WO2024026302A2

WO2024026302A2 - Compositions and methods for adeno-associated viral production

Info

Publication number: WO2024026302A2
Application number: PCT/US2023/070927
Authority: WO
Inventors: Jeremy J. GAM; Alec A.K. NIELSEN; Jeffrey B. MCMAHAN
Original assignee: Asimov Inc.
Priority date: 2022-07-26
Filing date: 2023-07-25
Publication date: 2024-02-01
Also published as: WO2024026302A3

Abstract

Disclosed herein are cell genetically engineered cells for AAV production. The genetically engineered cells comprise molecular systems for temporal control of expression of genes required for AAV production. Also disclosed herein are methods of using genetically engineered cells for AAV production.

Description

COMPOSITIONS AND METHODS FOR ADENO- ASSOCIATED VIRAL PRODUCTION

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63/369,476, filed July 26, 2022, the entire contents of which is hereby incorporated by reference in its entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (A121070010WO00-SEQ-CRP.xml; Size: 423,302 bytes; and Date of Creation: July 25, 2023) and is hereby incorporated by reference in its entirety.

FIELD

Described herein are Adeno- Associated Virus (AAV) production systems. Also described herein are engineered cells and kits comprising an AAV production system and methods of using the same for AAV production.

BACKGROUND OF INVENTION

AAV is a promising gene delivery modality for cell and gene therapy. AAV can be modified to carry therapeutic genetic payloads to cells within a subject. The production of AAV normally entails transient transfection of plasmids containing genes required for viral vector production into cell culture. However, transient transfection has several shortfalls. Large quantities of DNA and transfection reagent must be procured for the transfection process, which is costly. Also, poor transfection efficiency can result in minimal numbers of “transfected” cells and increased variation associated with transfection steps and viral production.

SUMMARY OF INVENTION

Described herein are AAV production systems that introduce inducible control of gene products required for AAV production including cytostatic or cytotoxic gene products. This inducible control can be mediated by recombinases. Each of the described AAV production systems can be integrated into the genome using random integration, targeted integration, or transposon-mediated integration.

In some aspects, the disclosure relates to Adeno-Associated Virus (AAV) production systems.

In some embodiments, an AAV production system comprises an expression control component and an AAV production component, wherein: (a) the expression control component comprises a polynucleic acid molecule encoding for a recombinase; and (b) the AAV production component comprises one or more polynucleic acid molecules collectively encoding for: Rep52 or Rep40; Rep78 or Rep68; E2A; E40rf6; VARNA; VP1; VP2; VP3; AAP; wherein a nucleic acid sequence comprising at least a portion of a nucleic acid sequence encoding for Rep52, Rep40, Rep78, Rep68, E2A, E40rf6, VARNA, VP1, VP2, VP3, or AAP is comprised within an invertible element, wherein the invertible element comprises, from 5’ to 3’: (i) a first recombinase attachment site; (ii) an internal sequence comprising the at least a portion of the nucleic acid sequence encoding for Rep52, Rep40, Rep78, Rep68, E2A, E40rf6, VARNA, VP1, VP2, VP3, or AAP; and (iii) a second recombinase attachment site; wherein the 5’ recombinase attachment site and the 3’ recombinase attachment site correspond to the recombinase of (a).

In some embodiments, an AAV production system comprises an expression control component and an AAV production component, wherein: (a) the expression control component comprises a polynucleic acid molecule encoding for a recombinase; and (b) the AAV production component comprises a polynucleic acid molecule having a first segment and a second segment, wherein the second segment is inverted relative to the first segment, and wherein: the first segment comprises: a promoter operably linked to a first portion of a nucleic acid sequence encoding for an AAV gene product; and the second segment comprises, from 5’ to 3’ : a first recombinase attachment site; a second portion of the nucleic acid sequence encoding for the first AAV gene product; and a second recombinase attachment site; and wherein the first recombinase attachment site and the second recombinase attachment site correspond to the same recombinase.

In some embodiments, the nucleic acid sequence encoding for the AAV gene product is comprised within the first portion of the nucleic acid sequence encoding for the AAV gene product and the second portion of the nucleic acid sequence encoding for the AAV gene product. In some embodiments, the second segment further comprises a nucleic acid encoding for at least a portion of an additional AAV gene product. In some embodiments, the polynucleic acid molecule having the first segment and the second segment further comprises a third segment, wherein the third segment is in the same orientation as the first segment, and wherein the polynucleic acid molecule comprises the structure: first segment - second segment - third segment, and wherein the third segment comprises a third portion of the nucleic acid sequence encoding for the AAV gene product. In some embodiments, the nucleic acid sequence encoding for the AAV gene product is comprised within the first portion of the nucleic acid sequence encoding for the AAV gene product, the second portion of the nucleic acid sequence encoding for the AAV gene product, and the third portion of the nucleic acid sequence encoding for the AAV gene product.

In some embodiments, the first recombinase attachment site comprises a nucleic acid sequence having at least 80% identity with the nucleic acid sequence of any one of SEQ ID NOs: 5-96.

In some embodiments, the second recombinase attachment site comprises a nucleic acid sequence having at least 80% identity with the nucleic acid sequence of any one of SEQ ID NOs: 5-96.

In some embodiments, the first segment comprises, from 5’ to 3’: the promoter operably linked to the first portion of the nucleic acid sequence encoding for the first AAV gene product; and an intron splice donor; and the second segment comprises, from 5’ to 3’: the first recombinase attachment site; an intron splice acceptor; and the second portion of the nucleic acid sequence encoding for the first AAV gene product; and the second recombinase attachment site. In some embodiments, the intron splice donor comprises a nucleic acid sequence having at least 80% identity with the nucleic acid sequence of any one of SEQ ID NOs: 1-2. In some embodiments, the intron splice acceptor comprises a nucleic acid sequence having at least 80% identity with the nucleic acid sequence of any one of SEQ ID NOs: 3-4. In some embodiments, the first segment comprises, from 5’ to 3’ : the promoter operably linked to the first portion of the nucleic acid sequence encoding for the first AAV gene product; the intron splice donor; and a nucleic acid sequence encoding for a stop codon.

In some embodiments, the AAV gene product is selected from the group consisting of Rep52, Rep40, Rep78, Rep68, E2A, E40rf6, VARNA, VP1, VP2, VP3, AAP, and MAAP.

In some embodiments, the recombinase of the expression control component is a PhiC31 recombinase, a Cre recombinase, a VCre recombinase, a Flp recombinase, a Bxbl recombinase, or a TP901 recombinase. In some embodiments, the recombinase comprises an amino acid sequence having at least 80% identity with the amino acid sequence of any one of SEQ ID NOs: 140-181. In some embodiments, the AAV production system comprises one or more polynucleic acid molecules collectively encoding for: Rep52 or Rep40; Rep78 or Rep68; E2A; E40rf6; VARNA; VP1; VP2; VP3; and AAP.

In some embodiments, an AAV production system comprises a polynucleic acid molecule having the structure: first segment - second segment - third segment, wherein the second segment is inverted relative to the first segment and the third segment, and wherein: (a) the first segment comprises, from 5’ to 3’: a promoter operably linked to a first portion of a nucleic acid sequence encoding for a first AAV gene product; and a first intron splice donor; (b) the second segment is an invertible element comprising, from 5’ to 3’: a first recombinase attachment site; a first intron splice acceptor; a second portion of the nucleic acid sequence encoding for the first AAV gene product; a promoter operably linked to a first portion of a nucleic acid sequence encoding for a second AAV gene product; and second splice intron donor; and a second recombinase attachment site; and (c) the third segment comprises, from 5’ to 3’: a second intron splice acceptor; and a second portion of the nucleic acid sequence encoding for the second AAV gene product; and wherein the first recombinase attachment site and the second recombinase attachment site correspond to the same recombinase.

In some embodiments, the first segment comprises, from 5’ to 3’ : the promoter operably linked to the first portion of the nucleic acid sequence encoding for the first AAV gene product; the first intron splice donor; and a nucleic acid sequence encoding for a stop codon; wherein the nucleic acid sequence encoding for the stop codon is in frame with the first portion of the nucleic acid sequence encoding for the first AAV gene product or the first portion of the nucleic acid sequence encoding for the second AAV gene product; the second segment comprises, from 5’ to 3’ : the first recombinase attachment site; the first intron splice acceptor; the second portion of the nucleic acid sequence encoding for the first AAV gene product; the promoter operably linked to the first portion of the nucleic acid sequence encoding for the second AAV gene product; the second intron splice donor; a nucleic acid sequence encoding for a stop codon; and the second recombinase attachment site; wherein the nucleic acid sequence encoding for the stop codon is in frame with the first portion of the nucleic acid sequence encoding for the first AAV product or the first portion of the nucleic acid sequence encoding for the second AAV gene product; or a combination thereof.

In some embodiments, an AAV production system comprises a polynucleic acid molecule having the structure: first segment - second segment - third segment - fourth segment, wherein the first segment and the second segment are inverted relative to the third segment and the fourth segment, wherein an invertible element comprises the second segment and the third segment, and wherein: (a) the third segment comprises, from 5’ to 3’ : a promoter operably linked to a first portion of a nucleic acid sequence encoding for a first AAV gene product; a first intron splice donor; and a first recombinase attachment site; (b) the first segment comprises, from 5’ to 3’: a first intron splice acceptor; and a second portion of the nucleic acid sequence encoding for the first AAV gene product; (c) the second segment comprises, from 5’ to 3’: a promoter operably linked to a first portion of a nucleic acid sequence encoding for a second AAV gene product; a second intron splice donor; and a second recombinase attachment site; and (d) the fourth segment comprises, from 5’ to 3’: a second intron splice acceptor; and a second portion of the nucleic acid sequence encoding for the second AAV gene product; and wherein the first recombinase attachment site and the second recombinase attachment site correspond to the same recombinase.

In some embodiments, the first segment comprises, from 5’ to 3’ : the first intron splice acceptor; a nucleic acid sequence encoding for a stop codon; and the second portion of the nucleic acid sequence encoding for the first AAV gene product; wherein the nucleic acid sequence encoding for the stop codon is in frame with the nucleic acid sequence encoding for the first portion of the second AAV gene product, but out of frame with the nucleic acid sequence encoding for the second portion of the first AAV gene product; the fourth segment comprises, from 5’ to 3’: the second intron splice acceptor; a nucleic acid sequence encoding for a stop codon; and the second portion of the nucleic acid sequence encoding for the second AAV gene product; wherein the nucleic acid sequence encoding for the stop codon is in frame with the nucleic acid sequence encoding for the first portion of the first AAV gene product, but out of frame with the nucleic acid sequence encoding for the second portion of the second AAV gene product; or a combination thereof.

In some embodiments, an AAV production system comprises a polynucleic acid molecule having the structure: first segment - second segment - third segment - fourth segment, wherein the first segment and the second segment are inverted relative to the third segment and the fourth segment, wherein an invertible element comprises the second segment and the third segment, and wherein: (a) the first segment comprises, from 5’ to 3’ : a promoter operably linked to a first portion of a nucleic acid sequence encoding for a first AAV gene product; and a first intron splice donor; (b) the third segment comprises, from 5’ to 3’: a first recombinase attachment site; a first intron splice acceptor; and a second portion of the nucleic acid sequence encoding for the first AAV gene product; (c) the fourth segment comprises, from 5’ to 3’: a promoter operably linked to a first portion of a nucleic acid sequence encoding for a second AAV gene product; and a second intron splice donor; and (d) the second segment comprises, from 5’ to 3’ : a second recombinase attachment site; a second intron splice acceptor; and a second portion of the nucleic acid sequence encoding for the second AAV gene product; and wherein the first recombinase attachment site and the second recombinase attachment site correspond to the same recombinase.

In some embodiments, the second segment comprises, from 5’ to 3’: the second recombinase attachment site; the second intron splice acceptor; a nucleic acid sequence encoding for a stop codon; and the second portion of the nucleic acid sequence encoding for the second AAV gene product; wherein the nucleic acid sequence encoding for the stop codon is in frame with the nucleic acid sequence encoding for the first portion of the first AAV gene product, but out of frame with the nucleic acid sequence encoding for the second portion of the second AAV gene product; the third segment comprises, from 5’ to 3’ : the first recombinase attachment site; the first intron splice acceptor; a nucleic acid sequence encoding for a stop codon; and the second portion of the nucleic acid sequence encoding for the first AAV gene product; wherein the nucleic acid sequence encoding for the stop codon is in frame with the nucleic acid sequence encoding for the first portion of the second AAV gene product, but out of frame with the nucleic acid sequence encoding for the second portion of the first AAV gene product; or a combination thereof.

In some embodiments, the first intron splice donor comprises a nucleic acid sequence having at least 80% identity with the nucleic acid sequence of any one of SEQ ID NOs: 1-2, the second intron splice donor comprises a nucleic acid sequence having at least 80% identity with the nucleic acid sequence of any one of SEQ ID NOs: 1-2, or a combination thereof.

In some embodiments, the first intron splice acceptor comprises a nucleic acid sequence having at least 80% identity with the nucleic acid sequence of any one of SEQ ID NOs: 3-4; the second intron splice acceptor comprises a nucleic acid sequence having at least 80% identity with the nucleic acid sequence of any one of SEQ ID NOs: 3-4, or a combination thereof. In some embodiments, the first AAV gene product is E2A and the second AAV gene product is E40rf6; the first AAV gene product is Rep52 and the second AAV gene product is VP1, VP2, VP3, or AAP; the first AAV gene product is Rep40 and the second AAV gene product is VP1, VP2, VP3, or AAP; the first AAV gene product is Rep78 and the second AAV gene product is VP1, VP2, VP3, or AAP; or the first AAV gene product is Rep68 and the second AAV gene product is VP1, VP2, VP3, or AAP.

In some embodiments an AAV production system further comprises an expression control component, wherein the expression control component comprises a polynucleic acid molecule encoding for a recombinase corresponding to the first recombination attachment site and the second recombination attachment site. In some embodiments, the recombinase is a PhiC31 recombinase, a Cre recombinase, a VCre recombinase, a Flp recombinase, a Bxbl recombinase, or a TP901 recombinase. In some embodiments, the recombinase comprises an amino acid sequence having at least 80% identity with the amino acid sequence of any one of SEQ ID NOs: 140-181.

In some embodiments, an AAV production system comprises one or more polynucleic acid molecules collectively encoding for: Rep52 or Rep40; Rep78 or Rep68; E2A; E40rf6; VARNA; VP1; VP2; VP3; and AAP.

In some aspects, the disclosure relates to engineered cells comprising an AAV production system (e.g., as described above).

In some embodiments, one or more polynucleic acid molecules of the AAV production component are stably integrated into the genome of the engineered cell.

In some embodiments, an engineered cell further comprises a transfer polynucleic acid molecule having a sequence encoding, from 5’ to 3’: (i) a nucleotide sequence of a 5’ inverted tandem repeat; (ii) a multiple cloning site; and (iii) a nucleotide sequence of a 3’ inverted tandem repeat. In some embodiments, the transfer polynucleic acid molecule is stably integrated into the genome of the engineered cell.

In some aspects, the disclosure relates to a kit comprising an AAV production system (e.g., as described above) and/or an engineered cell (e.g., as described above).

In some embodiments, a kit further comprises a transfer polynucleic acid molecule having a sequence encoding, from 5’ to 3’: (i) a nucleotide sequence of a 5’ inverted tandem repeat; (ii) a multiple cloning site; and (iii) a nucleotide sequence of a 3’ inverted tandem repeat. In some embodiments, the transfer polynucleic acid molecule is a plasmid or a vector.

In some aspects, the disclosure relates to methods of producing an AAV vector. In some embodiments, a method of producing an AAV vector comprises, expressing, in an engineered cell comprising an AAV production system, the recombinase of the expression control component, and Rep52 or Rep40, Rep78 or Rep68, E2A, E40rf6, VARNA, VP1, VP2, VP3, and AAP of the AAV production component.

In some aspects, the disclosure relates to a polynucleic acid molecule comprising a nucleic acid sequence having at least 80% identity with the nucleic acid sequence of any one of SEQ ID NOs: 111-139.

In some aspects, the disclosure relates to an engineered cell comprising a polynucleic acid molecule comprising a nucleic acid sequence having at least 80% identity with the nucleic acid sequence of any one of SEQ ID NOs: 111-139.

BRIEF DESCRIPTION OF DRAWINGS

FIGs. 1A-1C show plasmid schematics for helper gene control using a recombinase. The pHelper plasmid was modified to introduce synthetic introns into both E2A and E4orf6 (“E4”) helper gene coding sequences. Each intron contains recombinase attachment (e.g., att) sites (each represented by triangle, with the orientation indicated by the direction of the triangle) such that expression of the corresponding recombinase results in inversion of the sequence between the recombinase attachment sites. FIG. 1 A. Shows a head-to-tail helper gene orientation. The helper construct has convergent transcription and nonfunctional introns prior to recombinase inversion. Helper genes are both expressed in the forward orientation after recombination. Exemplary positions for the inclusion of stop codons are depicted with an “X.” FIG. IB. Shows a divergent gene orientation. The helper genes are spliced across the introns but translation of the downstream exon is out of frame, resulting in missense translation and early termination. Exemplary positions for the inclusion of stop codons are depicted with an “X.” As depicted, the 5’ portion of E4 is in frame with a stop codon positioned 3’ to an intron splice acceptor prior to recombination, and out of frame with the 3’ portion of E2A. Similarly, the 5’ portion of E2A is in frame with a stop codon positioned 3’ to an intron splice acceptor prior to recombination, and out of frame with the 3’ portion of E4. After recombination, the 5’ portion of E4 is positioned in frame with the 3’ portion of E4 (but out of frame with the stop codon 3’ to the intron splice acceptor). Similarly, after recombination, the 5’ portion of E2A is positioned in frame with the 3’ portion of E2A (but out of frame with the stop codon 3’ to the intron splice acceptor). FIG. 1C shows an additional architecture. Exemplary positions for the inclusion of stop codons are depicted with an “X.” As depicted, the 5’ portion of E4 is in frame with a stop codon positioned 3’ to an intron splice acceptor prior to recombination, and out of frame with the 3’ portion of E2A. Similarly, the 5’ portion of E2A is in frame with a stop codon positioned 3’ to an intron splice acceptor prior to recombination, and out of frame with the 3’ portion of E4. After recombination, the 5’ portion of E4 is positioned in frame with the 3’ portion of E4 (but out of frame with the stop codon 3’ to the intron splice acceptor). Similarly, after recombination, the 5’ portion of E2A is positioned in frame with the 3’ portion of E2A (but out of frame with the stop codon 3’ to the intron splice acceptor). SD: intron splice donor; SA: intron splice acceptor; promoters are indicated by the arrows (p40 is an exemplary promoter).

