, to provide at least twodifferent components into the cell. The two different components may then work together in the cell.

[0114] A polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same when comparing the two sequences. The term percent “sequence identity,” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent “sequence identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.

[0115] For sequence comparison, typically one sequence acts as a reference sequence (also called the subject sequence) to which test sequences (also called query sequences) are compared. The percent sequence identity is defined as a test sequence’s percent identity to a reference sequence. For example, when stated “Sequence A having a sequence identity of 50% to Sequence B,” Sequence A is the test sequence and Sequence B is the reference sequence. When using a sequence comparison algorithm, test and reference sequences are input into a computer program, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then aligns the sequences to achieve the maximum alignment, based on the designated program parameters, introducing gaps in the alignment if necessary. The percent sequence identity for the test sequence(s) relative to the reference sequence can then be determined from the alignment of the test sequence to the reference sequence. The equation for percent sequence identity from the aligned sequence is as follows:

[0116] [(Number of Identical Positions) / (Total Number of Positions in the Test Sequence)] × 100%

[0117] For purposes herein, percent identity and sequence similarity calculations are performed using the BLAST algorithm for sequence alignment, which is described in Altschul et al., J. Mol. Biol.215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ). The BLAST algorithm uses a test sequence (also called a query sequence) and a reference sequence (also called a subject sequence) to search against, or in some cases, a database of multiple reference sequences to search against. The BLAST algorithm performs sequencealignment by finding high-scoring alignment regions between the test and the reference sequences by scoring alignment of short regions of the test sequence (termed “words”) to the reference sequence. The scoring of each alignment is determined by the BLAST algorithm and takes factors into account, such as the number of aligned positions, as well as whether introduction of gaps between the test and the reference sequences would improve the alignment. The alignment scores for nucleic acids can be scored by set match / mismatch scores. For protein sequences, the alignment scores can be scored using a substitution matrix to evaluate the significance of the sequence alignment, for example, the similarity between aligned amino acids based on their evolutionary probability of substitution. For purposes herein, the substitution matrix used is the BLOSUM62 matrix. For purposes herein, the public default values of April 6, 2023 are used when using the BLASTN and BLASTP algorithms. The BLASTN and BLASTP algorithms then output a “Percent Identity” output value and a “Query Coverage” output value. The overall percent sequence identity as used herein can then be calculated from the BLASTN or BLASTP output values as follows:

[0118] Percent Sequence Identity = (“Percent Identity” output value) × (“Query Coverage” output value)

[0119] The following non-limiting examples illustrate the calculation of percent identity between two nucleic acids sequences. The percent identity is calculated as follows: [(number of identical nucleotide positions) / (total number of nucleotides in the test sequence)] × 100%. Percent identity is calculated to compare test sequence 1: AAAAAGGGGG (length = 10 nucleotides) to reference sequence 2: AAAAAAAAAA (length = 10 nucleotides). The percent identity between test sequence 1 and reference sequence 2 would be [(5) / (10)] ×100% = 50%. Test sequence 1 has 50% sequence identity to reference sequence 2. In another example, percent identity is calculated to compare test sequence 3: CCCCCGGGGGGGGGGCCCCC (length = 20 nucleotides) to reference sequence 4: GGGGGGGGGG (length = 10 nucleotides). The percent identity between test sequence 3 and reference sequence 4 would be [(10) / (20)] ×100% = 50%. Test sequence 3 has 50% sequence identity to reference sequence 4. In another example, percent identity is calculated to compare test sequence 5: GGGGGGGGGG (length = 10 nucleotides) to reference sequence 6: CCCCCGGGGGGGGGGCCCCC (length = 20 nucleotides). The percent identity between test sequence 5 and reference sequence 6 would be [(10) / (10)] ×100% = 100%. Test sequence 5 has 100% sequence identity to reference sequence 6.

[0120] The following non-limiting examples illustrate the calculation of percent identity between two protein sequences. The percent identity is calculated as follows: [(number of identical amino acid positions) / (total number of amino acids in the test sequence)] × 100%.Percent identity is calculated to compare test sequence 7: FFFFFYYYYY (length = 10 amino acids) to reference sequence 8: YYYYYYYYYY (length = 10 amino acids). The percent identity between test sequence 7 and reference sequence 8 would be [(5) / (10)] ×100% = 50%. Test sequence 7 has 50% sequence identity to reference sequence 8. In another example, percent identity is calculated to compare test sequence 9: LLLLLFFFFFYYYYYLLLLL (length = 20 amino acids) to reference sequence 10: FFFFFYYYYY (length = 10 amino acids). The percent identity between test sequence 9 and reference sequence 10 would be [(10) / (20)] ×100% = 50%. Test sequence 9 has 50% sequence identity to reference sequence 10. In another example, percent identity is calculated to compare test sequence 11: FFFFFYYYYY (length = 10 amino acids) to reference sequence 12: LLLLLFFFFFYYYYYLLLLL (length = 20 amino acids). The percent identity between test sequence 11 and reference sequence 12 would be [(10) / (10)] ×100% = 100%. Test sequence 11 has 100% sequence identity to reference sequence 12.

[0121] For purposes herein, reference to a polynucleotide sequence (e.g., a DNA sequence or an RNA sequence) also encompasses the reverse complement of the polynucleotide sequence. For example, a sequence of AAAAAGGGGG also encompasses a sequence of CCCCCTTTTT.

[0122] A "gene" refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated.

[0123] The term "host cell" denotes, for example, microorganisms, yeast cells, insect cells, and mammalian cells, that may be, or have been, used as recipients of an AAV vector system as described herein, or other transfer DNA. The term includes the progeny of the original cell which has been transfected. Thus, a "host cell" as used herein generally refers to a cell which has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to natural, accidental, or deliberate mutation. In some aspects, the disclosure provides transfected host cells.

[0124] The term "transfection" is used to refer to the uptake of foreign DNA by a cell, and a cell has been "transfected" when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous nucleic acids, such as a nucleotide integration vector and other nucleic acid molecules, into suitable host cells.

[0125] As used herein, the term "cell line" refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populationsderived from a single progenitor cell. It is further known in the art that spontaneous or induced changes may occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants.

[0126] The term "cell culture," refers to cells grown adherent or in suspension, bioreactors, roller bottles, hyperstacks, microspheres, macrospheres, flasks and the like, as well as the components of the supernatant or suspension itself, including but not limited to rAAV particles, cells, cell debris, cellular contaminants, colloidal particles, biomolecules, host cell proteins, nucleic acids, and lipids, and flocculants. Large scale approaches, such as bioreactors, including suspension cultures and adherent cells growing attached to microcarriers or macrocarriers in stirred bioreactors, are also encompassed by the term "cell culture." Cell culture procedures for both large and small-scale production of proteins are encompassed by the present disclosure.

[0127] As used herein, the terms "recombinant cell" refers to a cell into which an exogenous DNA segment, such as DNA segment that leads to the transcription of a biologically-active polypeptide or production of a biologically active nucleic acid such as an RNA, has been introduced.

[0128] As used herein, the term “intermediate cell line” refers to a cell line that contains the AAV rep and cap components integrated into the host cell genome or a cell line that contains the adenoviral helper functions integrated into the host cell genome.

[0129] As used herein, the term “packaging cell line” refers to a cell line that contains the AAV rep and cap components and the adenoviral helper functions integrated into the host cell genome. A payload construct must be added to the packaging cell line to generate rAAV virions.

[0130] As used herein, the term “production cell line” refers to a cell line that contains the AAV rep and cap components, the adenoviral helper functions, and a payload construct. The rep and cap components and the adenoviral helper functions are integrated into the host cell genome. The payload construct can be stably integrated into the host cell genome or transiently transfected. rAAV virions can be generated from the production cell line upon the introduction of one or more triggering agents in the absence of any plasmid or transfection agent.

[0131] As used herein, the term “downstream purification” refers to the process of separating rAAV virions from cellular and other impurities. Downstream purification processes include chromatography-based purification processes, such as ion exchange (IEX) chromatography and affinity chromatography.

[0132] The term “prepurification yield” refers to the rAAV yield prior to the downstream purification processes. The term “postpurification yield” refers to the rAAV yield after the downstream purification processes. rAAV yield can be measured as viral genome (vg) / L.

[0133] The encapsidation ratio of a population of rAAV virions can be measured as the ratio of rAAV viral particle (VP) to viral genome (VG). The rAAV viral particle includes empty capsids, partially full capsids (e.g., comprising a partial viral genome), and full capsids (e.g., comprising a full viral genome).

[0134] The F:E ratio of a population of rAAV virions can be measured as the ratio of rAAV full capsids to empty capsids. The rAAV full capsid particle includes partially full capsids (e.g., comprising a partial viral genome) and full capsids (e.g., comprising a full viral genome). The empty capsids lack a viral genome.

[0135] The potency or infectivity of a population of rAAV virions can be measured as the percentage of target cells infected by the rAAV virions at a multiplicity of infection (MOI; viral genomes / target cell). Exemplary MOI values are 1 × 101, 1 × 102, 2 × 103, 5 × 104, or 1 × 105vg / target cell. An MOI can be a value chosen from the range of 1 × 101to 1 × 105vg / target cell.

[0136] The term "expression vector or construct" or “synthetic construct” means any type of genetic construct containing a nucleic acid in which part or all of the nucleic acid encoding sequence is capable of being transcribed. In some embodiments, expression includes transcription of the nucleic acid, for example, to generate a biologically-active polypeptide product or functional RNA (e.g., guide RNA) from a transcribed gene.

[0137] The term “auxotrophic” or “auxotrophic selectable marker” as used herein refers to the usage of a medium lacking a supplement, such as a medium lacking an essential nutrient such as the purine precursors hypoxanthine and thymidine (HT), or the like, for selection of a functional enzyme which allows for growth in the medium lacking the essential nutrient, e.g., a functional dihydrofolate reductase or the like.

[0138] The terms “tetracycline” is used generically herein to refer to all antibiotics that are structurally and functionally related to tetracycline, including tetracycline, doxycycline, demeclocycline, minocycline, sarecycline, oxytetracycline, omadacycline, or eravacycline.

[0139] The terms “constitutive” or “constitutive expression” are used interchangeably herein. They refer to genes that are transcribed in an ongoing manner. Such gene are driven by a constitutive promoter. In some embodiments, the terms refer to the expression of a therapeutic payload or a nucleic acid sequence that is not conditioned on addition of an triggering agent to the cell culture medium. A constitutive promoter is capable of directing continuous gene expression in a cell. Constitutive promoters regulate expression of basal genes, like housekeeping genes. In contrast, an inducible promoter directs gene expression in the presenceof an external stimulus. Thus, an inducible promoter can be controlled by providing or withdrawing the stimulus.

[0140] As used herein, the term “polynucleotide payload” refers to a polynucleotide sequence that is packaged into a rAAV virion for delivery by the rAAV virion into a cell. A polynucleotide payload is flanked by AAV inverted terminal repeats (ITRs). Upon delivery to a cell, the polynucleotide payload may be available to the cell as a DNA (e.g., a homology region for homology-directed repair), transcribed into an RNA (e.g., a guide RNA (gRNA), a tRNA, a suppressor tRNA, a siRNA, a miRNA, an mRNA, a shRNA, a circular RNA, an antisense oligonucleotide (ASO)), or transcribed and translated into a polypeptide (e.g., an antibody, a hormone, a site-specific endonuclease, a reporter gene, a component of a CRISPR / Cas system, an adenosine deaminase acting on RNA (ADAR) enzyme, a transcriptional activator, a transcriptional repressor, a ribozyme, or a DNAzyme.

[0141] "Recombinant," as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature. A recombinant virus is a viral particle comprising a recombinant polynucleotide. The terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct.

[0142] A "control element" or "control sequence" is a nucleotide sequence involved in an interaction of molecules that contributes to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, speed, or specificity of the process, and may be enhancing or inhibitory in nature. Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers. A promoter is a DNA region capable under certain conditions of binding RNA polymerase and initiating transcription of a coding region usually located downstream (in the 3' direction) from the promoter. A promoter is usually upstream of a gene whose expression is controlled by the promoter.

[0143] "Operatively linked" or "operably linked" refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a promoter is operatively linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and coding region so long as this functional relationship is maintained.

[0144] "Heterologous" means derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared. For example, a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species is aheterologous polynucleotide. Additional sequences or sequence motifs operably linked to a sequence where it is not naturally found are also heterologous; such sequences or sequence motifs include polyA signal sequences, introns, and / or any other relevant sequence. Thus, for example, an rAAV that includes a heterologous nucleic acid encoding a heterologous payload is an rAAV that includes a nucleic acid not normally included in a naturally occurring, wild-type AAV, and the encoded heterologous payload is a payload not normally encoded by a naturally- occurring, wild-type AAV. As another example, a Capsid proteins coding sequence operably linked to a heterologous polyA signal sequence refers to a AAV Cap coding sequence operably linked to a sequence not native to AAV.

[0145] A cell is said to be "stably" altered, transduced, genetically modified, or transformed with a genetic sequence if the sequence is available to perform its function during extended culture of the cell in vitro. Generally, such a cell is "heritably" altered (genetically modified) in that a genetic alteration is introduced which is also inheritable by progeny of the altered cell. For example, a gene integrated into the nuclear genome of the cell and is available to perform its function during extended culture of the cell in vitro. A gene integrated into the nuclear genome of the cell is inheritable by progeny of the cell.

[0146] A cell is said to be "stably" altered, transduced, genetically modified, or transformed with a genetic sequence if the sequence is available to perform its function during extended culture of the cell in vitro. Generally, such a cell is "heritably" altered (genetically modified) in that a genetic alteration is introduced which is also inheritable by progeny of the altered cell. For example, a gene integrated into the nuclear genome of the cell and is available to perform its function during extended culture of the cell in vitro. A gene integrated into the nuclear genome of the cell is inheritable by progeny of the cell.

[0147] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component. Polypeptides such as anti-angiogenic polypeptides, neuroprotective polypeptides, and the like, when discussed in the context of delivering a payload to a mammalian subject, and compositions therefor, refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, which retains the desired biochemical function of the intact protein. Similarly, references to nucleic acids encoding anti-angiogenic polypeptides, nucleic acids encoding neuroprotective polypeptides, and other such nucleic acids for use in delivery of a payload to a mammalian subject (which may be referred to as "transgenes" to be delivered to a recipient cell), includepolynucleotides encoding the intact polypeptide or any fragment or genetically engineered derivative possessing the desired biochemical function.

[0148] An "isolated" plasmid, nucleic acid, vector, virus, virion, host cell, or other substance refers to a preparation of the substance devoid of at least some of the other components that may also be present where the substance or a similar substance naturally occurs or is initially prepared from. Thus, for example, an isolated substance may be prepared by using a purification technique to enrich it from a source mixture. Enrichment may be measured on an absolute basis, such as weight per volume of solution, or it may be measured in relation to a second, potentially interfering substance present in the source mixture. Increasing enrichments of the embodiments of this invention are increasingly more isolated. An isolated plasmid, nucleic acid, vector, virus, host cell, or other substance is in some cases purified, e.g., from about 80% to about 90% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99%, or more, pure.

[0149] The terms "treatment", "treating", "treat" and the like are used herein to generally refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and / or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and / or adverse effect attributable to the disease. The term “treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease and / or symptom(s) from occurring in a subject who may be predisposed to the disease or symptom(s) but has not yet been diagnosed as having it; (b) inhibiting the disease and / or symptom(s), i.e., arresting development of a disease and / or the associated symptoms; or (c) relieving the disease and the associated symptom(s), i.e., causing regression of the disease and / or symptom(s). Those in need of treatment may include those already afflicted (e.g., those with a neurological disorder) as well as those in which prevention is desired (e.g., those with increased susceptibility to a neurological disorder; those suspected of having a neurological disorder; those having one or more risk factors for a neurological disorder, etc.).

[0150] A "therapeutically effective amount" or "efficacious amount" means the amount of a compound that, when administered to a mammal or other subject for treating a disease, is sufficient, in combination with another agent, or alone in one or more doses, to effect such treatment for the disease. The "therapeutically effective amount" will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0151] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, human and non-human primates, including simians and humans; mammalian sport animals (e.g., horses, camels, etc.); mammalian farmanimals (e.g., sheep, goats, cows, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.). In some cases, the individual is a human.

[0152] The terms “v1.1 system” and “v1.2” system are interchangeable and are used herein to refer to the same version of system of polynucleotides.

[0153] The terms "hybridize" and "hybridization" refer to the formation of complexes between nucleotide sequences which are sufficiently complementary to form complexes via Watson Crick base pairing.

[0154] The term "homologous region" refers to a region of a nucleic acid with homology to another nucleic acid region. Thus, whether a "homologous region" is present in a nucleic acid molecule is determined with reference to another nucleic acid region in the same or a different molecule. Further, since a nucleic acid is often double-stranded, the term "homologous, region," as used herein, refers to the ability of nucleic acid molecules to hybridize to each other. For example, a single-stranded nucleic acid molecule may have two homologous regions which are capable of hybridizing to each other. Thus, the term "homologous region" includes nucleic acid segments with complementary sequences. Homologous regions may vary in length but will typically be between 4 and 500 nucleotides (e.g., from about 4 to about 40, from about 40 to about 80, from about 80 to about 120, from about 120 to about 160, from about 160 to about 200, from about 200 to about 240, from about 240 to about 280, from about 280 to about 320, from about 320 to about 360, from about 360 to about 400, from about 400 to about 440, etc.).

[0155] As used herein, the terms "complementary" or "complementarity" refers to polynucleotides that are able to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in an anti-parallel orientation between polynucleotide strands. Complementary polynucleotide strands may base pair in a Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in the art are aware, when using RNA as opposed to DNA, uracil (U) rather than thymine (T) is the base that is considered to be complementary to adenosine. However, when a uracil is denoted in the context of the present invention, the ability to substitute a thymine is implied, unless otherwise stated. "Complementarity" may exist between two RNA strands, two DNA strands, or between an RNA strand and a DNA strand. It is generally understood that two or more polynucleotides may be "complementary" and able to form a duplex despite having less than perfect or less than 100% complementarity. Two sequences are "perfectly complementary" or "100% complementary" if at least a contiguous portion of each polynucleotide sequence, comprising a region of complementarity, perfectly base pairs with the other polynucleotide without any mismatches or interruptions within such region. Two or more sequences are considered "perfectly complementary" or "100%complementary" even if either or both polynucleotides contain additional non-complementary sequences as long as the contiguous region of complementarity within each polynucleotide is able to perfectly hybridize with the other. "Less than perfect" complementarity refers to situations where less than all of the contiguous nucleotides within such region of complementarity are able to base pair with each other. Determining the percentage of complementarity between two polynucleotide sequences is a matter of ordinary skill in the art. As used herein, the term "recombination site" denotes a region of a nucleic acid molecule comprising a binding site or sequence-specific motif recognized by a site-specific recombinase that binds at the target site and catalyzes recombination of specific sequences of DNA at the target site. Site-specific recombinases catalyze recombination between two such target sites. The relative orientation of the target sites determines the outcome of recombination. For example, translocation occurs if the recombination sites are on separate DNA molecules. DNA between two recombination sites oriented in the same direction on the same DNA molecule will be excised as a circular loop of DNA. DNA between two recombination sites that are orientated in the opposite direction on the same DNA molecule will be inverted.

[0156] The terms "v1.1 system" and "v1.2 system” are interchangeable and are used herein to refer to the same version of system of polynucleotides. 1.2. Overview

[0157] To solve the problems associated with low rAAV titers, disclosed herein are polynucleotide constructs and cells, such as, cell lines stably integrated with the polynucleotide constructs that enable increased production of recombinant AAV (rAAV) virions. FIG.10 shows that in a v1.0 system, the production of payload sequences is higher than capsid production, indicating that capsid production could be a rate limiting step for producing encapsidated payloads, i.e., rAAV. FIG.10 demonstrates that increased encaspidated payload titer, i.e., increased rAAV titer was acheived by increasing total capsid production. Thus, increased production of rAAV is achieved by providing an overall increase in the levels of capsid proteins which in turn increases production of rAAV. Disclosed herein are various methods and constructs for increasing capsid production and therefore also increasing the titer of encapsidated genomes. In one embodiment, the increase in levels of capsid proteins is achieved by including a strong polyadenylation (polyA) signal sequence 3' to the sequence encoding the capsid proteins. In another embodiment, the increase in levels of capsid proteins is achieved by using an inducible promoter to drive expression of capsid proteins. In a further embodiment, the increase in levels of AAV capsid proteins is achieved by including a strong polyadenylation(polyA) signal sequence 3’ to the sequence encoding the AAV capsid proteins and by using an inducible promoter to drive expression of capsid proteins which inducible promoter is stronger than the native promoter controlling expression of AAV capsid proteins. In another embodiment, the increase in levels of capsid proteins is achieved by using two separate polynucleotide constructs each encoding AAV capsid proteins, one under the control of a native promoter and another under the control of an inducible promoter. One or more of these embodiments can also be combined to increase capsid proteins expression and enable increased production of recombinant AAV (rAAV). These embodiments are further described in detail below. 1.3. Polynucleotides Encoding AAV Capsid Proteins AAV Cap Encoding Sequence linked to PolyA Signal Sequence

[0158] In some aspects, polynucleotides comprising (i) a sequence encoding AAV Cap proteins and (ii) a polyadenylation signal sequence. In some embodiments, the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence. In some embodiments, the polyadenylation signal sequence is 3' of the sequence encoding AAV Cap proteins are provided. In certain embodiments, the polynucleotides comprise (i) a sequence encoding AAV Cap proteins and (ii) a polyadenylation signal sequence, wherein the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence and is a 3' of the sequence encoding AAV Cap proteins. Suitable polyadenylation signals are further described below in the section “Polyadenylation Signals”. Polyadenylation Signals

[0159] Polyadenylation (polyA) signal sequences generally include a short sequence that triggers polyadenylation of an mRNA. In certain instances, RNA stability, expression, and / or function can be enhanced with additional sequences surrounding a shorter sequence. Various polyA signaling sequences can be used for the coding sequences of various embodiments.

[0160] In some embodiments, the polyadenylation signal sequence is a SV40 polyadenylation signal sequence (SV40 polyA). In other embodiments, the polyadenylation signal sequence is a bovine growth hormone polyadenylation signal sequence (bGH polyA). In still other embodiments, the polyadenylation signal sequence is a Rabbit Beta Globin polyadenylation signal sequence.

[0161] In some embodiments, the polyadenylation signal sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 152 (SV40 poly A). In certain embodiments, the polyadenylation signal sequence has sequence of SEQ ID NO: 152 (SV40 poly A). In certain embodiments, the polyadenylation signal sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 151 (bGH polyA). In certain embodiments, the polyadenylation signal sequence has sequence of SEQ ID NO: 151 (bGH polyA). In some embodiments, the polyadenylation signal sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 170 (Rabbit Beta Globin PolyA). In certain embodiments, the polyadenylation signal sequence has sequence of SEQ ID NO: 170 (Rabbit Beta Globin PolyA).

[0162] In some embodiments, the polyadenylation signal sequence is any polyadenylation signal sequence that encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence. In some embodiments, the native AAV Cap polyadenylation signal sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 162. In certain embodiments, the native AAV Cap polyadenylation signal sequence has sequence of SEQ ID NO: 162.

[0163] In some embodiments, the stronger polyadenylation signal enhances RNA processing, RNA stability, RNA translation efficiency, or any combination thereof.

[0164] In some embodiments, the polynucleotide described herein is not flanked by inverted terminal repeat sequences.

[0165] Additional details regarding polyadenylation signaling sequences are disclosed in one or more of: US Pat. Nos.11,793,180, 11,752,181, 10,912,826, 8,975,391, 7,557,197, and 5,122,458; US Pat. Pub. Nos.2023 / 0332169, 2023 / 0330265, and 2023 / 0323390; or Gene Volume 231, Issues 1–2, 29 April 1999, Pages 77-86, Mol Cell Biol.1989 Oct; 9(10): 4248– 4258, and Nucleic Acids Research, Volume 15, Issue 23, 10 December 1987, Pages 9627–9640; the disclosures of which are hereby incorporated by reference in their entireties. AAV Cap proteins

[0166] In some embodiments, the AAV Cap proteins comprise VP1, VP2, and VP3.

[0167] In some embodiments, a serotype of the AAV Cap proteins is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20,AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16 and AAVhu68. In some embodiments, the serotype is an AAV5 Cap protein which comprises one or more mutations or insertions. In some embodiments, the AAV Cap proteins encode for AAV5 Cap proteins. In some embodiments, the AAV Cap proteins encode for AAV9 Cap proteins. In some embodiments, the serotype is an AAV9 Cap protein which comprises one or more mutations or insertions. In some embodiments, the AAV Cap proteins encode for PhP.EB Cap proteins. In some embodiments, the serotype is an PhP.EB Cap protein which comprises one or more mutations or insertions. In some embodiments, the AAV Cap proteins encode for AAV8 Cap proteins. In some embodiments, the serotype is an AAV8 Cap protein which comprises one or more mutations or insertions. In some embodiments, the AAV Cap proteins encode for AAV2 proteins. In some embodiments, the serotype is an AAV2 Cap protein which comprises one or more mutations or insertions. In some embodiments, the AAV Cap proteins encode for AAV6 Cap proteins. In some embodiments, the serotype is an AAV6 Cap protein which comprises one or more mutations or insertions. AAV Cap Encoding Sequence linked to Inducible Promoter

[0168] In some aspects, polynucleotides comprising (i) a sequence encoding AAV Cap proteins operably linked to an inducible promoter and (ii) a polyadenylation signal sequence, wherein the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence and is 3' of the sequence encoding AAV Cap proteins are provided. Any suitable inducible promoter that is stronger than the native p40 promoter may be used for increasing AAV Cap proteins expression. Suitable inducible promoters are further described below in the section "Inducible Promoters". Inducible Promoters

[0169] In certain aspects, the inducible promoters are selected on the basis of the regulatory sequence that allows for control of the promoter. The regulatory sequence may be operably linked to the promoter and positioned upstream of the promoter. Such regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. The regulatory sequence used to control expression may beendogenous or exogenous to the host cell. In some embodiments, bacterial gene control elements in combination with viral transactivator proteins are used to provide mammalian inducible expression. Examples of mammalian-compatible regulatory sequences include those capable of controlling an engineered promoter to adjust transcription in response to antibiotics including, without limitation, tetracyclines, streptogramins, and macrolides. For example, inclusion of a bacterial tetracycline response element (TRE) in a construct allows mammalian expression to be induced by tetracycline or a derivative thereof (e.g., doxycycline). See, e.g., Weber et al. (2004) Methods Mol. Biol.267:451-66, Das et al. (2016) Curr. Gene Ther.16(3):156-67, Chruscicka et al. (2015) J. Biomol. Screen.20(3):350-8, Yarranton (1992) Curr. Opin. Biotechnol.3(5):506- 11, Gossen & Bujard (1992) Proc. Natl.Acad. Sci. U.S.A.89(12):5547-51, Gossen et al. (1995) Science 268(5218):1766-9; herein incorporated by reference.

[0170] In some embodiments, the inducible promoter is a tetracycline-inducible promoter. In other embodiments, the inducible promoter is an ecdysone-inducible promoter. In still other embodiments, the inducible promoter is a cumate-inducible promoter.

[0171] In some embodiments, the inducible promoter comprises a tetracycline-responsive promoter element (TRE). In certain embodiments, the TRE comprises Tet operator (tetO) sequence concatemers fused to a minimal promoter. In some embodiments, the minimal promoter is a human cytomegalovirus promoter. In some embodiments, the TRE comprises seven repeats of a 19 base pair operator sequence (tetO). In further embodiments, the TRE comprises seven repeats of a 19 base pair operator sequence upstream of a minimal human cytomegalovirus (CMV) promoter.

[0172] In some embodiments, the inducible promoter is a Tet-On promoter.

[0173] In some embodiments, the inducible promoter comprises a first inducible promoter.

[0174] In some embodiments, the polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148. In certain embodiments, the polynucleotide has sequence of SEQ ID NO: 148. In some embodiments, the polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 163. In certain embodiments, the polynucleotide has sequence of SEQ ID NO: 163.

[0175] In some embodiments, the Cap coding sequence is operably linked to a promoter.

[0176] In some embodiments, the AAV Cap proteins comprise VP1, VP2, and VP3. In some embodiments, the sequence coding for VP1, the sequence coding for VP2, and the sequence coding for VP3 are operably linked to a promoter. In some embodiments, a single construct or separate constructs comprise these sequences, in any combination. In someembodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter comprises a tetracycline-inducible promoter, a cumate-inducible promoter, or a cumate-inducible promoter. In some embodiments, the promoter is a constitutive promoter, wherein the sequences coding for the one or more cap proteins are downstream of an excisable element (e.g., a sequence flanked by recombination sites and comprising a stop signal) and constitutive promoter, wherein upon excision of the excisable element (e.g., by a recombinase), the sequences coding for the one or more cap proteins are operably linked to the constitutive promoter. In some embodiments, the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter.

[0177] The Cap protein encoding sequence provided in a separate polynucleotide construct can be expressed under the control of an inducible promoter. The Cap coding sequence may include the sequence coding for VP1, the sequence coding for VP2, and the sequence coding for VP3 are operably linked to the inducible promoter.

[0178] In various embodiments, the Cap protein encoding sequence provided in a separate polynucleotide construct or in the same polynucleotide comprising the Rep coding sequence can be expressed under the control of an inducible promoter. In various embodiments, the Cap protein encoding sequence may be operably linked to a polyadenylation (polyA) signal sequence. The polyA signal sequence may be a polyA signal sequence functional in the cells used for producing the rAAV. In some instances, the polyA signal sequence may be a bovine Growth Hormone polyA (bGH-PolyA) signal sequence, a SV40 polyA signal sequence, or a Rabbit Beta Globin PolyA signal sequence. In some embodiments, the polyadenylation signal sequence is any polyadenylation signal sequence that encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence, wherein the native AAV Cap polyadenylation signal sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 162.

[0179] The bGH-PolyA signal sequence may include a nucleotide sequence that has at least 70%, 75%, 80% 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity or 100% sequence identity to nucleotide sequence set forth in SEQ ID NO: 151.

[0180] In certain cases, the SV40 polyA signal sequence may include a nucleotide sequence having at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or 100% sequence identity to nucleotide sequence set forth in SEQ ID NO: 152.

[0181] In some embodiments, the SV40 polyA sequence is shorter than SEQ ID NO: 152. In some embodiments, the SV40 polyA sequence is longer than SEQ ID NO: 152.

[0182] In certain cases, the Rabbit Beta Globin signal sequence may include a nucleotide sequence having at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or 100% sequence identity to nucleotide sequence set forth in SEQ ID NO: 170.

[0183] In various embodiments, the Cap protein is selected from the capsid of an avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV, and modifications, derivatives, or pseudotypes thereof.

[0184] In some embodiments, the capsid is a capsid selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4- 1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16 or AAVhu68 (described in WO2020 / 033842, incorporated herein by reference in its entirety). The hu68 capsid is described in WO 2018 / 160582, incorporated herein by reference in its entirety.

[0185] In some embodiments, the capsid is a derivative, modification, or pseudotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV 13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16 or AAVhu68.

[0186] In some embodiments, capsid protein is a chimera of capsid proteins from two or more serotype selected from AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16 (described in WO2020 / 033842, incorporated herein by reference in its entirety). In certain embodiments, the capsid is an rh32.33 capsid, described in US Pat. No.8,999,678, incorporated herein by reference in its entirety.

[0187] In particular embodiments, the capsid is an AAV1 capsid. In particular embodiments, the capsid is an AAV5 capsid. In particular embodiments, the capsid is an AAV9 capsid. AAV Cap and AAV Rep Encoding Polynucleotide

[0188] In some aspects, polynucleotides comprising: (i) a first sequence encoding AAV Rep proteins operably linked to one or more promoters; and (ii) a second sequence encoding the polynucleotide comprising a sequence encoding AAV Cap proteins operably linked to an inducible promoter and a polyadenylation signal sequence are provided. In some embodiments, the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence and is a 3’ of the sequence encoding AAV Cap proteins. The second sequence encoding the polynucleotide comprising a sequence encoding AAV Cap proteins operably linked to an inducible promoter and a polyadenylation signal sequence is described in section “AAV Cap Encoding Sequence linked to PolyA Signal Sequence” above.

[0189] In some embodiments, transcription of the first sequence is driven by native AAV promoters. In other embodiments, optionally, transcription of the first sequence is driven by the P5 and P19 native AAV promoters.

[0190] In some embodiments, the one or more promoters comprise P5 and P19 native promoters. A First Sequence Encoding AAV Rep Proteinsnce encoding AAV Rep proteins has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 160 or 161. In certain embodiments, the first sequence encoding AAV Rep proteins has sequence of SEQ ID NO: 160 or 161. In other embodiments, the first sequence encoding AAV Rep proteins has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136. In certain embodiments, the first sequence encoding AAV Rep proteins has sequence of SEQ ID NO: 136. In other embodiments, the first sequence encoding AAV Rep proteins has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136 but lacks the sequence of SEQ ID NO: 145 downstream of the sequence corresponding to the Cap sequence of SEQ ID NO: 136. In certain embodiments, the first sequence encoding AAV Rep proteins has sequence of SEQ ID NO: 136 but lacks the sequenceof SEQ ID NO: 145 downstream of the sequence corresponding to the Cap sequence of SEQ ID NO: 136.

[0192] In some embodiments, the first sequence encoding AAV Rep proteins is separated from the second sequence by an intervening sequence. In some embodiments, the intervening sequence comprises a transcriptional blocking element (TBE). In other embodiments, optionally, a sequence of the TBE has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 169. In certain embodiments, a sequence of the TBE has sequence of SEQ ID NO: 169.

[0193] In some embodiments, the first sequence encoding AAV Rep proteins comprises: (i) a first part of an AAV Rep proteins coding sequence, (ii) an excisable element, and (iii) a second part of the AAV Rep proteins coding sequence. In certain embodiments, the excisable element comprises a first recombination site, a coding sequence encoding a stop signaling sequence, a second recombination site, wherein the first and second recombination sites flank the coding sequence encoding a stop signaling sequence and wherein the first recombination site and the second recombination site are oriented in the same direction.

[0194] In some embodiments, the first sequence encoding AAV Rep proteins comprises from 5' to 3': one or more promoters operably linked to a sequence comprising a first part of an AAV Rep coding sequence, a 5' splice site, a first part of an intron, a first recombination site, a first 3' splice site, a coding sequence comprising a stop signaling sequence, a second recombination site, a second part of the intron, a second 3' splice site, and a sequence comprising a second part of the AAV Rep coding sequence. In certain embodiments, the first recombination site, the first 3' splice site, the coding sequence comprising the stop signaling sequence, and the second recombination site form an excisable element. In certain embodiments, the first recombination site and the second recombination site are oriented in the same direction. In certain embodiments, the one or more promoters are not operably linked to the sequence comprising the second part of the AAV Rep coding sequence. In certain embodiments, the first and second recombination sites are recombined by the inducible recombinase in the presence of a first triggering agent and a second triggering agent resulting in excision of the excisable element. In certain embodiments, the first part of the AAV Rep coding sequence and the first part of the intron are joined to the second part of the intron and the second part of the AAV Rep coding sequence to form a complete AAV Rep coding sequence, allowing expression of AAV Rep proteins.

[0195] In some embodiments, the coding sequence encoding the stop signaling sequence of the first sequence encodes for from 5' to 3': an exon and the stop signaling sequence. In someembodiments, the coding sequence encoding the stop signaling sequence of the first sequence further comprises a sequence encoding a protein marker, wherein the sequence encoding the protein marker is in-frame with the stop signaling sequence.

[0196] In some embodiments, the polynucleotide of the present disclosure further comprises a first constitutive promoter operably linked to a sequence encoding a first selectable marker or a first portion or a second portion of a split selectable marker.

[0197] In some embodiments, the polynucleotide of the present disclosure is a polynucleotide construct. In some embodiments, both the first sequence encoding AAV Rep proteins and the second sequence encoding AAV Cap proteins are on single construct.

[0198] In some embodiments, the first sequence encoding AAV Rep proteins and the second sequence encoding AAV Cap proteins are on separate constructs.

[0199] In some embodiments, the polynucleotide further comprises a selectable marker operably linked to a promoter. In certain embodiments, optionally the promoter is a constitutive promoter.

[0200] In some aspects, polynucleotides comprising: a first sequence encoding AAV Rep proteins and a second sequence encoding AAV Cap proteins, wherein the first sequence is operably linked to one or more promoters and the second sequence is operably linked to an inducible promoter are provided.

[0201] In some embodiments, the one or more promoters comprise P5 and P19 native promoters and the inducible promoter comprises a first inducible promoter.

[0202] In some embodiments, the first inducible promoter comprises a tetracycline-responsive promoter element (TRE). In some embodiments, the TRE comprises Tet operator (tetO) sequence concatemers fused to a minimal promoter. In some embodiments, the minimal promoter is a human cytomegalovirus promoter.

[0203] In some embodiments, the first sequence and the one or more promoters operably linked to the first sequence are separated from the second sequence and the inducible promoter operably linked to the second sequence by an intervening sequence. In some embodiments, the intervening sequence comprises a transcriptional blocking element (TBE).

[0204] In some embodiments, the inducible promoter is a Tet-On promoter.

[0205] In some embodiments, the first sequence encoding AAV Rep proteins comprises: (i) a first part of an AAV Rep proteins coding sequence, (ii) an excisable element comprising a first recombination site, a coding sequence encoding a stop signaling sequence, a second recombination site, wherein the first and second recombination sites flank the coding sequence encoding a stop signaling sequence and wherein the first recombination site and the second recombination site are oriented in the same direction, and (iii) a second part of the AAV Rep proteins coding sequence.

[0206] In some embodiments, the coding sequence encoding the stop signaling sequence of the first sequence further comprises a sequence encoding a protein marker, wherein the sequence encoding the protein marker is in-frame with the stop signaling sequence.

[0207] In some embodiments, the polypeptides further comprise a first constitutive promoter operably linked to a sequence encoding a first selectable marker or a first portion or a second portion of a split selectable marker. 1.4. Polynucleotide System

[0208] In some aspects, systems of polynucleotides comprising: a) a first polynucleotide comprising a sequence encoding AAV Rep proteins operably linked to one or more promoters; and b) a second polynucleotide comprising a sequence of polynucleotides comprising (i) a sequence encoding AAV Cap proteins and (ii) a polyadenylation signal sequence; and one or more of: c) a third polynucleotide comprising a sequence encoding one or more adenoviral helper proteins; and d) a fourth polynucleotide comprising a sequence encoding a payload are provided. In some embodiments, the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence.

[0209] In some embodiments, the polyadenylation signal sequence is 3' of the sequence encoding AAV Cap proteins are provided. The b) a second polynucleotide comprising a sequence of polynucleotides comprising (i) a sequence encoding AAV Cap proteins and (ii) a polyadenylation signal sequence is described in section “AAV Cap Encoding Sequence linked to PolyA Signal Sequence” above. First Polynucleotide encoding AAV Rep Protein

[0210] In some embodiments, transcription of a first polynucleotide is driven by native AAV promoters. In other embodiments, optionally, transcription of the first polynucleotide is driven by the P5 and P19 native AAV promoters.

[0211] In some embodiments, the one or more promoters comprise a P5 native AAV promoter and a P19 native AAV promoter.

[0212] In some embodiments, the first polynucleotide comprising a sequence encoding AAV Rep proteins operably linked to one or more promoters comprises: (i) a first part of an AAV Rep proteins coding sequence, (ii) an excisable element comprising a first recombination site, a coding sequence encoding a stop signaling sequence, a second recombination site, wherein the first and second recombination sites flank the coding sequence encoding a stop signalingsequence and wherein the first recombination site and the second recombination site are oriented in the same direction, and (iii) a second part of the AAV Rep proteins coding sequence.

[0213] In some embodiments, the first polynucleotide comprising a sequence encoding AAV Rep proteins operably linked to one or more promoters comprises from 5' to 3': one or more promoters operably linked to a first sequence comprising a first part of an AAV Rep coding sequence, a 5' splice site, a first part of an intron, a first recombination site, a first 3' splice site, a coding sequence comprising a stop signaling sequence, a second recombination site, a second part of the intron, a second 3' splice site, and a second sequence comprising a second part of the AAV Rep coding sequence, allowing expression of AAV Rep proteins. In certain embodiments, the first recombination site, the first 3' splice site, the coding sequence comprising the stop signaling sequence, and the second recombination site form an excisable element. In certain embodiments, the first recombination site and the second recombination site are oriented in the same direction. In certain embodiments, the one or more promoters are not operably linked to the second sequence comprising the second part of the AAV Rep coding sequence. In certain embodiments, the first and second recombination sites are recombined by the inducible recombinase in the presence of a first triggering agent and a second triggering agent resulting in excision of the excisable element. In certain embodiments, the first part of the AAV Rep coding sequence and the first part of the intron are joined to the second part of the intron and the second part of the AAV Rep coding sequence to form a complete AAV Rep coding sequence, allowing expression of AAV Rep proteins.

[0214] In some embodiments, the coding sequence encoding the stop signaling sequence of the first polynucleotide encodes for from 5' to 3': an exon and the stop signaling sequence. In some embodiments, the coding sequence encoding the stop signaling sequence of the first polynucleotide further comprises a sequence encoding a protein marker. In certain embodiments, the sequence encoding the protein marker is in-frame with the stop signaling sequence.

[0215] In some embodiments, the first polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 160 or 161.

[0216] In certain embodiments, a set of polynucleotides comprises: (i) a first polynucleotide encoding AAV Rep proteins; (ii) a second polynucleotide encoding the AAV Cap proteins; and (iii) a third polynucleotide encoding one or more adenoviral helper proteins wherein when the one or more polynucleotides are integrated into the nuclear genome of a cell, e.g., a mammalian cell, the AAV Rep proteins, the AAV Cap proteins, and / or the one or more adenoviral helper proteins are conditionally expressible and thereby conditionally produce recombinant AAV (rAAV) virions.

[0217] In some embodiments, the first polynucleotide further comprises a sequence encoding AAV Cap proteins. In some embodiments, transcription of the sequence encoding the AAV Cap proteins on the first polynucleotide is driven by a native AAV Cap proteins promoter. In certain embodiments, the native AAV Cap proteins promoter is a P40 native AAV promoter. In some embodiments, the Rep coding sequence is 5’ to the Cap coding sequence. In certain embodiments, the Cap coding sequence is operatively linked to an endogenous P40 promoter.

[0218] In some embodiments, the first polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136. In certain embodiments, the first polynucleotide has sequence of SEQ ID NO: 136.

[0219] In some embodiments, the first polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136 but lacks the sequence of SEQ ID NO: 145 downstream of the sequence corresponding to the Cap sequence of SEQ ID NO: 136. In certain embodiments, the first polynucleotide has sequence of SEQ ID NO: 136 but lacks the sequence of SEQ ID NO: 145 downstream of the sequence corresponding to the Cap sequence of SEQ ID NO: 136.

[0220] In certain embodiments, a set of polynucleotides comprises: (i) a first polynucleotide encoding AAV Rep proteins and AAV Cap proteins; (ii) a second polynucleotide encoding the AAV Cap proteins; and (iii) a third polynucleotide encoding one or more adenoviral helper proteins wherein when the one or more polynucleotides are integrated into the nuclear genome of a cell, e.g., a mammalian cell, the AAV Rep proteins, the AAV Cap proteins, and / or the one or more adenoviral helper proteins are conditionally expressible and thereby conditionally produce recombinant AAV (rAAV) virions.

[0221] The Rep sequence can encode Rep from any desired AAV serotype. In some embody

[0222] ments, the encoded Rep protein is drawn from the same serotype as the Cap protein. In some embodiments, the encoded Rep protein is drawn from a different serotype from the Cap protein. In particular embodiments, the encoded Rep protein includes, but is not limited to, a Rep protein from AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10 and AAV-11, or chimeric combinations thereof.

[0223] The nucleotide sequences of the genomes of the AAV serotypes are known. For example, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV- 2 is provided in GenBank Accession No. NC_001401 and Srivastava et al., J. Virol, 45: 555-564 (1983); the complete genome of AAV- 3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBankAccession No. NC_001829; the AAV-5 genome is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC_00 1862; at least portions of AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively (see also U.S. Patent Nos.7,282,199 and 7,790,449 relating to AAV-8); the AAV-9 genome is provided in Gao et al. Virol, 78: 6381-6388 (2004); the AAV-10 genome is provided in Mol Ther, 13(1): 67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2): 375-383 (2004).

[0224] In the exemplary embodiments, prior to the cell being contacted with the first triggering agent and the second triggering agent, the Rep coding sequence is interrupted by an excisable element. Addition of both the first triggering agent and the second triggering agent are required for excision of the excisable element. In some embodiments, the excisable element is inserted at CAG-G, CAG-A, AAG-G, AAG-A, wherein the dash (-) indicates the point of insertion of the excisable element, in the Rep coding sequence, and the excisable element is inserted downstream of the p19 promoter. In some embodiments, the excisable element is inserted at CAG-G, CAG-A, AAG-G, AAG-A, wherein the dash (-) indicates the point of insertion of the excisable element, in the Rep coding sequence, and the excisable element is inserted downstream of the p19 promoter and upstream of the p40 promoter.

[0225] In certain embodiments, the excisable element comprises, from 5’ to 3’, a first spacer segment, a second spacer segment, and a third spacer segment.

[0226] In particular embodiments, the first spacer segment comprises a 5’ splice site (5’SS) 5’ to the first spacer element. In some embodiments, the first spacer segment comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 1.

[0227] In some embodiments, the second spacer segment comprises a polynucleotide encoding a detectable protein marker flanked by lox sites. In certain embodiments, the detectable protein marker is a fluorescent protein. In particular embodiments, the fluorescent protein is a green or blue fluorescent protein (GFP of BFP). In specific embodiments, the GFP is EGFP. In particular embodiments, the fluorescent protein is a blue fluorescent protein (BFP). Screening for the fluorescent marker can be used to confirm integration of the construct into the cell genome, and can subsequently be used to confirm excision of the intervening spacer segment. In some embodiments, the second spacer segment further comprises a polyA signal sequence. In certain embodiments, the poly A signal sequence comprises a rabbit beta globin (RBG) polyA signal sequence. In some embodiments, the second spacer segment further comprises a first 3’ splice site (3’SS) between the first lox site and the polynucleotide encoding the protein marker.

[0228] In some embodiments, the second spacer segment comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 2.

[0229] In some embodiments, the third spacer segment further comprises a second 3’ splice site (3’SS). In particular embodiments, the second 3’ splice site is positioned 3’ to the second lox site.

[0230] In some embodiments, the third spacer segment comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 3.

[0231] In various embodiments, the Rep coding sequences are operatively linked to an endogenous P5 promoter. In various embodiments, the Rep coding sequences are operatively linked to an endogenous P19 promoter.

[0232] In some embodiments, the Rep coding sequences are operably linked to an inducible promoter. In some embodiments, the inducible promoter comprises a tetracycline-inducible promoter, a cumate-inducible promoter, or a cumate-inducible promoter. In some embodiments, the Rep coding sequences are operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter. Second Polynucleotide Encoding Inducible Cap Proteins

[0233] In some embodiments, the second polynucleotide comprises a sequence encoding AAV Cap proteins operably linked to an inducible promoter. In some embodiments, the second polynucleotide comprises (i) a sequence encoding AAV Cap proteins operably linked to an inducible promoter and (ii) a polyadenylation signal sequence, wherein the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence and is 3' of the sequence encoding AAV Cap proteins are provided. The second polynucleotide comprising a sequence of polynucleotides comprising (i) a sequence encoding AAV Cap proteins and optionally, (ii) a polyadenylation signal sequence, is described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above. Any suitable inducible promoter that is stronger than the native p40 promoter may be used for increasing AAV Cap proteins expression. Suitable inducible promoters are further described above in the section "Inducible Promoters".

[0234] In some embodiments, the first polynucleotide and second polynucleotide are in a first polynucleotide construct. For example, a first polynucleotide construct comprises the first polynucleotide and the second polynucleotide. In some embodiments, a first polynucleotide construct comprises the Rep coding sequence is linked to native promoters and the Cap coding sequence is linked to an inducible promoter and the Rep coding sequence and the native promoters are separated from the Cap coding sequence and the inducible promoter by anintervening sequence, such as, a TBE. For example, see the configuration of Construct 1 in FIG. 2A.

[0235] In certain embodiments, the TBE comprises a nucleotide sequence at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 169.

[0236] In some embodiments, the first polynucleotide lacks a sequence encoding AAV Cap proteins and the second polynucleotide comprises a sequence encoding AAV Cap proteins.

[0237] In some embodiments, the sequence encoding the AAV Cap proteins of the first polynucleotide is substantially identical to the sequence encoding the AAV Cap proteins in the first polynucleotide. Third Polynucleotide Encoding Adenoviral Helper Proteins

[0238] In some embodiments, the third polynucleotide comprising the sequence encoding one or more adenoviral helper proteins comprises: a second inducible promoter operably linked to a self-excising element; the self-excising element comprising a third recombination site and a fourth recombination site flanking a sequence encoding an inducible recombinase; a first constitutive promoter operably linked to a sequence encoding an activator. In some embodiments, the third recombination site and the fourth recombination site are oriented in the same direction. In some embodiments, the second inducible promoter is not operably linked to the sequence encoding the one or more adenoviral helper proteins. In some embodiments, the third polynucleotide constitutively expresses the activator and the activator is unable to activate the first inducible promoter or the second inducible promoter in absence of a first triggering agent. In some embodiments, in absence of activation of the first inducible promoter and the second inducible promoter, detectable levels of the Rep proteins from the first polynucleotide or if present the Cap proteins from the first polynucleotide, the Cap proteins from the second polynucleotide, the inducible recombinase, and the one or more adenoviral helper proteins are not expressed, and wherein the inducible recombinase is activated in the presence of a second triggering agent. In some embodiments, a second polynucleotide construct comprises the third polynucleotide as described herein.

[0239] In some embodiments, the third polynucleotide comprising the sequence encoding one or more adenoviral helper proteins comprises: (i) a first sequence comprising from 5' to 3': a second inducible promoter operably linked to a sequence encoding an inducible recombinase; a self-excising element comprising a third recombination site, the sequence encoding the inducible recombinase, and a fourth recombination site; and a sequence encoding one or more adenoviral helper proteins, wherein the second inducible promoter is not operably linked to the sequence encoding the one or more adenoviral helper proteins; (ii) a second sequence comprising a first constitutive promoter operably linked to a sequence encoding an activator. In some embodiments, the third recombination site and the fourth recombination site are oriented in the same direction. In some embodiments, the cell constitutively expresses the activator, and the activator is unable to activate the second inducible promoter in absence of a first triggering agent. In some embodiments, in the presence of the first triggering agent, the activator activates the second inducible promoter resulting in expression of the inducible recombinase, and the inducible recombinase is expressed. In some embodiments, in the presence of a second triggering agent, the inducible recombinase translocates to a nucleus of the cell and causes recombination between the third recombination site and the fourth recombination site resulting in excision of the self-excising element, thereby operably linking the second inducible promoter to the sequence encoding the one or more adenoviral helper proteins and allowing expression of the one or more adenoviral helper proteins.

[0240] In some embodiments, the one or more adenoviral helper proteins comprise one or more of adenovirus E1A protein, E1B protein, E2A protein, and E4 protein. In certain embodiments, the one or more adenoviral helper proteins comprises E2A protein and E4 protein.

[0241] In some embodiments, the third polynucleotide comprising the sequence encoding for one or more AAV helper proteins comprises a bicistronic open reading frame encoding two AAV helper proteins. In certain embodiments, the SEQ ID NO: 30 comprises the third polynucleotide.

[0242] In some embodiments, the one or more adenoviral helper proteins are separated by a bicistronic open reading frame. In certain embodiments, the bicistronic open reading frame comprises an internal ribosome entry site (IRES) or a peptide 2A (P2A) sequence.