FIG. 2 shows a plasmid schematic for Rep and Cap gene control using a recombinase. Similar to the head-to-tail helper construct, the pRepCap plasmid is modified to introduce synthetic introns bearing recombinase attachment sites (e.g., att sites) into both Rep and Cap helper gene coding sequences and inverting the sequence between the recombinase attachment sites. Exemplary positions for the inclusion of stop codons are depicted with an “X.” Expression of the recombinase restores the proper orientation and splicing of Rep and Cap. SD: intron splice donor; SA: intron splice acceptor; recombinase attachment site are represented by triangles (the orientation of the site is represented by the direction of the triangle); promoters are indicated by the arrows (p40 is an exemplary promoter).

FIG. 3 shows infectious titers generated by recombinase inversion AAV plasmids. Two sets of constructs were tested: 1) a divergent helper system based on AAV2, with pRepCap containing an attenuated p5 promoter with spacer (gray circle) and miR-342 expression; and 2) an Ad5 pHelper system with head-to-tail orientation and pRepCap containing a wild type p5 promoter without spacer. Both systems contain PhiC31 att sites and are either cotransfected with or without a PhiC31 expression plasmid. Individual pHelper and pRepCap inverted plasmids were tested in combination with each other, or with wild type pHelper/pRepCap plasmids. Pairs of columns are as follows: 1) inverted pHelper + wt pRepCap [divergent design], 2) wt pHelper + inverted pRepCap [divergent design], 3) inverted pHelper + inverted pRepCap [divergent design], 4) inverted pHelper + wt pRepCap [head-to-tail design], 5) wt pHelper + inverted pRepCap [head-to-tail design], 6) inverted pHelper + inverted pRepCap [head-to-tail design], 7) transfection with ITR-containing transfer plasmid only, 8) wt pHelper + wt pRepCap based on divergent plasmids, 9) wt pHelper + wt pRepCap based on head-to-tail plasmids. DETAILED DESCRIPTION OF INVENTION

Viral vectors are a promising gene delivery modality for cell and gene therapy. The production of viral vectors normally entails transient transfection of plasmids into cell culture. However, stable integration of genes necessary to produce therapeutic viral vectors into the genome offers several advantages compared to traditional production via transient transfection. Since cells amplify the viral genes during their own cell division, large quantities of DNA and transfection reagent no longer need to be procured for the transfection process, reducing costs. Also, since the DNA is already within the nucleus, viral titers may be higher and more consistent due to minimal numbers of “untransfected” cells and reduced variation associated with transfection steps. The simpler production process also saves scientist time.

However, several genes required for adeno-associated viral (AAV) vector production have been demonstrated by others to be cytostatic or cytotoxic, namely Rep, E2A and E4. The cytotoxic and cytostatic nature of these proteins has hampered the development of stable AAV producer cell lines in the widely used HEK293 cell line, since the native expression of adenovirus El genes in HEK293 cells upregulates expression of these toxic genes. Cells stably transfected with these genes fail to survive selection steps or have silenced expression, resulting in an inability to produce relevant quantities of AAV.

I. Adeno-Associated Virus Production Systems

In some aspects, the disclosure relates to adeno-associated virus (AAV) production systems. In some embodiments, AAV production systems allow for inducible control of a gene product(s) required for AAV production, including a product(s) that is cytotoxic or cytostatic to a cell. This inducible control can be mediated by recombinases. Because recombinases remove or invert sequences to produce a close to wild type final sequence, expression of viral genes can be controlled by native viral promoters and other native sequences, resulting in near-native expression levels. For some designs, part of the viral coding sequences themselves can be inverted, resulting in near zero expression of the full coding sequence. The possibility for near-zero background expression in the absence of recombinase and near-native expression in presence of recombinase makes recombinases a promising technology for viral platforms which have complex and poorly characterized regulation. In contrast, systems that directly regulate viral genes with synthetic promoters (e.g., Tet-On or cumate) require significant tuning and may result in leaky expression in the off state.

An AAV production system, as described herein, may comprise one or more nucleic acid molecules comprising: (a) an AAV production component; (b) an expression control component; (c) a transcriptional activator; (d) a transfer polynucleic acid molecule; (e) a selection marker; or (f) a combination thereof. a. AAV Production Component

In some embodiments, an AAV production system comprises an AAV production component. An AAV production component, may comprise one or more nucleic acid molecules that collectively encode gene products required for generation of an AAV in a recombinant host cell (or an “engineered cell” as described herein). Exemplary AAV gene products include Rep52, Rep40, Rep78, Rep68, El, E2A, E40rf6, VARNA, CAP (VP1, VP2, VP3), AAP, and MAAP or functional variants thereof. The Rep gene products (comprising Rep52, Rep40, Rep78 and Rep68) are involved in AAV genome replication and packaging. The El genes upregulate transcription of several adenovirus and AAV genes. The E2A gene product is involved in aiding DNA synthesis processivity during AAV replication. The E40rf6 gene product supports AAV replication. The VARNA gene product plays a role in regulating translation. The CAP gene products (comprising VP1, VP2, VP3) encode viral capsid proteins. The AAP gene product plays a role in capsid assembly. MAAP is a protein residing in an alternate reading from of VP1 and appears to play a role in the viral capsid as described in Ogden et al. Science 366.6469 (2019): 1139-1143, which is incorporated by reference in its entirety.

In some embodiments, a nucleic acid sequence encoding an AAV gene product is operably linked to a promoter. As used herein, the term “promoter” refers to a nucleic acid sequence that is bound by proteins to initiate transcription of RNA from DNA. A promoter may be a constitutive promoter (i.e., an unregulated promoter that allows for continual transcription). Examples of constitutive promoters are known in the art and include, but are not limited to, cytomegalovirus (CMV) promoters, elongation factor 1 a (EFla) promoters, simian vacuolating virus 40 (SV40) promoters, ubiquitin-C (UBC) promoters, U6 promoters, p5 promoters, pl9 promoters, p40 promoters, E2A promoters, E4 promoters and phosphoglycerate kinase (PGK) promoters. See e.g., Ferreira et al. Proc. Natl. Acad. Sci. U.S.A. 2013 Jul; 110(28): 11284-89; Pub. No.: US 2014/377861 Al; Qin et al. PloS one 5.5 (2010): e!0611. - the entireties of which are incorporated herein by reference. Alternatively, a promoter may be an inducible promoter (i.e., only activates transcription under specific circumstances). An inducible promoter may be a chemically inducible be a chemically inducible promoter, a temperature inducible promoter, or a light inducible promoter. Additional types of inducible promoters are known to those having ordinary skill in the art. Examples of inducible promoters are known in the art and include, but are not limited to, tetracycline/doxycy cline inducible promoters, cumate inducible promoters, ABA inducible promoters, CRY2-CIB1 inducible promoters, DAPG inducible promoters, pTRE3G promoters, pTREtight promoters, the Gal4 UAS operator sequences and mifepristone inducible promoters, and a promoters containing at least one of VanR, TtgR, PhlF, or CymR operator sequences. See e.g., Stanton et al., ACS Synth. Biol. 2014 Dec 19; 3(12): 880-91; Liang et al., Sci. Signal. 2011 Mar 15; 4(164): rs2; Patent No.: US 7,745,592 B2; Patent No.: US 7,935,788 B2 - the entireties of which are incorporated herein by reference.

In some embodiments, an AAV production component comprises a nucleic acid sequence encoding a functional variant of Rep52 comprising at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 97, wherein the functional variant is capable of functioning in AAV genome replication. In some embodiments, the AAV production component comprises a nucleic acid sequence encoding a Rep52 polypeptide comprising the amino acid sequence of SEQ ID NO:

97 operably linked to a promoter (as described herein).

In some embodiments, an AAV production component comprises a nucleic acid sequence encoding a functional variant of Rep40 comprising at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 98, wherein the functional variant is capable of functioning in AAV genome replication. In some embodiments, the AAV production component comprises a nucleic acid sequence encoding a Rep40 polypeptide comprising the amino acid sequence of SEQ ID NO:

98 operably linked to a promoter (as described herein).

In some embodiments, an AAV production component comprises a nucleic acid sequence encoding a functional variant of Rep78 comprising at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 99, wherein the functional variant is capable of functioning in AAV genome replication. In some embodiments, the AAV production component comprises a nucleic acid sequence encoding a Rep78 polypeptide comprising the amino acid sequence of SEQ ID NO:

99 operably linked to a promoter (as described herein). In some embodiments, an AAV production component comprises a nucleic acid sequence encoding a functional variant of Rep68 comprising at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 100, wherein the functional variant is capable of functioning in AAV genome replication. In some embodiments, an AAV production component comprises a nucleic acid sequence encoding a Rep68 polypeptide comprising the amino acid sequence of SEQ ID NO: 100 operably linked to a promoter (as described herein).

In some embodiments, an AAV production component comprises a nucleic acid sequence encoding a functional variant of E2A comprising at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 101, wherein the functional variant is capable of aiding DNA synthesis processivity during AAV replication. In some embodiments, the AAV production component comprises a nucleic acid sequence encoding a E2A polypeptide comprising the amino acid sequence of SEQ ID NO: 101 operably linked to a promoter (as described herein).

In some embodiments, an AAV production component comprises a nucleic acid sequence encoding a functional variant of E4ORF6 comprising at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 102, wherein the functional variant is capable of supporting AAV replication. In some embodiments, the AAV production component comprises a nucleic acid sequence encoding a functional variant of E4ORF6 comprising at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 103, wherein the functional variant is capable of supporting AAV replication. In some embodiments, the AAV production component comprises a nucleic acid sequence encoding a E4ORF6 polypeptide comprising the amino acid sequence of SEQ ID NO: 102 operably linked to a promoter (as described herein). In some embodiments, the AAV production component comprises a nucleic acid sequence encoding SEQ ID NO: 103 operably linked to a promoter (as described herein).

In some embodiments, an AAV production component comprises a nucleic acid sequence encoding a functional variant of VARNA comprising at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 104, wherein the functional variant is capable regulating translation. In some embodiments, the AAV production component comprises a nucleic acid sequence encoding a VARNA of SEQ ID NO: 104 operably linked to a promoter (as described herein). In some embodiments, an AAV production component comprises a nucleic acid sequence encoding a functional variant of VP1 comprising at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 105, wherein the functional variant is capable of being incorporated into the AAV capsid. In some embodiments, the AAV production component comprises a nucleic acid sequence encoding a VP1 polypeptide comprising the amino acid sequence of SEQ ID NO: 105 operably linked to a promoter (as described herein).

In some embodiments, an AAV production component comprises a nucleic acid sequence encoding a functional variant of VP2 comprising at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 106, wherein the functional variant is capable of being incorporated into the AAV capsid. In some embodiments, the AAV production component comprises a nucleic acid sequence encoding a VP2 polypeptide comprising the amino acid sequence of SEQ ID NO: 106 operably linked to a promoter (as described herein).

In some embodiments, an AAV production component comprises a nucleic acid sequence encoding a functional variant of VP3 comprising at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 107, wherein the functional variant is capable of being incorporated into the AAV capsid. In some embodiments, the AAV production component comprises a nucleic acid sequence encoding a VP3 polypeptide comprising the amino acid sequence of SEQ ID NO: 107 operably linked to a promoter (as described herein).

In some embodiments, an AAV production component comprises a nucleic acid sequence encoding a functional variant of AAP comprising at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 108, wherein the functional variant is capable of regulating AAV capsid assembly. In some embodiments, the AAV production component comprises a nucleic acid sequence encoding an AAP polypeptide comprising the amino acid sequence of SEQ ID NO: 108 operably linked to a promoter (as described herein).

In some embodiments, an AAV production component comprises a nucleic acid sequence encoding a functional variant of MAAP comprising at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 229, wherein the functional variant is capable of regulating AAV capsid assembly. In some embodiments, the AAV production component comprises a nucleic acid sequence encoding an MAAP polypeptide comprising the amino acid sequence of SEQ ID NO: 229 operably linked to a promoter (as described herein).

In some embodiments, an AAV production component comprises a nucleic acid sequence encoding a polypeptide having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 109. In some embodiments, the AAV production component comprises a nucleic acid sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 109 operably linked to a promoter (as described herein).

In some embodiments, an AAV production component comprises a nucleic acid sequence encoding a polypeptide having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 110. In some embodiments, the AAV production component comprises a nucleic acid sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 110 operably linked to a promoter (as described herein).

In some embodiments, an AAV production component is (i.e., the gene products of the AAV component are) encoded on a single nucleic acid molecule. In other embodiments, multiple nucleic acid molecules collectively comprise the AAV production component (i.e., at least two of the gene products of the AAV production component are encoded on different nucleic acid molecules). For example, an AAV production component may comprise at least

2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 10, or at least 11 nucleic acid molecules. In some embodiments, an AAV production component comprises 2,

3, 4, 5, 6, 7, 8, 9, 10, or 11 nucleic acid molecules.

In some embodiments, an AAV production system comprises one or more nucleic acid sequences that collectively encode the gene products: Rep52 or Rep40; Rep78 or Rep68; E2A; E40rf6; VARNA; VP1; VP2; VP3; and AAP. In some embodiments, an AAV production system comprises one or more nucleic acid sequences that collectively encode the gene products: Rep52, Rep40, Rep78, Rep68, E2A, E40rf6, VARNA, VP1, VP2, VP3, and AAP. In some embodiments, the one or more nucleic acid molecules that collectively encode the gene products required for generation of an AAV are each operably linked to a promoter as described herein.

In some embodiments, an AAV production component comprises an invertible element. As used herein, the term “invertible element” refers to a nucleic acid sequence comprising an internal sequence flanked by a first recombinase attachment site and a second recombinase attachment site, wherein the first recombinase attachment site and the second recombinase attachment site are capable of being recombined by a recombinase, and wherein exposure to the recombinase results in the inversion of the invertible element. In some embodiments, an AAV production components comprise two or more invertible elements. For example, an AAV production system may comprise two, three, four, or five invertible elements.

In some embodiments, an invertible element comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of any one of SEQ ID NOs: 111-139. In some embodiments, the AAV production component comprises a polynucleic acid having the nucleic acid sequence of any one of SEQ ID NOs: 111-139.

1. Recombinase Attachment Sites

The invertible elements described herein comprise an internal sequence flanked by a pair of recombinase attachment sites that are capable of being bound and recombined by a recombinase. Numerous recombinase attachment sites have been described previously and are known to those having ordinary skill in the art. In some embodiments, an internal sequence is flanked by a first pair of recombinase attachments sites that is capable of being bound and recombined by a first recombinase (e.g., Cre) and a second pair of recombinase attachments sites that is capable of being bound and recombined by a second recombinase (e.g., Flp), which can - in some embodiments - render an inversion event irreversible (see e.g., blog.addgene.org/plasmids-101-flex-vectors).

In some embodiments, an internal sequence is flanked by a recombinase attachment site that comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of any one of SEQ ID NOs: 5-96. In some embodiments, a recombinase attachment site comprises the nucleic acid sequence of any one of SEQ ID NOs: 5-96.

In some embodiments, an internal sequence of an invertible element is flanked by recombinase attachment sites that are capable of being bound and recombined by a PhiC31 recombinase.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 5 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 21, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a PhiC31 recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 5 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 21.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 6 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 22, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a PhiC31 recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 6 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 22.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 7 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 23, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a PhiC31 recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 7 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 23.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 8 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 24, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a PhiC31 recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 8 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 24.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 9 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 25, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a PhiC31 recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 9 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 25.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 10 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 26, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a PhiC31 recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 10 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 26.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 11 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 27, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a PhiC31 recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 11 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 27.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 12 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 28, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a PhiC31 recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 12 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 28.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 13 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 29, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a PhiC31 recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 13 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 29.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 14 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 30, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a PhiC31 recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 14 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 30.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 15 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 31, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a PhiC31 recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 15 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 31.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 16 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 32, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a PhiC31 recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 16 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 32.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 17 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 33, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a PhiC31 recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 17 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 33.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 18 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 34, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a PhiC31 recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 18 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 34.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 19 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 35, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a PhiC31 recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 19 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 35.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 20 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 36, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a PhiC31 recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 20 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 36.

In some embodiments, an internal sequence of an invertible element is flanked by recombinase attachment sites that are capable of being bound and recombined by a Bxbl recombinase.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 37 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 53, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a Bxbl recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 37 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 53.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 38 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 54, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a Bxbl recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 38 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 54.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 39 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 55, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a Bxbl recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 39 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 55.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 40 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 56, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a Bxbl recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 40 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 56.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 41 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 57, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a Bxbl recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 41 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 57.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 42 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 58, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a Bxbl recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 42 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 58.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 43 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 59, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a Bxbl recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 43 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 59.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 44 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 60, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a Bxbl recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 44 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 60.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 45 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 61, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a Bxbl recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 45 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 61.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 46 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 62, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a Bxbl recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 46 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 62.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 47 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 63, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a Bxbl recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 47 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 63.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 48 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 64, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a Bxbl recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 48 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 64.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 49 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 65, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a Bxbl recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 49 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 65.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 50 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 66, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a Bxbl recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 50 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 66.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 51 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 67, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a Bxbl recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 51 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 67.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 52 and on the other end by a second recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 68, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a Bxbl recombinase. In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having nucleic acid sequence of SEQ ID NO: 52 and on the other end by a second recombinase attachment site having the nucleic acid sequence of SEQ ID NO: 68.

In some embodiments, an internal sequence of an invertible element is flanked by recombinase attachment sites that are capable of being bound and recombined by a Cre recombinase.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of any one of SEQ ID NOs: 69-77 and on the other end by a second recombinase attachment site that is substantially identical to the first recombinase attachment site but in a reverse complement orientation relative to the first recombinase attachment site, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a Cre recombinase. In some embodiments, the first recombinase attachment site has the nucleic acid sequence of any one of SEQ ID NOs: 69-77.