[0243] In some embodiments, the second inducible promoter operably linked to the self- excising element in the third polynucleotide is a tetracycline-inducible promoter, an ecdysone- inducible promoter, or a cumate-inducible promoter.

[0244] In some embodiments, the first inducible promoter and the second inducible promoter are the same. In some embodiments, the first inducible promoter and the second inducible promoter are a tetracycline-inducible promoter. In certain embodiments, the tetracycline- inducible promoter comprises a tetracycline-responsive promoter element (TRE). In certain embodiments, the TRE comprises Tet operator (tetO) sequence concatemers fused to a minimal promoter. In certain embodiments, the minimal promoter is a human cytomegalovirus promoter.

[0245] In some embodiments, the first constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter.

[0246] In some embodiments, the activator is reverse tetracycline-controlled transactivator (rTA) comprising a Tet Repressor binding protein (TetR) fused to a VP16 transactivation domain.

[0247] In some embodiments, a triggering agent for inducing the tetracycline-inducible promoter is tetracycline. In other embodiments, a triggering agent for inducing the tetracycline- inducible promoter is doxycycline.

[0248] In some embodiments, the inducible recombinase is fused to an estrogen response element (ER) and translocates to the nucleus in the presence of tamoxifen.

[0249] In some embodiments, the recombination sites in the first polynucleotide and the third polynucleotide are lox sites and the inducible recombinase is a Cre recombinase. In other embodiments, the recombination sites in the first polynucleotide and the third polynucleotide are flippase recognition target (FRT) sites and the inducible recombinase is a flippase (Flp) recombinase.

[0250] In some embodiments, presence of the triggering agent activates the activator for activation of the first inducible promoter to express AAV Cap proteins from the second polynucleotide encoding the AAV Cap proteins.

[0251] In some embodiments, presence of the triggering agent activates the activator for activation of the second inducible promoter to express the Rep proteins of the first polynucleotide, if present the Cap proteins of the first polynucleotide, the inducible recombinase, and the one or more adenoviral helper proteins.

[0252] In some embodiments, upon expression of the inducible recombinase, recombination between the first recombination site and the second recombination site in the first polynucleotide results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein of the first polynucleotide and if present the Cap Proteins of the first polynucleotide; and recombination between the third recombination site and the fourth recombination site in the third polynucleotide results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins.

[0253] In some embodiments, the third polynucleotide further comprises a third selectable marker operably linked to a third promoter. Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA

[0254] In some embodiments, the third polynucleotide further comprises a sequence encoding a viral associated RNA (VA-RNA). In certain embodiments, the VA-RNA is a mutated VA-RNA. In some embodiments, the VA-RNA is wild-type VA-RNA. In other embodiments, VA-RNA comprises one or more mutations in the VA-RNA internal promoter. In some embodiments, a second polynucleotide construct comprises the third polynucleotide as described herein.

[0255] In some embodiments, the sequence encoding the VA-RNA is operably linked to an inactive promoter comprising a first part of a second constitutive promoter and a second part of the second constitutive promoter separated by a second excisable element comprising a fifth recombination site and a sixth recombination site flanking a stuffer sequence, and excision of the second excisable element by the inducible recombinase generates a functional complete second constitutive promoter operably linked to the VA-RNA coding sequence to allow expression of the VA-RNA. In some embodiments, the fifth and sixth recombination sites are oriented in the same direction.

[0256] In some embodiments, the first part of the second constitutive promoter comprises a distal sequence element (DSE) of an RNA polymerase III promoter, and the second part of the second constitutive promoter comprises a proximal sequence element (PSE) of an RNA polymerase III promoter. In other embodiments, the first part of the second constitutive promoter comprises a distal sequence element (DSE) of a U6 promoter, and the second part of the second constitutive promoter comprises a proximal sequence element (PSE) of a U6 promoter. In still other embodiments, the first part of the second constitutive promoter comprises a distal sequence element (DSE) of a U7 promoter, and the second part of the second constitutive promoter comprises a proximal sequence element (PSE) of a U7 promoter.

[0257] In some embodiments, the set of nucleic acids or any of the recombinant nucleic acids as disclosed herein further comprises a nucleic acid sequence encoding a viral associated RNA (“VA-RNA”) sequence. In some embodiments, the third recombinant nucleic acid sequence comprises a nucleic acid sequence encoding a VA-RNA sequence. In some embodiments, the expression of VA-RNA is constitutive. In some embodiments, the expression of VA-RNA is inducible. In some embodiments, the VA-RNA sequence comprises one or more mutations in the VA-RNA internal promoter, preferably G16A and G60A. In some embodiments, the expression of VA-RNA is driven by a EF1alpha promoter, a U6 promoter, or a U7 promoter. Insome embodiments, the expression of VA-RNA is driven by a U6 promoter or a U7 promoter. In some embodiments, the U6 promoter or the U7 promoter comprises a) a first part of a U6 or U7 promoter sequence, b) a stuffer sequence, and c) a second part of a U6 or U7 promoter sequence, and wherein the stuffer sequence is capable of being excised by a Cre polypeptide.

[0258] In some embodiments, the VA-RNA comprises a G16A mutation or a G60A mutation, or a combination thereof.

[0259] In some embodiments, the third polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 153. In certain embodiments, the third polynucleotide has sequence of SEQ ID NO: 153. Fourth Polynucleotide Encoding Payload

[0260] In some embodiments, the payload of the fourth polynucleotide comprises a reporter gene, a therapeutic gene, or a transgene encoding a protein of interest. In certain embodiments, the payload of the fourth polynucleotide is progranulin. In some embodiments, a third polynucleotide construct comprises the fourth polynucleotide as described herein.

[0261] In some embodiments, the sequence encoding the payload of the fourth polynucleotide comprises a sequence encoding a reporter gene, a therapeutic gene, or a transgene encoding a protein of interest. In some embodiments, the sequence encoding the payload of the fourth polynucleotide is a sequence encoding progranulin. In some embodiments, the sequence encoding progranulin has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 166. In certain embodiments, the sequence encoding progranulin has sequence of SEQ ID NO: 166.

[0262] In some embodiments, the sequence encoding the payload comprises a sequence encoding a suppressor tRNA, a guide RNA, or a homology region for homology-directed repair.

[0263] In some embodiments, the fourth polynucleotide comprising the sequence encoding the payload comprises the sequence encoding the payload flanked by a 5' AAV inverted terminal repeat (5' ITR) and a 3' AAV inverted terminal repeat (3' ITR).

[0264] In some embodiments, the sequence encoding the payload is flanked by a 5' AAV inverted terminal repeat (5' ITR) and a 3' AAV inverted terminal repeat (3' ITR) has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 146. In certain embodiments, thesequence encoding the payload is flanked by a 5' AAV inverted terminal repeat (5' ITR) and a 3' AAV inverted terminal repeat (3' ITR) has sequence of SEQ ID NO: 146.

[0265] In some embodiments, the sequence encoding the payload has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In certain embodiments, the sequence encoding the payload has sequence of SEQ ID NO: 156.

[0266] In some embodiments, the sequence encoding the payload has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 158. In certain embodiments, the sequence encoding the payload has sequence of SEQ ID NO: 158. In some embodiments, the payload construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 147. Polynucleotide Encoding a Payload

[0267] In some embodiments, the fourth integrated synthetic construct (also referred to as Construct 4) comprises the coding sequence for an expressible payload and a third mammalian cell selection element. In the exemplary embodiments, the expressible payload is under the control of a constitutive promoter. This construct can be referred to as construct 4 or payload construct, interchangeably.

[0268] In some embodiments, the expressible payload encodes a guide RNA. In certain embodiments, the guide RNA directs RNA editing. In some embodiments, the guide RNA directs Cas-mediated DNA editing. In some embodiments, the guide RNA directs ADAR- mediated RNA editing. In some embodiments, the fourth integrated synthetic construct comprises a sequence encoding for any of the expressible payloads disclosed herein. For example, said sequence can encode for any therapeutic. For example, the therapeutic may be a transgene, a guide RNA, an antisense RNA, an oligonucleotide, an mRNA, a miRNA, a shRNA, a tRNA suppressor, a CRISPR-Cas protein, any gene editing enzyme, or any combination thereof. In some embodiments, the transgene encodes for progranulin. In some embodiments, the tRNA suppressor is capable of suppressing an opal stop codon. In some embodiments, the tRNA suppressor is capable of suppressing an ochre stop codon. In some embodiments, the tRNA suppressor is capable of suppressing an amber stop codon. In some embodiments, the fourth integrated synthetic construct comprises sequences encoding for more than one of the expressible payloads disclosed herein. For example, the fourth integrated synthetic construct comprises 2 gRNA, 3 gRNA, 4 gRNA, 5 gRNA, 6 gRNA, 7 gRNA, 8 gRNA, 9 gRNA, or 10 gRNA. These gRNAs can all be the same, all be different, or any combination of the same and different. For example, the fourth integrated synthetic construct comprises 2 suppressor tRNAs,3 suppressor tRNAs, 4 suppressor tRNAs, 5 suppressor tRNAs, 6 suppressor tRNAs, 7 suppressor tRNAs, 8 suppressor tRNAs, 9 suppressor tRNAs, or 10 suppressor tRNAs. These suppressor tRNAs can all be the same, all be different, or any combination of the same and different.

[0269] In some embodiments, the expressible payload encodes a protein. In certain embodiments, the expressible payload is an enzyme, useful for replacement gene therapy. In some embodiments, the protein is a therapeutic antibody. In some embodiments, the protein is a vaccine immunogen. In particular embodiments, the vaccine immunogen is a viral protein.

[0270] In some embodiments, the expressible payload is a homology construct for homologous recombination.

[0271] In various embodiments, the third mammalian cell selection element is an auxotrophic selection element.

[0272] In some embodiments, the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 33 or SEQ ID NO: 139. In some embodiments, the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 33 or SEQ ID NO: 139, wherein SEQ ID NO: 34 in SEQ ID NO: 33 or SEQ ID NO: 139 is replaced with a sequence of the payload of interest. In some embodiments, the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, or SEQ ID NO: 159. In some embodiments, the payload construct is a plasmid comprising at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 147, SEQ ID NO: 157, or SEQ ID NO: 159.

[0273] In some embodiments, the payload construct comprises a sequence of a payload flanked by ITR sequences. In some embodiments, expression of the sequence of the payload is driven by a constitutive promoter or an inducible promoter. In some embodiments, the promoter and sequence of the payload are flanked by ITR sequences. In some embodiments, the payload construct flanked by ITRs comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 146.

[0274] In some embodiments, the sequence of the payload comprises a polynucleotide sequence coding for a gene. In some embodiments, the gene codes for a selectable marker or detectable marker. In some embodiments, the gene codes for a therapeutic polypeptide or transgene. In some embodiments, the therapeutic polypeptide or transgene is progranulin. In some embodiments, the sequence of the payload comprises a polynucleotide sequence coding for a therapeutic polynucleotide. In some embodiments, the therapeutic polynucleotide is a tRNA suppressor or a guide RNA. In some embodiments, the tRNA suppressor is capable ofsuppressing an opal stop codon. In some embodiments, the tRNA suppressor is capable of suppressing an ochre stop codon. In some embodiments, the tRNA suppressor is capable of suppressing an amber stop codon. In some embodiments, the guide RNA is a polyribonucleotide capable of binding to a protein. In some embodiments, the protein is nuclease. In some embodiments, the protein is a Cas protein, an ADAR protein, or an ADAT protein. In some embodiments, the guide RNA, when bound to a target RNA, recruits an ADAR protein for editing of the target RNA. In some embodiments, the Cas protein is catalytically inactive Cas protein. In some embodiments, the payload construct is stably integrated into the genome of the cell. In some embodiments, a plurality of the payload construct are stably integrated into the genome of the cell. In some embodiments, the plurality of the payload constructs are separately stably integrated into the genome of the cell.

[0275] In some embodiments, the payload construct further comprises a sequence coding for a selectable marker or detectable marker outside of the ITR sequences. In some embodiments, expression of the selectable marker or detectable marker outside of the ITR sequences is driven by a promoter. The promoter can be a constitutive promoter or an inducible promoter. In some embodiments, the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter. In some embodiments, the inducible promoter is a tetracycline-inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter. In some embodiments, the selectable marker is a mammalian cell selection element (e.g., a third mammalian cell selection element). In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments, the selectable marker is DHFR Z- Nter or DHFR Z-Cter. In some embodiments, the DHFR Z-Nter comprises a sequence having atleast 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C- terminal intein of a split intein and the second auxotrophic selection element codes for an N- terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C- terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.

[0276] In some embodiments, the payload construct further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the payload construct further encodes a helper enzyme involvedin production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.

[0277] In some embodiments, a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90 – SEQ ID NO: 98, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101 – SEQ ID NO: 109.

[0278] . In some embodiments, the selectable marker is outside of the ITR sequences on the payload construct. In some embodiments, the selectable marker outside of the ITR sequences is a split intein linked to an N-terminus of the auxotrophic protein or split intein linked to a C- terminus of the auxotrophic protein. In some embodiments, the selectable marker outside of the ITR sequences is a leucine zipper linked to an N-terminus of the auxotrophic or leucine zipper linked to a C-terminus of the auxotrophic. In some embodiments, the selectable marker outside of the ITR sequences is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker outside of the ITR sequences is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the payload construct further comprises a spacer between the 5’ ITR and the promoter / selectable marker or promoter / detectable marker outside of the ITR sequences. In some embodiments, the payload construct further comprises a spacer between the 3’ ITR and the promoter / selectable marker or promoter / detectable marker outside of the ITR sequences. In some embodiments, the spacer ranges in length from 500 base pairs to 5000 base pairs, including any length within this range such as 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1250, 1500, 1750, 2000, 2225, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, or 5000 base pairs. In some embodiments, the spacer length is a sufficientlength for decreasing reverse packaging of the selectable marker or detectable marker that is outside the ITR sequences.

[0279] In some embodiments, the fourth integrated synthetic construct comprising the coding sequence for a payload and a selectable marker or detectable marker is further engineered to remove locations having the potential for Rep-mediated nicking. For example, a location having the potential for Rep-mediated nicking is a location having the sequence CAGTGAGCGAGCGAGCGCGCAG (SEQ ID NO: 87); a sequence comprising GAGC (SEQ ID NO: 88) repeats; or the sequence GATGGAGTTGGCCACTCCCTC (SEQ ID NO: 89). These sequences can be engineered to prevent binding of Rep proteins for Rep-mediated nicking. In some embodiments, the location having the potential for Rep-mediated nicking that is engineered to prevent binding of Rep proteins for Rep-mediated nicking is in a region within 100 nucleotides of an ITR sequence. In some embodiments, the location having the potential for Rep-mediated nicking that is engineered to prevent binding of Rep proteins for Rep-mediated nicking is in a region within 200 nucleotides of an ITR sequence. In some embodiments, the location having the potential for Rep-mediated nicking that is engineered to prevent binding of Rep proteins for Rep-mediated nicking is in a region within 300 nucleotides of an ITR sequence. In some embodiments, the location having the potential for Rep-mediated nicking that is engineered to prevent binding of Rep proteins for Rep-mediated nicking is in a region within 400 nucleotides of an ITR sequence. In some embodiments, the location having the potential for Rep-mediated nicking that is engineered to prevent binding of Rep proteins for Rep-mediated nicking is in a region within 500 nucleotides of an ITR sequence. In some embodiments, the location having the potential for Rep-mediated nicking that is engineered to prevent binding of Rep proteins for Rep-mediated nicking is in a region within 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1250, 1500, 1750, 2000, 2225, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, or 5000 nucleotides of an ITR sequence.

[0280] In some embodiments, a payload construct comprises a polynucleotide construct coding for a VA-RNA. In some embodiments, the VA-RNA is operably linked to a constitutive promoter or an inducible promoter. In some embodiments, the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter. In some embodiments, the inducible promoter is a tetracycline-inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter. In some embodiments, a payload construct comprises a polynucleotide construct coding for a VA-RNA, wherein a sequence coding for the VA-RNA comprises at least two mutations in an internal promoter. In some embodiments, a separate polynucleotide construct codes for a VA-RNA, wherein a sequence coding for the VA-RNA comprises at least two mutations in an internal promoter. In some embodiments, the sequence coding for the VA-RNAcomprises a sequence coding for a transcriptionally dead VA-RNA. In some embodiments, the sequence coding for the VA-RNA comprises a deletion of from about 5-10 nucleotides in the promoter region. In some embodiments, the sequence coding for the VA-RNA comprises at least one mutation. In some embodiments, the at least one mutation is in the A Box promoter region. In some embodiments, the at least one mutation is in the B Box promoter region. In some embodiments, the at least one mutation is G16A and G60A. In some embodiments, the expression of the VA-RNA is under the control of an RNA polymerase III promoter. In some embodiments, the expression of the VA-RNA is under the control of an interrupted RNA polymerase III promoter. In some embodiments, the expression of the VA-RNA is under the control of a U6 or U7 promoter. In some embodiments, the expression of the VA-RNA is under the control of an interrupted U6 or U7 promoter. In some embodiments, the polynucleotide construct comprises upstream of the VA-RNA gene sequence, from 5’ to 3’: a) a first part of a U6 or U7 promoter sequence; b) a first recombination site; c) a stuffer sequence; d) a second recombination site; e) a second part of a U6 or U7 promoter sequence. In some embodiments, the stuffer sequence is excisable by a recombinase. In some embodiments, the stuffer sequence comprises a sequence encoding a gene. In some embodiments, the stuffer sequence comprises a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a CMV promoter. In some embodiments, the gene encodes a detectable marker or a selectable marker. In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments, the selectable marker is DHFR Z-Nter or DHFR Z-Cter. In some embodiments, the DHFR Z- Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequenceidentity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N- terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.

[0281] In some embodiments, the stuffer sequence further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the stuffer sequence further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzymefacilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GT—CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.

[0282] In some embodiments, a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90 – SEQ ID NO: 98, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101 – SEQ ID NO: 109.

[0283] In some embodiments, the detectable marker comprises a luminescent marker or a fluorescent marker. In some embodiments, the fluorescent marker is GFP, EGFP, RFP, CFP, BFP, YFP, or mCherry. In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA-RNA or the VA-RNA construct further comprising a sequence coding for a recombinase. In some embodiments, the recombinase is exogenously provided. In some embodiments, the recombinase is a site-specific recombinase. In some embodiments, the recombinase is a Cre polypeptide or a Flippase polypeptide. In some embodiments, the Cre polypeptide is fused to a ligand binding domain. In some embodiments, the ligand binding domain is a hormone receptor. In some embodiments, the hormone receptor is an estrogen receptor. In some embodiments, the estrogen receptor comprises a point mutation. In some embodiments, the estrogen receptor is ERT2. In some embodiments, the recombinase is a Cre-ERT2 polypeptide. In some embodiments, the first recombination site is a first lox sequence and the second recombination site is a second lox sequence. In some embodiments, the first lox sequence is a first loxP site and the second lox sequence is a second loxP site. In some embodiments, the first recombination site is a first FRT site and the second recombination site is a second FRT site. In some embodiments, the construct comprising the VA-RNA as described herein further comprises a sequence coding for a selectable marker. In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%,90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C- terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments, the selectable marker is DHFR Z-Nter or DHFR Z-Cter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.

[0284] In some embodiments, the construct comprising VA-RNA further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the construct comprising the VA-RNA further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.

[0285] In some embodiments, a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90 – SEQ ID NO: 98, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101 – SEQ ID NO: 109.

[0286] In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA-RNA or the VA-RNA construct is in a vector. In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA-RNA or the VA-RNA construct is in a plasmid. In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA-RNA or the VA-RNA construct is in a bacterial artificial chromosome or yeast artificial chromosome. In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA-RNA or the VA-RNA construct is a synthetic nucleic acid construct. In some embodiments, an inducible helper construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 13 – SEQ ID NO: 19 or SEQ ID 23 – SEQ ID NO:26. In some embodiments, an inducible helper construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 13 – SEQ ID NO: 19 or SEQ ID 23 – SEQ ID NO: 26. In some embodiments, a VA-RNA construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 13 – SEQ ID NO: 19 or SEQ ID 23 – SEQ ID NO: 26. In some embodiments, a VA-RNA construct has a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 13 – SEQ ID NO: 19 or SEQ ID 23 – SEQ ID NO: 26. Payloads

[0287] Disclosed herein are payloads that may be encoded for by polynucleotide construct 4, which encodes for a payload. This fourth polynucleotide is referred to herein as a “payload construct” or “therapeutic payload.” Thus, disclosed herein are stable mammalian cell lines that encapsidate a payload. In some embodiments, the payload may be an expressible payload. In some embodiments, the polynucleotide may encode for any therapeutic. For example, the therapeutic may be a transgene, a guide RNA, an antisense RNA, an oligonucleotide, an mRNA, a miRNA, a shRNA, a tRNA suppressor, a CRISPR-Cas protein, any gene editing enzyme, or any combination thereof. In some embodiments, the payload is guide RNA, wherein the guide RNA, when bound to a target RNA, recruits an ADAR enzyme for editing of the target RNA. In some embodiments, the payload is progranulin. In some embodiments, the stable mammalian cell lines disclosed herein can conditionally produce rAAV virions that encapsidate more than one payload. Any combination of payloads disclosed herein is contemplated.

[0288] The sequence encoding a payload as disclosed herein encompasses any nucleotide sequence that is to be delivered to a cell. The nucleotide sequence may be utilized in the cell for, e.g., insertion of the nucleotide sequence or a part thereof. For example, the nucleotide sequence may be used to repair an endogenous DNA. In such a case, the nucleotide sequence itself is the payload being delivered by the rAAV to a cell.

[0289] In other cases, the polynucleotide payload is transcribed in the cell into an RNA which is not translated into a protein. In such a case, the RNA is the payload that is delivered by the polynucleotide payload present in the rAAV. In other cases, the polynucleotide payload is transcribed in the cell into an mRNA which is translated into a protein. In such a case, the protein is the payload that is delivered by the polynucleotide payload present in the rAAV.

[0290] The polynucleotide payload may include a promoter operably linked to a DNA sequence. The promoter may be any promoter that allows for transcription of the DNA in the cell. A payload disclosed herein may be a therapeutic payload.

[0291] The DNA sequence may be transcribed to produce RNA in the cell. The RNA may be mRNA. The RNA may be a guide RNA (gRNA), a tRNA, a suppressor tRNA, an mRNA, or a circular RNA. The RNA may be a regulatory RNA of interest such as, but not limited to, a microRNA (miRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a small nuclear RNA (snRNA), a long non-coding RNA (lncRNA), an antisense nucleic acid, and the like.

[0292] The polynucleotide payload may be a gene encoding a polypeptide, such as, an antibody, a hormone, a site-specific endonuclease, a reporter gene, a component of a CRISPR / Cas system, an adenosine deaminase acting on RNA (ADAR) enzyme, a transcriptional activator, a transcriptional repressor, a ribozyme, a DNAzyme, or any combination thereof.

[0293] A payload may include any one or combination of the following: a transgene, a tRNA suppressor, a guide RNA, or any other target binding / modifying oligonucleotide or derivative thereof, or payloads may include immunogens for vaccines, and elements for any gene editing machinery (DNA or RNA editing). Payloads may also include those that deliver a transgene encoding antibody chains or fragments that are amenable to viral vector-mediated expression (also referred to as “vectored or vectorized antibody” for gene delivery). See, e.g., Curr Opinion HIV AIDS.2015 May; 10(3): 190–197, describing vectored antibody gene delivery for the prevention or treatment of HIV infection. See also, U.S. Pat. No.10,780,182, which describes AAV delivery of trastuzumab (Herceptin) for treatment of HER2+ brain metastases. A payload disclosed herein may not be a therapeutic payload (e.g., a coding for a detectable marker such as GFP). In particular, in some instances the polynucleotide payload refers to a polynucleotide that may be a homology element for homology-directed repair, or polynucleotide transcribed into a guide RNA to be delivered for a variety of purposes. In some embodiments, the transgene refers to a nucleic acid sequence coding for expression of guide RNA for ADAR editing or ADAT editing. In some embodiments, the transgene refers to a transgene packaged for gene therapy. In some embodiments, the transgene refers to synthetic constructs packaged for vaccines. In certain aspects, a polynucleotide payload may be described as encoding an RNA, which is meant to refer to the RNA transcribed from the polynucleotide.

[0294] In certain examples, the sequence encoding the payload comprises two expressible sequences, wherein a first expressible sequence encodes for a first gRNA and a second expressible sequence encodes for a second gRNA. In some embodiments, the first gRNA and the second gRNA are different. In some embodiments, the first gRNA and the second gRNA are the same. In certain examples, the sequence encoding the payload comprises two or more expressible sequences. In some embodiments, the two or more expressible sequences encode fortwo or more gRNA. In some embodiments, the two or more gRNA are all different gRNA, all the same gRNA, or a combination of the same and different gRNA.

[0295] In some cases, the sequence encoding the payload comprises an expressible sequence encoding both a heterologous RNA and a heterologous polypeptide. In other cases, the expressible sequence encodes two or more heterologous payloads. Where the expressible sequence encodes two heterologous payloads, in some cases, the nucleotide sequences encoding the two heterologous payloads are operably linked to the same promoter. Where the expressible sequence encodes two heterologous payloads, in some cases, the nucleotide sequences encoding the two heterologous payloads are operably linked to two different promoters. In some cases, sequence encoding the payload comprises an expressible sequence encoding three heterologous payloads. Where the expressible sequence encodes three heterologous payloads, in some cases, the nucleotide sequences encoding the three heterologous payloads are operably linked to the same promoter. Where the expressible sequence encodes three heterologous payloads, in some cases, the nucleotide sequences encoding the three heterologous payloads are operably linked to two or three different promoters. In some cases, the fourth polynucleotide construct of the present disclosure comprises two or more expressible sequences, each comprising a nucleotide sequence encoding a heterologous payload.

[0296] In some embodiments, the expressible sequence encodes a polypeptide of interest. The polypeptide of interest may be any type of protein / peptide including, without limitation, an enzyme, an extracellular matrix protein, a receptor, transporter, ion channel, or other membrane protein, a hormone, a neuropeptide, an antibody, or a cytoskeletal protein; or a fragment thereof, or a biologically active domain of interest. In some cases, the payload is a therapeutic polypeptide, e.g., a polypeptide that provides clinical benefit.

[0297] Where the payload is an interfering RNA (RNAi), suitable RNAi include RNAi that decrease the level of an apoptotic or angiogenic factor in a cell. For example, an RNAi may be an shRNA or siRNA that reduces the level of a payload that induces or promotes apoptosis in a cell. A payload may be a gene whose gene product induces or promotes apoptosis are referred to herein as “pro-apoptotic genes” and the products of those genes (mRNA; protein) are referred to as “pro-apoptotic gene products.” Pro-apoptotic gene products include, e.g., Bax, Bid, Bak, and Bad gene products. See, e.g., U.S. Patent No.7,846,730. In another example, the RNAi specifically reduces the level of an RNA and / or a polypeptide product of a defective allele.

[0298] In some embodiments, the payload is an aptamer. In some cases, the aptamer is a therapeutic aptamer. For example, the aptamer may function as an antagonist by blocking interactions at a disease-associated target (e.g., receptor-ligand interactions). Alternatively, an aptamer may serve as an agonist for activating the function of a target receptor. Exemplaryaptamers of interest include aptamers against growth factor receptors and growth factors such as aptamers that bind to epidermal growth factor receptor (see, e.g., Wang et al. (2014) Biochem. Biophys. Res. Commun.453(4):681-5), transforming growth factor-beta type III receptor (see, e.g., Ohuchi et al. (2006) Biochimie 88(7):897-904.), vascular endothelial growth factor (VEGF) (see, e.g., Ng et al. (2006) Nat. Rev. Drug Discovery 5:123; and Lee et al. (2005) Proc. Natl. Acad. Sci. USA 102:18902) or platelet-derived growth factor (PDGF), e.g., E10030 (see, e.g., Ni and Hui (2009) Ophthalmologica 223:401; and Akiyama et al. (2006) J. Cell Physiol. 207:407).

[0299] In some embodiments, the expressible sequence encodes a sequence-specific endonuclease for use in genome editing. The sequence specific endonuclease may be used to create a double-stranded break at a specific site in the genome. The double stranded breaks may then be repaired by non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), or homology-directed repair (HDR) pathways. Desired genome edits may be introduced into the genome using donor DNA to repair double-strand breaks by homologous recombination. Various sequence-specific endonucleases may be used in genome editing for creation of double-strand breaks in DNA, including, without limitation, engineered zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, and clustered regularly interspaced short palindromic repeats (CRISPR) Cas9. See, e.g., Targeted Genome Editing Using Site-Specific Nucleases: ZFNs, TALENs, and the CRISPR / Cas9 System (T. Yamamoto ed., Springer, 2015); Genome Editing: The Next Step in Gene Therapy (Advances in Experimental Medicine and Biology, T. Cathomen, M. Hirsch, and M. Porteus eds., Springer, 2016); Aachen Press Genome Editing (CreateSpace Independent Publishing Platform, 2015); herein incorporated by reference. Precise control over the timing of production of the genome editing enzyme may be achieved by inducibly producing recombinant adenovirus associated virus (rAAV) virions with the vector system to allow turning on and off of expression as desired.

[0300] In some cases, a payload of interest is a site-specific endonuclease that provides for site- specific knock-down of gene function, e.g., where the endonuclease knocks out an allele associated with a disease. For example, in a case where a dominant allele encodes a defective copy of a gene, and the wild-type gene provides for normal function, a site-specific endonuclease may be targeted to the defective allele and knock out the defective allele. In some cases, a site-specific endonuclease is an RNA-guided endonuclease.

[0301] A site-specific nuclease may also be used to stimulate homologous recombination with a donor DNA that encodes a functional copy of the protein encoded by the defective allele. Thus, e.g., a subject rAAV virion may be used to deliver a site-specific endonuclease that knocks out adefective allele and also be used to deliver a functional copy of the defective allele, resulting in repair of the defective allele, thereby providing for production of a functional gene product.

[0302] In some cases, the payload is an RNA-guided endonuclease. In some cases, the payload is an RNA comprising a nucleotide sequence encoding an RNA-guided endonuclease. In some cases, the payload is a guide RNA, e.g., a single-guide RNA. In some cases, the payloads are: 1) a guide RNA; and 2) an RNA-guided endonuclease. The guide RNA may comprise: a) a protein- binding region that binds to the RNA-guided endonuclease; and b) a region that binds to a target nucleic acid. An RNA-guided endonuclease is also referred to herein as a “genome editing nuclease.”

[0303] Examples of RNA-guided endonucleases are CRISPR / Cas endonucleases (e.g., class 2 CRISPR / Cas endonucleases such as a type II, type V, or type VI CRISPR / Cas endonucleases). A suitable genome editing nuclease is a CRISPR / Cas endonuclease (e.g., a class 2 CRISPR / Cas endonuclease such as a type II, type V, or type VI CRISPR / Cas endonuclease). In some cases, a suitable RNA-guided endonuclease is a class 2 CRISPR / Cas endonuclease. In some cases, a suitable RNA-guided endonuclease is a class 2 type II CRISPR / Cas endonuclease (e.g., a Cas9 protein). In some cases, a genome targeting composition includes a class 2 type V CRISPR / Cas endonuclease (e.g., a Cpf1 protein, a C2c1 protein, or a C2c3 protein). In some cases, a suitable RNA-guided endonuclease is a class 2 type VI CRISPR / Cas endonuclease (e.g., a C2c2 protein; also referred to as a “Cas13a” protein). Also suitable for use is a CasX protein. Also suitable for use is a CasY protein.

[0304] In some cases, the genome-editing endonuclease is a Type II CRISPR / Cas endonuclease. In some cases, the genome-editing endonuclease is a Cas9 polypeptide. The Cas9 protein is guided to a target site (e.g., stabilized at a target site) within a target nucleic acid sequence (e.g., a chromosomal sequence or an extrachromosomal sequence, e.g., an episomal sequence, a minicircle sequence, a mitochondrial sequence, a chloroplast sequence, etc.) by virtue of its association with the protein-binding segment of the Cas9 guide RNA. In some cases, the Cas9 polypeptide used in a composition or method of the present disclosure is a Staphylococcus aureus Cas9 (saCas9) polypeptide. In some cases, a suitable Cas9 polypeptide is a high-fidelity (HF) Cas9 polypeptide. Kleinstiver et al. (2016) Nature 529:490. In some cases, a suitable Cas9 polypeptide exhibits altered PAM specificity. See, e.g., Kleinstiver et al. (2015) Nature 523:481. In some cases, the genome-editing endonuclease is a type V CRISPR / Cas endonuclease. In some cases a type V CRISPR / Cas endonuclease is a Cpf1 protein. In some cases, the genome-editing endonuclease is a CasX or a CasY polypeptide. CasX and CasY polypeptides are described in Burstein et al. (2017) Nature 542:237.

[0305] In some cases, a genome editing nuclease is a fusion protein that is fused to a heterologous polypeptide (also referred to as a “fusion partner”). In some cases, a genome editing nuclease is fused to an amino acid sequence (a fusion partner) that provides for subcellular localization, i.e., the fusion partner is a subcellular localization sequence (e.g., one or more nuclear localization signals (NLSs) for targeting to the nucleus, two or more NLSs, three or more NLSs, etc.).

[0306] Also suitable for use is an RNA-guided endonuclease with reduced enzymatic activity. Such an RNA-guided endonuclease is referred to as a “dead” RNA-guided endonuclease; for example, a Cas9 polypeptide that comprises certain amino acid substitutions such that it exhibits substantially no endonuclease activity, but such that it still binds to a target nucleic acid when complexed with a guide RNA, is referred to as a “dead” Cas9 or “dCas9.” In some cases, a “dead” Cas9 protein has a reduced ability to cleave both the complementary and the non- complementary strands of a double stranded target nucleic acid. For example, a “nuclease defective” Cas9 lacks a functioning RuvC domain (i.e., does not cleave the non-complementary strand of a double stranded target DNA) and lacks a functioning HNH domain (i.e., does not cleave the complementary strand of a double stranded target DNA). Such a Cas9 protein has a reduced ability to cleave a target nucleic acid (e.g., a single stranded or double stranded target nucleic acid) but retains the ability to bind a target nucleic acid. A Cas9 protein that may not cleave target nucleic acid (e.g., due to one or more mutations, e.g., in the catalytic domains of the RuvC and HNH domains) is referred to as a “nuclease defective Cas9”, “dead Cas9” or simply “dCas9.” Other residues may be mutated to achieve the above effects (i.e. inactivate one or the other nuclease portions).

[0307] In some cases, the genome-editing endonuclease is an RNA-guided endonuclease (and its corresponding guide RNA) known as Cas9-synergistic activation mediator (Cas9-SAM). The RNA-guided endonuclease (e.g., Cas9) of the Cas9-SAM system is a “dead” Cas9 fused to a transcriptional activation domain (wherein suitable transcriptional activation domains include, e.g., VP64, p65, MyoD1, HSF1, RTA, and SET7 / 9) or a transcriptional repressor domain (where suitable transcriptional repressor domains include, e.g., a KRAB domain, a NuE domain, an NcoR domain, a SID domain, and a SID4X domain). The guide RNA of the Cas9-SAM system comprises a loop that binds an adapter protein fused to a transcriptional activator domain (e.g., VP64, p65, MyoD1, HSF1, RTA, or SET7 / 9) or a transcriptional repressor domain (e.g., a KRAB domain, a NuE domain, an NcoR domain, a SID domain, or a SID4X domain). For example, in some cases, the guide RNA is a single-guide RNA comprising an MS2 RNA aptamer inserted into one or two loops of the sgRNA; the dCas9 is a fusion polypeptidecomprising dCas9 fused to VP64; and the adaptor / functional protein is a fusion polypeptide comprising: i) MS2; ii) p65; and iii) HSF1. See, e.g., U.S. Patent Publication No.2016 / 0355797.

[0308] Also suitable for use is a chimeric polypeptide comprising: a) a dead RNA-guided endonuclease; and b) a heterologous fusion polypeptide. Examples of suitable heterologous fusion polypeptides include a polypeptide having, e.g., methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, DNA integration activity, or nucleic acid binding activity.

[0309] A nucleic acid that binds to a class 2 CRISPR / Cas endonuclease (e.g., a Cas9 protein; a type V or type VI CRISPR / Cas protein; a Cpf1 protein; etc.) and targets the complex to a specific location within a target nucleic acid is referred to herein as a “guide RNA” or “CRISPR / Cas guide nucleic acid” or “CRISPR / Cas guide RNA.” A guide RNA provides target specificity to the complex (the RNP complex) by including a targeting segment, which includes a guide sequence (also referred to herein as a targeting sequence), which is a nucleotide sequence that is complementary to a sequence of a target nucleic acid.

[0310] In some cases, a guide RNA includes two separate nucleic acid molecules: an “activator” and a “targeter” and is referred to herein as a “dual guide RNA”, a “double-molecule guide RNA”, a “two-molecule guide RNA”, or a “dgRNA.” In some cases, the guide RNA is one molecule (e.g., for some class 2 CRISPR / Cas proteins, the corresponding guide RNA is a single molecule; and in some cases, an activator and targeter are covalently linked to one another, e.g., via intervening nucleotides), and the guide RNA is referred to as a “single guide RNA”, a “single-molecule guide RNA,” a “one-molecule guide RNA”, or simply “sgRNA.”

[0311] In some cases, the guide RNA is at least partially complementary to a target RNA sequence and is capable of recruiting an ADAR enzyme for RNA editing of the target RNA sequence.

[0312] Where the payload is an RNA-guided endonuclease or is both an RNA-guided endonuclease and a guide RNA, the payload may modify a target nucleic acid. In some cases, e.g., where a target nucleic acid comprises a deleterious mutation in a defective allele (e.g., a deleterious mutation in a neural cell target nucleic acid), the RNA-guided endonuclease / guide RNA complex, together with a donor nucleic acid comprising a nucleotide sequence that corrects the deleterious mutation (e.g., a donor nucleic acid comprising a nucleotide sequence that encodes a functional copy of the protein encoded by the defective allele), may be used to correct the deleterious mutation, e.g., via homology-directed repair (HDR).

[0313] In some cases, the payloads are an RNA-guided endonuclease and 2 separate sgRNAs, where the 2 separate sgRNAs provide for deletion of a target nucleic acid via non-homologous end joining (NHEJ).

[0314] In some cases, the payloads are: i) an RNA-guided endonuclease; and ii) one guide RNA. In some cases, the guide RNA is a single-molecule (or “single guide”) guide RNA (an “sgRNA”). In some cases, the guide RNA is a dual-molecule (or “dual-guide”) guide RNA (“dgRNA”).

[0315] In some cases, the payloads are: i) an RNA-guided endonuclease; and ii) 2 separate sgRNAs, where the 2 separate sgRNAs provide for deletion of a target nucleic acid via non- homologous end joining (NHEJ). In some cases, the guide RNAs are sgRNAs. In some cases, the guide RNAs are dgRNAs.

[0316] In some cases, the payloads are: i) a Cpf1 polypeptide; and ii) a guide RNA precursor; in these cases, the precursor may be cleaved by the Cpf1 polypeptide to generate 2 or more guide RNAs.

[0317] The payloads as described herein may be flanked by ITRs. Fifth Polynucleotide Encoding VA-RNA

[0318] In some embodiments, systems of polynucleotides comprising a) a first polynucleotide comprising a sequence encoding AAV Rep proteins operably linked to one or more promoters; and b) a second polynucleotide comprising a sequence of polynucleotides comprising (i) a sequence encoding AAV Cap proteins and (ii) a polyadenylation signal sequence; and one or more of: c) a third polynucleotide comprising a sequence encoding one or more adenoviral helper proteins; and d) a fourth polynucleotide comprising a sequence encoding a payload further comprise e) a fifth polynucleotide comprising a sequence encoding a viral associated RNA (VA-RNA). In certain embodiments, the VA-RNA is a mutated VA-RNA. In some embodiments, a second polynucleotide construct or a fourth comprises the fifth polynucleotide as described herein.

[0319] In some embodiments, the VA-RNA is wild-type VA-RNA or VA-RNA comprising one or more mutations in the VA-RNA internal promoter.

[0320] In some embodiments, the sequence encoding the VA-RNA is operably linked to an inactive promoter comprising a first part of a constitutive promoter and a second part of the constitutive promoter separated by a second excisable element comprising a fifth recombination site and a sixth recombination site flanking a stuffer sequence, the fifth and sixth recombination sites are oriented in the same direction, and excision of the second excisable element by theinducible recombinase generates a functional complete constitutive promoter operably linked to the VA-RNA coding sequence to allow expression of the VA-RNA.

[0321] In some embodiments, the first part of the constitutive promoter comprises a distal sequence element (DSE) of an RNA polymerase III promoter, and the second part of the constitutive promoter comprises a proximal sequence element (PSE) of an RNA polymerase III promoter. In other embodiments, the first part of the constitutive promoter comprises a distal sequence element (DSE) of a U6 promoter, and the second part of the constitutive promoter comprises a proximal sequence element (PSE) of a U6 promoter. In still other embodiments, the first part of the constitutive promoter comprises a distal sequence element (DSE) of a U7 promoter, and the second part of the constitutive promoter comprises a proximal sequence element (PSE) of a U7 promoter.

[0322] In some embodiments, the VA-RNA comprises a G16A mutation. In other embodiments, the VA-RNA comprises a G60A mutation. In still other embodiments, the VA-RNA comprises a G16A mutation and a G60A mutation. Selectable Markers for Polynucleotides

[0323] In some embodiments, a) the first polynucleotide further comprises a sequence encoding a first selectable marker operably linked to a first promoter.

[0324] In some embodiments, b) the second polynucleotide further comprises a sequence encoding a second selectable marker operably linked to a second promoter.

[0325] In some embodiments, c) the third polynucleotide further comprises a sequence encoding a third selectable marker operably linked to a third promoter.

[0326] In some embodiments, d) the fourth polynucleotide further comprises a sequence encoding a fourth selectable marker operably linked to a fourth promoter.

[0327] In some embodiments, e) the fifth polynucleotide further comprises a sequence encoding a fifth selectable marker operably linked to a fifth promoter.

[0328] In some embodiments, the system of polynucleotides comprises any combinations of the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, and the fifth selectable marker.

[0329] In some embodiments, any combinations of the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, and the fifth selectable marker are different selectable markers; the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, the fifth selectablemarker, or any combinations thereof are the same selectable marker but as a different split portion of the selectable marker; or any combinations thereof.

[0330] In some embodiments, any combinations of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter are the same constitutive promoter or different constitutive promoters.

[0331] In some embodiments, the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, and the fifth selectable marker, or any combination thereof is an antibiotic resistance gene. In certain embodiments, the antibiotic resistance gene is a blasticidin resistance gene, a hygromycin resistance gene, or a puromycin resistance gene.

[0332] In some embodiments, the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, and the fifth selectable marker, or any combination thereof is a first split portion of an antibiotic resistance gene. In certain embodiments, the first split portion of the antibiotic resistance gene is a first split portion of the blasticidin resistance gene.

[0333] In some embodiments, the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, and the fifth selectable marker, or any combination thereof is a second split portion of an antibiotic resistance gene. In certain embodiments, the second split portion of the antibiotic resistance gene is a second split portion of the blasticidin resistance gene.

[0334] In some embodiments, the first promoter, the second promoter, the third promoter, the fourth promoter, the fifth promoter, or any combination thereof, is an EF1alpha promoter or an attenuated version thereof. In some embodiments, the attenuated version comprises a mutation in the TATA box. In certain embodiments, the attenuated EF1alpha promoter has weaker promoter activity than an EF1alpha promoter.

[0335] In some embodiments, the fourth polynucleotide comprising the sequence encoding the payload further comprises a spacer between the 5' ITR and the sequence encoding the fourth selectable marker or a spacer between the sequence encoding the fourth selectable marker and the 3' ITR, or a combination thereof. In some embodiments, (i) the first polynucleotide further comprises a constitutive promoter operably linked to a sequence encoding a first portion of a split selectable marker; (ii) the second polynucleotide further comprises a sequence encoding a selectable marker operably linked to a constitutive promoter; (iii) the third polynucleotide further comprises a sequence encoding a selectable marker operably linked to a constitutive promoter; and / or (iv) the fourth polynucleotide further comprises a constitutive promoter operably linked to a sequence encodinga selectable marker or a second part of the split selectable marker. In some embodiments, the constitutive promoter is an EF1 alpha promoter and / or the split selectable marker is a split antibiotic resistance protein. In some embodiments, the constitutive promoter is an EF1 alpha promoter and / or the selectable marker is a first antibiotic resistance protein. In some embodiments, the constitutive promoter is an CMV promoter and / or the selectable marker is a second antibiotic resistance protein. In some embodiments, the constitutive promoter is an EF1 alpha promoter.

[0336] In some embodiments, the fourth polynucleotide comprising the sequence encoding the payload further comprises a spacer between the 5' ITR and the sequence encoding the selectable marker or a spacer between the sequence encoding the fourth selectable marker and the 3' ITR, or a combination thereof. In some embodiments, the spacer ranges in length from 500 base pairs to 5000 base pairs. Selectable Marker

[0337] In some embodiments, suitable markers include genes which confer resistance to antibiotics or toxins, or sensitivity, or impart color, or change the antigenic characteristics when cells, which have been transfected with the nucleic acid constructs, are grown in an appropriate selective medium. Exemplary selectable marker genes include, without limitation, the neomycin resistance gene (neo encoding aminoglycoside phosphotransferase (APH)) that allows selection in mammalian cells by conferring resistance to G418 (Geneticin), the hygromycin-B resistance gene (hygB encoding hygromycin-B-phosphotransferase (HPH)) that confers resistance to hygromycin-B, the puromycin resistance gene (pac encoding puromycin-N-acetyltransferase) that confers resistance to puromycin, Zeocin resistance gene (Sh bla encodes a protein that binds to Zeocin) that prevents Zeocin from binding DNA and damaging it, and the blasticidin resistance gene (BSD) that confers resistance to blasticidin. In addition, dihydrofolate reductase (DHFR)-based methotrexate (MTX) selection or glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection may be used in mammalian cells. Other suitable markers and selection methods are known to those of skill in the art.

[0338] In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split selectable marker that allows for selection of cells retaining two different polynucleotides using a single selective pressure. In some embodiments, the antibiotic resistance protein is split into two portions that can associate to form a functional antibiotic resistance protein. A first portion of the antibiotic resistance protein is encoded by a first polynucleotide and a second portion of the antibiotic resistance protein is encoded by a second polynucleotide. In some embodiments, a split intervening proteins (inteins) system thatpermits stable retention of two integrated nucleic acid constructs under a single selective pressure is used. Inteins auto catalyze a protein splicing reaction that results in excision of the intein and joining of the flanking amino acids (extein sequences) via a peptide bond. Inteins exist in nature as a single domain within a host protein or, less frequently, in a split form. For split inteins, the two separate polypeptide fragments of the intein must associate in order for protein trans-splicing to occur to excise the intein. Split intein systems are described in: Cheriyan et al, J. Biol. Chem 288: 6202-6211 (2013); Stevens et al, PNAS 114: 8538-8543 (2017); Jillette et al., Nat Comm 10: 4968 (2019); US 2020 / 0087388 A1; and US 2020 / 0263197 A1.In some embodiments, a split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, a first portion of the antibiotic resistance protein is the N- terminal portion which is fused to a N-terminal intein at the C-terminus and a second portion of the antibiotic resistance protein is the C-terminal portion which is fused to a C-terminal intein at the N-terminus. When both portions are present, the N-terminal intein associates with the C- terminal intein resulting in excision of the inteins and splicing of the C-terminus of the N- terminal portion of the antibiotic resistance protein to the N-terminus of the C-terminal portion of the antibiotic resistance protein, thereby forming a functional antibiotic resistance proteins.

[0339] In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter and DHFR Z-Nter. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein.

[0340] In certain embodiments, a split auxotrophic selection system that permits stable retention of two integrated nucleic acid constructs under a single selective pressure can be used. One construct encodes the N-terminal fragment of mammalian dihydrofolate reductase (DHFR) fused to a leucine zipper peptide (“Nter-DHFR”). This N-terminal fragment is enzymaticallynonfunctional. The other construct encodes the C-terminal fragment of DHFR fused to a leucine zipper peptide (“Cter-DHFR”). This C-terminal fragment is enzymatically nonfunctional. When both fragments are concurrently expressed in the cell, a functional DHFR enzyme complex is formed through association of the leucine zipper peptides. Both constructs can be stably retained in the genome of a DHFR null cell by growth in a medium lacking hypoxanthine and thymidine. Embodiments of Polynucleotide System

[0341] In some embodiments, the system of polynucleotide comprises the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above; the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above; and the third polynucleotide described in sections “Third Polynucleotide Encoding Adenoviral Helper Proteins” or “Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA” above.

[0342] In some embodiments, the system of polynucleotide comprises the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above; the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above; and the fourth polynucleotide described in section “Fourth Polynucleotide Encoding Payload” above.

[0343] In some embodiments, the system of polynucleotide comprises the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above; the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above; the third polynucleotide described in sections “Third Polynucleotide Encoding Adenoviral Helper Proteins” or “Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA” above; and the fourth polynucleotide described in section “Fourth Polynucleotide Encoding Payload” above.

[0344] In some embodiments, the system of polynucleotide comprises the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above; the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above; the third polynucleotide described in sections “Third Polynucleotide Encoding Adenoviral Helper Proteins” or “Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA” above; and the fifth polynucleotide described in section “Fifth Polynucleotide Encoding VA-RNA” above.

[0345] In some embodiments, the system of polynucleotide comprises the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above; the secondpolynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above; the fourth polynucleotide described in section “Fourth Polynucleotide Encoding Payload” above; and the fifth polynucleotide described in section “Fifth Polynucleotide Encoding VA-RNA” above.

[0346] In some embodiments, the system of polynucleotide comprises the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above; the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above; and the fifth polynucleotide described in section “Fifth Polynucleotide Encoding VA- RNA” above.

[0347] In some embodiments, the system of polynucleotide comprises the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above; the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above; the third polynucleotide described in sections “Third Polynucleotide Encoding Adenoviral Helper Proteins” or “Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA” above; the fourth polynucleotide described in section “Fourth Polynucleotide Encoding Payload” above; and the fifth polynucleotide described in section “Fifth Polynucleotide Encoding VA-RNA” above.

[0348] In some aspects, systems of polynucleotides comprising: (a) the polynucleotide of any one of of the embodiments disclosed here, wherein the polynucleotide is a first polynucleotide; and one or more of: b) a second polynucleotide comprising a sequence encoding AAV Cap proteins; c) a third polynucleotide comprising a sequence encoding one or more adenoviral helper proteins; and d) a fourth polynucleotide comprising a sequence encoding a payload are provided.

[0349] In some embodiments, the sequence encoding the AAV Cap proteins in the second polynucleotide is substantially identical to the sequence encoding the AAV Cap proteins in the first polynucleotide.

[0350] In some embodiments, the second polynucleotide comprises an inducible promoter operably linked to the sequence encoding the AAV Cap proteins.

[0351] In some embodiments, the inducible promoter is a second inducible promoter.

[0352] In some embodiments, the second polynucleotide further comprises a selectable marker operably linked to a promoter.

[0353] In some embodiments, the selectable marker is a second selectable marker and the promoter is a constitutive promoter, optionally wherein the constitutive promoter is an EF1alpha promoter or an attenuated version thereof, wherein the attenuated version comprises a mutationin the TATA box, optionally wherein the attenuated EF1alpha promoter has weaker promoter activity than an EF1alpha promoter.