In some embodiments, an internal sequence of an invertible element is flanked by recombinase attachment sites that are capable of being bound and recombined by a VCre recombinase.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of any one of SEQ ID NOs: 78-83 and on the other end by a second recombinase attachment site that is substantially identical to the first recombinase attachment site but in a reverse complement orientation relative to the first recombinase attachment site, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a Cre recombinase. In some embodiments, the first recombinase attachment site has the nucleic acid sequence of any one of SEQ ID NOs: 78-83.

In some embodiments, an internal sequence of an invertible element is flanked by recombinase attachment sites that are capable of being bound and recombined by a Flp recombinase.

In some embodiments, an internal sequence of an invertible element is flanked on one end by a first recombinase attachment site having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of any one of SEQ ID NOs: 84-96 and on the other end by a second recombinase attachment site that is substantially identical to the first recombinase attachment site but in a reverse complement orientation relative to the first recombinase attachment site, wherein the first and the second recombinase attachment sites are capable of being bound and recombined by a Flp recombinase. In some embodiments, the first recombinase attachment site has the nucleic acid sequence of any one of SEQ ID NOs: 84-96.

2. Internal Sequences

The invertible elements described herein comprise an internal sequence. In some embodiments, an internal sequence comprises the nucleic acid sequence encoding for one or more AAV gene product(s) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 AAV gene product(s)).

In some embodiments, an internal sequence comprises a first portion of a nucleic acid sequence encoding for an AAV gene product, but not a second portion or a third (and remaining) portion of a nucleic acid sequence encoding the AAV gene product, wherein the invertible element is flanked by the second portion and the third (and remaining) portion, and wherein the first portion is in an inverted orientation relative to the second portion and the third portion.

In some embodiments, the internal sequence comprises a first portion of a nucleic acid sequence encoding for Rep52, but not a second portion of the nucleic acid sequence encoding for Rep52 or a third (and remaining) portion of a nucleic acid sequence encoding from Rep52, wherein the invertible element is flanked on one end by the second portion and on the other end by the third (and remaining) portion, and wherein the first portion is in an inverted orientation relative to the second portion and the third portion. In some embodiments, the first portion, the second portion, and the third portion of the nucleic acid sequence encoding for Rep52 collectively comprise a nucleic acid sequence encoding a polypeptide having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 97. In some embodiments, the second portion or the third portion of the nucleic acid sequence encoding for Rep52 is operably linked to a promoter (as described herein).

In some embodiments, the internal sequence comprises a first portion of a nucleic acid sequence encoding for Rep40, but not a second portion of the nucleic acid sequence encoding for Rep40 or a third (and remaining) portion of a nucleic acid sequence encoding from Rep40, wherein the invertible element is flanked on one end by the second portion and on the other end by the third (and remaining) portion, and wherein the first portion is in an inverted orientation relative to the second portion and the third portion. In some embodiments, the first portion, the second portion, and the third portion of the nucleic acid sequence encoding for Rep40 collectively comprise a nucleic acid sequence encoding a polypeptide having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 98. In some embodiments, the second portion or the third portion of the nucleic acid sequence encoding for Rep40 is operably linked to a promoter (as described herein).

In some embodiments, the internal sequence comprises a first portion of a nucleic acid sequence encoding for Rep78, but not a second portion of the nucleic acid sequence encoding for Rep78 or a third (and remaining) portion of a nucleic acid sequence encoding from Rep78, wherein the invertible element is flanked on one end by the second portion and on the other end by the third (and remaining) portion, and wherein the first portion is in an inverted orientation relative to the second portion and the third portion. In some embodiments, the first portion, the second portion, and the third portion of the nucleic acid sequence encoding for Rep78 collectively comprise a nucleic acid sequence encoding a polypeptide having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 99. In some embodiments, the second portion or the third portion of the nucleic acid sequence encoding for Rep78 is operably linked to a promoter (as described herein).

In some embodiments, the internal sequence comprises a first portion of a nucleic acid sequence encoding for Rep68, but not a second portion of the nucleic acid sequence encoding for Rep68 or a third (and remaining) portion of a nucleic acid sequence encoding from Rep68, wherein the invertible element is flanked on one end by the second portion and on the other end by the third (and remaining) portion, and wherein the first portion is in an inverted orientation relative to the second portion and the third portion. In some embodiments, the first portion, the second portion, and the third portion of the nucleic acid sequence encoding for Rep68 collectively comprise a nucleic acid sequence encoding a polypeptide having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 100. In some embodiments, the second portion or the third portion of the nucleic acid sequence encoding for Rep68 is operably linked to a promoter (as described herein).

In some embodiments, the internal sequence comprises a first portion of a nucleic acid sequence encoding for E2A, but not a second portion of the nucleic acid sequence encoding for E2A or a third (and remaining) portion of a nucleic acid sequence encoding from E2A, wherein the invertible element is flanked on one end by the second portion and on the other end by the third (and remaining) portion, and wherein the first portion is in an inverted orientation relative to the second portion and the third portion. In some embodiments, the first portion, the second portion, and the third portion of the nucleic acid sequence encoding for E2A collectively comprise a nucleic acid sequence encoding a polypeptide having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 101. In some embodiments, the second portion or the third portion of the nucleic acid sequence encoding for E2A is operably linked to a promoter (as described herein).

In some embodiments, the internal sequence comprises a first portion of a nucleic acid sequence encoding for E4ORF6, but not a second portion of the nucleic acid sequence encoding for E4ORF6 or a third (and remaining) portion of a nucleic acid sequence encoding from E4ORF6, wherein the invertible element is flanked on one end by the second portion and on the other end by the third (and remaining) portion, and wherein the first portion is in an inverted orientation relative to the second portion and the third portion. In some embodiments, the first portion, the second portion, and the third portion of the nucleic acid sequence encoding for E4ORF6 collectively comprise a nucleic acid sequence encoding a polypeptide having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NOs: 102 or 103. In some embodiments, the second portion or the third portion of the nucleic acid sequence encoding for E4ORF6 is operably linked to a promoter (as described herein).

In some embodiments, the internal sequence comprises a first portion of a nucleic acid sequence encoding for VARNA, but not a second portion of the nucleic acid sequence encoding for VARNA or a third (and remaining) portion of a nucleic acid sequence encoding from VARNA, wherein the invertible element is flanked on one end by the second portion and on the other end by the third (and remaining) portion, and wherein the first portion is in an inverted orientation relative to the second portion and the third portion. In some embodiments, the first portion, the second portion, and the third portion of the nucleic acid sequence encoding for VARNA collectively comprise a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 104. In some embodiments, the second portion or the third portion of the nucleic acid sequence encoding for VARNA is operably linked to a promoter (as described herein).

In some embodiments, the internal sequence comprises a first portion of a nucleic acid sequence encoding for VP1, but not a second portion of the nucleic acid sequence encoding for VP 1 or a third (and remaining) portion of a nucleic acid sequence encoding from VP1, wherein the invertible element is flanked on one end by the second portion and on the other end by the third (and remaining) portion, and wherein the first portion is in an inverted orientation relative to the second portion and the third portion. In some embodiments, the first portion, the second portion, and the third portion of the nucleic acid sequence encoding for VP1 collectively comprise a nucleic acid sequence encoding a polypeptide having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 105. In some embodiments, the second portion or the third portion of the nucleic acid sequence encoding for VP1 is operably linked to a promoter (as described herein).

In some embodiments, the internal sequence comprises a first portion of a nucleic acid sequence encoding for VP2, but not a second portion of the nucleic acid sequence encoding for VP2 or a third (and remaining) portion of a nucleic acid sequence encoding from VP2, wherein the invertible element is flanked on one end by the second portion and on the other end by the third (and remaining) portion, and wherein the first portion is in an inverted orientation relative to the second portion and the third portion. In some embodiments, the first portion, the second portion, and the third portion of the nucleic acid sequence encoding for VP2 collectively comprise a nucleic acid sequence encoding a polypeptide having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 106. In some embodiments, the second portion or the third portion of the nucleic acid sequence encoding for VP2 is operably linked to a promoter (as described herein).

In some embodiments, the internal sequence comprises a first portion of a nucleic acid sequence encoding for VP3, but not a second portion of the nucleic acid sequence encoding for VP3 or a third (and remaining) portion of a nucleic acid sequence encoding from VP3, wherein the invertible element is flanked on one end by the second portion and on the other end by the third (and remaining) portion, and wherein the first portion is in an inverted orientation relative to the second portion and the third portion. In some embodiments, the first portion, the second portion, and the third portion of the nucleic acid sequence encoding for VP3 collectively comprise a nucleic acid sequence encoding a polypeptide having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 107. In some embodiments, the second portion or the third portion of the nucleic acid sequence encoding for VP3 is operably linked to a promoter (as described herein).

In some embodiments, the internal sequence comprises a first portion of a nucleic acid sequence encoding for AAP, but not a second portion of the nucleic acid sequence encoding for AAP or a third (and remaining) portion of a nucleic acid sequence encoding from AAP, wherein the invertible element is flanked on one end by the second portion and on the other end by the third (and remaining) portion, and wherein the first portion is in an inverted orientation relative to the second portion and the third portion. In some embodiments, the first portion, the second portion, and the third portion of the nucleic acid sequence encoding for AAP collectively comprise a nucleic acid sequence encoding a polypeptide having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 108. In some embodiments, the second portion or the third portion of the nucleic acid sequence encoding for AAP is operably linked to a promoter (as described herein).

In some embodiments, the internal sequence comprises a first portion of a nucleic acid sequence encoding for MAAP, but not a second portion of the nucleic acid sequence encoding for MAAP or a third (and remaining) portion of a nucleic acid sequence encoding from MAAP, wherein the invertible element is flanked on one end by the second portion and on the other end by the third (and remaining) portion, and wherein the first portion is in an inverted orientation relative to the second portion and the third portion. In some embodiments, the first portion, the second portion, and the third portion of the nucleic acid sequence encoding for MAAP collectively comprise a nucleic acid sequence encoding a polypeptide having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 229. In some embodiments, the second portion or the third portion of the nucleic acid sequence encoding for MAAP is operably linked to a promoter (as described herein). In some embodiments, the internal sequence comprises a first portion of a nucleic acid sequence encoding a first AAV gene product, but not a second (and remaining) portion of a nucleic acid sequence encoding the AAV gene product, wherein the first portion is in an inverted orientation relative to the second (and remaining) portion. In such embodiments, the internal sequence may further comprise the nucleic acid sequence encoding for one or more additional AAV gene product(s) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 AAV gene product(s)).

In some embodiments, the internal sequence comprises a first portion of a nucleic acid sequence encoding for Rep52, but not a second (and remaining) portion of the nucleic acid sequence encoding for Rep52, wherein the invertible element is flanked by the second (and remaining) portion, and wherein the first portion is in an inverted orientation relative to the second portion. In some embodiments, the first portion and the second portion of the nucleic acid sequence encoding for Rep52 collectively comprise a nucleic acid sequence encoding a polypeptide having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 97. In some embodiments, the first portion or the second portion of the nucleic acid sequence encoding for Rep52 is operably linked to a promoter (as described herein). In some embodiments, the internal sequence further comprises a nucleic acid sequence encoding for one or more additional AAV gene product(s) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 AAV gene product(s)). For example, in some embodiments, the internal sequence further comprises a nucleic acid sequence encoding for Rep40 (or a portion thereof), a nucleic acid sequence encoding for Rep78 (or a portion thereof), a nucleic acid sequence encoding for Rep68 (or a portion thereof), a nucleic acid sequence encoding for E2A (or a portion thereof), a nucleic acid sequence for E4ORF6 (or a portion thereof), a nucleic acid sequence encoding for VARNA (or a portion thereof), a nucleic acid sequence encoding for VP1 (or a portion thereof), a nucleic acid sequence encoding for VP2 (or a portion thereof), a nucleic acid sequence encoding for VP3 (or a portion thereof), a nucleic acid sequence encoding for AAP (or a portion thereof), a nucleic acid sequence encoding for MAAP (or a portion thereof), or any combination thereof.

In some embodiments, the internal sequence comprises a first portion of a nucleic acid sequence encoding for Rep40, but not a second (and remaining) portion of the nucleic acid sequence encoding for Rep40, wherein the invertible element is flanked by the second (and remaining) portion, and wherein the first portion is in an inverted orientation relative to the second portion. In some embodiments, the first portion and the second portion of the nucleic acid sequence encoding for Rep40 collectively comprise a nucleic acid sequence encoding a polypeptide having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 98. In some embodiments, the first portion or the second portion of the nucleic acid sequence encoding for Rep40 is operably linked to a promoter (as described herein). In some embodiments, the internal sequence further comprises a nucleic acid sequence encoding for one or more additional AAV gene product(s) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 AAV gene product(s)). For example, in some embodiments, the internal sequence further comprises a nucleic acid sequence encoding for Rep52 (or a portion thereof), a nucleic acid sequence encoding for Rep78 (or a portion thereof), a nucleic acid sequence encoding for Rep68 (or a portion thereof), a nucleic acid sequence encoding for E2A (or a portion thereof), a nucleic acid sequence for E4ORF6 (or a portion thereof), a nucleic acid sequence encoding for VARNA (or a portion thereof), a nucleic acid sequence encoding for VP1 (or a portion thereof), a nucleic acid sequence encoding for VP2 (or a portion thereof), a nucleic acid sequence encoding for VP3 (or a portion thereof), a nucleic acid sequence encoding for AAP (or a portion thereof), a nucleic acid sequence encoding for MAAP (or a portion thereof), or any combination thereof.

In some embodiments, the internal sequence comprises a first portion of a nucleic acid sequence encoding for Rep78, but not a second (and remaining) portion of the nucleic acid sequence encoding for Rep78, wherein the invertible element is flanked by the second (and remaining) portion, and wherein the first portion is in an inverted orientation relative to the second portion. In some embodiments, the first portion and the second portion of the nucleic acid sequence encoding for Rep78 collectively comprise a nucleic acid sequence encoding a polypeptide having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 99. In some embodiments, the first portion or the second portion of the nucleic acid sequence encoding for Rep78 is operably linked to a promoter (as described herein). In some embodiments, the internal sequence further comprises a nucleic acid sequence encoding for one or more additional AAV gene product(s) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 AAV gene product(s)). For example, in some embodiments, the internal sequence further comprises a nucleic acid sequence encoding for Rep52 (or a portion thereof), a nucleic acid sequence encoding for Rep40 (or a portion thereof), a nucleic acid sequence encoding for Rep68 (or a portion thereof), a nucleic acid sequence encoding for E2A (or a portion thereof), a nucleic acid sequence for E4ORF6 (or a portion thereof), a nucleic acid sequence encoding for VARNA (or a portion thereof), a nucleic acid sequence encoding for VP1 (or a portion thereof), a nucleic acid sequence encoding for VP2 (or a portion thereof), a nucleic acid sequence encoding for VP3 (or a portion thereof), a nucleic acid sequence encoding for AAP (or a portion thereof), a nucleic acid sequence encoding for MAAP (or a portion thereof), or any combination thereof.

In some embodiments, the internal sequence comprises a first portion of a nucleic acid sequence encoding for Rep68, but not a second (and remaining) portion of the nucleic acid sequence encoding for Rep68, wherein the invertible element is flanked by the second (and remaining) portion, and wherein the first portion is in an inverted orientation relative to the second portion. In some embodiments, the first portion and the second portion of the nucleic acid sequence encoding for Rep68 collectively comprise a nucleic acid sequence encoding a polypeptide having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 100. In some embodiments, the first portion or the second portion of the nucleic acid sequence encoding for Rep68 is operably linked to a promoter (as described herein). In some embodiments, the internal sequence further comprises a nucleic acid sequence encoding for one or more additional AAV gene product(s) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 AAV gene product(s)). For example, in some embodiments, the internal sequence further comprises a nucleic acid sequence encoding for Rep52 (or a portion thereof), a nucleic acid sequence encoding for Rep40 (or a portion thereof), a nucleic acid sequence encoding for Rep78 (or a portion thereof), a nucleic acid sequence encoding for E2A (or a portion thereof), a nucleic acid sequence for E4ORF6 (or a portion thereof), a nucleic acid sequence encoding for VARNA (or a portion thereof), a nucleic acid sequence encoding for VP1 (or a portion thereof), a nucleic acid sequence encoding for VP2 (or a portion thereof), a nucleic acid sequence encoding for VP3 (or a portion thereof), a nucleic acid sequence encoding for AAP (or a portion thereof), a nucleic acid sequence encoding for MAAP (or a portion thereof), or any combination thereof.

In some embodiments, the internal sequence comprises a first portion of a nucleic acid sequence encoding for E2A, but not a second (and remaining) portion of the nucleic acid sequence encoding for E2A, wherein the invertible element is flanked by the second (and remaining) portion, and wherein the first portion is in an inverted orientation relative to the second portion. In some embodiments, the first portion and the second portion of the nucleic acid sequence encoding for E2A collectively comprise a nucleic acid sequence encoding a polypeptide having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 101. In some embodiments, the first portion or the second portion of the nucleic acid sequence encoding for E2A is operably linked to a promoter (as described herein). In some embodiments, the internal sequence further comprises a nucleic acid sequence encoding for one or more additional AAV gene product(s) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 AAV gene product(s)). For example, in some embodiments, the internal sequence further comprises a nucleic acid sequence encoding for Rep52 (or a portion thereof), a nucleic acid sequence encoding for Rep40 (or a portion thereof), a nucleic acid sequence encoding for Rep78 (or a portion thereof), a nucleic acid sequence encoding for Rep68 (or a portion thereof), a nucleic acid sequence for E4ORF6 (or a portion thereof), a nucleic acid sequence encoding for VARNA (or a portion thereof), a nucleic acid sequence encoding for VP1 (or a portion thereof), a nucleic acid sequence encoding for VP2 (or a portion thereof), a nucleic acid sequence encoding for VP3 (or a portion thereof), a nucleic acid sequence encoding for AAP (or a portion thereof), a nucleic acid sequence encoding for MAAP (or a portion thereof), or any combination thereof.