[0354] In some embodiments, the third polynucleotide comprising the sequence encoding one or more adenoviral helper proteins comprises: an inducible promoter operably linked to a self- excising element, wherein the inducible promoter is a third inducible promoter; the self-excising element comprising a third recombination site and a fourth recombination site flanking a sequence encoding an inducible recombinase, wherein the third recombination site and the fourth recombination site are oriented in the same direction, wherein the third inducible promoter is not operably linked to the sequence encoding the one or more AAV helper proteins; a constitutive promoter operably linked to a sequence encoding an activator,

[0355] wherein a cell comprising the third polynucleotide constitutively expresses the activator and the activator is unable to activate the first inducible promoter, if present the second inducible promoter, or the third inducible promoter in absence of a first triggering agent; wherein in absence of activation of the first inducible promoter, if present the second inducible promoter, and the third inducible promoter, the cell does not express detectable levels of the Rep proteins or the Cap proteins from the first polynucleotide, if present the Cap proteins from the second polynucleotide, the inducible recombinase, and the one or more AAV helper proteins, and wherein the inducible recombinase is activated in the presence of a second triggering agent.

[0356] In some embodiments, the one or more helper proteins comprise one or more of adenovirus E1A protein, E1B protein, E2A protein, and E4 protein, and optionally comprises E2A protein and E4 protein.

[0357] In some embodiments, the fourth polynucleotide comprising the sequence encoding the payload further comprises: a selectable marker or a second part or a first part of the split selectable marker operably linked to a constitutive promoter and the sequence encoding the payload is flanked by a 5' AAV inverted terminal repeat (5' ITR) and a 3' AAV inverted terminal repeat (3' ITR); optionally, wherein the sequence encoding the payload is flanked by a 5' AAV inverted terminal repeat (5' ITR) and a 3' AAV inverted terminal repeat (3' ITR) has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 146 or SEQ ID NO: 147.

[0358] In some embodiments, the fourth polynucleotide comprising the sequence encoding the payload further comprises further comprises a spacer between the 5' ITR and the sequence encoding the selectable marker or a spacer between the sequence encoding the selectable marker and the 3' ITR, or a combination thereof; optionally wherein SEQ ID NO: 146 or SEQ ID NO: 147 comprises the fourth polynucleotide.

[0359] In some embodiments, the third polynucleotide comprises a sequence encoding a viral associated RNA (VA-RNA); optionally, wherein the VA-RNA is a mutated VA-RNA.

[0360] In some embodiments, the VA-RNA is wild-type VA-RNA or VA-RNA comprising one or more mutations in the VA-RNA internal promoter.

[0361] In some embodiments, the sequence encoding the VA-RNA is operably linked to an inactive promoter comprising a first part of a constitutive promoter and a second part of the constitutive promoter separated by a second excisable element comprising a fifth recombination site and a sixth recombination site flanking a stuffer sequence, the fifth and sixth recombination sites are oriented in the same direction, and excision of the second excisable element by the inducible recombinase generates a functional complete constitutive promoter operably linked to the VA-RNA coding sequence to allow expression of the VA-RNA.

[0362] In some embodiments, the first part of the constitutive promoter comprises a distal sequence element (DSE) of an RNA polymerase III promoter, and the second part of the constitutive promoter comprises a proximal sequence element (PSE) of an RNA polymerase III promoter, or the first part of the constitutive promoter comprises a distal sequence element (DSE) of a U6 promoter, and the second part of the constitutive promoter comprises a proximal sequence element (PSE) of a U6 promoter, or the first part of the constitutive promoter comprises a distal sequence element (DSE) of a U7 promoter, and the second part of the constitutive promoter comprises a proximal sequence element (PSE) of a U7 promoter.

[0363] In some embodiments, the VA-RNA comprises a G16A mutation or a G60A mutation, or a combination thereof.

[0364] In some embodiments, (i) the first polynucleotide comprising the sequence encoding the first sequence encoding AAV Rep proteins and the second sequence encoding AAV Cap proteins comprises a constitutive promoter operably linked to a sequence encoding a first portion of a split selectable marker, optionally, wherein the constitutive promoter is an EF1 alpha promoter and / or the split selectable marker is a split antibiotic resistance protein; (ii) if present, the second polynucleotide comprising the sequence encoding the AAV Cap proteins comprises a sequence encoding a selectable marker operably linked to a constitutive promoter, optionally wherein the constitutive promoter is an EF1 alpha promoter and / or the selectable marker is a first antibiotic resistance protein; (iii) the third polynucleotide comprising the sequence encoding one or more adenoviral helper proteins comprises a sequence encoding a selectable marker operably linked to a constitutive promoter, optionally, wherein the constitutive promoter is an CMV promoter and / or the selectable marker is a second antibiotic resistance protein; and / or (iv) the fourth polynucleotide comprising the sequence encoding the payload comprises a constitutive promoter operably linked to a sequence encoding a selectable marker or a secondpart of the split selectable marker, optionally, wherein the constitutive promoter is an EF1 alpha promoter. 1.5. Polynucleotide Construct System

[0365] In some aspects, systems of polynucleotide constructs comprising: a) a first polynucleotide construct comprising the sequence of the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above and the sequence of the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above; and one or more of: b) a second polynucleotide construct comprising the sequence of the third polynucleotide described in sections “Third Polynucleotide Encoding Adenoviral Helper Proteins” and “Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA” above; c) a third polynucleotide construct comprising the sequence of the fourth polynucleotide described in section “Fourth Polynucleotide Encoding Payload” above are provided. In some embodiments, the system of polynucleotide further comprises d) a fourth polynucleotide construct comprising the sequence of the fifth polynucleotide described in section “Fifth Polynucleotide Encoding VA-RNA” above are provided.

[0366] In some aspects, systems of polynucleotide constructs comprising: a) a first polynucleotide construct comprising the sequence of the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above and the sequence of the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above; and b) a second polynucleotide construct comprising the sequence of the third polynucleotide described in sections “Third Polynucleotide Encoding Adenoviral Helper Proteins” and “Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA” above are provided. In some embodiments, the system of polynucleotide further comprises d) a fourth polynucleotide construct comprising the sequence of the fifth polynucleotide described in section “Fifth Polynucleotide Encoding VA-RNA” above.

[0367] In some aspects, systems of polynucleotide constructs comprising: a) a first polynucleotide construct comprising the sequence of the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above and the sequence of the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above; and b) a third polynucleotide construct comprising the sequence of the fourth polynucleotide described in section “Fourth Polynucleotide Encoding Payload” above are provided. In some embodiments, the system of polynucleotide further comprises d) a fourthpolynucleotide construct comprising the sequence of the fifth polynucleotide described in section “Fifth Polynucleotide Encoding VA-RNA” above.

[0368] In some aspects, systems of polynucleotide constructs comprising: a) a first polynucleotide construct comprising the sequence of the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above and the sequence of the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above; and b) a second polynucleotide construct comprising the sequence of the third polynucleotide described in sections “Third Polynucleotide Encoding Adenoviral Helper Proteins” and “Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA” above; c) a third polynucleotide construct comprising the sequence of the fourth polynucleotide described in section “Fourth Polynucleotide Encoding Payload” above are provided. In some embodiments, the system of polynucleotide further comprises d) a fourth polynucleotide construct comprising the sequence of the fifth polynucleotide described in section “Fifth Polynucleotide Encoding VA-RNA” above.

[0369] In some embodiments, the sequence of the first polynucleotide is separated from the sequence of the second polynucleotide by an intervening sequence. In certain embodiments, the intervening sequence comprises a transcriptional blocking element (TBE).

[0370] In some embodiments, the first polynucleotide construct comprises a sequence encoding a single selectable marker.

[0371] In some embodiments, (i) the first polynucleotide construct further comprises a constitutive promoter operably linked to a sequence encoding a first portion of a split selectable marker. In certain embodiments, the constitutive promoter is an EF1 alpha promoter and / or first portion of the split selectable marker is a first portion of a split of a first antibiotic resistance protein.

[0372] In some embodiments, (ii) the second polynucleotide construct further comprises a sequence encoding a selectable marker operably linked to a constitutive promoter. In certain embodiments, the constitutive promoter is an CMV promoter and / or the selectable marker is a second antibiotic resistance protein.

[0373] In some embodiments, (iv) the third polynucleotide construct further comprises a constitutive promoter operably linked to a sequence encoding a selectable marker or a second part of the split selectable marker. In certain embodiments, the constitutive promoter is an EF1 alpha promoter and / or the second part of the split selectable maker is a second portion of the first antibiotic resistance protein.

[0374] In some embodiments, (i) the first polynucleotide construct further comprises a constitutive promoter operably linked to a sequence encoding a first portion of a split selectablemarker, optionally, wherein the constitutive promoter is an EF1 alpha promoter and / or first portion of the split selectable marker is a first portion of a split of a first antibiotic resistance protein; (ii) the second polynucleotide construct further comprises a sequence encoding a selectable marker operably linked to a constitutive promoter, optionally, wherein the constitutive promoter is an CMV promoter and / or the selectable marker is a second antibiotic resistance protein; and / or (iv) the third polynucleotide construct further comprises a constitutive promoter operably linked to a sequence encoding a selectable marker or a second part of the split selectable marker, optionally, wherein the constitutive promoter is an EF1 alpha promoter and / or the second part of the split selectable maker is a second portion of the first antibiotic resistance protein.

[0375] In some embodiments, the first antibiotic resistance protein is a blasticidin resistance protein.

[0376] In some embodiments, the second antibiotic resistance protein is a puromycin resistance protein. AAV Rep Cap Construct(s) and Cap Construct

[0377] Disclosed herein are polynucleotide constructs encoding for a AAV Rep protein and Cap protein, where Cap protein is expressed from a native promoter and polynucleotide constructs encoding for the Cap protein, where Cap protein is expressed from an inducible promoter.

[0378] Disclosed herein is a polynucleotide construct encoding for a Rep and Cap protein, where Cap protein is expressed from an inducible promoter.

[0379] Disclosed herein is a polynucleotide construct encoding for a Rep and Cap protein, where Cap protein is expressed from an inducible promoter and lacks expression from a native promoter.

[0380] Provided herein is a first polynucleotide construct, which encodes for Rep and Cap proteins and comprises an excisable element within the Rep coding sequence. This first polynucleotide construct (Construct 1) is also referred to as a Rep Cap construct, and / or “AAV Rep Cap Construct.” In some embodiments, the elements of this first polynucleotide construct can be in one or more separate constructs. For example, the AAV Rep Cap Construct is Construct 1 of FIG.2A.

[0381] Provided herein is a second polynucleotide construct, which encodes for the Cap proteins under the control of an inducible promoter. The second polynucleotide construct (Construct 2) is also referred to as a “Cap construct,” and / or “AAV Cap Construct.” For example, the AAV Cap Construct is Construct 2 of FIG.1A. FIG.1A shows two separate polynucleotide constructs thateach express AAV Capsid proteins, thereby resulting in a higher AAV capsid proteins level as compared to those expressed by cells having a single polynucleotide construct for encoding AAV Capsid proteins. These cells are capable of conditionally producing recombinant AAV (rAAV) virions within which are packaged an expressible payload.

[0382] In some embodiments, the level of AAV Capsids produced by the cells having two separate polynucleotide constructs expressing the AAV Capsids is at least 2-fold higher, e.g., at least 5-fold to at least 100-fold higher than level of AAV Capsids produced by cells not having two separate polynucleotide constructs expressing the AAV Capsids. For example, cells with two separate polynucleotide constructs encoding AAV Capsid proteins produce a level of AAV Capsids that is at least 10-fold higher, 20-fold higher, 30-fold higher, 35-fold higher, 50-fold higher, or 100-fold higher than the level of AAV Capsids produced by cells with only a single polynucleotide construct encoding the AAV Capsids.

[0383] In some embodiments, the percent encapsidation of a payload in virus particles produced by the cells having two separate polynucleotide constructs expressing the AAV Capsids is at least 2-fold higher, e.g., at least 5-fold to at least 20-fold higher than the percent encapsidation of a payload in virus particles produced by cells not having two separate polynucleotide constructs expressing the AAV Capsids.

[0384] In some embodiments, the Cap coding sequence is operably linked to a promoter. In some embodiments, the sequence coding for VP1, the sequence coding for VP2, and the sequence coding for VP3 are operably linked to a promoter. In some embodiments, a single construct or separate constructs comprise these sequences, in any combination. In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter comprises a tetracycline-inducible promoter, a cumate-inducible promoter, or a cumate-inducible promoter. In some embodiments, the promoter is a constitutive promoter, wherein the sequences coding for the one or more cap proteins are downstream of an excisable element (e.g., a sequence flanked by recombination sites and comprising a stop signal) and constitutive promoter, wherein upon excision of the excisable element (e.g., by a recombinase), the sequences coding for the one or more cap proteins are operably linked to the constitutive promoter. In some embodiments, the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter.

[0385] The Cap protein encoding sequence provided in a separate polynucleotide construct can be expressed under the control of an inducible promoter. The Cap coding sequence may include the sequence coding for VP1, the sequence coding for VP2, and the sequence coding for VP3 are operably linked to the inducible promoter.

[0386] In various embodiments, the Cap protein encoding sequence provided in a separate polynucleotide construct or in the same polynucleotide comprising the Rep coding sequence can be expressed under the control of an inducible promoter. In various embodiments, the Cap protein encoding sequence may be operably linked to a polyadenylation signal sequence. The polyA signal sequence may be a polyA signal sequence functional in the cells used for producing the rAAV. In some instances, the polyA signal sequence may be a bovine Growth Hormone polyA (bGH-PolyA) signal sequence, a SV40 polyA signal sequence, or a Rabbit Beta Globin PolyA signal sequence.

[0387] The bGH-PolyA signal sequence may include a nucleotide sequence that has at least 70%, 75%, 80% 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity or 100% sequence identity to nucleotide sequence set forth in SEQ ID NO: 151.

[0388] In certain cases, the SV40 polyA signal sequence may include a nucleotide sequence having at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or 100% sequence identity to nucleotide sequence set forth in SEQ ID NO: 152.

[0389] In some embodiments, the SV40 polyA sequence is shorter than SEQ ID NO: 152. In some embodiments, the SV40 polyA sequence is longer than SEQ ID NO: 152.

[0390] In certain cases, the Rabbit Beta Globin signal sequence may include a nucleotide sequence having at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or 100% sequence identity to nucleotide sequence set forth in SEQ ID NO: 170.

[0391] In various embodiments, the Cap protein is selected from the capsid of an avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV, and modifications, derivatives, or pseudotypes thereof.

[0392] In some embodiments, the capsid is a capsid selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4- 1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16 or AAVhu68 (described in WO2020 / 033842, incorporated herein by reference in its entirety). The hu68 capsid is described in WO 2018 / 160582, incorporated herein by reference in its entirety.

[0393] In some embodiments, the capsid is a derivative, modification, or pseudotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12,AAV 13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16 or AAVhu68.

[0394] In some embodiments, capsid protein is a chimera of capsid proteins from two or more serotype selected from AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16 (described in WO2020 / 033842, incorporated herein by reference in its entirety). In certain embodiments, the capsid is an rh32.33 capsid, described in US Pat. No.8,999,678, incorporated herein by reference in its entirety.

[0395] In particular embodiments, the capsid is an AAV1 capsid. In particular embodiments, the capsid is an AAV5 capsid. In particular embodiments, the capsid is an AAV9 capsid.

[0396] In various embodiments, the first integrated construct further comprises a first mammalian cell selection element.

[0397] In some embodiments, an inducible Rep and Cap construct is as shown in FIG.1A, FIG. 2A, or FIG.3A. In some embodiments, the inducible polynucleotide construct comprises one or more promoters operably linked to a sequence comprising a first part of an AAV Rep coding sequence, a 5’ splice site, a first part of an intron, a first recombination site, a first 3’ splice site, a coding sequence comprising a stop signaling sequence, a second recombination site, a second part of the intron, a second 3’ splice site, and a sequence comprising a second part of the AAV Rep coding sequence, wherein the first recombination site, the first 3’ splice site, the coding sequence comprising the stop signaling sequence, and the second recombination site form an excisable element, wherein the first recombination site and the second recombination site are oriented in the same direction, and wherein the one or more promoters are not operably linked to the sequence comprising the second part of the AAV Rep coding sequence. Without excision of the excisable element, Rep is minimally expressed or not expressed at all. In some embodiments, the Rep polypeptide is a wildtype Rep polypeptide. In other embodiments, the Rep polypeptide is a mutant Rep polypeptide. In some embodiments, the Cap polypeptide is a wildtype Cappolypeptide. In other embodiments, the Cap polypeptide is a mutant Cap polypeptide. The intron may be spliced out by endogenous cellular machinery.

[0398] In some embodiments, the excisable element is excised by a recombinase. A recombinase can be Cre. Cre may be provided as any form of exogenous Cre, such as Cre vesicles. Cre may also be encoded for by a third polynucleotide construct or by any separate polynucleotide construct. In some embodiments, a construct encoding for adenoviral helper proteins also encodes for Cre. In some embodiments, the third polynucleotide construct is also inducible, for example, as described below. In some embodiments, a construct encoding for Rep / Cap proteins also encodes for Cre.

[0399] In some embodiments, expression of the Rep and Cap are driven by native promoters, including P5, P19, P40, or any combination thereof. In some embodiments, expression of the Rep and Cap are driven by inducible promoters. In some embodiments, expression of the Rep and Cap are driven by constitutive promoters. In some embodiments the exon of the excisable element may be any detectable marker. For example, detectable markers contemplated herein include luminescent markers, fluorescent markers, or radiolabels. Fluorescent markers include, but are not limited to, EGFP, GFP, BFP, RFP, or any combination thereof.

[0400] In some embodiments, the Rep Cap construct is a polynucleotide construct comprising: a) a sequence of a first part of a Rep gene; b) sequence of a second part of the Rep gene; c) a sequence of a Cap gene; and d) an excisable element positioned between the first part of the sequence of Rep gene and the second part of the sequence of the Rep gene. In some embodiments, the excisable element comprises a stop signaling sequence. In some embodiments, the excisable element comprises a rabbit beta globin intron. In some embodiments, the excisable element comprises an exon. In some embodiments, the excisable element comprises an intron and an exon. In some embodiments, the excisable element comprises an intron. In some embodiments, two splice sites are positioned between the sequence of the first part of the Rep gene and the sequence of the second part of the Rep gene. In some embodiments, the two splice sites are a 5’ splice site and a 3’ splice site. In some embodiments, the 5’ splice site is a rabbit beta globin 5’ splice site. In some embodiments, the 3’ splice site is a rabbit beta globin 3’ splice site. In some embodiments, three splice sites are positioned between the sequence of the first part of the Rep gene and the sequence of the second part of the Rep gene. In some embodiments, the three splice sites are a 5’ splice site, a first 3’ splice site, and a second 3’ splice site. In some embodiments, a first 3’ splice site is a duplicate of the second 3’ splice site. In some embodiments, the first 3’ splice site is a rabbit beta globin 3’ splice site. In some embodiments, the second 3’ splice site is a rabbit beta globin 3’ splice site. In some embodiments, the excisable element comprises a recombination site. In someembodiments, the recombination site is a lox site or FRT site. In some embodiments, the lox site is a loxP site. In some embodiments, the excisable element comprises from 5’ to 3’: a) the 5’ splice site; b) a first recombination site; c) the first 3’ splice site; d) a stop signaling sequence; e) a second recombination site; and f) the second 3’ splice site. In some embodiments, the excisable element comprises from 5’ to 3’: a) the 5’ splice site; b) a first spacer segment; c) a second spacer segment comprising: i) a first recombination site; ii) the first 3’ splice site; iv) a stop signaling sequence; and v) a second recombination site; and d) a third spacer segment comprising the second 3’ splice site. A recombinase recombines the first and second recombination sites. A transcript produced from the recombined sequence includes an intron flanked by the 5’ splice site and the second 3 splice site and is processed by the endogenous cellular machinery to produce a mRNA in which the intron has been spliced out. In some embodiments, the first spacer sequence comprises an intron. In some embodiments, the first spacer segment comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the second spacer segment comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the third spacer segment comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 3. In some embodiments, the third spacer segment comprises an intron. In some embodiments, the first spacer segment and the third spacer segment are capable of being excised by endogenous cellular machinery. In some embodiments, the second spacer segment comprises an exon. In some embodiments, the second spacer segment further comprises a polyA sequence. In some embodiments, the polyA sequence is 3’ of the exon. In some embodiments, the polyA sequence comprises a rabbit beta globin (RBG) polyA sequence. The polynucleotide construct of any one of the embodiments disclosed herein, wherein the second spacer segment comprises from 5’ to 3’: a) a first recombination site; b) the first 3’ splice site; c) an exon; d) a stop signaling sequence; and e) a second recombination site. In some embodiments, the first recombination site is a first lox sequence and the second recombination site is a second lox sequence. In some embodiments, the first lox sequence is a first loxP sequence and a second lox sequence is a second loxP sequence. In some embodiments, the first recombination site is a first FRT site and the second recombination site is a second FRT site. In some embodiments, the stop signaling sequence is a termination codon of the exon or a polyA sequence. In some embodiments, the polyA sequence comprises a rabbit beta globin (RBG) polyA sequence. In some embodiments, the exon encodes a detectable marker or a selectable marker. In some embodiments, the detectable marker comprises a luminescent marker or a fluorescent marker. In some embodiments, the fluorescent marker is GFP, EGFP, RFP, CFP, BFP, YFP, or mCherry. Insome embodiments, the second spacer segment is excisable by a recombinase. In some embodiments, the recombinase is a site-specific recombinase. In some embodiments, the recombinase is a Cre polypeptide or a Flippase polypeptide. In some embodiments, the Cre polypeptide is fused to a ligand binding domain. In some embodiments, the ligand binding domain is a hormone receptor. In some embodiments, the hormone receptor is an estrogen receptor. In some embodiments, the estrogen receptor comprises a point mutation. In some embodiments, the estrogen receptor is ERT2. In some embodiments, the recombinase is a ER2 Cre polypeptide. In some embodiments, the recombinase is encoded by a third polynucleotide construct or exogenously provided. In some embodiments, the Rep gene codes for Rep polypeptides. In some embodiments, the Cap gene codes for Cap polypeptides. In some embodiments, transcription of the Rep gene and the Cap gene are driven by native promoters. In some embodiments, the native promoters comprise P5, P19, and P40. In some embodiments, transcription of the Rep gene and / or the Cap gene are driven by inducible promoters. In some embodiments, the Rep polypeptides are wildtype Rep polypeptides. In some embodiments, the Rep polypeptides comprise Rep78, Rep68, Rep52, and Rep40. In some embodiments, a truncated replication associated protein comprising a polypeptide expressed from the sequence of first part of a Rep gene and the exon is capable of being expressed in the absence of the recombinase. In some embodiments, the Cap polypeptides are wildtype Cap polypeptides. In some embodiments, the Cap polypeptides are AAV capsid proteins. In some embodiments, the AAV capsid proteins comprise VP1, VP2, and VP3. In some embodiments, a serotype of the AAV capsid proteins is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, and AAVhu68.

[0401] In some embodiments, the Rep Cap construct further comprises a sequence coding for a selectable marker. In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments, the selectable marker is DHFR Z- Nter or DHFR Z-Cter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C- terminal intein of a split intein and the second auxotrophic selection element codes for an N- terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C- terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.

[0402] In some embodiments, the Rep Cap construct further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the Rep Cap construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the Rep Cap construct further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.

[0403] In some embodiments, a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90 – SEQ ID NO: 98, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, a selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101 – SEQ ID NO: 109.

[0404] In some embodiments, the Rep Cap construct is in a vector. In some embodiments, the Rep Cap construct is in a plasmid. In some embodiments, the Rep Cap construct is in a bacterial artificial chromosome or yeast artificial chromosome. In some embodiments, the Rep Cap construct is a synthetic nucleic acid construct. In some embodiments, the Rep Cap construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 1 – SEQ ID NO: 3, SEQ ID 6 – SEQ ID NO: 8, SEQ ID NO: 32, SEQ ID NO: 90 – SEQ ID NO: 99, SEQ ID NO: 101 – SEQ ID NO: 109, SEQ ID NO: 112 – SEQ ID NO: 131, or SEQ ID NO: 136 – SEQ ID NO: 138, or any combination thereof. In some embodiments, the Rep Cap construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 1 – SEQ ID NO: 3, SEQ ID 6 – SEQ ID NO: 8,SEQ ID NO: 32, SEQ ID NO: 90 – SEQ ID NO: 99, SEQ ID NO: 101 – SEQ ID NO: 109, SEQ ID NO: 112 – SEQ ID NO: 131, or SEQ ID NO: 136 – SEQ ID NO: 138, or any combination thereof. In some embodiments, the Rep Cap construct comprises SEQ ID NO: 145 downstream of the sequence encoding the AAV Cap proteins. In some embodiments, the Rep Cap construct lacks SEQ ID NO: 145 downstream of the sequence encoding the AAV Cap proteins. In some embodiments, the Rep Cap construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 1 – SEQ ID NO: 3, SEQ ID 6 – SEQ ID NO: 8, SEQ ID NO: 32, SEQ ID NO: 90 – SEQ ID NO: 99, SEQ ID NO: 101 – SEQ ID NO: 109, SEQ ID NO: 112 – SEQ ID NO: 131, or SEQ ID NO: 136 – SEQ ID NO: 138, but wherein the Rep Cap construct lacks SEQ ID NO: 145 downstream of the sequence encoding the AAV Cap proteins.

[0405] In some embodiments, the Rep Cap construct further comprises a sequence coding for VA-RNA. In some embodiments, a sequence coding for VA-RNA is in separate construct or in any separate construct coding for an element of the Rep Cap construct. In some embodiments, a payload construct comprises a polynucleotide construct coding for a VA-RNA. In some embodiments, the VA-RNA is operably linked to a constitutive promoter or an inducible promoter. In some embodiments, the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter. In some embodiments, the inducible promoter is a tetracycline- inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter. In some embodiments, the sequence coding for VA-RNA is a transcriptionally dead sequence. In some embodiments, the sequence coding for VA-RNA comprises at least two mutations in the internal promoter. In some embodiments, the expression of the VA-RNA is under the control of an RNA polymerase III promoter. In some embodiments, the expression of the VA-RNA is under the control of an interrupted RNA polymerase III promoter. In some embodiments, the expression of the VA-RNA is under the control of a U6 or U7 promoter. In some embodiments, the expression of the VA-RNA is under the control of an interrupted U6 or U7 promoter. In some embodiments, the polynucleotide construct comprises upstream of the sequence coding for VA- RNA gene sequence, from 5’ to 3’: a) a first part of a U6 or U7 promoter sequence; b) a first recombination site; c) a stuffer sequence; d) a second recombination site; e) a second part of a U6 or U7 promoter sequence. In some embodiments, the stuffer sequence is excisable by the recombinase. In some embodiments, the stuffer sequence comprises a sequence encoding a gene. In some embodiments, the stuffer sequence comprises a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a CMV promoter.