In some embodiments, the internal sequence comprises a first portion of a nucleic acid sequence encoding for E4ORF6, but not a second (and remaining) portion of the nucleic acid sequence encoding for E4ORF6, wherein the invertible element is flanked by the second (and remaining) portion, and wherein the first portion is in an inverted orientation relative to the second portion. In some embodiments, the first portion and the second portion of the nucleic acid sequence encoding for E4ORF6 collectively comprise a nucleic acid sequence encoding a polypeptide having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NOs: 102 or 103. In some embodiments, the first portion or the second portion of the nucleic acid sequence encoding for E4ORF6 is operably linked to a promoter (as described herein). In some embodiments, the internal sequence further comprises a nucleic acid sequence encoding for one or more additional AAV gene product(s) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 AAV gene product(s)). For example, in some embodiments, the internal sequence further comprises a nucleic acid sequence encoding for Rep52 (or a portion thereof), a nucleic acid sequence encoding for Rep40 (or a portion thereof), a nucleic acid sequence encoding for Rep78 (or a portion thereof), a nucleic acid sequence encoding for Rep68 (or a portion thereof), a nucleic acid sequence for E2A (or a portion thereof), a nucleic acid sequence encoding for VARNA (or a portion thereof), a nucleic acid sequence encoding for VP1 (or a portion thereof), a nucleic acid sequence encoding for VP2 (or a portion thereof), a nucleic acid sequence encoding for VP3 (or a portion thereof), a nucleic acid sequence encoding for AAP (or a portion thereof), a nucleic acid sequence encoding for MAAP (or a portion thereof), or any combination thereof.

In some embodiments, the internal sequence comprises a first portion of a nucleic acid sequence encoding for VARNA, but not a second (and remaining) portion of the nucleic acid sequence encoding for VARNA, wherein the invertible element is flanked by the second (and remaining) portion, and wherein the first portion is in an inverted orientation relative to the second portion. In some embodiments, the first portion and the second portion of the nucleic acid sequence encoding for VARNA collectively comprise a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 104. In some embodiments, the first portion or the second portion of the nucleic acid sequence encoding for VARNA is operably linked to a promoter (as described herein). In some embodiments, the internal sequence further comprises a nucleic acid sequence encoding for one or more additional AAV gene product(s) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 AAV gene product(s)). For example, in some embodiments, the internal sequence further comprises a nucleic acid sequence encoding for Rep52 (or a portion thereof), a nucleic acid sequence encoding for Rep40 (or a portion thereof), a nucleic acid sequence encoding for Rep78 (or a portion thereof), a nucleic acid sequence encoding for Rep68 (or a portion thereof), a nucleic acid sequence for E2A (or a portion thereof), a nucleic acid sequence encoding for E4ORF6 (or a portion thereof), a nucleic acid sequence encoding for VP1 (or a portion thereof), a nucleic acid sequence encoding for VP2 (or a portion thereof), a nucleic acid sequence encoding for VP3 (or a portion thereof), a nucleic acid sequence encoding for AAP (or a portion thereof), a nucleic acid sequence encoding for MAAP (or a portion thereof), or any combination thereof.

In some embodiments, the internal sequence comprises a first portion of a nucleic acid sequence encoding for VP1, but not a second (and remaining) portion of the nucleic acid sequence encoding for VP1, wherein the invertible element is flanked by the second (and remaining) portion, and wherein the first portion is in an inverted orientation relative to the second portion. In some embodiments, the first portion and the second portion of the nucleic acid sequence encoding for VP1 collectively comprise a nucleic acid sequence encoding a polypeptide having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 105. In some embodiments, the first portion or the second portion of the nucleic acid sequence encoding for VP1 is operably linked to a promoter (as described herein). In some embodiments, the internal sequence further comprises a nucleic acid sequence encoding for one or more additional AAV gene product(s) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 AAV gene product(s)). For example, in some embodiments, the internal sequence further comprises a nucleic acid sequence encoding for Rep52 (or a portion thereof), a nucleic acid sequence encoding for Rep40 (or a portion thereof), a nucleic acid sequence encoding for Rep78 (or a portion thereof), a nucleic acid sequence encoding for Rep68 (or a portion thereof), a nucleic acid sequence for E2A (or a portion thereof), a nucleic acid sequence encoding for VARNA (or a portion thereof), a nucleic acid sequence encoding for VARNA (or a portion thereof), a nucleic acid sequence encoding for VP2 (or a portion thereof), a nucleic acid sequence encoding for VP3 (or a portion thereof), a nucleic acid sequence encoding for AAP (or a portion thereof), a nucleic acid sequence encoding for MAAP (or a portion thereof), or any combination thereof.

In some embodiments, the internal sequence comprises a first portion of a nucleic acid sequence encoding for VP2, but not a second (and remaining) portion of the nucleic acid sequence encoding for VP2, wherein the invertible element is flanked by the second (and remaining) portion, and wherein the first portion is in an inverted orientation relative to the second portion. In some embodiments, the first portion and the second portion of the nucleic acid sequence encoding for VP2 collectively comprise a nucleic acid sequence encoding a polypeptide having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 106. In some embodiments, the first portion or the second portion of the nucleic acid sequence encoding for VP2 is operably linked to a promoter (as described herein). In some embodiments, the internal sequence further comprises a nucleic acid sequence encoding for one or more additional AAV gene product(s) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 AAV gene product(s)). For example, in some embodiments, the internal sequence further comprises a nucleic acid sequence encoding for Rep52 (or a portion thereof), a nucleic acid sequence encoding for Rep40 (or a portion thereof), a nucleic acid sequence encoding for Rep78 (or a portion thereof), a nucleic acid sequence encoding for Rep68 (or a portion thereof), a nucleic acid sequence for E2A (or a portion thereof), a nucleic acid sequence encoding for VARNA (or a portion thereof), a nucleic acid sequence encoding for VARNA (or a portion thereof), a nucleic acid sequence encoding for VP1 (or a portion thereof), a nucleic acid sequence encoding for VP3 (or a portion thereof), a nucleic acid sequence encoding for AAP (or a portion thereof), a nucleic acid sequence encoding for MAAP (or a portion thereof), or any combination thereof. In some embodiments, the internal sequence comprises a first portion of a nucleic acid sequence encoding for VP3, but not a second (and remaining) portion of the nucleic acid sequence encoding for VP3, wherein the invertible element is flanked by the second (and remaining) portion, and wherein the first portion is in an inverted orientation relative to the second portion. In some embodiments, the first portion and the second portion of the nucleic acid sequence encoding for VP3 collectively comprise a nucleic acid sequence encoding a polypeptide having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 107. In some embodiments, the first portion or the second portion of the nucleic acid sequence encoding for VP3 is operably linked to a promoter (as described herein). In some embodiments, the internal sequence further comprises a nucleic acid sequence encoding for one or more additional AAV gene product(s) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 AAV gene product(s)). For example, in some embodiments, the internal sequence further comprises a nucleic acid sequence encoding for Rep52 (or a portion thereof), a nucleic acid sequence encoding for Rep40 (or a portion thereof), a nucleic acid sequence encoding for Rep78 (or a portion thereof), a nucleic acid sequence encoding for Rep68 (or a portion thereof), a nucleic acid sequence for E2A (or a portion thereof), a nucleic acid sequence encoding for VARNA (or a portion thereof), a nucleic acid sequence encoding for VARNA (or a portion thereof), a nucleic acid sequence encoding for VP1 (or a portion thereof), a nucleic acid sequence encoding for VP2 (or a portion thereof), a nucleic acid sequence encoding for AAP (or a portion thereof), a nucleic acid sequence encoding for MAAP (or a portion thereof), or any combination thereof.

In some embodiments, the internal sequence comprises a first portion of a nucleic acid sequence encoding for AAP, but not a second (and remaining) portion of the nucleic acid sequence encoding for AAP, wherein the invertible element is flanked by the second (and remaining) portion, and wherein the first portion is in an inverted orientation relative to the second portion. In some embodiments, the first portion and the second portion of the nucleic acid sequence encoding for AAP collectively comprise a nucleic acid sequence encoding a polypeptide having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 108. In some embodiments, the first portion or the second portion of the nucleic acid sequence encoding for AAP is operably linked to a promoter (as described herein). In some embodiments, the internal sequence further comprises a nucleic acid sequence encoding for one or more additional AAV gene product(s) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 AAV gene product(s)). For example, in some embodiments, the internal sequence further comprises a nucleic acid sequence encoding for Rep52 (or a portion thereof), a nucleic acid sequence encoding for Rep40 (or a portion thereof), a nucleic acid sequence encoding for Rep78 (or a portion thereof), a nucleic acid sequence encoding for Rep68 (or a portion thereof), a nucleic acid sequence for E2A (or a portion thereof), a nucleic acid sequence encoding for VARNA (or a portion thereof), a nucleic acid sequence encoding for VARNA (or a portion thereof), a nucleic acid sequence encoding for VP1 (or a portion thereof), a nucleic acid sequence encoding for VP2 (or a portion thereof), a nucleic acid sequence encoding for VP1 (or a portion thereof), a nucleic acid sequence encoding for MAAP (or a portion thereof), or any combination thereof.

In some embodiments, the internal sequence comprises a first portion of a nucleic acid sequence encoding for MAAP, but not a second (and remaining) portion of the nucleic acid sequence encoding for MAAP, wherein the invertible element is flanked by the second (and remaining) portion, and wherein the first portion is in an inverted orientation relative to the second portion. In some embodiments, the first portion and the second portion of the nucleic acid sequence encoding for MAAP collectively comprise a nucleic acid sequence encoding a polypeptide having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 229. In some embodiments, the first portion or the second portion of the nucleic acid sequence encoding for MAAP is operably linked to a promoter (as described herein). In some embodiments, the internal sequence further comprises a nucleic acid sequence encoding for one or more additional AAV gene product(s) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 AAV gene product(s)). For example, in some embodiments, the internal sequence further comprises a nucleic acid sequence encoding for Rep52 (or a portion thereof), a nucleic acid sequence encoding for Rep40 (or a portion thereof), a nucleic acid sequence encoding for Rep78 (or a portion thereof), a nucleic acid sequence encoding for Rep68 (or a portion thereof), a nucleic acid sequence for E2A (or a portion thereof), a nucleic acid sequence encoding for VARNA (or a portion thereof), a nucleic acid sequence encoding for VARNA (or a portion thereof), a nucleic acid sequence encoding for VP1 (or a portion thereof), a nucleic acid sequence encoding for VP2 (or a portion thereof), a nucleic acid sequence encoding for VP1 (or a portion thereof), a nucleic acid sequence encoding for AAP (or a portion thereof), or any combination thereof.

In some embodiments, the internal sequence comprises: (1) a first portion of a nucleic acid sequence encoding a first AAV gene product, but not a second (and remaining) portion of a nucleic acid sequence encoding the first AAV gene product, wherein the first portion of the nucleic acid sequence encoding the first AAV gene product is in an inverted orientation relative to the second (and remaining) portion of the nucleic acid sequence encoding the first AAV gene product; and (2) a first portion of a nucleic acid sequence encoding a second AAV gene product, but not a second (and remaining) portion of a nucleic acid sequence encoding the second AAV gene product, wherein the first portion of the nucleic acid sequence encoding the second AAV gene product is in an inverted orientation relative to the second (and remaining) portion of the nucleic acid sequence encoding the second AAV gene product. In such embodiments, the internal sequence may further comprise the nucleic acid sequence encoding for one or more additional AAV gene product(s) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 AAV gene product(s)), wherein the nucleic acid sequence encoding for the one or more additional AAV gene product(s) is positioned between the first portion of the nucleic acid sequence encoding the first AAV gene product and the first portion of the nucleic acid sequence encoding the second AAV.

In some embodiments, the internal sequence comprises: (1) a first portion of a nucleic acid sequence encoding Rep52, but not a second (and remaining) portion of a nucleic acid sequence encoding Rep52, wherein the first portion of the nucleic acid sequence encoding the first Rep52 is in an inverted orientation relative to the second (and remaining) portion of the nucleic acid sequence encoding Rep52 (as described above); and (2) a first portion of a nucleic acid sequence encoding a second AAV gene product (e.g., Rep40, Rep78, Rep68, E2A, E40RF6, VARNA, VP1, VP2, VP3, AAP, or MAAP), but not a second (and remaining) portion of a nucleic acid sequence encoding the second AAV gene product, wherein the first portion of the nucleic acid sequence encoding the second AAV gene product is in an inverted orientation relative to the second (and remaining) portion of the nucleic acid sequence encoding the second AAV gene product.

In some embodiments, the internal sequence comprises: (1) a first portion of a nucleic acid sequence encoding Rep40, but not a second (and remaining) portion of a nucleic acid sequence encoding Rep40, wherein the first portion of the nucleic acid sequence encoding the first Rep40 is in an inverted orientation relative to the second (and remaining) portion of the nucleic acid sequence encoding Rep40 (as described above); and (2) a first portion of a nucleic acid sequence encoding a second AAV gene product (e.g., Rep52, Rep78, Rep68, E2A, E40RF6, VARNA, VP1, VP2, VP3, AAP, or MAAP), but not a second (and remaining) portion of a nucleic acid sequence encoding the second AAV gene product, wherein the first portion of the nucleic acid sequence encoding the second AAV gene product is in an inverted orientation relative to the second (and remaining) portion of the nucleic acid sequence encoding the second AAV gene product.

In some embodiments, the internal sequence comprises: (1) a first portion of a nucleic acid sequence encoding Rep78, but not a second (and remaining) portion of a nucleic acid sequence encoding Rep78, wherein the first portion of the nucleic acid sequence encoding the first Rep78 is in an inverted orientation relative to the second (and remaining) portion of the nucleic acid sequence encoding Rep78 (as described above); and (2) a first portion of a nucleic acid sequence encoding a second AAV gene product (e.g., Rep52, Rep40, Rep68, E2A, E40RF6, VARNA, VP1, VP2, VP3, AAP, or MAAP), but not a second (and remaining) portion of a nucleic acid sequence encoding the second AAV gene product, wherein the first portion of the nucleic acid sequence encoding the second AAV gene product is in an inverted orientation relative to the second (and remaining) portion of the nucleic acid sequence encoding the second AAV gene product.

In some embodiments, the internal sequence comprises: (1) a first portion of a nucleic acid sequence encoding Rep68, but not a second (and remaining) portion of a nucleic acid sequence encoding Rep68, wherein the first portion of the nucleic acid sequence encoding the first Rep68 is in an inverted orientation relative to the second (and remaining) portion of the nucleic acid sequence encoding Rep68 (as described above); and (2) a first portion of a nucleic acid sequence encoding a second AAV gene product (e.g., Rep52, Rep40, Rep78, E2A, E40RF6, VARNA, VP1, VP2, VP3, AAP, or MAAP), but not a second (and remaining) portion of a nucleic acid sequence encoding the second AAV gene product, wherein the first portion of the nucleic acid sequence encoding the second AAV gene product is in an inverted orientation relative to the second (and remaining) portion of the nucleic acid sequence encoding the second AAV gene product.

In some embodiments, the internal sequence comprises: (1) a first portion of a nucleic acid sequence encoding E2A, but not a second (and remaining) portion of a nucleic acid sequence encoding E2A, wherein the first portion of the nucleic acid sequence encoding the first E2A is in an inverted orientation relative to the second (and remaining) portion of the nucleic acid sequence encoding E2A (as described above); and (2) a first portion of a nucleic acid sequence encoding a second AAV gene product (e.g., Rep52, Rep40, Rep68, Rep78, E40RF6, VARNA, VP1, VP2, VP3, AAP, or MAAP), but not a second (and remaining) portion of a nucleic acid sequence encoding the second AAV gene product, wherein the first portion of the nucleic acid sequence encoding the second AAV gene product is in an inverted orientation relative to the second (and remaining) portion of the nucleic acid sequence encoding the second AAV gene product.

In some embodiments, the internal sequence comprises: (1) a first portion of a nucleic acid sequence encoding E4ORF6, but not a second (and remaining) portion of a nucleic acid sequence encoding E4ORF6, wherein the first portion of the nucleic acid sequence encoding the first E4ORF6 is in an inverted orientation relative to the second (and remaining) portion of the nucleic acid sequence encoding E4ORF6 (as described above); and (2) a first portion of a nucleic acid sequence encoding a second AAV gene product (e.g., Rep52, Rep40, Rep68, Rep78, E2A, VARNA, VP1, VP2, VP3, AAP, or MAAP), but not a second (and remaining) portion of a nucleic acid sequence encoding the second AAV gene product, wherein the first portion of the nucleic acid sequence encoding the second AAV gene product is in an inverted orientation relative to the second (and remaining) portion of the nucleic acid sequence encoding the second AAV gene product.

In some embodiments, the internal sequence comprises: (1) a first portion of a nucleic acid sequence encoding VARNA, but not a second (and remaining) portion of a nucleic acid sequence encoding VARNA, wherein the first portion of the nucleic acid sequence encoding the first VARNA is in an inverted orientation relative to the second (and remaining) portion of the nucleic acid sequence encoding VARNA (as described above); and (2) a first portion of a nucleic acid sequence encoding a second AAV gene product (e.g., Rep52, Rep40, Rep68, Rep78, E2A, E4ORF6, VP1, VP2, VP3, AAP, or MAAP), but not a second (and remaining) portion of a nucleic acid sequence encoding the second AAV gene product, wherein the first portion of the nucleic acid sequence encoding the second AAV gene product is in an inverted orientation relative to the second (and remaining) portion of the nucleic acid sequence encoding the second AAV gene product.

In some embodiments, the internal sequence comprises: (1) a first portion of a nucleic acid sequence encoding VP1, but not a second (and remaining) portion of a nucleic acid sequence encoding VP1, wherein the first portion of the nucleic acid sequence encoding the first VP1 is in an inverted orientation relative to the second (and remaining) portion of the nucleic acid sequence encoding VP1 (as described above); and (2) a first portion of a nucleic acid sequence encoding a second AAV gene product (e.g., Rep52, Rep40, Rep68, Rep78, E2A, E4ORF6, VARNA, VP2, VP3, AAP, or MAAP), but not a second (and remaining) portion of a nucleic acid sequence encoding the second AAV gene product, wherein the first portion of the nucleic acid sequence encoding the second AAV gene product is in an inverted orientation relative to the second (and remaining) portion of the nucleic acid sequence encoding the second AAV gene product.

In some embodiments, the internal sequence comprises: (1) a first portion of a nucleic acid sequence encoding VP2, but not a second (and remaining) portion of a nucleic acid sequence encoding VP2, wherein the first portion of the nucleic acid sequence encoding the first VP2 is in an inverted orientation relative to the second (and remaining) portion of the nucleic acid sequence encoding VP2 (as described above); and (2) a first portion of a nucleic acid sequence encoding a second AAV gene product (e.g., Rep52, Rep40, Rep68, Rep78, E2A, E4ORF6, VARNA, VP1, VP3, AAP, or MAAP), but not a second (and remaining) portion of a nucleic acid sequence encoding the second AAV gene product, wherein the first portion of the nucleic acid sequence encoding the second AAV gene product is in an inverted orientation relative to the second (and remaining) portion of the nucleic acid sequence encoding the second AAV gene product.

In some embodiments, the internal sequence comprises: (1) a first portion of a nucleic acid sequence encoding VP3, but not a second (and remaining) portion of a nucleic acid sequence encoding VP3, wherein the first portion of the nucleic acid sequence encoding the first VP3 is in an inverted orientation relative to the second (and remaining) portion of the nucleic acid sequence encoding VP2 (as described above); and (2) a first portion of a nucleic acid sequence encoding a second AAV gene product (e.g., Rep52, Rep40, Rep68, Rep78, E2A, E4ORF6, VARNA, VP1, VP2, AAP, or MAAP), but not a second (and remaining) portion of a nucleic acid sequence encoding the second AAV gene product, wherein the first portion of the nucleic acid sequence encoding the second AAV gene product is in an inverted orientation relative to the second (and remaining) portion of the nucleic acid sequence encoding the second AAV gene product.

In some embodiments, the internal sequence comprises: (1) a first portion of a nucleic acid sequence encoding AAP, but not a second (and remaining) portion of a nucleic acid sequence encoding AAP, wherein the first portion of the nucleic acid sequence encoding the first AAP is in an inverted orientation relative to the second (and remaining) portion of the nucleic acid sequence encoding AAP (as described above); and (2) a first portion of a nucleic acid sequence encoding a second AAV gene product (e.g., Rep52, Rep40, Rep68, Rep78, E2A, E4ORF6, VARNA, VP1, VP2, VP3, or MAAP), but not a second (and remaining) portion of a nucleic acid sequence encoding the second AAV gene product, wherein the first portion of the nucleic acid sequence encoding the second AAV gene product is in an inverted orientation relative to the second (and remaining) portion of the nucleic acid sequence encoding the second AAV gene product.

In some embodiments, the internal sequence comprises: (1) a first portion of a nucleic acid sequence encoding MAAP, but not a second (and remaining) portion of a nucleic acid sequence encoding MAAP, wherein the first portion of the nucleic acid sequence encoding the first MAAP is in an inverted orientation relative to the second (and remaining) portion of the nucleic acid sequence encoding MAAP (as described above); and (2) a first portion of a nucleic acid sequence encoding a second AAV gene product (e.g., Rep52, Rep40, Rep68, Rep78, E2A, E4ORF6, VARNA, VP1, VP2, VP3, or AAP), but not a second (and remaining) portion of a nucleic acid sequence encoding the second AAV gene product, wherein the first portion of the nucleic acid sequence encoding the second AAV gene product is in an inverted orientation relative to the second (and remaining) portion of the nucleic acid sequence encoding the second AAV gene product.

In some embodiments, wherein the internal sequence comprises:(l) a first portion of a nucleic acid sequence encoding a first AAV gene product, but not a second (and remaining) portion of a nucleic acid sequence encoding the first AAV gene product, wherein the first portion of the nucleic acid sequence encoding the first AAV gene product is in an inverted orientation relative to the second (and remaining) portion of the nucleic acid sequence encoding the first AAV gene product and (2) a first portion of a nucleic acid sequence encoding a second AAV gene product, but not a second (and remaining) portion of a nucleic acid sequence encoding the second AAV gene product, wherein the first portion of the nucleic acid sequence encoding the second AAV gene product is in an inverted orientation relative to the second (and remaining) portion of the nucleic acid sequence encoding the second AAV gene product (as described above), the internal sequence further comprises the nucleic acid sequence encoding for one or more additional AAV gene product(s) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 AAV gene product(s)), wherein the nucleic acid sequence encoding for the one or more additional AAV gene product(s) is flanked on one end by the nucleic acid sequence encoding the first portion of the first AAV gene product, and on the other end by the nucleic acid sequence encoding the first portion of the second AAV gene product.

In some embodiments, wherein the internal sequence comprises (1) a first portion of a nucleic acid sequence encoding a first AAV gene product, but not a second (and remaining) portion of a nucleic acid sequence encoding the first AAV gene product, wherein the first portion of the nucleic acid sequence encoding the first AAV gene product is in an inverted orientation relative to the second (and remaining) portion of the nucleic acid sequence encoding the first AAV gene product and (2) a first portion of a nucleic acid sequence encoding a second AAV gene product, but not a second (and remaining) portion of a nucleic acid sequence encoding the second AAV gene product, wherein the first portion of the nucleic acid sequence encoding the second AAV gene product is in an inverted orientation relative to the second (and remaining) portion of the nucleic acid sequence encoding the second AAV gene product (as described above), the first portion of the first AAV gene product is operably linked to a promoter (as described herein). In some embodiments, the second portion of the first AAV gene product is operably linked to a promoter (as described herein). In some embodiment, the first portion of the second AAV gene product is operably linked to a promoter (as described herein). In some embodiments, the second portion of the second AAV gene product is operably linked to a promoter (as described herein). In some embodiments, the first portion of the first AAV gene product is operably linked to a first promoter, and the first or second portion of the second AAV gene product is operably linked to a second promoter.

3. Intron Splice Donors/Acceptors

In some embodiments, one or more of the polynucleic acids of an AAV production component comprises a nucleic acid sequence of an intron splice donor, a nucleic acid sequence of an intron splice acceptor, or a combination thereof. Numerous intron splice donor and intron splice acceptors sequences are known to those having ordinary skill in the art.

In some embodiments, an intron splice donor comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of any one of SEQ ID NOs: 1-2.

In some embodiments, an intron splice acceptor comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of any one of SEQ ID NOs: 3-4.

In some embodiments, an invertible element comprises a nucleic acid sequence of an intron splice acceptor. In some embodiments, an invertible element comprises a nucleic acid sequence of an intron splice donor.

In some embodiments, an invertible element comprises a nucleic acid sequence of an intron splice acceptor and a nucleic acid sequence of an intron splice donor. In some embodiments the nucleic acid sequence of the intron splice acceptor and the nucleic acid sequence of the intron splice donor are in the same orientation. In some embodiments, an invertible element comprises a nucleic acid sequence of a first intron splice acceptor and a nucleic acid sequence of a second intron splice acceptor. In some embodiments the nucleic acid sequence of the first intron splice acceptor and the nucleic acid sequence of the second intron splice acceptor are in opposite orientations.

In some embodiments, an invertible element comprises a nucleic acid sequence of a first intron splice donor and a nucleic acid sequence of a second intron splice donor. In some embodiments the nucleic acid sequence of the first intron splice donor and the nucleic acid sequence of the second intron splice donor are in opposite orientations.

4. Exemplary Architecture 1

In some embodiments, an AAV production system comprises an AAV production component comprising a polynucleic acid molecule having a first segment and a second segment, wherein the second segment is inverted relative to the first segment, and wherein: (a) the first segment comprises, from 5’ to 3’: a first portion of a nucleic acid sequence encoding for a first AAV gene product; and (b) the second segment comprises, from 5’ to 3’ : a first recombinase attachment site; a second portion of the nucleic acid sequence encoding for the first AAV gene product; and a second recombinase attachment site; and wherein the first recombinase attachment site and the second recombinase attachment site correspond to the same recombinase. In some embodiments, the first portion of the nucleic acid sequence encoding for the first AAV gene product is operably linked to a promoter. In some embodiments, the second portion of the nucleic acid sequence encoding for the first AAV gene product is operably linked to a promoter.

In some embodiments, the nucleic acid sequence encoding for the AAV gene product is comprised within the first portion of the nucleic acid sequence encoding for the AAV gene product and the second portion of the nucleic acid sequence encoding for the AAV gene product. In some embodiments, the first segment further comprises a nucleic acid encoding for at least a portion of an additional AAV gene product (e.g., at least a portion of one or more of Rep52, Rep40, Rep78, Rep68, E2A, E40rf6, VARNA, VP1, VP2, VP3, AAP, and MAAP). In some embodiments, the second segment further comprises a nucleic acid encoding for at least a portion of an additional AAV gene product (e.g., at least a portion of one or more of Rep52, Rep40, Rep78, Rep68, E2A, E40rf6, VARNA, VP1, VP2, VP3, AAP, and MAAP).

In some embodiments, the polynucleic acid molecule having the first segment and the second segment further comprises a third segment, wherein the third segment is in the same orientation as the first segment, and wherein the polynucleic acid molecule comprises the structure: first segment - second segment - third segment, and wherein the third segment comprises a third portion of the nucleic acid sequence encoding for the AAV gene product. In some embodiments, the nucleic acid sequence encoding for the AAV gene product is comprised within the first portion of the nucleic acid sequence encoding for the AAV gene product, the second portion of the nucleic acid sequence encoding for the AAV gene product, and the third portion of the nucleic acid sequence encoding for the AAV gene product.

In some embodiments, the first recombinase attachment site comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity with the nucleic acid sequence of any one of SEQ ID NOs: 5-96. In some embodiments, the second recombinase attachment site comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity with the nucleic acid sequence of any one of SEQ ID NOs: 5-96.

In some embodiments, the first segment comprises, from 5’ to 3’ : a promoter operably linked to the first portion of the nucleic acid sequence encoding for the first AAV gene product; and an intron splice donor; and the second segment comprises, from 5’ to 3’ : the first recombinase attachment site; an intron splice acceptor; and the second portion of the nucleic acid sequence encoding for the first AAV gene product; and the second recombinase attachment site. In some embodiments, the first segment comprises, from 5’ to 3’: the promoter operably linked to the first portion of the nucleic acid sequence encoding for the first AAV gene product; the intron splice donor; and a nucleic acid sequence encoding for a stop codon (or a tandem repeat of at least two or at least three stop codons), wherein the nucleic acid sequence encoding for the stop codon is in frame with the first portion of the nucleic acid sequence encoding for the first AAV gene product. In some embodiments, the second segment comprises, from 5’ to 3’ : the first recombinase attachment site; the intron splice acceptor; the second portion of the nucleic acid sequence encoding for the first AAV gene product; a nucleic acid sequence encoding for a stop codon (or a tandem repeat of at least two or at least three stop codons), wherein the nucleic acid sequence encoding for the stop codon is in frame with the first portion of the nucleic acid sequence encoding for the first AAV gene product; and the second recombinase attachment site. In some embodiments, the first segment comprises, from 5’ to 3’: an intron splice acceptor; the first portion of the nucleic acid sequence encoding for the first AAV gene product; and the second segment comprises, from 5’ to 3’ : the first recombinase attachment site; a promoter operably linked to the second portion of the nucleic acid sequence encoding for the first AAV gene product; an intron splice donor; and the second recombinase attachment site. In some embodiments, the first segment comprises, from 5’ to 3’: a nucleic acid sequence encoding for a stop codon (or a tandem repeat of at least two or at least three stop codons), wherein the nucleic acid sequence encoding for the stop codon is in frame with the second portion of the nucleic acid sequence encoding for the first AAV gene product; the intron splice acceptor; the first portion of the nucleic acid sequence encoding for the first AAV gene product. In some embodiments, the second segment comprises, from 5’ to 3’ : the first recombinase attachment site; the promoter operably linked to the second portion of the first AAV gene product; the intron splice donor; a nucleic acid sequence encoding for a stop codon (or a tandem repeat of at least two or at least three stop codons), wherein the nucleic acid sequence encoding for the stop codon is in frame with the second portion of the nucleic acid sequence encoding for the first AAV gene product; and the second recombinase attachment site.

In some embodiments, the intron splice donor comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity with the nucleic acid sequence of any one of SEQ ID NOs: 1-2. In some embodiments, the intron splice acceptor comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity with the nucleic acid sequence of any one of SEQ ID NOs: 3-4.

In some embodiments, the first AAV gene product is selected from the group consisting of Rep52, Rep40, Rep78, Rep68, E2A, E40rf6, VARNA, VP1, VP2, VP3, AAP, and MAAP.

5. Exemplary Architecture 2

In some embodiments, an AAV production system comprises a polynucleic acid molecule having the structure: first segment - second segment - third segment, wherein the second segment is inverted relative to the first segment and the third segment, and wherein:

(a) the first segment comprises, from 5’ to 3’: a promoter operably linked to a first portion of a nucleic acid sequence encoding for a first AAV gene product; and a first intron splice donor;

(b) the second segment is an invertible element comprising, from 5’ to 3’: a first recombinase attachment site; a first intron splice acceptor; a second portion of the nucleic acid sequence encoding for the first AAV gene product; a promoter operably linked to a first portion of a nucleic acid sequence encoding for a second AAV gene product; and second splice intron donor; and a second recombinase attachment site; and (c) the third segment comprises, from 5’ to 3’ : a second intron splice acceptor; and a second portion of the nucleic acid sequence encoding for the second AAV gene product; and wherein the first recombinase attachment site and the second recombinase attachment site correspond to the same recombinase.

In some embodiments, the first segment comprises, from 5’ to 3’: the promoter operably linked to the first portion of the nucleic acid sequence encoding for the first AAV gene product; the first intron splice donor; and a nucleic acid sequence encoding for a stop codon (or a tandem repeat of at least two or at least three stop codons), wherein the nucleic acid sequence encoding for the stop codon is in frame with the first portion of the nucleic acid sequence encoding for the first AAV gene product and/or the first portion of the nucleic acid sequence encoding for the second AAV gene product.

In some embodiments, the second segment comprises, from 5’ to 3’: the first recombinase attachment site; the first intron splice acceptor; the second portion of the nucleic acid sequence encoding for the first AAV gene product; the promoter operably linked to the first portion of the nucleic acid sequence encoding for the second AAV gene product; the second splice intron donor; a nucleic acid sequence encoding for a stop codon (or a tandem repeat of at least two or at least three stop codons), wherein the nucleic acid sequence encoding for the stop codon is in frame with the first portion of the nucleic acid sequence encoding for the first AAV gene product and/or the first portion of the nucleic acid sequence encoding for the second AAV gene product; and a second recombinase attachment site.

In some embodiments, the first intron splice donor comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity with the nucleic acid sequence of any one of SEQ ID NOs: 1-2, the second intron splice donor comprises a nucleic acid sequence having at least 80% identity with the nucleic acid sequence of any one of SEQ ID NOs: 1-2, or a combination thereof. In some embodiments, the first intron splice acceptor comprises a nucleic acid sequence having at least 80% identity with the nucleic acid sequence of any one of SEQ ID NOs: 3-4; the second intron splice acceptor comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity with the nucleic acid sequence of any one of SEQ ID NOs: 3-4, or a combination thereof.

In some embodiments, the first AAV gene product is E2A, and the second AAV gene product is E40rf6. In some embodiments, the first AAV gene product is E40rf6, and the second AAV gene product is E2A.

In some embodiments, the first AAV gene product is Rep52 and the second AAV gene product is VP1, VP2, VP3, or AAP. In some embodiments, the second AAV gene product is VP1. In some embodiments, the second gene product is VP2. In some embodiments, the second gene product is VP3. In some embodiments, the second gene product is AAP.

In some embodiments, the first AAV gene product is Rep40 and the second AAV gene product is VP1, VP2, VP3, or AAP. In some embodiments, the second AAV gene product is VP1. In some embodiments, the second gene product is VP2. In some embodiments, the second gene product is VP3. In some embodiments, the second gene product is AAP.

In some embodiments, the first AAV gene product is Rep78 and the second AAV gene product is VP1, VP2, VP3, or AAP. In some embodiments, the second AAV gene product is VP1. In some embodiments, the second gene product is VP2. In some embodiments, the second gene product is VP3. In some embodiments, the second gene product is AAP.

In some embodiments, the first AAV gene product is Rep68 and the second AAV gene product is VP1, VP2, VP3, or AAP. In some embodiments, the second AAV gene product is VP1. In some embodiments, the second gene product is VP2. In some embodiments, the second gene product is VP3. In some embodiments, the second gene product is AAP.

In some embodiments, the first AAV gene product is VP1 and the second AAV gene product is Rep52, Rep40, Rep68, Rep78, or AAP. In some embodiments, the second AAV gene product is Rep52. In some embodiments, the second gene product is Rep40. In some embodiments, the second gene product is Rep68. In some embodiments, the second gene product is Rep78. In some embodiments, the second gene product is AAP.

In some embodiments, the first AAV gene product is VP2 and the second AAV gene product is Rep52, Rep40, Rep68, Rep78, or AAP. In some embodiments, the second AAV gene product is Rep52. In some embodiments, the second gene product is Rep40. In some embodiments, the second gene product is Rep68. In some embodiments, the second gene product is Rep78. In some embodiments, the second gene product is AAP. In some embodiments, the first AAV gene product is VP3 and the second AAV gene product is Rep52, Rep40, Rep68, Rep78, or AAP. In some embodiments, the second AAV gene product is Rep52. In some embodiments, the second gene product is Rep40. In some embodiments, the second gene product is Rep68. In some embodiments, the second gene product is Rep78. In some embodiments, the second gene product is AAP.

In some embodiments, the first segment further comprises a nucleic acid sequence encoding for one or more additional AAV gene product (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional AAV gene products).

In some embodiments, the second segment further comprises a nucleic acid sequence encoding for one or more additional AAV gene product (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional AAV gene products).

In some embodiments, the third segment further comprises a nucleic acid sequence encoding for one or more additional AAV gene product (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional AAV gene products).

In some embodiments, an AAV production system comprises a polynucleic acid molecule as depicted in FIG. 1 A.

In some embodiments, an AAV production system comprises a polynucleic acid molecule as depicted in FIG. 2.