[0406] In some embodiments the gene codes for a selectable marker. In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectablemarker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C- terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments, the selectable marker is DHFR Z-Nter or DHFR Z-Cter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaEintein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.

[0407] In some embodiments, the stuffer sequence further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the stuffer sequence further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.

[0408] In some embodiments, a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90 – SEQ ID NO: 98, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101 – SEQ ID NO: 109.

[0409] A major advantage of the inducible polynucleotide constructs disclosed herein encoding for Rep and Cap include that upon stable integration into a mammalian cell line, expression of Rep and Cap is inducible even in the absence of a transfection agent or a plasmid. In some embodiments, the stable cell line populations disclosed herein are homogeneous. For example, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the stable cell population comprises the stably integrated polynucleotide construct encoding for Rep and Cap proteins.

[0410] The second polynucleotide construct may include a Cap protein encoding sequence identical or substantially identical to the Cap protein encoding sequence present in the Rep / Cap encoding polynucleotide construct. The second polynucleotide construct may include a Cap protein encoding sequence identical or substantially identical to the Cap protein encoding sequence present in the Rep / Cap encoding polynucleotide construct, but the Cap protein encoding sequence is driven by an inducible promoter. The second polynucleotide construct may be referred to as an inducible Cap construct. The inducible promoter may be any inducible promoter that is described herein in the below Adenoviral Helper Construct(s) section and is activated by any triggering agent and activator as described herein in the below Adenoviral Helper Construct(s) section. In some embodiments, the inducible promoter is a Tet inducible promoter. In some embodiments, the Tet inducible promoter is activated when bound to Tet responsive activator protein, e.g., Tet-on 3G, in the presence of a trigger agent, such as doxycycline. In some embodiments, a polynucleotide encoding a sequence comprising an inducible promoter Cap protein sequence may have at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 148. The second polynucleotide construct may be provided as a plasmid. The plasmid may have at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 149. The second polynucleotide construct may be provided as a vector. Adenoviral Helper Construct(s)

[0411] In some aspects, provided herein is a third polynucleotide construct (also referred to as Construct 3), which encodes for one or more adenoviral helper proteins. Construct 3 is also referred to as Ad helper / Cre construct. This third polynucleotide construct is also referred to as an inducible helper construct (e.g., Adenoviral Helper Construct provides one or more helper proteins selected from E1A, E1B, E2A and E4 absent from a host cell) to be used in production of rAAV virions. In some embodiments, the sequence encoding E4 is a sequence encoding E4orf6. In some embodiments, the elements of an inducible helper construct (e.g., one or more helper proteins selected from E1A, E1B, E2A and E4 absent from a host cell) are in one or more separate constructs to be used in production of rAAV virions. In some embodiments, the host cell provides, one, two, or three of the four helper proteins. For example, for a host cell expressing E1A and E1B, the adenoviral helper construct provides E2A and E4. For a host cell expressing E2A and E4, the adenoviral helper construct provides E1A and E1B. For a host cell expressing E1B, the adenoviral helper construct provides E1A, E2A and E4. For a host cell expressing E2A, the adenoviral helper construct provides E1B, E1A and E4. For a host cellexpressing E4, the adenoviral helper construct provides E1B, E2A and E1A. For a host cell expressing E1A, E2A and E4, the adenoviral helper construct provides E1B. For a host cell expressing E1B, E1A and E4, the adenoviral helper construct provides E2A. For a host cell expressing E1B, E2A and E1A, the adenoviral helper construct provides E4. In some embodiments, E4 is E4orf6.

[0412] In some embodiments, the sequences coding for E1A, E1B, E2A, and E4 are operably linked to separate promoters. In some embodiments, the sequences coding for E1A, E1B, E2A, and E4 are operably linked to one promoter. In some embodiments, the sequences coding for E1A, E1B, E2A, and E4 are operably linked, in any combination, to one promoter or separate promoters. The separate promoters can be the same promoters or different promoters. A combination of the separate promoters and the one promoter can be the same promoters or different promoters. The one promoter can be a native promoter, a constitutive promoter, or an inducible promoter. The separate promoters can be native promoters, constitutive promoters, inducible promoters, or any combination thereof. In some embodiments, the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter. In some embodiments, the inducible promoter is a tetracycline-inducible promoter, a cumate-inducible promoter, or an ecdysone-inducible promoter. For example, some the sequence coding for E1A and E1B are separated by an IRES sequence of P2A sequence and are operably linked to one promoter. In some embodiments, the sequence coding for E1A and E2A are separated by an IRES sequence of P2A sequence and are operably linked to one promoter. In some embodiments, the sequence coding for E1A and E4 are separated by an IRES sequence of P2A sequence and are operably linked to one promoter. In some embodiments, the sequence coding for E1B and E2A are separated by an IRES sequence of P2A sequence and are operably linked to one promoter. In some embodiments, the sequence coding for E1B and E4 are separated by an IRES sequence of P2A sequence and are operably linked to one promoter. In some embodiments, the sequence coding for E2A and E4 are separated by an IRES sequence of P2A sequence and are operably linked to one promoter. In some embodiments, the sequences coding for the helper proteins are in different orientations. In some embodiments, the sequences coding for the helper proteins are bidirectional. In some embodiments, the E1A is operably linked to a natural or constitutive promoter, E1B is operably linked to a natural or constitutive promoter, and E2A and E4 are downstream of an excisable element (e.g., a sequence flanked by recombination sites and comprising a stop signal) and constitutive promoter, wherein upon excision of the excisable element (e.g., by a recombinase), E2A and E4 are operably linked to the constitutive promoter. In some embodiments, the E1A is operably linked to a natural or constitutive promoter, E1B is operably linked to a natural or constitutive promoter, and E2A and E4 are downstream of anexcisable element (e.g., a sequence flanked by recombination sites and comprising a stop signal) and inducible promoter, wherein upon excision of the excisable element (e.g., by a recombinase), E2A and E4 are operably linked to the inducible promoter. In some embodiments, the E2A is operably linked to a natural or constitutive promoter, E4 is operably linked to a natural or constitutive promoter, and E1A and E1B are downstream of an excisable element (e.g., a sequence flanked by recombination sites and comprising a stop signal) and constitutive promoter, wherein upon excision of the excisable element (e.g., by a recombinase), E1A and E1B are operably linked to the constitutive promoter. In some embodiments, the E2A is operably linked to a natural or constitutive promoter, E4 is operably linked to a natural or constitutive promoter, and E1A and E1B are downstream of an excisable element (e.g., a sequence flanked by recombination sites and comprising a stop signal) and inducible promoter, wherein upon excision of the excisable element (e.g., by a recombinase), E1A and E1B are operably linked to the inducible promoter.

[0413] In certain embodiments, the adenoviral helper construct provides inducible production of one or more of the helper proteins. In some embodiments, an adenoviral helper protein further comprises a protein tag. A protein tag can be a FLAG tag. In some embodiments, E2A is a FLAG tagged E2A. In some embodiments, E4 is a FLAG tagged E4. A protein tag, such as a FLAG tag, can be used to screen for or to confirm integration of the third polynucleotide construct and expression of the adenoviral helper protein from the third polynucleotide construct in a cell after induction.

[0414] In some embodiments, the third integrated synthetic construct comprises conditionally expressible recombinase and conditionally expressible adenovirus helper proteins. In some embodiments, the third synthetic construct comprises conditionally expressible recombinase and conditionally expressible adenovirus helper proteins. In some embodiments, the one or more separate integrated constructs comprises conditionally expressible recombinase and conditionally expressible adenovirus helper proteins. In some embodiments, the one or more separate constructs comprises conditionally expressible recombinase and conditionally expressible adenovirus helper proteins. In some embodiments, the third integrated synthetic construct comprises conditionally expressible Cre recombinase and conditionally expressible adenovirus helper proteins. In the exemplary embodiments illustrated in FIG.1A and FIG.2A, prior to the cell being contacted with at least a third triggering agent, the third integrated construct comprises, from 5’ to 3’: an inducible promoter, a Cre coding sequence, a first polyA sequence, adenoviral helper protein coding sequences, a second polyA sequence, a constitutive promoter, a coding sequence for a protein that is responsive to the first triggering agent, and a second mammalian cell selection element.

[0415] In typical embodiments, the Cre coding sequence is operatively linked to the inducible promoter. Various inducible promoters suitable for controlling expression of the Cre coding sequence and the Cap proteins coding sequences are described throughout the specification, e.g., in the section “Inducible Promoters”. In various embodiments, the inducible promoter comprises an element responsive to the third triggering agent. In some embodiments, the inducible promoter contains a regulatory sequence that allows for control of the promoter. The regulatory sequence can be operably linked to the promoter and positioned upstream of the promoter. Such regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. The regulatory sequence used to control expression may be endogenous or exogenous to the host cell. In some embodiments, bacterial gene control elements in combination with viral transactivator proteins are used to provide mammalian inducible expression. Examples of mammalian-compatible regulatory sequences include those capable of controlling an engineered promoter to adjust transcription in response to antibiotics including, without limitation, tetracyclines, streptogramins, and macrolides. For a description of various inducible expression systems, see, e.g., Weber et al. (2004) Methods Mol. Biol.267:451-66, Das et al. (2016) Curr. Gene Ther.16(3):156-67, Chruscicka et al. (2015) J. Biomol. Screen.20(3):350-8, Yarranton (1992) Curr. Opin. Biotechnol.3(5):506-11, Gossen & Bujard (1992) Proc. Natl.Acad. Sci. U.S.A.89(12):5547-51, Gossen et al. (1995) Science 268(5218):1766-9; herein incorporated by reference.

[0416] In some embodiments, a bacterial tetracycline response element (TRE) is included in a construct to allow mammalian expression to be induced by tetracycline or a derivative thereof (e.g., doxycycline). In certain embodiments, the inducible promoter comprises a plurality of tetracycline (Tet) operator elements capable of binding to a Tet responsive activator protein in the presence of a tetracycline. In some embodiments, the plurality of tetracycline (Tet) operator elements form a Tetracycline Responsive element (TRE). In some embodiments, the TRE comprises seven repeats of a 19 base pair operator sequence. In further embodiments, the TRE comprises seven repeats of a 19 base pair operator sequence upstream of a minimal CMV promoter sequence.

[0417] In some embodiments, a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the amino acid sequence:

[0418] MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLD ALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYE TLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTDSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPADALDDFD LDMLPADALDDFDLDMLPG (SEQ ID NO:40).

[0419] In some embodiments, a Tet responsive activator protein is encoded by a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the nucleotide sequence:

[0420] ATGTCTAGACTGGACAAGAGCAAAGTCATAAACTCTGCTCTGGAATTACTCA ATGGAGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGCTCAAAAGCTGGGAGTT GAGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCCTGCTCGATGCCCT GCCAATCGAGATGCTGGACAGGCATCATACCCACTTCTGCCCCCTGGAAGGCGAGT CATGGCAAGACTTTCTGCGGAACAACGCCAAGTCATTCCGCTGTGCTCTCCTCTCAC ATCGCGACGGGGCTAAAGTGCATCTCGGCACCCGCCCAACAGAGAAACAGTACGA AACCCTGGAAAATCAGCTCGCGTTCCTGTGTCAGCAAGGCTTCTCCCTGGAGAACG CACTGTACGCTCTGTCCGCCGTGGGCCACTTTACACTGGGCTGCGTATTGGAGGAAC AGGAGCATCAAGTAGCAAAAGAGGAAAGAGAGACACCTACCACCGATTCTATGCC CCCACTTCTGAGACAAGCAATTGAGCTGTTCGACCGGCAGGGAGCCGAACCTGCCT TCCTTTTCGGCCTGGAACTAATCATATGTGGCCTGGAGAAACAGCTAAAGTGCGAA AGCGGCGGGCCGGCCGACGCCCTTGACGATTTTGACTTAGACATGCTCCCAGCCGA TGCCCTTGACGACTTTGACCTTGATATGCTGCCTGCTGACGCTCTTGACGATTTTGAC CTTGACATGCTCCCCGGGTAA (SEQ ID NO:69).

[0421] In some embodiments, a Tet responsive activator protein is encoded by a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the nucleotide sequence:

[0422] ATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTA ATGGGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTA GAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTT ACCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAA GCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTC ATCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAA ACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCA TTATATGCACTCAGCGCTGTGGGGCACTTTACTTTAGGTTGCGTATTGGAAGAACAA GAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTGATAGTATGCCGCC ATTATTACGACAAGCTATCGAATTATTTGATCGCCAAGGTGCAGAGCCAGCCTTCTT ATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGTG GGTCCGCGTACAGCCGCGCGCGTACGAAAAACAATTACGGGTCTACCATCGAGGGC CTGCTCGATCTCCCGGACGACGACGCCCCCGAAGAGGCGGGGCTGGCGGCTCCGCGCCTGTCCTTTCTCCCCGCGGGACACACGCGCAGACTGTCGACGGCCCCCCCGACCGA TGTCAGCCTGGGGGACGAGCTCCACTTAGACGGCGAGGACGTGGCGATGGCGCATG CCGACGCGCTAGACGATTTCGATCTGGACATGTTGGGGGACGGGGATTCCCCGGGT CCGGGATTTACCCCCCACGACTCCGCCCCCTACGGCGCTCTGGATATGGCCGACTTC GAGTTTGAGCAGATGTTTACCGATGCCCTTGGAATTGACGAGTACGGTGGGTAG (SEQ ID NO: 70).

[0423] In some embodiments, a Tet responsive activator protein is encoded by a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the nucleotide sequence:

[0424] ATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTA ATGGGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTA GAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTT ACCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAA GCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTC ATCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAA ACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCA TTATATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGCGTATTGGAAGATCAA GAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTGATAGTATGCCGCC ATTATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGCAGAGCCAGCCTTCTT ATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGTG GGTCCGCGTACAGCCGCGCGCGTACGAAAAACAATTACGGGTCTACCATCGAGGGC CTGCTCGATCTCCCGGACGACGACGCCCCCGAAGAGGCGGGGCTGGCGGCTCCGCG CCTGTCCTTTCTCCCCGCGGGACACACGCGCAGACTGTCGACGGCCCCCCCGACCGA TGTCAGCCTGGGGGACGAGCTCCACTTAGACGGCGAGGACGTGGCGATGGCGCATG CCGACGCGCTAGACGATTTCGATCTGGACATGTTGGGGGACGGGGATTCCCCGGGT CCGGGATTTACCCCCCACGACTCCGCCCCCTACGGCGCTCTGGATATGGCCGACTTC GAGTTTGAGCAGATGTTTACCGATGCCCTTGGAATTGACGAGTACGGTGGGTAG (SEQ ID NO: 71).

[0425] In some embodiments, a Tet responsive activator protein is encoded by a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the nucleotide sequence:

[0426] ATGTCTAGATTAGATAAAAGTAAAGTGATTAACGGCGCATTAGAGCTGCTTA ATGGGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTA GAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTT ACCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAAGCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTC ATCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAA ACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCA TTATATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGCGTATTGGAAGATCAA GAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTGATAGTATGCCGCC ATTATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGCAGAGCCAGCCTTCTT ATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGTG GGTCCGCGTACAGCCGCGCGCGTACGAAAAACAATTACGGGTCTACCATCGAGGGC CTGCTCGATCTCCCGGACGACGACGCCCCCGAAGAGGCGGGGCTGGCGGCTCCGCG CCTGTCCTTTCTCCCCGCGGGACACACGCGCAGACTGTCGACGGCCCCCCCGACCGA TGTCAGCCTGGGGGACGAGCTCCACTTAGACGGCGAGGACGTGGCGATGGCGCATG CCGACGCGCTAGACGATTTCGATCTGGACATGTTGGGGGACGGGGATTCCCCGGGT CCGGGATTTACCCCCCACGACTCCGCCCCCTACGGCGCTCTGGATATGGCCGACTTC GAGTTTGAGCAGATGTTTACCGATGCCCTTGGAATTGACGAGTACGGTGGGTAG (SEQ ID NO: 72).

[0427] In some embodiments, a Tet ...

Claims

WHAT IS CLAIMED IS:

1. A polynucleotide comprising: (i) a sequence encoding AAV Cap proteins operably linked to an inducible promoter; and (ii) a polyadenylation signal sequence, wherein the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence and is a 3’ of the sequence encoding AAV Cap proteins.

2. The polynucleotide of claim 1, wherein the polyadenylation signal sequence is a SV40 polyadenylation signal sequence, a bovine growth hormone polyadenylation signal sequence, or a Rabbit Beta Globin polyadenylation signal sequence.

3. The polynucleotide of claim 1 or 2, wherein the polyadenylation signal sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 152, has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 151, or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

170.

4. The polynucleotide construct of claim 1, wherein the native AAV Cap polyadenylation signal sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

162.

5. The polynucleotide of any one of claims 1-4, wherein the polynucleotide is not flanked by inverted terminal repeat sequences.

6. The polynucleotide of claim 1, wherein the stronger polyadenylation signal enhances RNA processing, RNA stability, RNA translation efficiency, or any combination thereof.

7. The polynucleotide of claim 1, wherein the inducible promoter is a tetracycline- inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter.

8. The polynucleotide of claim 1, wherein the inducible promoter comprises a tetracycline- responsive promoter element (TRE).

9. The polynucleotide of claim 8, wherein the TRE comprises Tet operator (tetO) sequence concatemers fused to a minimal promoter.

10. The polynucleotide of claim 9, wherein the minimal promoter is a human cytomegalovirus promoter.

11. The polynucleotide of claim 1, wherein the inducible promoter is a Tet-On promoter.

12. The polynucleotide of claim 1, wherein the inducible promoter comprises a first inducible promoter.

13. The polynucleotide of claim 1, wherein the polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

163.

14. The polynucleotide of claim 1, wherein the AAV Cap proteins comprise VP1, VP2, and VP3.

15. The polynucleotide of claim 1, wherein the AAV Cap proteins encode for AAV5 Cap proteins, AAV9 Cap proteins, PhP.EB Cap proteins, AAV8 Cap proteins, AAV2 proteins, or AAV6 Cap proteins.

16. A polynucleotide comprising: a) a first sequence encoding AAV Rep proteins operably linked to one or more promoters; and b) a second sequence encoding the polynucleotide of any one of claims 1-15.

17. The polynucleotide of claim 16, wherein transcription of the first sequence is driven by native AAV promoters; optionally, wherein transcription of the first sequence is driven by the P5 and P19 native AAV promoters.

18. The polynucleotide of claim 16, wherein the one or more promoters comprise P5 and P19 native promoters.

19. The polynucleotide of claim 16, wherein the first sequence has: a) at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 160 or 161; b) at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136; or c) at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136 but lacks the sequence of SEQ ID NO: 145 downstream of the sequence corresponding to the Cap sequence of SEQ ID NO:

136.

20. The polynucleotide of any one of claims 16-19, wherein the first sequence is separated from the second sequence by an intervening sequence.

21. The polynucleotide of claim 20, wherein the intervening sequence comprises a transcriptional blocking element (TBE); optionally, wherein a sequence of the TBE has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

169.

22. The polynucleotide of any one of claims 16-21, wherein the first sequence encoding AAV Rep proteins comprises: a. a first part of an AAV Rep proteins coding sequence, b. an excisable element comprising a first recombination site, a coding sequence encoding a stop signaling sequence, a second recombination site, wherein the first and second recombination sites flank the coding sequence encoding a stop signaling sequence and wherein the first recombination site and the second recombination site are oriented in the same direction, and c. a second part of the AAV Rep proteins coding sequence.

23. The polynucleotide of any one of claims 16-22, the first sequence encoding AAV Rep proteins comprises from 5’ to 3’:one or more promoters operably linked to a sequence comprising a first part of an AAV Rep coding sequence, a 5’ splice site, a first part of an intron, a first recombination site, a first 3’ splice site, a coding sequence comprising a stop signaling sequence, a second recombination site, a second part of the intron, a second 3’ splice site, and a sequence comprising a second part of the AAV Rep coding sequence, wherein the first recombination site, the first 3’ splice site, the coding sequence comprising the stop signaling sequence, and the second recombination site form an excisable element, wherein the first recombination site and the second recombination site are oriented in the same direction, and wherein the one or more promoters are not operably linked to the sequence comprising the second part of the AAV Rep coding sequence, wherein the first and second recombination sites are recombined by the inducible recombinase in the presence of a first triggering agent and a second triggering agent resulting in excision of the excisable element, and the first part of the AAV Rep coding sequence and the first part of the intron are joined to the second part of the intron and the second part of the AAV Rep coding sequence to form a complete AAV Rep coding sequence, allowing expression of AAV Rep proteins.

24. The system of polynucleotides of claim 23, wherein the coding sequence encoding the stop signaling sequence of the first sequence encodes for, from 5’ to 3’: an exon and the stop signaling sequence.

25. The polynucleotide of claim 23, wherein the coding sequence encoding the stop signaling sequence of the first sequence further comprises a sequence encoding a protein marker, wherein the sequence encoding the protein marker is in-frame with the stop signaling sequence.

26. The polynucleotide of any one of claims 16-25, further comprising a first constitutive promoter operably linked to a sequence encoding a first selectable marker or a first portion or a second portion of a split selectable marker.

27. The polynucleotide of any one of claims 16-26, wherein the polynucleotide is a polynucleotide construct.

28. The polynucleotide of any one of claims 16-27, wherein the polynucleotide further comprises a selectable marker operably linked to a promoter; optionally wherein the promoter is a constitutive promoter.

29. A system of polynucleotides comprising: a) a first polynucleotide comprising a sequence encoding AAV Rep proteins operably linked to one or more promoters; and b) a second polynucleotide comprising the sequence of the polynucleotide of any one of claims 1-15; and one or more of: c) a third polynucleotide comprising a sequence encoding one or more adenoviral helper proteins; and d) a fourth polynucleotide comprising a sequence encoding a payload.

30. The system of polynucleotides of claim 29, wherein transcription of the first polynucleotide is driven by native AAV promoters; optionally, wherein transcription of the first polynucleotide is driven by the P5 and P19 native AAV promoters.

31. The system of polynucleotides of claim 29, wherein the one or more promoters comprise a P5 native AAV promoter and a P19 native AAV promoter.

32. The system of polynucleotides of any one of claims 29-31, wherein the first polynucleotide comprising a sequence encoding AAV Rep proteins operably linked to one or more promoters comprises: a) a first part of an AAV Rep proteins coding sequence, b) an excisable element comprising a first recombination site, a coding sequence encoding a stop signaling sequence, a second recombination site, wherein the first and second recombination sites flank the coding sequence encoding a stop signaling sequence and wherein the first recombination site and the second recombination site are oriented in the same direction, and c) a second part of the AAV Rep proteins coding sequence.

33. The system of polynucleotides of any one of claims 29-32, wherein the first polynucleotide comprising a sequence encoding AAV Rep proteins operably linked to one or more promoters comprises from 5’ to 3’:one or more promoters operably linked to a first sequence comprising a first part of an AAV Rep coding sequence, a 5’ splice site, a first part of an intron, a first recombination site, a first 3’ splice site, a coding sequence comprising a stop signaling sequence, a second recombination site, a second part of the intron, a second 3’ splice site, and a second sequence comprising a second part of the AAV Rep coding sequence, wherein the first recombination site, the first 3’ splice site, the coding sequence comprising the stop signaling sequence, and the second recombination site form an excisable element, wherein the first recombination site and the second recombination site are oriented in the same direction, and wherein the one or more promoters are not operably linked to the second sequence comprising the second part of the AAV Rep coding sequence, wherein the first and second recombination sites are recombined by the inducible recombinase in the presence of a first triggering agent and a second triggering agent resulting in excision of the excisable element, and the first part of the AAV Rep coding sequence and the first part of the intron are joined to the second part of the intron and the second part of the AAV Rep coding sequence to form a complete AAV Rep coding sequence, allowing expression of AAV Rep proteins.

34. The system of polynucleotides of claim 32 or 33, wherein the coding sequence encoding the stop signaling sequence of the first polynucleotide encodes for, from 5’ to 3’: an exon and the stop signaling sequence.

35. The system of polynucleotides of any one of claims 32-34, wherein the coding sequence encoding the stop signaling sequence of the first polynucleotide further comprises a sequence encoding a protein marker, wherein the sequence encoding the protein marker is in-frame with the stop signaling sequence.

36. The system of polynucleotides of any one of claims 29-35, wherein the first polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 160 or 161.

37. The system of polynucleotides of any one of claims 29-36, the first polynucleotide further comprises a sequence encoding AAV Cap proteins.

38. The system of polynucleotides of claim 37, wherein the sequence encoding the AAV Cap proteins in the second polynucleotide is substantially identical to the sequence encoding the AAV Cap proteins in the first polynucleotide.

39. The system of polynucleotides of claim 37, wherein transcription of the sequence encoding the AAV Cap proteins on the first polynucleotide is driven by a native AAV Cap proteins promoter; optionally, wherein the native AAV Cap proteins promoter is a P40 native AAV promoter.

40. The system of polynucleotides of any one of claims 29-39, wherein the first polynucleotide has: a) at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136; or b) at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136 but lacks the sequence of SEQ ID NO: 145 downstream of the sequence corresponding to the Cap sequence of SEQ ID NO:

136.