6. Exemplary Architecture 3

In some embodiments, an AAV production system comprises a polynucleic acid molecule having the structure: first segment - second segment - third segment - fourth segment, wherein the first segment and the second segment are inverted relative to the third segment and the fourth segment, wherein an invertible element comprises the second segment and the third segment, and wherein: (a) the third segment comprises, from 5’ to 3’ : a promoter operably linked to a first portion of a nucleic acid sequence encoding for a first AAV gene product; a first intron splice donor; and a first recombinase attachment site; (b) the first segment comprises, from 5’ to 3’ : a first intron splice acceptor; and a second portion of the nucleic acid sequence encoding for the first AAV gene product; (c) the second segment comprises, from 5’ to 3’: a promoter operably linked to a first portion of a nucleic acid sequence encoding for a second AAV gene product; a second intron splice donor; and a second recombinase attachment site; and (d) the fourth segment comprises, from 5’ to 3’: a second intron splice acceptor; and a second portion of the nucleic acid sequence encoding for the second AAV gene product; and wherein the first recombinase attachment site and the second recombinase attachment site correspond to the same recombinase.

In some embodiments, the first segment comprises, from 5’ to 3’: the first intron splice acceptor; a nucleic acid sequence encoding for a stop codon (or a tandem repeat of at least two or at least three stop codons); and the second portion of the nucleic acid sequence encoding for the first AAV gene product; wherein the nucleic acid sequence encoding for the stop codon is in frame with the nucleic acid sequence encoding for the first portion of the second AAV gene product, but out of frame with the nucleic acid sequence encoding for the second portion of the first AAV gene product.

In some embodiments, the fourth segment comprises, from 5’ to 3’ : the second intron splice acceptor; a nucleic acid sequence encoding for a stop codon (or a tandem repeat of at least two or at least three stop codons); and the second portion of the nucleic acid sequence encoding for the second AAV gene product; wherein the nucleic acid sequence encoding for the stop codon is in frame with the nucleic acid sequence encoding for the first portion of the first AAV gene product, but out of frame with the nucleic acid sequence encoding for the second portion of the second AAV gene product.

In some embodiments, the first intron splice donor comprises a nucleic acid sequence having at least 80% identity with the nucleic acid sequence of any one of SEQ ID NOs: 1-2, the second intron splice donor comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity with the nucleic acid sequence of any one of SEQ ID NOs: 1-2, or a combination thereof. In some embodiments, the first intron splice acceptor comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity with the nucleic acid sequence of any one of SEQ ID NOs: 3-4; the second intron splice acceptor comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity with the nucleic acid sequence of any one of SEQ ID NOs: 3-4, or a combination thereof. In some embodiments, the first AAV gene product is E2A, and the second AAV gene product is E40rf6. In some embodiments, the first AAV gene product is E40rf6, and the second AAV gene product is E2A.

In some embodiments, the first AAV gene product is VP2 and the second AAV gene product is Rep52, Rep40, Rep68, Rep78, or AAP. In some embodiments, the second AAV gene product is Rep52. In some embodiments, the second gene product is Rep40. In some embodiments, the second gene product is Rep68. In some embodiments, the second gene product is Rep78. In some embodiments, the second gene product is AAP.

In some embodiments, the first AAV gene product is VP3 and the second AAV gene product is Rep52, Rep40, Rep68, Rep78, or AAP. In some embodiments, the second AAV gene product is Rep52. In some embodiments, the second gene product is Rep40. In some embodiments, the second gene product is Rep68. In some embodiments, the second gene product is Rep78. In some embodiments, the second gene product is AAP. In some embodiments, the first segment further comprises a nucleic acid sequence encoding for one or more additional AAV gene product (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional AAV gene products).

In some embodiments, the fourth segment further comprises a nucleic acid sequence encoding for one or more additional AAV gene product (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional AAV gene products).

In some embodiments, an AAV production system comprises a polynucleic acid molecule as depicted in FIG. IB.

7. Exemplary Architecture 4

In some embodiments, the second segment comprises, from 5’ to 3’: the second recombinase attachment site; the second intron splice acceptor; a nucleic acid sequence encoding for a stop codon; and the second portion of the nucleic acid sequence encoding for the second AAV gene product; wherein the nucleic acid sequence encoding for the stop codon is in frame with the nucleic acid sequence encoding for the first portion of the first AAV gene product, but out of frame with the nucleic acid sequence encoding for the second portion of the second AAV gene product.

In some embodiments, the third segment comprises, from 5’ to 3’: the first recombinase attachment site; the first intron splice acceptor; a nucleic acid sequence encoding for a stop codon; and the second portion of the nucleic acid sequence encoding for the first AAV gene product; wherein the nucleic acid sequence encoding for the stop codon is in frame with the nucleic acid sequence encoding for the first portion of the second AAV gene product, but out of frame with the nucleic acid sequence encoding for the second portion of the first AAV gene product.

In some embodiments, the first intron splice donor comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity with the nucleic acid sequence of any one of SEQ ID NOs: 1-2, the second intron splice donor comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity with the nucleic acid sequence of any one of SEQ ID NOs: 1-2, or a combination thereof. In some embodiments, the first intron splice acceptor comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity with the nucleic acid sequence of any one of SEQ ID NOs: 3-4; the second intron splice acceptor comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity with the nucleic acid sequence of any one of SEQ ID NOs: 3-4, or a combination thereof.

In some embodiments, the first AAV gene product is VP3 and the second AAV gene product is Rep52, Rep40, Rep68, Rep78, or AAP. In some embodiments, the second AAV gene product is Rep52. In some embodiments, the second gene product is Rep40. In some embodiments, the second gene product is Rep68. In some embodiments, the second gene product is Rep78. In some embodiments, the second gene product is AAP.

In some embodiments, the first segment further comprises a nucleic acid sequence encoding for one or more additional AAV gene product (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional AAV gene products). In some embodiments, the second segment further comprises a nucleic acid sequence encoding for one or more additional AAV gene product (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional AAV gene products).

In some embodiments, an AAV production system comprises a polynucleic acid molecule as depicted in FIG. 1C. b. The Expression Control Component

In some embodiments, an AAV production system comprises one or more polynucleic acid molecules collectively comprising an expression control component. An expression control component is used to control expression of one or more genes required for AAV production. In some embodiments, the expression control component comprises a recombinase (or a polynucleic acid molecule encoding the same). Exemplary recombinases, and their corresponding recombinase attachment sites, are known to those having skill in the art and include, but are not limited to, PhiC31 recombinases, Bxbl recombinases, Cre recombinases, VCre recombinases, Flp recombinases, TP901 recombinases, R4 recombinases, and Dre recombinases.

In some embodiments, an expression control component comprises two or more recombinases (or polynucleic acid molecules encoding the same).

In some embodiments, an expression control component comprises a polynucleic acid molecule comprising a nucleic acid sequence encoding for a recombinase operably linked to a promoter (as described herein), such as an inducible promoter.

In some embodiments, an expression control component comprises a PhiC31 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, a PhiC31 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 140. In some embodiments, a Phi3Cl recombinase comprises the amino acid sequence SEQ ID NO: 140. In some embodiments, a nucleic acid sequence encoding for a PhiC31 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 182. In some embodiments, a nucleic acid sequence encoding for a PhiC31 recombinase comprises the nucleic acid sequence of SEQ ID NO: 182. In some embodiments, an expression control component comprises a polynucleic acid encoding for a PhiC31 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises a Cre recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, a Cre recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 141. In some embodiments, a Cre recombinase comprises the amino acid sequence SEQ ID NO: 141. In some embodiments, a nucleic acid sequence encoding for a Cre recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 183. In some embodiments, a nucleic acid sequence encoding for a Cre recombinase comprises the nucleic acid sequence of SEQ ID NO: 183. In some embodiments, an expression control component comprises a polynucleic acid encoding for a Cre recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises a VCre recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, a VCre recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 142. In some embodiments, a VCre recombinase comprises the amino acid sequence SEQ ID NO: 142. In some embodiments, a nucleic acid sequence encoding for a VCre recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 184. In some embodiments, a nucleic acid sequence encoding for a VCre recombinase comprises the nucleic acid sequence of SEQ ID NO: 184. In some embodiments, an expression control component comprises a polynucleic acid encoding for a VCre recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises a Flp recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, a Flp recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 143. In some embodiments, a Flp recombinase comprises the amino acid sequence SEQ ID NO: 143. In some embodiments, a nucleic acid sequence encoding for a Flp recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 185. In some embodiments, a nucleic acid sequence encoding for a Flp recombinase comprises the nucleic acid sequence of SEQ ID NO: 185. In some embodiments, an expression control component comprises a polynucleic acid encoding for a Flp recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises a Bxbl recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, a Bxbl recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 144. In some embodiments, a Bxbl recombinase comprises the amino acid sequence SEQ ID NO: 144. In some embodiments, a nucleic acid sequence encoding for a Bxbl recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 186. In some embodiments, a nucleic acid sequence encoding for a Bxbl recombinase comprises the nucleic acid sequence of SEQ ID NO: 186. In some embodiments, an expression control component comprises a polynucleic acid encoding for a Bxbl recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises a TP901 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, a TP901 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 145. In some embodiments, a TP901 recombinase comprises the amino acid sequence SEQ ID NO: 145. In some embodiments, a nucleic acid sequence encoding for a TP901 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 187. In some embodiments, a nucleic acid sequence encoding for a TP901 recombinase comprises the nucleic acid sequence of SEQ ID NO: 187. In some embodiments, an expression control component comprises a polynucleic acid encoding for a TP901 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises a R4 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, a R4 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 146. In some embodiments, a R4 recombinase comprises the amino acid sequence SEQ ID NO: 146. In some embodiments, a nucleic acid sequence encoding for a R4 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 188. In some embodiments, a nucleic acid sequence encoding for a R4 recombinase comprises the nucleic acid sequence of SEQ ID NO: 188. In some embodiments, an expression control component comprises a polynucleic acid encoding for a R4 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises a Dre recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, a Dre recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 147. In some embodiments, a Dre recombinase comprises the amino acid sequence SEQ ID NO: 147. In some embodiments, a nucleic acid sequence encoding for a Dre recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 189. In some embodiments, a nucleic acid sequence encoding for a Dre recombinase comprises the nucleic acid sequence of SEQ ID NO: 189. In some embodiments, an expression control component comprises a polynucleic acid encoding for a Dre recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Inti recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Inti recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 148. In some embodiments, an Inti recombinase comprises the amino acid sequence SEQ ID NO: 148. In some embodiments, a nucleic acid sequence encoding for an Inti recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 190. In some embodiments, a nucleic acid sequence encoding for an Inti recombinase comprises the nucleic acid sequence of SEQ ID NO: 190. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Inti recombinase operably linked to a promoter (as described herein). In some embodiments, an expression control component comprises an Int2 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Int2 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 149. In some embodiments, an Int2 recombinase comprises the amino acid sequence SEQ ID NO: 149. In some embodiments, a nucleic acid sequence encoding for an Int2 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 191. In some embodiments, a nucleic acid sequence encoding for an Int2 recombinase comprises the nucleic acid sequence of SEQ ID NO: 191. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Int2 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Int3 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Int3 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 150. In some embodiments, an Int3 recombinase comprises the amino acid sequence SEQ ID NO: 150. In some embodiments, a nucleic acid sequence encoding for an Int3 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 192. In some embodiments, a nucleic acid sequence encoding for an Int3 recombinase comprises the nucleic acid sequence of SEQ ID NO: 192. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Int3 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Int4 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Int4 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 151. In some embodiments, an Int4 recombinase comprises the amino acid sequence SEQ ID NO: 151. In some embodiments, a nucleic acid sequence encoding for an Int4 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 193. In some embodiments, a nucleic acid sequence encoding for an Int4 recombinase comprises the nucleic acid sequence of SEQ ID NO: 193. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Int4 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Int5 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Int5 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 152. In some embodiments, an Int5 recombinase comprises the amino acid sequence SEQ ID NO: 152. In some embodiments, a nucleic acid sequence encoding for an Int5 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 194. In some embodiments, a nucleic acid sequence encoding for an Int5 recombinase comprises the nucleic acid sequence of SEQ ID NO: 194. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Int5 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Int6 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Int6 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 153. In some embodiments, an Int6 recombinase comprises the amino acid sequence SEQ ID NO: 153. In some embodiments, a nucleic acid sequence encoding for an Int6 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 195. In some embodiments, a nucleic acid sequence encoding for an Int6 recombinase comprises the nucleic acid sequence of SEQ ID NO: 195. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Int6 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Int7 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Int7 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 154. In some embodiments, an Int7 recombinase comprises the amino acid sequence SEQ ID NO: 154. In some embodiments, a nucleic acid sequence encoding for an Int7 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 196. In some embodiments, a nucleic acid sequence encoding for an Int7 recombinase comprises the nucleic acid sequence of SEQ ID NO: 196. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Int7 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Int8 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Int8 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 155. In some embodiments, an Int8 recombinase comprises the amino acid sequence SEQ ID NO: 155. In some embodiments, a nucleic acid sequence encoding for an Int8 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 197. In some embodiments, a nucleic acid sequence encoding for an Int8 recombinase comprises the nucleic acid sequence of SEQ ID NO: 197. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Int8 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Int9 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Int9 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 156. In some embodiments, an Int9 recombinase comprises the amino acid sequence SEQ ID NO: 156. In some embodiments, a nucleic acid sequence encoding for an Int9 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 198. In some embodiments, a nucleic acid sequence encoding for an Int9 recombinase comprises the nucleic acid sequence of SEQ ID NO: 198. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Int9 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an IntlO recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an IntlO recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 157. In some embodiments, an IntlO recombinase comprises the amino acid sequence SEQ ID NO: 157. In some embodiments, a nucleic acid sequence encoding for an Inti 0 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 199. In some embodiments, a nucleic acid sequence encoding for an IntlO recombinase comprises the nucleic acid sequence of SEQ ID NO: 199. In some embodiments, an expression control component comprises a polynucleic acid encoding for an IntlO recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Inti 1 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Inti 1 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 158. In some embodiments, an Inti 1 recombinase comprises the amino acid sequence SEQ ID NO: 158. In some embodiments, a nucleic acid sequence encoding for an Inti 1 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 200. In some embodiments, a nucleic acid sequence encoding for an Inti 1 recombinase comprises the nucleic acid sequence of SEQ ID NO: 200. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Inti 1 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Intl2 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Intl2 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 159. In some embodiments, an Intl2 recombinase comprises the amino acid sequence SEQ ID NO: 159. In some embodiments, a nucleic acid sequence encoding for an Intl2 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 201. In some embodiments, a nucleic acid sequence encoding for an Intl2 recombinase comprises the nucleic acid sequence of SEQ ID NO: 201. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Intl2 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Inti 3 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Intl3 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 160. In some embodiments, an Intl3 recombinase comprises the amino acid sequence SEQ ID NO: 160. In some embodiments, a nucleic acid sequence encoding for an Inti 3 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 202. In some embodiments, a nucleic acid sequence encoding for an Intl3 recombinase comprises the nucleic acid sequence of SEQ ID NO: 202. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Inti 3 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Intl4 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Intl4 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 161. In some embodiments, an Intl4 recombinase comprises the amino acid sequence SEQ ID NO: 161. In some embodiments, a nucleic acid sequence encoding for an Intl4 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 203. In some embodiments, a nucleic acid sequence encoding for an Intl4 recombinase comprises the nucleic acid sequence of SEQ ID NO: 203. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Intl4 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Inti 5 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Inti 5 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 162. In some embodiments, an Intl5 recombinase comprises the amino acid sequence SEQ ID NO: 162. In some embodiments, a nucleic acid sequence encoding for an Inti 5 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 204. In some embodiments, a nucleic acid sequence encoding for an Inti 5 recombinase comprises the nucleic acid sequence of SEQ ID NO: 204. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Inti 5 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Inti 6 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Inti 6 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 163. In some embodiments, an Intl6 recombinase comprises the amino acid sequence SEQ ID NO: 163. In some embodiments, a nucleic acid sequence encoding for an Inti 6 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 205. In some embodiments, a nucleic acid sequence encoding for an Intl6 recombinase comprises the nucleic acid sequence of SEQ ID NO: 205. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Inti 6 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Inti 7 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Intl7 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 164. In some embodiments, an Intl7 recombinase comprises the amino acid sequence SEQ ID NO: 164. In some embodiments, a nucleic acid sequence encoding for an Inti 7 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 206. In some embodiments, a nucleic acid sequence encoding for an Intl7 recombinase comprises the nucleic acid sequence of SEQ ID NO: 206. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Inti 7 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Inti 8 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Inti 8 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 165. In some embodiments, an Intl8 recombinase comprises the amino acid sequence SEQ ID NO: 165. In some embodiments, a nucleic acid sequence encoding for an Inti 8 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 207. In some embodiments, a nucleic acid sequence encoding for an Inti 8 recombinase comprises the nucleic acid sequence of SEQ ID NO: 207. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Inti 8 recombinase operably linked to a promoter (as described herein). In some embodiments, an expression control component comprises an Inti 9 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Intl9 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 166. In some embodiments, an Intl9 recombinase comprises the amino acid sequence SEQ ID NO: 166. In some embodiments, a nucleic acid sequence encoding for an Inti 9 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 208. In some embodiments, a nucleic acid sequence encoding for an Intl9 recombinase comprises the nucleic acid sequence of SEQ ID NO: 208. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Inti 9 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Int20 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Int20 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 167. In some embodiments, an Int20 recombinase comprises the amino acid sequence SEQ ID NO: 167. In some embodiments, a nucleic acid sequence encoding for an Int20 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 209. In some embodiments, a nucleic acid sequence encoding for an Int20 recombinase comprises the nucleic acid sequence of SEQ ID NO: 209. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Int20 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Int21 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Int21 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 168. In some embodiments, an Int21 recombinase comprises the amino acid sequence SEQ ID NO: 168. In some embodiments, a nucleic acid sequence encoding for an Int21 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 210. In some embodiments, a nucleic acid sequence encoding for an Int21 recombinase comprises the nucleic acid sequence of SEQ ID NO: 210. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Int21 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Int22 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Int22 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 169. In some embodiments, an Int22 recombinase comprises the amino acid sequence SEQ ID NO: 169. In some embodiments, a nucleic acid sequence encoding for an Int22 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 211. In some embodiments, a nucleic acid sequence encoding for an Int22 recombinase comprises the nucleic acid sequence of SEQ ID NO: 211. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Int22 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Int23 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Int23 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 170. In some embodiments, an Int23 recombinase comprises the amino acid sequence SEQ ID NO: 170. In some embodiments, a nucleic acid sequence encoding for an Int23 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 212. In some embodiments, a nucleic acid sequence encoding for an Int23 recombinase comprises the nucleic acid sequence of SEQ ID NO: 212. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Int23 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Int24 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Int24 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 171. In some embodiments, an Int24 recombinase comprises the amino acid sequence SEQ ID NO: 171. In some embodiments, a nucleic acid sequence encoding for an Int24 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 213. In some embodiments, a nucleic acid sequence encoding for an Int24 recombinase comprises the nucleic acid sequence of SEQ ID NO: 213. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Int24 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Int25 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Int25 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 172. In some embodiments, an Int25 recombinase comprises the amino acid sequence SEQ ID NO: 172. In some embodiments, a nucleic acid sequence encoding for an Int25 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 214. In some embodiments, a nucleic acid sequence encoding for an Int25 recombinase comprises the nucleic acid sequence of SEQ ID NO: 214. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Int25 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Int26 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Int26 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 173. In some embodiments, an Int26 recombinase comprises the amino acid sequence SEQ ID NO: 173. In some embodiments, a nucleic acid sequence encoding for an Int26 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 215. In some embodiments, a nucleic acid sequence encoding for an Int26 recombinase comprises the nucleic acid sequence of SEQ ID NO: 215. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Int26 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Int27 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Int27 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 174. In some embodiments, an Int27 recombinase comprises the amino acid sequence SEQ ID NO: 174. In some embodiments, a nucleic acid sequence encoding for an Int27 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 216. In some embodiments, a nucleic acid sequence encoding for an Int27 recombinase comprises the nucleic acid sequence of SEQ ID NO: 216. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Int27 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Int28 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Int28 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 175. In some embodiments, an Int28 recombinase comprises the amino acid sequence SEQ ID NO: 175. In some embodiments, a nucleic acid sequence encoding for an Int28 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 217. In some embodiments, a nucleic acid sequence encoding for an Int28 recombinase comprises the nucleic acid sequence of SEQ ID NO: 217. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Int28 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Int29 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Int29 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 176. In some embodiments, an Int29 recombinase comprises the amino acid sequence SEQ ID NO: 176. In some embodiments, a nucleic acid sequence encoding for an Int29 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 218. In some embodiments, a nucleic acid sequence encoding for an Int29 recombinase comprises the nucleic acid sequence of SEQ ID NO: 218. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Int29 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Int30 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Int30 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 177. In some embodiments, an Int30 recombinase comprises the amino acid sequence SEQ ID NO: 177. In some embodiments, a nucleic acid sequence encoding for an Int30 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 219. In some embodiments, a nucleic acid sequence encoding for an Int30 recombinase comprises the nucleic acid sequence of SEQ ID NO: 219. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Int30 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Int31 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Int31 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 178. In some embodiments, an Int31 recombinase comprises the amino acid sequence SEQ ID NO: 178. In some embodiments, a nucleic acid sequence encoding for an Int31 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 220. In some embodiments, a nucleic acid sequence encoding for an Int31 recombinase comprises the nucleic acid sequence of SEQ ID NO: 220. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Int31 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Int32 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Int32 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 179. In some embodiments, an Int32 recombinase comprises the amino acid sequence SEQ ID NO: 179. In some embodiments, a nucleic acid sequence encoding for an Int32 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 221. In some embodiments, a nucleic acid sequence encoding for an Int32 recombinase comprises the nucleic acid sequence of SEQ ID NO: 221. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Int32 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Int33 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Int33 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 180. In some embodiments, an Int33 recombinase comprises the amino acid sequence SEQ ID NO: 180. In some embodiments, a nucleic acid sequence encoding for an Int33 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 222. In some embodiments, a nucleic acid sequence encoding for an Int33 recombinase comprises the nucleic acid sequence of SEQ ID NO: 222. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Int33 recombinase operably linked to a promoter (as described herein).

In some embodiments, an expression control component comprises an Int34 recombinase (or a polynucleic acid molecule encoding the same). In some embodiments, an Int34 recombinase comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 181. In some embodiments, an Int34 recombinase comprises the amino acid sequence SEQ ID NO: 181. In some embodiments, a nucleic acid sequence encoding for an Int34 recombinase comprises a nucleic acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleic acid sequence of SEQ ID NO: 223. In some embodiments, a nucleic acid sequence encoding for an Int34 recombinase comprises the nucleic acid sequence of SEQ ID NO: 223. In some embodiments, an expression control component comprises a polynucleic acid encoding for an Int34 recombinase operably linked to a promoter (as described herein). c. Transcriptional activator

In some embodiments, an AAV production system further comprises a transcriptional activator (or a polynucleic acid molecule encoding the same). As used herein, the term “transcriptional activator” refers to a transcription factor that binds to and regulates expression of an inducible promoter of an AAV production system (e.g., an inducible promoter operably linked to a nucleic acid sequence encoding for an AAV gene product, an inducible promoter operably linked to a nucleic acid encoding for a recombinase, etc.). Exemplary transcriptional activators, and their corresponding promoter recognition sites, are known to those having skill in the art and include, but are not limited to, TetOn-3 G, TetOn- VI 6, TetOff- Advanced, VanR-VP16, TtgR-VP16, PhlF-VP16, and the cumate cTA and rcTA. In some embodiments, the transcriptional activator is operably linked to a promoter (as described herein). In some embodiments, the transcriptional activator binds to its corresponding promoter recognition site when exposed to a small molecule inducer. In some embodiments, the small molecule inducer is selected from the group consisting of doxycycline, vanillate, phloretin, rapamycin, abscisic acid, gibberellic acid acetoxymethyl ester, and cumate.

In some embodiments, an AAV production system comprises two or more recombinases (or polynucleic acid molecules encoding the same).

In some embodiments, an AAV production system comprises a TetOff- Advanced transcriptional activator (or a polynucleic acid molecule encoding the same). In some embodiments, TetOff- Advanced comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 224. In some embodiments, TetOff- Advanced comprises the amino acid sequence SEQ ID NO: 224. In some embodiments, an AAV production system comprises a polynucleic acid encoding for a TetOff- Advanced transcriptional activator operably linked to a promoter (as described herein).

In some embodiments, an AAV production system comprises a VanR-VP16 transcriptional activator (or a polynucleic acid molecule encoding the same). In some embodiments, VanR-VP16 comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 225. In some embodiments, VanR-VP16 comprises the amino acid sequence SEQ ID NO: 225. In some embodiments, an AAV production system comprises a polynucleic acid encoding for a VanR-VP16 transcriptional activator operably linked to a promoter (as described herein).

In some embodiments, an AAV production system comprises a TtgR-VP16 transcriptional activator (or a polynucleic acid molecule encoding the same). In some embodiments, TtgR-VP16 comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 226. In some embodiments, TtgR-VP16 comprises the amino acid sequence SEQ ID NO: 226. In some embodiments, an AAV production system comprises a polynucleic acid encoding for a TtgR-VP16 transcriptional activator operably linked to a promoter (as described herein).

In some embodiments, an AAV production system comprises a PhlF-VP16 transcriptional activator (or a polynucleic acid molecule encoding the same). In some embodiments, PhlF-VP16 comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 227. In some embodiments, PhlF-VP16 comprises the amino acid sequence SEQ ID NO: 227. In some embodiments, an AAV production system comprises a polynucleic acid encoding for a PhlF-VP16 transcriptional activator operably linked to a promoter (as described herein).

In some embodiments, an AAV production system comprises a cTA transcriptional activator (or a polynucleic acid molecule encoding the same). In some embodiments, cTA comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 228. In some embodiments, cTA comprises the amino acid sequence SEQ ID NO: 228. In some embodiments, an AAV production system comprises a polynucleic acid encoding for a cTA transcriptional activator operably linked to a promoter (as described herein). d. Transfer Polynucleic Acid Molecule

In some embodiments, an AAV production system further comprises a transfer polynucleic acid molecule. In some embodiments, a transfer polynucleic acid molecule comprises, from 5’ to 3’: (i) a nucleic acid sequence of a 5’ inverted tandem repeat; (ii) a central nucleic acid; and (iii) a nucleic acid sequence of a 3’ inverted tandem repeat. In some embodiments, the nucleic acid sequence is a plasmid or a vector.

In some embodiments, a central nucleic acid of the transfer polynucleic acid molecule comprises a multiple cloning site. Exemplary multiple cloning sites are known to those having ordinary skill in the art. A multiple cloning site can be used for cloning a payload molecule (or gene of interest) - or an expression cassette encoding a payload molecule - into the transfer nucleic acid molecule prior to the generation of viral vectors in a host cell.

In some embodiments, a central nucleic acid of the transfer polynucleic acid molecule comprises a gene product of interest. a. Selection Marker

A polynucleic acid of the AAV production system may further comprise a nucleic acid sequence encoding for a selection marker. As used herein, the term “selection marker” or refers to a protein that - when introduced into or expressed in a cell - confers a trait that is suitable for selection. As used herein, the term “selection cassette” refers to a nucleic acid sequence encoding a selection marker operably linked to a promoter (as described herein) and a terminator. A selection marker may be a fluorescent protein. Examples of fluorescent proteins are known in the art (e.g., TagBFP, EBFP2, EGFP, EYFP, mK02, or Sirius). See e.g., Patent No.: US 5,874,304; Patent No.: EP 0969284 Al; Pub. No.: US 2010/167394 A - the entireties of which are incorporated here by reference.

Alternatively, or in addition, a selection marker may be an antibiotic resistance protein. Examples of antibiotic resistance proteins are known in the art (e.g., facilitating puromycin, hygromycin, neomycin, zeocin, blasticidin, or phleomycin selection). See e.g., Pub. No.: WO 1997/15668 A2; Pub. No.: WO 1997/43900 Al - the entireties of which are incorporated here by reference.

Alternatively, or in addition, a selection marker may be an auxotrophic selection marker (e.g., glutamine synthetase). e. Percent Identity

As used herein, the term “percent identity” (or “% identity”) refers to a relationship between the sequences of two polypeptides or polynucleotides, as determined by sequence comparison (alignment). In some embodiments, identity is determined across the entire length of a sequence. In some embodiments, identity is determined over a region of a sequence. Identity of related polypeptides or nucleic acid sequences can be readily calculated by those having ordinary skill in the art. For example, the percent identity of two sequences e.g., nucleic acid or amino acid sequences) may be determined using BLAST®, NBLAST®, XBLAST®, Gapped BLAST®, and Clustal Omega programs, using default parameters of the respective programs. In some embodiments, the identity of two polypeptides is determined by aligning the two amino acid sequences, calculating the number of identical amino acids, and dividing by the length of one of the amino acid sequences. In some embodiments, the identity of two nucleic acids is determined by aligning the two nucleotide sequences and calculating the number of identical nucleotides and dividing by the length of one of the nucleic acids.

II. Engineered Cells for AAV Production

In aspects, the disclosure relates to engineered cells for AAV production. In some embodiments, the engineered cell may comprise any part (and any combination of parts) of the AAV production systems described herein.

For example, an engineered cell may comprise at least a portion of the AAV production component. For example, and as described above, an AAV production component may comprise multiple nucleic acid molecules. In such embodiments, an engineered cell comprises one or more of said multiple nucleic acid molecules - each of which may be located extra-chromosomally or stably integrated into the genome of the engineered cell. In some embodiments, an engineered cell comprises the entire AAV production component.

Alternatively, or in addition, an engineered cell may comprise the expression control component of the AAV production system. In such embodiments, an engineered cell comprises one or more of said multiple nucleic acid molecules - each of which may be located extra-chromosomally or stably integrated into the genome of the engineered cell. In some embodiments, an engineered cell comprises the entire expression control component.

Alternatively, or in addition, an engineered cell may comprise a polynucleic acid molecule comprising the nucleic acid sequence of a transcriptional activator, which may be located extra-chromosomally or stably integrated into the genome of the engineered cell.

Alternatively, or in addition, an engineered cell may comprise a transfer polynucleic acid molecule, which may be located extra-chromosomally or stably integrated into the genome of the engineered cell.

In some embodiments, an AAV production system comprises: (a) an engineered cell comprising an AAV production component comprising one or more heterologous nucleic acid molecules that collectively encode the genes required for AAV production and (b) an expression control component capable of controlling expression of at least one gene required for AAV production.

As used herein, the term “stably integrated” refers to an exogenous nucleic acid sequence, nucleic acid molecule, construct, gene, or nucleic acid sequence that has been inserted into the genome of and organism (e.g. the engineered cell as described herein) and is passed on to future generations after cell division. It is to be understood that any nucleic acid sequence, nucleic acid molecule, construct, gene or nucleic acid sequence described herein may be stably integrated. In some embodiments, any nucleic acid sequence, nucleic acid molecule, construct gene or nucleic acid sequence may be integrated into the genome using random integration, targeted integration, or transposon-mediated integration. It is to be understood that any of the stably integrated nucleic acid molecules described herein may comprise IR/DR sequences that are capable of binding the Sleeping Beauty transposase. Stable integration using the Sleeping Beauty transposase is described in Mates, Lajos, et al. Nature genetics 41.6 (2009): 753-761 which is incorporated by reference in its entirety. In some embodiments, a IR/DR sequence comprises a Sleeping Beauty 100X (SB100X) IR/DR. An engineered cell described herein may further comprise a landing pad. As used herein, the term “landing pad” refers to a heterologous nucleic acid molecule sequence that facilitates the targeted insertion of a “payload” sequence into a specific locus (or multiple loci) of the cell’s genome. Accordingly, the landing pad is integrated into the genome of the cell. A fixed integration site is desirable to reduce the variability between experiments that may be caused by positional epigenetic effects or proximal regulatory elements. The ability to control payload copy number is also desirable to modulate expression levels of the payload without changing any genetic components.

In some embodiments, the landing pad is located at a safe harbor site in the genome of the engineered cell. As used herein, the term “safe harbor site” refers to a location in the genome where genes or genetic elements can be introduced without disrupting the expression or regulation of adjacent genes and/or adjacent genomic elements do not disrupt expression or regulation of the introduced genes or genetic elements. Examples of safe harbor sites are known to those having skill in the art and include, but are not limited to, AAVS1, ROSA26, COSMIC, Hl 1, CCR5, and LiPS-A3S. See e.g., Gaidukov et al., Nucleic Acids Res. 2018 May 4; 46(8): 4072-4086; Patent No.: US 8,980,579 B2; Patent No.: US 10,017,786 B2; Patent No.: US 9,932,607 B2; Pub. No.: US 2013/280222 A; Pub. No.: WO 2017/180669 Al - the entireties of which are incorporated herein. In some embodiments, the safe harbor site is a known site. In other embodiments, the safe harbor site is a previously undisclosed site. See “Methods of Identifying High-Expressing Genomic Loci and Uses Thereof’ herein. In some embodiments, an engineered cell described herein comprises a landing pad that is integrated at a safe harbor locus selected from the group consisting of AAVS1, ROSA26, COSMIC, Hl l, CCR5, and LiPS-A3S.

In some embodiments, the engineered cell is derived from a HEK293 cell. In some embodiments, the engineered HEK293 cell comprises a landing pad that is integrated at a safe harbor locus selected from the group consisting of AAVS1, ROSA26, COSMIC, Hl l, CCR5, and LiPS-A3S.

Each of the landing pads described herein comprises at least one recombination site. Recombination sites for various integrases have been identified previously. For example, a landing pad may comprise recombination sites corresponding to a Bxbl integrase, lambdaintegrase, Cre recombinase, Flp recombinase, gamma-delta resolvase, Tn3 resolvase, q>C31 integrase, or R4 integrase. Exemplary recombination site sequences are known in the art (e.g., attP, attB, attR, attL, Lox, and Frt).

The landing pads described herein may comprise one or more expression cassettes. III. Kits

In some aspects, the disclosure relates to kits comprising an AAV production system described herein and/or an engineered cell described herein.

In some embodiments, a kit comprises one or more nucleic acid molecules collectively comprising an AAV production system.

In some embodiments, the kit further comprises a small molecule inducer corresponding to a chemically inducible promoter of the AAV production system. In some embodiments, a small molecule inducer is doxycycline, vanillate, phloretin, rapamycin, abscisic acid, gibberellic acid acetoxymethyl ester, and cumate.

In some embodiments, a kit comprises a nucleic acid molecule comprising a nucleic acid sequence of a transcriptional activator operably linked to a nucleic acid sequence of a promoter, wherein the transcriptional activator, when expressed in the presence of the small molecule inducer, binds to a chemically inducible promoter of the AAV production system, optionally wherein an engineered cell comprises the nucleic acid molecule comprising the nucleic acid sequence of the transcriptional activator. In some embodiments, the transcriptional activator is selected from the group consisting of TetOn-3G, TetOn-V16, T etOff- Advanced, VanR-VP16, TtgR-VP16, PhlF-VP16, and the cumate cTA and rcTA.

In some embodiments, the kit may further comprise instructions for use of the cells.

IV. Methods of using engineered cells for AAV production

In some aspects, the present disclosure provides methods for producing AAV using an AAV production system described herein that comprises: (a) an AAV production component collectively encode gene products required for generation of an AAV in a recombinant host cell; and (b) an expression control component. In some embodiments, the method of AAV production comprises transfecting or stably integrating into an engineered cell any combination of the one or more nucleic acid molecules collectively comprising the AAV production component and the expression control component as described herein. In some embodiments, the method of AAV production further comprises transfecting a nucleic acid molecule comprising a payload for AAV delivery (e.g. a therapeutic DNA sequence) as described above. In some embodiments, the method comprises growing the engineered cell to a confluency that is optimal for AAV production. An optimal confluency may be dependent, for example, on the type of cell the engineered cell is derived from. The skilled person will know or be able to determine the optimal confluency for AAV production. In some embodiments, the method comprises harvesting the AAV produced from the culture of engineered cells using methods that are well known to those of skill in the art.