41. The system of polynucleotides of any one of claims 29-40, wherein the third polynucleotide comprising the sequence encoding one or more adenoviral helper proteins comprises: a second inducible promoter operably linked to a self-excising element; the self-excising element comprising a third recombination site and a fourth recombination site flanking a sequence encoding an inducible recombinase, wherein the third recombination site and the fourth recombination site are oriented in the same direction, wherein the second inducible promoter is not operably linked to the sequence encoding the one or more adenoviral helper proteins; a first constitutive promoter operably linked to a sequence encoding an activator, wherein the third polynucleotide constitutively expresses the activator and the activator is unable to activate the first inducible promoter or the second inducible promoter in absence of a first triggering agent; wherein in absence of activation of the first inducible promoter and the second inducible promoter, detectable levels of the Rep proteins from the first polynucleotide or if present the Cap proteins from the first polynucleotide, the Cap proteins from thesecond polynucleotide, the inducible recombinase, and the one or more adenoviral helper proteins are not expressed, and wherein the inducible recombinase is activated in the presence of a second triggering agent.

42. The system of polynucleotides of any one of claims 29-41, wherein the third polynucleotide comprising the sequence encoding one or more adenoviral helper proteins comprises: a first sequence comprising from 5’ to 3’: a second inducible promoter operably linked to a sequence encoding an inducible recombinase; a self-excising element comprising a third recombination site, the sequence encoding the inducible recombinase, and a fourth recombination site, wherein the third recombination site and the fourth recombination site are oriented in the same direction; and a sequence encoding one or more adenoviral helper proteins, wherein the second inducible promoter is not operably linked to the sequence encoding the one or more adenoviral helper proteins; a second sequence comprising a first constitutive promoter operably linked to a sequence encoding an activator, wherein the cell constitutively expresses the activator and the activator is unable to activate the second inducible promoter in absence of a first triggering agent, wherein in the presence of the first triggering agent, the activator activates the second inducible promoter resulting in expression of the inducible recombinase, and the inducible recombinase is expressed and wherein in the presence of a second triggering agent, the inducible recombinase translocates to a nucleus of the cell and causes recombination between the third recombination site and the fourth recombination site resulting in excision of the self-excising element, thereby operably linking the second inducible promoter to the sequence encoding the one or more adenoviral helper proteins and allowing expression of the one or more adenoviral helper proteins 43. The system of polynucleotides of claim 41 or 42, wherein the one or more adenoviral helper proteins comprise one or more of adenovirus E1A protein, E1B protein, E2A protein, and E4 protein, and optionally comprises E2A protein and E4 protein.

44. The system of polynucleotides of any one of claims 29-43, wherein the third polynucleotide comprising the sequence encoding for one or more AAV helper proteins comprises a bicistronic open reading frame encoding two AAV helper proteins; optionally, wherein the SEQ ID NO: 30 comprises the third polynucleotide.

45. The system of polynucleotides of any one of claims 41-44, wherein the one or more adenoviral helper proteins are separated by a bicistronic open reading frame; optionally, wherein the bicistronic open reading frame comprises an internal ribosome entry site (IRES) or a peptide 2A (P2A) sequence.

46. The system of polynucleotides of any one of claims 41-45, wherein the second inducible promoter operably linked to the self-excising element in the third polynucleotide is a tetracycline-inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter; optionally, wherein the first inducible promoter and the second inducible promoter are the same; further optionally, wherein the first inducible promoter and the second inducible promoter are a tetracycline-inducible promoter.

47. The system of polynucleotides of claim 46, wherein the tetracycline-inducible promoter comprises a tetracycline-responsive promoter element (TRE).

48. The system of polynucleotides of claim 47, wherein the TRE comprises Tet operator (tetO) sequence concatemers fused to a minimal promoter.

49. The system of polynucleotides of claim 48, wherein the minimal promoter is a human cytomegalovirus promoter.

50. The system of polynucleotides of any one of claims 41-49, wherein the first constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter.

51. The system of polynucleotides of any one of claims 41-50, wherein the activator is reverse tetracycline-controlled transactivator (rTA) comprising a Tet Repressor binding protein (TetR) fused to a VP16 transactivation domain.

52. The system of polynucleotides of claim 47, wherein a triggering agent for inducing the tetracycline-inducible promoter is tetracycline or doxycycline.

53. The system of polynucleotides of any one of claims 41-53, wherein the inducible recombinase is fused to an estrogen response element (ER) and translocates to the nucleus in the presence of tamoxifen.

54. The system of polynucleotides of any one of claims 41-53, wherein the recombination sites in the first polynucleotide and the third polynucleotide are lox sites and the inducible recombinase is a cre recombinase or wherein the recombination sites in the first polynucleotide and the third polynucleotide are flippase recognition target (FRT) sites and the inducible recombinase is a flippase (Flp) recombinase.

55. The system of polynucleotides of any one of claims 41-54, wherein presence of the triggering agent activates the activator for activation of the first inducible promoter to express AAV Cap proteins from the second polynucleotide encoding the AAV Cap proteins.

56. The system of polynucleotides of any one of claims 41-55, wherein presence of the triggering agent activates the activator for activation of the second inducible promoter to express the AAV Rep proteins of the first polynucleotide, if present the AAV Cap proteins of the first polynucleotide, the inducible recombinase, and the one or more adenoviral helper proteins.

57. The system of polynucleotides of any one of claims 41-56, wherein upon expression of the inducible recombinase, recombination between the first recombination site and the second recombination site in the first polynucleotide results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein of the first polynucleotide and if present the AAV Cap Proteins of the first polynucleotide; and recombination between the third recombination site and the fourth recombination site in the third polynucleotide results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins.

58. The system of polynucleotides of any one of claims 29-57, wherein the third polynucleotide further comprises a third selectable marker operably linked to a third promoter.

59. The system of polynucleotides of any one of claims 29-58, wherein the third polynucleotide further comprises a sequence encoding a viral associated RNA (VA- RNA); optionally, wherein the VA-RNA is a mutated VA-RNA.

60. The system of polynucleotides of claim 59, wherein the VA-RNA is wild-type VA- RNA or VA-RNA comprising one or more mutations in the VA-RNA internal promoter.

61. The system of polynucleotides of claim 59 or 60, wherein the sequence encoding the VA-RNA is operably linked to an inactive promoter comprising a first part of a second constitutive promoter and a second part of the second constitutive promoter separated by a second excisable element comprising a fifth recombination site and a sixth recombination site flanking a stuffer sequence, the fifth and sixth recombination sites are oriented in the same direction, and excision of the second excisable element by the inducible recombinase generates a functional complete second constitutive promoter operably linked to the VA-RNA coding sequence to allow expression of the VA-RNA.

62. The system of polynucleotides of claim 61, wherein the first part of the second constitutive promoter comprises a distal sequence element (DSE) of an RNA polymerase III promoter, and the second part of the second constitutive promoter comprises a proximal sequence element (PSE) of an RNA polymerase III promoter, or the first part of the second constitutive promoter comprises a distal sequence element (DSE) of a U6 promoter, and the second part of the second constitutive promoter comprises a proximal sequence element (PSE) of a U6 promoter, or the first part of the second constitutive promoter comprises a distal sequence element (DSE) of a U7 promoter, and the second part of the second constitutive promoter comprises a proximal sequence element (PSE) of a U7 promoter.

63. The system of polynucleotides of any one of claims 59-62, wherein the VA-RNA comprises a G16A mutation or a G60A mutation, or a combination thereof.

64. The system of polynucleotides of any one of claims 59-63, wherein the third polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

153.

65. The system of polynucleotides of any one of claims 29-64, wherein the payload of the fourth polynucleotide comprises a reporter gene, a therapeutic gene, or a transgene encoding a protein of interest; optionally, wherein the payload of the fourth polynucleotide is progranulin.

66. The system of polynucleotides of any one of claims 29-65, wherein the sequence encoding the payload of the fourth polynucleotide comprises a sequence encoding a reporter gene, a therapeutic gene, or a transgene encoding a protein of interest; optionally, wherein the sequence encoding the payload of the fourth polynucleotide is a sequence encoding progranulin; further optionally, wherein the sequence encoding progranulin has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

166.

67. The system of polynucleotides of any one of claims 29-66, wherein the sequence encoding the payload comprises a sequence encoding a suppressor tRNA, a guide RNA, or a homology region for homology-directed repair.

68. The system of polynucleotides of any one of claims 29-67, wherein the fourth polynucleotide comprising the sequence encoding the payload comprises the sequence encoding the payload flanked by a 5’ AAV inverted terminal repeat (5’ ITR) and a 3’ AAV inverted terminal repeat (3’ ITR).

69. The system of polynucleotides of any one of claims 29-68, wherein the sequence encoding the payload is flanked by a 5’ AAV inverted terminal repeat (5’ ITR) and a 3’ AAV inverted terminal repeat (3’ ITR) has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 146; has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156; or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 158; optionally, wherein a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the SEQ ID NO: 147, SEQ ID NO: 157, or SEQ ID NO: 159 comprises the sequence encoding the payload is flanked by a 5’ AAV inverted terminal repeat (5’ ITR) and a 3’ AAV inverted terminal repeat (3’ ITR).

70. The system of polynucleotides of any one of claims 29-69 further comprising a fifth polynucleotide comprising a sequence encoding a viral associated RNA (VA-RNA); optionally, wherein the VA-RNA is a mutated VA-RNA.

71. The system of polynucleotides of claim 70, wherein the VA-RNA is wild-type VA- RNA or VA-RNA comprising one or more mutations in the VA-RNA internal promoter.

72. The system of polynucleotides of claim 70 or 71, wherein the sequence encoding the VA-RNA is operably linked to an inactive promoter comprising a first part of a constitutive promoter and a second part of the constitutive promoter separated by a second excisable element comprising a fifth recombination site and a sixth recombination site flanking a stuffer sequence, the fifth and sixth recombination sites are oriented in the same direction, and excision of the second excisable element by the inducible recombinase generates a functional complete constitutive promoter operably linked to the VA-RNA coding sequence to allow expression of the VA-RNA.

73. The system of polynucleotides of claim 72, wherein the first part of the constitutive promoter comprises a distal sequence element (DSE) of an RNA polymerase III promoter, and the second part of the constitutive promoter comprises a proximal sequence element (PSE) of an RNA polymerase III promoter, orthe first part of the constitutive promoter comprises a distal sequence element (DSE) of a U6 promoter, and the second part of the constitutive promoter comprises a proximal sequence element (PSE) of a U6 promoter, or the first part of the constitutive promoter comprises a distal sequence element (DSE) of a U7 promoter, and the second part of the constitutive promoter comprises a proximal sequence element (PSE) of a U7 promoter.

74. The system of polynucleotides of any one of claims 70-73, wherein the VA-RNA comprises a G16A mutation or a G60A mutation, or a combination thereof.

75. The system of polynucleotides of any one of claims 70-74, wherein: a) the first polynucleotide further comprises a sequence encoding a first selectable marker operably linked to a first promoter, b) the second polynucleotide further comprises a sequence encoding a second selectable marker operably linked to a second promoter, c) the third polynucleotide further comprises a sequence encoding a third selectable marker operably linked to a third promoter, d) the fourth polynucleotide further comprises a sequence encoding a fourth selectable marker operably linked to a fourth promoter, e) the fifth polynucleotide further comprises a sequence encoding a fifth selectable marker operably linked to a fifth promoter, or g) any combination of thereof; optionally, wherein any combinations of the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, and the fifth selectable marker are different selectable markers; the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, the fifth selectable marker, or any combinations thereof are the same selectable marker but as a different split portion of the selectable marker; or any combinations thereof; further optionally, wherein any combinations of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter are the same constitutive promoter or different constitutive promoters.

76. The system of polynucleotides of claim 75, wherein the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, and the fifth selectable marker, or any combination thereof is an antibiotic resistance gene;optionally, wherein the antibiotic resistance gene is a blasticidin resistance gene, a hygromycin resistance gene, or a puromycin resistance gene.

77. The system of polynucleotides of claim 75, wherein the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, and the fifth selectable marker, or any combination thereof is a first split portion of an antibiotic resistance gene; optionally, wherein the first split portion of the antibiotic resistance gene is a first split portion of the blasticidin resistance gene.

78. The system of polynucleotides of claim 75, wherein the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, and the fifth selectable marker, or any combination thereof is a second split portion of an antibiotic resistance gene; optionally, wherein the second split portion of the antibiotic resistance gene is a second split portion of the blasticidin resistance gene.

79. The system of polynucleotides of claim 75, wherein the first promoter, the second promoter, the third promoter, the fourth promoter, the fifth promoter, or any combination thereof, is an EF1alpha promoter or an attenuated version thereof, wherein the attenuated version comprises a mutation in the TATA box, optionally wherein the attenuated EF1alpha promoter has weaker promoter activity than an EF1alpha promoter.

80. The system of polynucleotides of any one of claims 29-79, wherein the fourth polynucleotide comprising the sequence encoding the payload further comprises a spacer between the 5’ ITR and the sequence encoding the fourth selectable marker or a spacer between the sequence encoding the fourth selectable marker and the 3’ ITR, or a combination thereof.

81. The system of polynucleotides of any one of claims 29-80, wherein: (i) the first polynucleotide further comprises a constitutive promoter operably linked to a sequence encoding a first portion of a split selectable marker, optionally, wherein the constitutive promoter is an EF1 alpha promoter and / or the split selectable marker is a split antibiotic resistance protein; (ii) the second polynucleotide further comprises a sequence encoding a selectable marker operably linked to a constitutive promoter, optionallywherein the constitutive promoter is an EF1 alpha promoter and / or the selectable marker is a first antibiotic resistance protein; (iii) the third polynucleotide further comprises a sequence encoding a selectable marker operably linked to a constitutive promoter, optionally, wherein the constitutive promoter is an CMV promoter and / or the selectable marker is a second antibiotic resistance protein; and / or (iv) the fourth polynucleotide further comprises a constitutive promoter operably linked to a sequence encoding a selectable marker or a second part of the split selectable marker, optionally, wherein the constitutive promoter is an EF1 alpha promoter.

82. The system of polynucleotides of any one of claims 29-81, wherein the fourth polynucleotide comprising the sequence encoding the payload further comprises a spacer between the 5’ ITR and the sequence encoding the selectable marker or a spacer between the sequence encoding the fourth selectable marker and the 3’ ITR, or a combination thereof.

83. The system of polynucleotides of claim 82, wherein the spacer ranges in length from 500 base pairs to 5000 base pairs.

84. The system of polynucleotides of any one of claims 29-83, comprising: a) the first polynucleotide of any one of claims 16-40 and 75-83; b) the second polynucleotide of any one of claims 1-15, 29 and 75-83; and c) the third polynucleotide of any one of claims 41-64 and 75-83.

85. The system of polynucleotides of any one of claims 29-83, comprising: a) the first polynucleotide of any one of claims 16-40 and 75-83; b) the second polynucleotide of any one of claims 1-15, 29 and 75-83; and c) the fourth polynucleotide of any one of claims 65-69 and 75-83.

86. The system of polynucleotides of any one of claims 29-83, comprising: a) the first polynucleotide of any one of claims 6-40 and 75-83; b) the second polynucleotide of any one of claims 1-15, 29 and 75-83; c) the third polynucleotide of any one of claims 41-64 and 75-83; d) the fourth polynucleotide of any one of claims 65-69 and 75-83.

87. The system of polynucleotides of any one of claims 29-83, comprising: a) the first polynucleotide of any one of claims 16-40 and 75-83; b) the second polynucleotide of any one of claims 1-15, 29 and 75-83; c) the third polynucleotide of any one of claims 41-64 and 75-83; and d) the fifth polynucleotide of any one of claims 70-83.

88. The system of polynucleotides of any one of claims, comprising: a) the first polynucleotide of any one of claims 16-40 and 75-83; b) the second polynucleotide of any one of claims 1-15, 29 and 75-83; c) the fourth polynucleotide of any one of claims 65-69 and 75-83; and d) the fifth polynucleotide of any one of claims 70-83.

89. The system of polynucleotides of any one of claims 16-40 and 75-83, comprising: a) the first polynucleotide of any one of claims 16-40 and 75-83; b) the second polynucleotide of any one of claims 1-15, 29 and 75-83; and c) the fifth polynucleotide of any one of claims 70-83.

90. The system of polynucleotides of any one of claims 16-40 and 75-83, comprising: a) the first polynucleotide of any one of claims 16-40 and 75-83; b) the second polynucleotide of any one of claims 1-15, 29 and 75-83; c) the third polynucleotide of any one of claims 41-64 and 75-83; d) the fourth polynucleotide of any one of claims 65-69 and 75-83; and e) the fifth polynucleotide of any one of claims 70-83.

91. A system of polynucleotide constructs comprising: a) a first polynucleotide construct comprising the sequence of the first polynucleotide of any one of claims 16-40 and 75-83 and the sequence of the second polynucleotide of any one of claims 1-15, 29 and 75-83; and one or more of: b) a second polynucleotide construct comprising the sequence of the third polynucleotide of any one of claims 41-64 and 75-83; c) a third polynucleotide construct comprising the sequence of the fourth polynucleotide of any one of claims 65-69 and 75-83.

92. The system of polynucleotide constructs of claim 91, comprising: a) a first polynucleotide construct comprising the sequence of the first polynucleotide of any one of claims 16-40 and 75-83 and the sequence of the second polynucleotide of any one of claims 1-15, 29 and 75-83; and b) a second polynucleotide construct comprising the sequence of the third polynucleotide of any one of claims 41-64 and 75-83.

93. The system of polynucleotide constructs of claim 91, comprising: a) a first polynucleotide construct comprising the sequence of the first polynucleotide of any one of claims 16-40 and 75-83 and the sequence of the second polynucleotide of any one of claims 1-15, 29 and 75-83; and b) a third polynucleotide construct comprising the sequence of the fourth polynucleotide of any one of claims 65-69 and 75-83.

94. The system of polynucleotide constructs of claim 91, comprising: a) a first polynucleotide construct comprising the sequence of the first polynucleotide of any one of claims 16-40 and 75-83 and the sequence of the second polynucleotide of any one of claims 1-15, 29 and 75-83; and b) a second polynucleotide construct comprising the sequence of the third polynucleotide of any one of claims 41-64 and 75-83; c) a third polynucleotide construct comprising the sequence of the fourth polynucleotide of any one of claims 65-69 and 75-83.

95. The system of polynucleotide constructs of any one of claims 91-94, further comprising d) a fourth polynucleotide construct comprising the sequence of the fifth polynucleotide of any one of claims 70-83.

96. The system of polynucleotide constructs of any one of claims 91-95, wherein the sequence of the first polynucleotide is separated from the sequence of the second polynucleotide by an intervening sequence.

97. The system of polynucleotide constructs of claim 96, wherein the intervening sequence comprises a transcriptional blocking element (TBE).

98. The system of polynucleotide constructs of any one of claims 91-97, wherein the first polynucleotide construct comprises a sequence encoding a single selectable marker.

99. The system of polynucleotide constructs of any one of claims 91-98, wherein: (i) the first polynucleotide construct further comprises a constitutive promoter operably linked to a sequence encoding a first portion of a split selectable marker, optionally, wherein the constitutive promoter is an EF1 alpha promoter and / or first portion of the split selectable marker is a first portion of a split of a first antibiotic resistance protein; (ii) the second polynucleotide construct further comprises a sequence encoding a selectable marker operably linked to a constitutive promoter, optionally, wherein the constitutive promoter is an CMV promoter and / or the selectable marker is a second antibiotic resistance protein; and / or (iv) the third polynucleotide construct further comprises a constitutive promoter operably linked to a sequence encoding a selectable marker or a second part of the split selectable marker, optionally, wherein the constitutive promoter is an EF1 alpha promoter and / or the second part of the split selectable maker is a second portion of the first antibiotic resistance protein.

100. The system of polynucleotide constructs of claim 99, wherein the first antibiotic resistance protein is a blasticidin resistance protein and the second antibiotic resistance protein is a puromycin resistance protein.

101. A vector comprising the polynucleotide of any one of claims 1-100.

102. A vector system comprising: a) a first vector comprising the sequence of the first polynucleotide of any one of claims 16-40 and 75-83; b) a second vector comprising the sequence of the second polynucleotide of any one of claims 1-15, 29 and 75-83; c) a third vector comprising the sequence of the third polynucleotide of any one of claims 41-64 and 75-83; and d) a fourth vector comprising the sequence of the fourth polynucleotide of any one of claims 65-69 and 75-83.

103. The vector system of claim 102, further comprising d) a fifth vector comprising the sequence of the fifth polynucleotide of any one of claims 70-83.

104. The vector system of any one of claims 102 or 103, wherein the first vector is a first plasmid, the second vector is a second plasmid, the third vector is a third plasmid, and the fourth vector is a fourth plasmid.

105. The vector system of claim 103 or 104, wherein the fifth vector is a fifth plasmid.

106. The vector system of any one of claims 102-105, wherein the first plasmid has least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32 or SEQ ID NO:

160.

107. The vector system of any one of claims 102-105, wherein the second plasmid has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

149.

108. The vector system of any one of claims 102-105, wherein the third plasmid has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30 or SEQ ID NO:

154.

109. The vector system of any one of claims 102-105, wherein the fourth plasmid has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 157 or SEQ ID NO:

159.

110. A vector system comprising: a) a first vector comprising the sequence of the first polynucleotide of any one of claims 16-40 and 75-83 and the sequence of the second polynucleotide of any one of claims 1- 15, 29 and 75-83 or the first polynucleotide construct of any one of claims 91-100; and one or more of:b) a second vector comprising the sequence of the third polynucleotide of any one of claims 41-64 and 75-83 or the second polynucleotide construct of any one of claims 1- 15, 29 and 75-83; and c) a third vector comprising the sequence of the fourth polynucleotide of any one of claims 65-69 and 75-83 or the third polynucleotide construct of any one of claims 41-64 and 75-83.

111. The vector system of claim 110, comprising: a) a first vector comprising the sequence of the first polynucleotide of any one of claims 16-40 and 75-83 and the sequence of the second polynucleotide of any one of claims 1- 15, 29 and 75-83 or the first polynucleotide construct of any one of claims 91-100; and b) a second vector comprising the sequence of the third polynucleotide of any one of claims 41-64 and 75-83 or the second polynucleotide construct of any one of claims 91- 100.

112. The vector system of claim 110 or 111, comprising: a) a first vector comprising the sequence of the first polynucleotide of any one of claims 16-40 and 75-83 and the sequence of the second polynucleotide of any one of claims 1- 15, 29 and 75-83 or the first polynucleotide construct of any one of claims 91-100; and b) a third vector comprising the sequence of the fourth polynucleotide of any one of claims 65-69 and 75-83 or the third polynucleotide construct of any one of claims 91- 100.

113. The vector system of any one of claims 110-112, further comprising a fourth vector comprising the sequence of the fifth polynucleotide of any one of claims 70-83 or the fourth polynucleotide construct of any one of claims 91-100.

114. The vector system of any one of claims 110-113, wherein the first vector is a first plasmid, the second vector is a second plasmid, and the third vector is a third plasmid.

115. The vector system of claim 113 or 114, wherein the fourth vector is a fourth plasmid.

116. The vector system of claim 114, wherein the first plasmid comprises a nucleic acid sequence having least 70%, at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

160.

117. The vector system of claim 114, wherein the second plasmid has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30 or SEQ ID NO:

154.

118. The vector system of claim 114, wherein the third plasmid has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 157 or SEQ ID NO:

159.

119. A plasmid comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

164.

120. A plasmid comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

149.

121. A plasmid comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

148.

122. A plasmid comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

164.

123. A plasmid comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148 and a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 164 or 165.

124. A cell comprising the polynucleotide of any one of claims 1-28.

125. A cell comprising the polynucleotide system of any one of claims 29-100.

126. The cell of claim 125, wherein one or more of the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, and the fifth polynucleotide are integrated into the nuclear genome of the cell.

127. The cell of claim 126, wherein the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions.

128. The cell of claim 126, wherein the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, the VA-RNA, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, the VA-RNA, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions.

129. The cell of any one of claims 125-128, wherein the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, and the fifth polynucleotide are, in any combination, on one or more polynucleotide constructs.

130. A cell comprising the polynucleotide construct system of any one of claims 91- 100.

131. The cell of claim 130, wherein one or more of the first polynucleotide construct, the second polynucleotide construct, the third polynucleotide construct, and the fourth polynucleotide are integrated into the nuclear genome of the cell.

132. The cell of claim 131, wherein the first polynucleotide construct, the second polynucleotide construct, and the third polynucleotide construct are integrated into thenuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions.

133. The cell of claim 131, wherein the first polynucleotide construct, the second polynucleotide construct, and the third polynucleotide construct are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, the VA-RNA, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, the VA-RNA, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions.

134. A cell comprising the vector system of any of claims 102-118.

135. The cell of claim 134, wherein one or more of the first vector, the second vector, the third vector, and the fourth vector are integrated into the nuclear genome of the cell.

136. The cell of claim 135, wherein the first vector, the second vector, and the third vector are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions.

137. The cell of claim 135, wherein the first vector, the second vector, and the third vector, are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, the VA-RNA, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, the VA-RNA, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions.

138. The cell of any one of claims 124-137, wherein the cell comprises a polynucleotide comprising a sequence encoding an adenovirus E1A protein, a polynucleotide comprising a sequence encoding an E1B protein, a polynucleotidecomprising a sequence encoding an E2A protein, a polynucleotide comprising a sequence encoding an E4 protein, or any combination thereof.

139. The cell of any one of claims 124-138, wherein the third polynucleotide comprising the sequence encoding one or more adenoviral helper proteins comprises the polynucleotide comprising the sequence encoding the adenovirus E1A protein, the polynucleotide comprising the sequence encoding the E1B protein, the polynucleotide comprising the sequence encoding the E2A protein, the polynucleotide comprising the sequence encoding the E4 protein, or any combination thereof; optionally, wherein the second polynucleotide construct comprises the polynucleotide comprising the sequence encoding the adenovirus E1A protein, the polynucleotide comprising the sequence encoding the E1B protein, the polynucleotide comprising the sequence encoding the E2A protein, the polynucleotide sequence comprising the encoding the E4 protein, or any combination thereof.

140. The cell of any one of claims 124-139, wherein the cell comprises an adenovirus E1A protein and E1B protein, and the one or more adenoviral helper proteins expressed by the third polynucleotide are an adenovirus E2A protein and E4 protein or wherein the cell comprises an adenovirus E2A protein and E4 protein, and the one or more AAV helper proteins expressed by the second polynucleotide construct are an adenovirus E1A protein and E1B protein.

141. The cell of any one of claims 124-140, wherein the cell constitutively expresses any two proteins from a group consisting of an adenovirus E1A protein, an adenovirus E1B protein, an adenovirus E2A protein, and adenovirus E4 protein, from a nucleic acid integrated in the nuclear genome; optionally, wherein the two proteins are the adenovirus E1A protein and the adenovirus E1B protein or the adenovirus E2A protein and the adenovirus E4 protein.

142. The cell of any one of claims 124-141, wherein the third polynucleotide comprising the sequence encoding for one or more adenoviral helper proteins comprises a bicistronic open reading frame encoding two adenoviral helper proteins; optionally, wherein the third polynucleotide comprises SEQ ID NO: 30.

143. The cell of any one of claims 126-142, wherein the two adenoviral helper proteins are any two proteins from a group consisting of an adenovirus E1A protein, an adenovirus E1B protein, an adenovirus E2A protein, and adenovirus E4 protein; optionally, wherein the any two proteins are E2A and E4 or E1A and E1B.

144. The cell of claim 142, wherein the bicistronic open reading frame comprises an internal ribosome entry site (IRES) or a peptide 2A (P2A) sequence.

145. The cell of any one of claims 126-144, wherein the cell is a mammalian cell.

146. The cell of claim 145, wherein the mammalian cell is a HEK293 cell.

147. The cell of claim 146, wherein the HEK293 cell is DHFR-deficient or GS- deficient.

148. The cell of any one of claims 126-147, wherein the cell expresses adenoviral helper proteins E1A and E1B.

149. The cell of any one of claims 126-148, wherein upon expression of the inducible recombinase, recombination between the first recombination site and the second recombination site in the first polynucleotide construct results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein of the first polynucleotide; and recombination between the third recombination site and the fourth recombination site in the third polynucleotide results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins.

150. The cell of any one of claims 126-149, wherein upon expression of the inducible recombinase, recombination between the first recombination site and the second recombination site in the first polynucleotide construct results in excision of theexcisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein of the first polynucleotide construct; and recombination between the third recombination site and the fourth recombination site in the second polynucleotide construct results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins.

151. The cell of any one of claims 126-150, wherein after induction, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147; has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156; or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

155.

152. The cell of any one of claims 126-151, wherein in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147; has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156; or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

161.

153. The cell of any one of claims 126-151, wherein in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147; has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156; or has at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

165.

154. The cell of any one of claims 126-151, wherein in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147; has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156; or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

164.

155. The cell of any one of claims 126-151, wherein in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147; has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156; or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148 or SEQ ID NO:

163.

156. The cell of any one of claims 126-151, wherein in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147; has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156; or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156 or SEQ ID NO:

158.

157. A method of generating a cell line for inducibly producing recombinant AAV (rAAV) virions comprising a payload, the method comprising: introducing into a cell the third polynucleotide of any one of claims 41-64 and 75-83; selecting for a cell expressing a selectable marker of the third polynucleotide;introducing into the cell expressing the selectable marker of the third polynucleotide, the first polynucleotide of any one of claims 16-40 and 75-83, the second polynucleotide of any one of claims 1-15, 29 and 75-83, and the fourth polynucleotide of any one of claims 65-69 and 75-83; selecting for a cell expressing the selectable marker of the third polynucleotide, a selectable marker of the first polynucleotide, a selectable marker of the second polynucleotide, and a selectable marker of the fourth polynucleotide; expanding the cell expressing the selectable marker of the third polynucleotide, the selectable marker of the first polynucleotide, the selectable marker of the second polynucleotide, and the selectable marker of the fourth polynucleotide into a plurality of cells, thereby generating the plurality of cells as the cell line for inducibly producing the rAAV virions.

158. The method of claim 157, further comprising contacting a cell of the cell line to a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter resulting in expression of the recombinase, wherein recombination between the first recombination site and the second recombination site in the first polynucleotide results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein, wherein the one or more promoters are operably linked to the complete AAV Rep proteins coding sequence to allow expression of an AAV Rep protein and, if present in the first polynucleotide, expression of an AAV Cap protein; and recombination between the third recombination site and the fourth recombination site in the third polynucleotide results in excision of the self-excising element comprising the sequence encoding the second inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins; and wherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of an AAV Cap protein of the second polynucleotide.

159. A method of generating a cell line for inducibly producing recombinant AAV (rAAV) virions comprising a payload, the method comprising: introducing into a cell the second polynucleotide construct of any one of claims 91-100; selecting for a cell expressing a selectable marker of the second polynucleotide construct; introducing into the cell expressing the selectable marker of the second polynucleotide construct, the first polynucleotide construct of any one of claims 91-100, and the third polynucleotide construct of any one of claims 91-100; selecting for a cell expressing the selectable marker of the second polynucleotide construct, a selectable marker of the first polynucleotide construct, and a selectable marker of the third polynucleotide construct; expanding the cell expressing the selectable marker of the second polynucleotide construct, the selectable marker of the first polynucleotide construct, and the selectable marker of the third polynucleotide construct into a plurality of cells, thereby generating the plurality of cells as the cell line for inducibly producing the rAAV virions.

160. The method of claim 159, further comprising contacting a cell of the cell line to a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter resulting in expression of the recombinase, wherein recombination between the first recombination site and the second recombination site in the second polynucleotide construct results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein, wherein the one or more promoters are operably linked to the complete AAV Rep proteins coding sequence to allow expression of an AAV Rep protein and, if present in the second polynucleotide construct, expression of an AAV Cap protein; and recombination between the third recombination site and the fourth recombination site in the second polynucleotide construct results in excision of the self-excising element comprising the sequence encoding the second inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins; andwherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of an AAV Cap protein of the second polynucleotide construct.

161. A method of generating a cell line for inducibly producing recombinant AAV (rAAV) virions comprising a payload, the method comprising: introducing into a cell the third vector of any one of claims 102-118; selecting for a cell expressing a selectable marker of the third vector; introducing into the cell expressing the selectable marker of the third vector, the first vector of any one of claims 102-118, the second vector of any of any one of claims 102- 118, and the fourth vector of any one of claims 102-118; selecting for a cell expressing the selectable marker of the third vector, a selectable marker of the first vector, a selectable marker of the vector polynucleotide, and a selectable marker of the fourth vector; expanding the cell expressing the selectable marker of the third vector, the selectable marker of the first vector, the selectable marker of the second vector, and the selectable marker of the fourth vector into a plurality of cells, thereby generating the plurality of cells as the cell line for inducibly producing the rAAV virions.

162. The method of claim 161, further comprising contacting a cell of the cell line to a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter resulting in expression of the recombinase, wherein recombination between the first recombination site and the second recombination site in the first vector results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein, wherein the one or more promoters are operably linked to the complete AAV Rep proteins coding sequence to allow expression of an AAV Rep protein and, if present in the first polynucleotide, expression of an AAV Cap protein; and recombination between the third recombination site and the fourth recombination site in the third vector results in excision of the self-excising element comprising the sequence encoding the second inducible recombinase, wherein the second inducible promoterbecomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins; and wherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of an AAV Cap protein of the second vector.

163. A method of generating a cell line for inducibly producing recombinant AAV (rAAV) virions comprising a payload, the method comprising: introducing into a cell the second vector of any one of claims 102-118; selecting for a cell expressing a selectable marker of the second vector; introducing into the cell expressing the selectable marker of the second vector, the first vector of any one of claims 102-118, and the third vector of any one of claims 102-118; selecting for a cell expressing the selectable marker of the second vector, a selectable marker of the first vector, and a selectable marker of the third vector; expanding the cell expressing the selectable marker of the second vector, the selectable marker of the first vector, and the selectable marker of the third vector into a plurality of cells, thereby generating the plurality of cells as the cell line for inducibly producing the rAAV virions.

164. The method of claim 163, further comprising contacting a cell of the cell line to a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter resulting in expression of the recombinase, wherein recombination between the first recombination site and the second recombination site in the second vector results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein, wherein the one or more promoters are operably linked to the complete AAV Rep proteins coding sequence to allow expression of an AAV Rep protein and, if present in the second polynucleotide construct, expression of an AAV Cap protein; and recombination between the third recombination site and the fourth recombination site in the second vector results in excision of the self-excising element comprising the sequence encoding the second inducible recombinase, wherein the second induciblepromoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins; and wherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of an AAV Cap protein of the second vector.

165. A method for generating a recombinant adenovirus associated virus (rAAV) virion comprising a sequence encoding a payload, the method comprising contacting the cell according to any one of claims 124-156 to a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter of the third polynucleotide resulting in expression of the inducible recombinase, wherein recombination between the first recombination site and the second recombination site in the second polynucleotide by the inducible recombinase results in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein and, if present, an AAV Cap protein, and recombination between the third recombination site and the fourth recombination site in the third polynucleotide by the recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins; wherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of the AAV Cap proteins of the second polynucleotide; wherein the expression of the one or more AAV helper proteins results in expression of the one or more Rep proteins and the AAV Cap proteins, thereby generating an rAAV virion comprising the sequence encoding the payload.

166. The method of claim 165, wherein the generated rAAV is at least 35-fold higher than rAAV generated from a cell lacking the second polynucleotide.

167. The method of claim 165, wherein the generated rAAV titer is at least 35-fold higher than rAAV generated titer from a cell lacking the second polynucleotide.

168. A method for generating a recombinant adenovirus associated virus (rAAV) virion comprising a sequence encoding a payload, the method comprising contacting the cell according to any one of claims 124-156 to a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter of the second polynucleotide construct resulting in expression of the inducible recombinase, wherein recombination between the first recombination site and the second recombination site in the first polynucleotide construct by the recombinase results in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein and, if present, an AAV Cap protein, and recombination between the third recombination site and the fourth recombination site in the second polynucleotide construct by the inducible recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins; wherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of the AAV Cap proteins of the first polynucleotide construct; wherein the expression of the one or more adenoviral helper proteins results in expression of the one or more Rep proteins and the AAV Cap proteins, thereby generating an rAAV virion comprising the sequence encoding the payload.

169. The method of claim 168, wherein the generated rAAV is at least 35-fold higher than rAAV generated from a cell lacking the second polynucleotide of the first polynucleotide construct.

170. The method of claim 168, wherein the generated rAAV titer is at least 35-fold higher than rAAV generated titer from a cell lacking the second polynucleotide of the first polynucleotide construct.

171. A method for generating a recombinant adenovirus associated virus (rAAV) virion comprising a sequence encoding a payload, the method comprising contacting the cell according to any one of claims 124-156 to a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter of the third vector resulting in expression of the inducible recombinase, wherein recombination between the first recombination site and the second recombination site in the second vector by the inducible recombinase results in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein and, if present, an AAV Cap protein, and recombination between the third recombination site and the fourth recombination site in the third vector by the inducible recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins; wherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of the AAV Cap proteins of the second vector; wherein the expression of the one or more adenoviral helper proteins results in expression of the one or more Rep proteins and the AAV Cap proteins, thereby generating an rAAV virion comprising the sequence encoding the payload.

172. The method of claim 171, wherein the generated rAAV is at least 35-fold higher than rAAV generated from a cell lacking the second vector.

173. The method of claim 171, wherein the generated rAAV titer is at least 35-fold higher than rAAV generated titer from a cell lacking the second vector.

174. A method for generating a recombinant adenovirus associated virus (rAAV) virion comprising a sequence encoding a payload, the method comprising contacting thecell according to any one of claims 124-156 to a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter of the second vector resulting in expression of the inducible recombinase, wherein recombination between the first recombination site and the second recombination site in the first vector by the inducible recombinase results in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein and, if present, an AAV Cap protein, and recombination between the third recombination site and the fourth recombination site in the second vector by the inducible recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins; wherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of the AAV Cap proteins of the first vector; wherein the expression of the one or more adenoviral helper proteins results in expression of the one or more Rep proteins and the AAV Cap proteins, thereby generating an rAAV virion comprising the sequence encoding the payload.

175. The method of claim 174, wherein the generated rAAV is at least 35-fold higher than rAAV generated from a cell lacking the second polynucleotide of the first vector.

176. The method of claim 174, wherein the generated rAAV titer is at least 35-fold higher than rAAV generated titer from a cell lacking the second polynucleotide of the first vector.

177. A method for producing recombinant AAV, the method comprising performing transfection of a cell with the polynucleotides of the system of any one of claims 29- 100, the polynucleotide constructs of the system of any one of claims 91-100, or the vectors of the system of any one of claims 102-118 and contacting the cell to a first triggering agent and a second triggering agent.

178. The method of claim 177, wherein the polynucleotides of the system of any one of claims 29-100, the polynucleotide constructs of the system of any one of claims 91- 100, or the vectors of the system of any one of claims 102-118 are integrated into a nuclear genome of the cell.

179. The method of claim 177, wherein the polynucleotides of the system of any one of claims 29-100, the polynucleotide constructs of the system of any one of claims 91- 100, or the vectors of the system of any one of claims 102-118 are not integrated into a nuclear genome of the cell.

180. The method of any one of claims 177-179, wherein one or more of the polynucleotides of the system of any one of claims 29-100, of the polynucleotide constructs of the system of any one of claims 91-100, or of the vectors of the system of any one of claims 102-118 are integrated into a nuclear genome of the cell and one or more of the polynucleotides of the system of any one of claims 29-100, of the polynucleotide constructs of the system of any one of claims 91-100, or of the vectors of the system of any one of claims 102-118 are not integrated into the nuclear genome of the cell.

181. A method for inducibly producing recombinant AAV, the method comprising: culturing a cell comprising the polynucleotides of the system of any one of claims 29- 100 integrated into a nuclear genome of the cell in the presence of a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent, the activator activates the second inducible promoter of the third polynucleotide resulting in expression of the inducible recombinase, wherein in the presence of the second triggering agent the inducible recombinase translocates to the nucleus and catalyzes recombination between the first recombination site and the second recombination site in the first polynucleotide resulting in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein, and recombination between the third recombination site and the fourth recombination site in the third polynucleotide by the inducible recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase,wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins; wherein the first triggering agent and the activator activate the first inducible promoter operably linked to the AAV Cap proteins coding sequence; wherein the expression of the one or more adenoviral helper proteins, one or more Rep proteins and the AAV Cap proteins results in generation of rAAV virions comprising the sequence encoding the payload.

182. A method for inducibly producing recombinant AAV, the method comprising: culturing a cell comprising the polynucleotide constructs of the system of any one of claims 91-100 integrated into a nuclear genome of the cell in the presence of a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent, the activator activates the second inducible promoter of the second polynucleotide construct resulting in expression of the inducible recombinase, wherein in the presence of the second triggering agent the inducible recombinase translocates to the nucleus and catalyzes recombination between the first recombination site and the second recombination site in the first polynucleotide construct resulting in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein, and recombination between the third recombination site and the fourth recombination site in the second polynucleotide construct by the inducible recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins; wherein the first triggering agent and the activator activate the first inducible promoter operably linked to the AAV Cap proteins coding sequence; wherein the expression of the one or more adenoviral helper proteins, one or more Rep proteins, and the AAV Cap proteins results in generation of rAAV virions comprising the sequence encoding the payload.

183. A method for inducibly producing recombinant AAV, the method comprising: culturing a cell comprising the vectors of the system of any one of claims 102-118 integrated into a nuclear genome of the cell in the presence of a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent, the activator activates the second inducible promoter of the third vector resulting in expression of the inducible recombinase, wherein in the presence of the second triggering agent the inducible recombinase translocates to the nucleus and catalyzes recombination between the first recombination site and the second recombination site in the first vector resulting in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein, and recombination between the third recombination site and the fourth recombination site in the third vector by the inducible recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins; wherein the first triggering agent and the activator activate the first inducible promoter operably linked to the AAV Cap proteins coding sequence; wherein the expression of the one or more adenoviral helper proteins, one or more Rep proteins and the AAV Cap proteins results in generation of rAAV virions comprising the sequence encoding the payload.

184. A method for inducibly producing recombinant AAV, the method comprising: culturing a cell comprising the vectors of the system of any one of claims 102-118 integrated into a nuclear genome of the cell in the presence of a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent, the activator activates the second inducible promoter of the second vector resulting in expression of the inducible recombinase, wherein in the presence of the second triggering agent the inducible recombinase translocates to the nucleus and catalyzes recombination between the first recombination site and the second recombination site in the first vector resulting in excision of the excisable element, and the first part of the AAV Rep coding sequence andthe second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein, and recombination between the third recombination site and the fourth recombination site in the second vector by the inducible recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins; wherein the first triggering agent and the activator activate the first inducible promoter operably linked to the AAV Cap proteins coding sequence; wherein the expression of the one or more adenoviral helper proteins, one or more Rep proteins, and the AAV Cap proteins results in generation of rAAV virions comprising the sequence encoding the payload.

185. A method of generating a cell for inducibly producing recombinant AAV (rAAV) comprising a payload, the method comprising: (i) introducing into cells the third polynucleotide of any one of claims 41-64 and 75-83; (ii) selecting for cells expressing a selectable marker encoded by third polynucleotide; (iii) introducing into the cells selected in (ii) the first polynucleotide and the fourth polynucleotide of any one of claims 65-69 and 75-83, wherein the first polynucleotide encodes a first part of a split selectable marker and the fourth polynucleotide encodes a second part of a split selectable marker; (iv) selecting for cells expressing an active selectable marker formed from the first part and the second part of the split selectable marker; and (v) expanding the cells expressing the selectable marker encoded by the third polynucleotide and the selectable marker encoded by the first polynucleotide and the fourth polynucleotide, thereby generating the cells for inducibly producing the rAAV virions.

186. The method of claim 185, further comprising: (iii) introducing into the cells selected in (iv) or the cells expanded in (v), the second polynucleotide of any one of claims 1-15 and 75-83; and (iv) selecting for cells expressing a selectable marker encoded by the second polynucleotide.

187. A method of generating a cell for inducibly producing recombinant AAV (rAAV) comprising a payload, the method comprising: (i) introducing into cells the second polynucleotide construct of any one of claims 91- 100; (ii) selecting for cells expressing a selectable marker encoded by second polynucleotide construct; (iii) introducing into the cells selected in (ii) the first polynucleotide construct and the third polynucleotide construct of any one of claims 91-100, wherein the first polynucleotide construct encodes a first part of a split selectable marker and the third polynucleotide construct encodes a second part of a split selectable marker; (iv) selecting for cells expressing an active selectable marker formed from the first part and the second part of the split selectable marker; and (v) expanding the cells expressing the selectable marker encoded by the second polynucleotide construct and the selectable marker encoded by the first polynucleotide construct and the third polynucleotide construct, thereby generating the cells for inducibly producing the rAAV virions.

188. A method of generating a cell for inducibly producing recombinant AAV (rAAV) comprising a payload, the method comprising: (i) introducing into cells the third vector of any one of claims 102-118; (ii) selecting for cells expressing a selectable marker encoded by third vector; (iii) introducing into the cells selected in (ii) the first vector and the fourth vector of any one of claims 102-118, wherein the first vector encodes a first part of a split selectable marker and the fourth vector encodes a second part of a split selectable marker; (iv) selecting for cells expressing an active selectable marker formed from the first part and the second part of the split selectable marker; and (v) expanding the cells expressing the selectable marker encoded by the third vector and the selectable marker encoded by the first vector and the fourth vector, thereby generating the cells for inducibly producing the rAAV virions.

189. The method of claim 188, further comprising: (vi) introducing into the cells selected in (iv) or the cells expanded in (v), the second vector of any one of claims 102-118; and (vii) selecting for cells expressing a selectable marker encoded by the second vector.

190. A method of generating a cell for inducibly producing recombinant AAV (rAAV) comprising a payload, the method comprising: (i) introducing into cells the second vector of any one of claims 102-118; (ii) selecting for cells expressing a selectable marker encoded by second vector; (iii) introducing into the cells selected in (ii) the first vector and the third vector of any one of claims 102-118, wherein the first vector encodes a first part of a split selectable marker and the vector encodes a second part of a split selectable marker; (iv) selecting for cells expressing an active selectable marker formed from the first part and the second part of the split selectable marker; and (v) expanding the cells expressing the selectable marker encoded by the second vector and the selectable marker encoded by the first vector and the third vector, thereby generating the cells for inducibly producing the rAAV virions.

191. A method of generating a cell for inducibly producing recombinant AAV (rAAV) virions comprising a payload, the method comprising: introducing into a cell a first polynucleotide comprising: a first sequence comprising from 5’ to 3’: a first inducible promoter operably linked to a sequence encoding an inducible recombinase; a self-excising element comprising a first recombination site, the sequence encoding the inducible recombinase, and a second recombination site, wherein the first recombination site and the second recombination site are oriented in the same direction; and a sequence encoding one or more adenoviral helper proteins, wherein the first inducible promoter is not operably linked to the sequence encoding the one or more adenoviral helper proteins; a second sequence comprising a first constitutive promoter operably linked to a sequence encoding an activator, wherein the cell constitutively expresses the activator and the activator is unable to activate the first inducible promoter in absence of a first triggering agent, wherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of the inducible recombinase, and the inducible recombinase is expressed and wherein in the presence of a second triggering agent, the inducible recombinase translocates to a nucleus of the cell and causes recombination between the first recombination site and the second recombination site resulting in excision of the self-excising element, thereby operably linking the first inducible promoter to thesequence encoding the one or more adenoviral helper proteins and allowing expression of the one or more adenoviral helper proteins; and a third sequence comprising a second constitutive promoter operably linked to a sequence encoding a first selectable marker, wherein the cell constitutively expresses the first selectable marker, selecting for a cell expressing the first selectable marker; introducing a second polynucleotide and a third polynucleotide into the cell expressing the first selectable marker, the second polynucleotide comprising: a first sequence encoding AAV Cap proteins operably linked to a second inducible promoter; and from 5’ to 3’: one or more promoters operably linked to a second sequence comprising a first part of an AAV Rep coding sequence, a 5’ splice site, a first part of an intron, a third recombination site, a first 3’ splice site, a coding sequence comprising a stop signaling sequence, a fourth recombination site, a second part of the intron, a second 3’ splice site, and a third sequence comprising a second part of the AAV Rep coding sequence, wherein the third recombination site, the first 3’ splice site, the coding sequence comprising the stop signaling sequence, and the fourth recombination site form an excisable element, wherein the third recombination site and the fourth recombination site are oriented in the same direction, and wherein the one or more promoters are not operably linked to the third sequence comprising the second part of the AAV Rep coding sequence, wherein the third and fourth recombination sites are recombined by the inducible recombinase in the presence of the first triggering agent and the second triggering agent resulting in excision of the excisable element, and the first part of the AAV Rep coding sequence and the first part of the intron are joined to the second part of the intron and the second part of the AAV Rep coding sequence to form a complete AAV Rep coding sequence, allowing expression of AAV Rep proteins; and a third constitutive promoter operably linked to a sequence encoding a first portion of a second selectable marker, the third polynucleotide comprising a sequence encoding the payload and a fourth constitutive promoter operably linked to a second portion of the second selectable marker, wherein the sequence encoding the payload is flanked by AAV inverted terminal repeats (ITRs);selecting for a cell expressing the first selectable marker and the second selectable marker, thereby generating the cell for inducibly producing recombinant AAV (rAAV) virions comprising the payload.

192. The method of claim 191, further comprising contacting the cell with the first triggering agent and the second triggering agent for inducibly producing recombinant AAV (rAAV) virions comprising the payload.

193. The method of claim 191 or 192, wherein the coding sequence encoding the stop signaling sequence of the second polynucleotide encodes for from 5’ to 3’: an exon and the stop signaling sequence.

194. The method of any one of claims 191-193, wherein the first sequence encoding AAV Cap proteins operably linked to the second inducible promoter of the second polynucleotide further comprises a polyadenylation signal.

195. The method of claim 194, wherein the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence.

196. The polynucleotide of claim 194 or 195, wherein the polyadenylation signal sequence is a 3’ of the sequence encoding AAV Cap proteins.

197. The method of any one of claims 194-196, wherein the polyadenylation signal sequence is a SV40 polyadenylation signal sequence or a bovine growth hormone polyadenylation signal sequence.

198. The method of any one of claims 194-197, wherein the polyadenylation signal sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 152 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 151.

199. The method of claim 195, wherein the native AAV Cap polyadenylation signal sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

162.

200. The method of any one of claims 191-199, wherein the first sequence encoding AAV Cap proteins operably linked to the second inducible promoter of the second polynucleotide is not flanked by inverted terminal repeat sequences.

201. The method of claim 195, wherein the stronger polyadenylation signal enhances RNA processing, RNA stability, RNA translation efficiency, or any combination thereof.

202. The method of any one of claims 191-201, wherein the first inducible promoter is a tetracycline-inducible promoter, an ecdysone-inducible promoter, or a cumate- inducible promoter and second inducible promoter is a tetracycline-inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter; optionally, wherein the first inducible promoter and the second inducible promoter are the same.

203. The method of any one of claims 191-202, wherein the first inducible promoter comprises a tetracycline-responsive promoter element (TRE) and the second inducible promoter comprises a tetracycline-responsive promoter element (TRE).

204. The method of claim 203, wherein the TRE comprises Tet operator (tetO) sequence concatemers fused to a minimal promoter.

205. The method of claim 204, wherein the minimal promoter is a human cytomegalovirus promoter.

206. The method of any one of claims 191-203, wherein the first inducible promoter is a Tet-On promoter and the second inducible promoter is a Tet-On promoter.

207. The method of any one of claims 191-206, wherein the first sequence encoding AAV Cap proteins operably linked to the second inducible promoter of the second polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

163.

208. The method of any one of claims 191-207, wherein the AAV Cap proteins comprise VP1, VP2, and VP3.

209. The method of any one of claims 157-208, wherein the first triggering agent is tetracycline and the second triggering agent is tamoxifen.

210. The method of any one of claims 157-208, wherein the first triggering agent is doxycycline and the second triggering agent is tamoxifen.

211. The rAAV virion produced by the method of any one of claims 157-210.

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