EXAMPLES

Viral vectors are a promising gene delivery modality for cell and gene therapy. The production of viral vectors normally entails transient transfection of plasmids into cell culture. However, stable integration of genes necessary to produce therapeutic viral vectors into the genome offers several advantages compared to traditional production via transient transfection. Since cells amplify the viral genes during their own cell division, large quantities of DNA and transfection reagent no longer need to be procured for the transfection process, reducing costs. Also since the DNA is already within the nucleus, viral titers may be higher and more consistent due to minimal numbers of “untransfected” cells and reduced variation associated with transfection steps. The simpler production process also saves scientist time.

The following designs introduce inducible control of cytostatic or cytotoxic genes using recombinases. Each of the described constructs can be integrated into the genome using random integration, targeted integration, or transposon-mediated integration.

Example 1: AAV Expression control with a Recombinase

Description of approach and genetic schematic:

Recombinase inversion-based control of AAV production relies on the use of a recombinase (typically a serine recombinase like PhiC31 or Bxbl) to invert the DNA between two recombinase attachment (e.g., att) sites, restoring expression of AAV-associated genes which are initially in an incorrect layout. Prior to recombinase expression, genes are oriented in such a way that expression of AAV-associated genes is disrupted. Recombinase attachment sites are added within the coding sequence of the AAV-associated genes, since inversion within the coding region should significantly disrupt AAV production. Recombinase attachment sites are placed within synthetic introns, such that after induction with recombinase, the recombinase attachment sequences are spliced out of the mRNA transcripts. Otherwise, the recombinase attachment site sequences would introduce additional amino acids into the AAV proteins.

Preliminary data and experiment description:

Adherent HEK293FT cells were co-transfected with EGFP-expressing transfer plasmid, modified pRepCap, and modified pHelper (FIG. 1). Control samples containing ‘wild type’ AAV2 pRepCap and pHelper plasmids or completely untransfected were also prepared. Small molecules Shieldl or trimethoprim (TMP) were added to cells at the time of transfection at 1 uM or 10 uM concentration respectively. 48-72 hours after transfection, AAV was harvested by three freeze thaw cycles in a dry ice isopropanol bath. Virus stock was serially diluted 1-, 10- and 100-fold and 10 uL of resulting viral stocks was transduced by addition to 5e4 HEK293FT cells plated in a 96-well plate. 48-72 hours after transduction, transduced cells were harvested and percentage of EGFP positive cells was determined by flow cytometry and used to calculate transducing units per mL (TU/mL) (FIG. 3).

Table 1: Exemplary Intron Splice Donors/Acceptors

Table 2: Exemplary Recombinase Attachment Sites

Table 3: Exemplary AAV Gene Product Amino Acid Sequences

Table 4: Exemplary Invertible Element Sequences

Table 5: Exemplary Recombinase Amino Acid Sequences

Table 6: Exemplary Recombinase Nucleic Acid Sequences

Table 7: Exemplary Transcriptional Activator Amino Acid Sequences

Claims

CLAIMS What is claimed is:

1. An Adeno- Associated Virus (AAV) production system comprising an expression control component and an AAV production component, wherein:

(a) the expression control component comprises a polynucleic acid molecule encoding for a recombinase; and

(b) the AAV production component comprises one or more polynucleic acid molecules collectively encoding for: Rep52 or Rep40; Rep78 or Rep68; E2A; E40rf6; VARNA; VP1; VP2; VP3; AAP; wherein a nucleic acid sequence comprising at least a portion of a nucleic acid sequence encoding for Rep52, Rep40, Rep78, Rep68, E2A, E40rf6, VARNA, VP1, VP2, VP3, or AAP is comprised within an invertible element, wherein the invertible element comprises, from 5’ to 3’: (i) a first recombinase attachment site; (ii) an internal sequence comprising the at least a portion of the nucleic acid sequence encoding for Rep52, Rep40, Rep78, Rep68, E2A, E40rf6, VARNA, VP1, VP2, VP3, or AAP; and (iii) a second recombinase attachment site; wherein the 5’ recombinase attachment site and the 3’ recombinase attachment site correspond to the recombinase of (a).

2. An Adeno-Associated Virus (AAV) production system comprising an expression control component and an AAV production component, wherein:

(b) the AAV production component comprises a polynucleic acid molecule having a first segment and a second segment, wherein the second segment is inverted relative to the first segment, and wherein: the first segment comprises: a promoter operably linked to a first portion of a nucleic acid sequence encoding for an AAV gene product; and the second segment comprises, from 5’ to 3’: a first recombinase attachment site; a second portion of the nucleic acid sequence encoding for the first AAV gene product; and a second recombinase attachment site; and wherein the first recombinase attachment site and the second recombinase attachment site correspond to the same recombinase.

3. The AAV production system of claim 2, wherein the nucleic acid sequence encoding for the AAV gene product is comprised within the first portion of the nucleic acid sequence encoding for the AAV gene product and the second portion of the nucleic acid sequence encoding for the AAV gene product.

4. The AAV production system of claim 3, wherein the second segment further comprises a nucleic acid encoding for at least a portion of an additional AAV gene product.

5. The AAV production system of claim 2, wherein the polynucleic acid molecule having the first segment and the second segment further comprises a third segment, wherein the third segment is in the same orientation as the first segment, and wherein the polynucleic acid molecule comprises the structure: first segment - second segment - third segment, and wherein the third segment comprises a third portion of the nucleic acid sequence encoding for the AAV gene product.

6. The AAV production system of claim 5, wherein the nucleic acid sequence encoding for the AAV gene product is comprised within the first portion of the nucleic acid sequence encoding for the AAV gene product, the second portion of the nucleic acid sequence encoding for the AAV gene product, and the third portion of the nucleic acid sequence encoding for the AAV gene product.

7. The AAV production system of any one of claims 2-6, wherein the first recombinase attachment site comprises a nucleic acid sequence having at least 80% identity with the nucleic acid sequence of any one of SEQ ID NOs: 5-96.

8. The AAV production system of any one of claims 2-7, wherein the second recombinase attachment site comprises a nucleic acid sequence having at least 80% identity with the nucleic acid sequence of any one of SEQ ID NOs: 5-96.

9. The AAV production system of any one of claims 2-8, wherein: the first segment comprises, from 5’ to 3’: the promoter operably linked to the first portion of the nucleic acid sequence encoding for the first AAV gene product; and an intron splice donor; and the second segment comprises, from 5’ to 3’: the first recombinase attachment site; an intron splice acceptor; and the second portion of the nucleic acid sequence encoding for the first AAV gene product; and the second recombinase attachment site.

10. The AAV production system of claim 9, wherein the intron splice donor comprises a nucleic acid sequence having at least 80% identity with the nucleic acid sequence of any one of SEQ ID NOs: 1-2.

11. The AAV production system of claim 9 or claim 10, wherein the intron splice acceptor comprises a nucleic acid sequence having at least 80% identity with the nucleic acid sequence of any one of SEQ ID NOs: 3-4.

12. The AAV production system of any one of claims 9-11, wherein the first segment comprises, from 5’ to 3’ : the promoter operably linked to the first portion of the nucleic acid sequence encoding for the first AAV gene product; the intron splice donor; and a nucleic acid sequence encoding for a stop codon.

13. The AAV production system of any one of claims 2-12, wherein the AAV gene product is selected from the group consisting of Rep52, Rep40, Rep78, Rep68, E2A, E40rf6, VARNA, VP1, VP2, VP3, AAP, and MAAP.

14. The AAV production system of any one of claims 2-13, wherein the recombinase of the expression control component is a PhiC31 recombinase, a Cre recombinase, a VCre recombinase, a Flp recombinase, a Bxbl recombinase, or a TP901 recombinase.

15. The AAV production system of claims 2-14, wherein the recombinase comprises an amino acid sequence having at least 80% identity with the amino acid sequence of any one of SEQ ID NOs: 140-181.

16. The AAV production system of any one of claims 2-15, wherein the AAV production system comprises one or more polynucleic acid molecules collectively encoding for: Rep52 or Rep40; Rep78 or Rep68; E2A; E40rf6; VARNA; VP1; VP2; VP3; and AAP.

17. An Adeno- Associated Virus (AAV) production system comprising a polynucleic acid molecule having the structure: first segment - second segment - third segment, wherein the second segment is inverted relative to the first segment and the third segment, and wherein:

(a) the first segment comprises, from 5’ to 3’ : a promoter operably linked to a first portion of a nucleic acid sequence encoding for a first AAV gene product; and a first intron splice donor;

(b) the second segment is an invertible element comprising, from 5’ to 3’ : a first recombinase attachment site; a first intron splice acceptor; a second portion of the nucleic acid sequence encoding for the first AAV gene product; a promoter operably linked to a first portion of a nucleic acid sequence encoding for a second AAV gene product; and second splice intron donor; and a second recombinase attachment site; and

(c) the third segment comprises, from 5’ to 3’ : a second intron splice acceptor; and a second portion of the nucleic acid sequence encoding for the second AAV gene product; and wherein the first recombinase attachment site and the second recombinase attachment site correspond to the same recombinase.

18. The AAV production system of claim 17, wherein: the first segment comprises, from 5’ to 3’: the promoter operably linked to the first portion of the nucleic acid sequence encoding for the first AAV gene product; the first intron splice donor; and a nucleic acid sequence encoding for a stop codon; wherein the nucleic acid sequence encoding for the stop codon is in frame with the first portion of the nucleic acid sequence encoding for the first AAV gene product or the first portion of the nucleic acid sequence encoding for the second AAV gene product; the second segment comprises, from 5’ to 3’: the first recombinase attachment site; the first intron splice acceptor; the second portion of the nucleic acid sequence encoding for the first AAV gene product; the promoter operably linked to the first portion of the nucleic acid sequence encoding for the second AAV gene product; the second intron splice donor; a nucleic acid sequence encoding for a stop codon; and the second recombinase attachment site; wherein the nucleic acid sequence encoding for the stop codon is in frame with the first portion of the nucleic acid sequence encoding for the first AAV product or the first portion of the nucleic acid sequence encoding for the second AAV gene product; or a combination thereof.

19. An Adeno- Associated Virus (AAV) production system comprising a polynucleic acid molecule having the structure: first segment - second segment - third segment - fourth segment, wherein the first segment and the second segment are inverted relative to the third segment and the fourth segment, wherein an invertible element comprises the second segment and the third segment, and wherein:

(a) the third segment comprises, from 5’ to 3’ : a promoter operably linked to a first portion of a nucleic acid sequence encoding for a first AAV gene product; a first intron splice donor; and a first recombinase attachment site;

(b) the first segment comprises, from 5’ to 3’ : a first intron splice acceptor; and a second portion of the nucleic acid sequence encoding for the first AAV gene product;

(c) the second segment comprises, from 5’ to 3’ : a promoter operably linked to a first portion of a nucleic acid sequence encoding for a second AAV gene product; a second intron splice donor; and a second recombinase attachment site; and

(d) the fourth segment comprises, from 5’ to 3’ : a second intron splice acceptor; and a second portion of the nucleic acid sequence encoding for the second AAV gene product; and wherein the first recombinase attachment site and the second recombinase attachment site correspond to the same recombinase.

20. The AAV production system of claim 19, wherein: the first segment comprises, from 5’ to 3’: the first intron splice acceptor; a nucleic acid sequence encoding for a stop codon; and the second portion of the nucleic acid sequence encoding for the first AAV gene product; wherein the nucleic acid sequence encoding for the stop codon is in frame with the nucleic acid sequence encoding for the first portion of the second AAV gene product, but out of frame with the nucleic acid sequence encoding for the second portion of the first AAV gene product; the fourth segment comprises, from 5’ to 3’ : the second intron splice acceptor; a nucleic acid sequence encoding for a stop codon; and the second portion of the nucleic acid sequence encoding for the second AAV gene product; wherein the nucleic acid sequence encoding for the stop codon is in frame with the nucleic acid sequence encoding for the first portion of the first AAV gene product, but out of frame with the nucleic acid sequence encoding for the second portion of the second AAV gene product; or a combination thereof.

21. An Adeno-Associated Virus (AAV) production system comprising a polynucleic acid molecule having the structure: first segment - second segment - third segment - fourth segment, wherein the first segment and the second segment are inverted relative to the third segment and the fourth segment, wherein an invertible element comprises the second segment and the third segment, and wherein:

(b) the third segment comprises, from 5’ to 3’ : a first recombinase attachment site; a first intron splice acceptor; and a second portion of the nucleic acid sequence encoding for the first AAV gene product;

(c) the fourth segment comprises, from 5’ to 3’ : a promoter operably linked to a first portion of a nucleic acid sequence encoding for a second AAV gene product; and a second intron splice donor; and

(d) the second segment comprises, from 5’ to 3’ : a second recombinase attachment site; a second intron splice acceptor; and a second portion of the nucleic acid sequence encoding for the second AAV gene product; and wherein the first recombinase attachment site and the second recombinase attachment site correspond to the same recombinase.

22. The AAV production system of claim 21, wherein: the second segment comprises, from 5’ to 3’: the second recombinase attachment site; the second intron splice acceptor; a nucleic acid sequence encoding for a stop codon; and the second portion of the nucleic acid sequence encoding for the second AAV gene product; wherein the nucleic acid sequence encoding for the stop codon is in frame with the nucleic acid sequence encoding for the first portion of the first AAV gene product, but out of frame with the nucleic acid sequence encoding for the second portion of the second AAV gene product; the third segment comprises, from 5’ to 3’ : the first recombinase attachment site; the first intron splice acceptor; a nucleic acid sequence encoding for a stop codon; and the second portion of the nucleic acid sequence encoding for the first AAV gene product; wherein the nucleic acid sequence encoding for the stop codon is in frame with the nucleic acid sequence encoding for the first portion of the second AAV gene product, but out of frame with the nucleic acid sequence encoding for the second portion of the first AAV gene product; or a combination thereof.

23. The AAV production system of any one of claims 17-22, wherein the first recombinase attachment site comprises a nucleic acid sequence having at least 80% identity with the nucleic acid sequence of any one of SEQ ID NOs: 5-96.

24. The AAV production system of any one of claims 17-23, wherein the second recombinase attachment site comprises a nucleic acid sequence having at least 80% identity with the nucleic acid sequence of any one of SEQ ID NOs: 5-96.

25. The AAV production system of any one of claims 17-24, wherein the first intron splice donor comprises a nucleic acid sequence having at least 80% identity with the nucleic acid sequence of any one of SEQ ID NOs: 1-2, the second intron splice donor comprises a nucleic acid sequence having at least 80% identity with the nucleic acid sequence of any one of SEQ ID NOs: 1-2, or a combination thereof.

26. The AAV production system of any one of claims 17-25, wherein the first intron splice acceptor comprises a nucleic acid sequence having at least 80% identity with the nucleic acid sequence of any one of SEQ ID NOs: 3-4; the second intron splice acceptor comprises a nucleic acid sequence having at least 80% identity with the nucleic acid sequence of any one of SEQ ID NOs: 3-4, or a combination thereof.

27. The AAV production system of any one of claims 17-26, wherein:

- the first AAV gene product is E2A and the second AAV gene product is E40rf6;

- the first AAV gene product is Rep52 and the second AAV gene product is VP1, VP2, VP3, or AAP;

- the first AAV gene product is Rep40 and the second AAV gene product is VP1, VP2, VP3, or AAP;

- the first AAV gene product is Rep78 and the second AAV gene product is VP1, VP2, VP3, or AAP; or

- the first AAV gene product is Rep68 and the second AAV gene product is VP1, VP2, VP3, or AAP.

28. The AAV production system of any one of claims 17-27, further comprising an expression control component, wherein the expression control component comprises a polynucleic acid molecule encoding for a recombinase corresponding to the first recombination attachment site and the second recombination attachment site.

29. The AAV production system of claim 28, wherein the recombinase is a PhiC31 recombinase, a Cre recombinase, a VCre recombinase, a Flp recombinase, a Bxbl recombinase, or a TP901 recombinase.

30. The AAV production system of claim 28 or claim 29, wherein the recombinase comprises an amino acid sequence having at least 80% identity with the amino acid sequence of any one of SEQ ID NOs: 140-181.

31. The AAV production system of any one of claims 28-30, wherein the AAV production system comprises one or more polynucleic acid molecules collectively encoding for: Rep52 or Rep40; Rep78 or Rep68; E2A; E40rf6; VARNA; VP1; VP2; VP3; and AAP.

32. An engineered cell comprising an AAV production system according to any one of claims 1-31.

33. The engineered cell of claim 32, wherein one or more polynucleic acid molecules of the AAV production component are stably integrated into the genome of the engineered cell.

34. The engineered cell of claim 32 or claim 33, further comprising a transfer polynucleic acid molecule having a sequence encoding, from 5’ to 3’: (i) a nucleotide sequence of a 5’ inverted tandem repeat; (ii) a multiple cloning site; and (iii) a nucleotide sequence of a 3’ inverted tandem repeat.

35. The engineered cell of claim 34, wherein the transfer polynucleic acid molecule is stably integrated into the genome of the engineered cell.

36. A kit comprising an Adeno-Associated Virus production system of any one of claims 1-31.

37. The kit of claim 36, further comprising a transfer polynucleic acid molecule having a sequence encoding, from 5’ to 3’ : (i) a nucleotide sequence of a 5’ inverted tandem repeat; (ii) a multiple cloning site; and (iii) a nucleotide sequence of a 3’ inverted tandem repeat.

38. The kit of claim 37, wherein the transfer polynucleic acid molecule is a plasmid or a vector.

39. A kit comprising the engineered cell of claim 32 or claim 33 and a transfer polynucleic acid molecule, wherein the transfer polynucleic acid molecule has a sequence encoding, from 5’ to 3’: (i) a nucleotide sequence of a 5’ inverted tandem repeat; (ii) a multiple cloning site; and (iii) a nucleotide sequence of a 3’ inverted tandem repeat.

40. A kit comprising the engineered cell of claim 34 or claim 35.

41. A method of producing an AAV vector, comprising expressing, in an engineered cell comprising an AAV production system according to any one of claims 1, 13-15, and 31, the recombinase of the expression control component and Rep52 or Rep40, Rep78 or Rep68, E2A, E40rf6, VARNA, VP1, VP2, VP3, and AAP of the AAV production component.

42. A polynucleic acid molecule comprising a nucleic acid sequence having at least 80% identity with the nucleic acid sequence of any one of SEQ ID NOs: 111-139.

43. An engineered cell comprising the polynucleic acid of claim 42.