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US-20260125702-A1 - COMPOSITIONS AND METHODS FOR ADENO-ASSOCIATED VIRAL PRODUCTION

US20260125702A1US 20260125702 A1US20260125702 A1US 20260125702A1US-20260125702-A1

Abstract

Disclosed herein are 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.

Inventors

  • Jeremy J. Gam
  • Alec A.K. NIELSEN

Assignees

  • ASIMOV INC.

Dates

Publication Date
20260507
Application Date
20231012

Claims (20)

  1. 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; E4Orf6; VARNA; VP1; VP2; VP3; and AAP; at least one of which comprises a nucleic acid sequence of an excisable element, wherein the excisable element comprises, from 5′ to 3′: (i) a first intron splice donor; (ii) a first recombinase site; (iii) a first intron splice acceptor; (iv) an exon comprising a stop codon; (v) a second intron splice donor; (vi) a second recombinase site; (vii) a second intron splice acceptor; wherein the first recombinase site and the second recombinase site correspond to the recombinase of (a).
  2. 2 . The AAV production system of claim 1 , 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.
  3. 3 . The AAV production system of claim 1 or claim 2 , 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.
  4. 4 . The AAV production system of any one of claims 1-3 , 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.
  5. 5 . The AAV production system of any one of claims 1-4 , wherein 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.
  6. 6 . The AAV production system of any one of claims 1-5 , 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.
  7. 7 . The AAV production system of any one of claims 1-6 , wherein 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.
  8. 8 . The AAV production system of any one of claims 1-7 , wherein the first intron splice acceptor of the excisable element is stronger than the second intron splice acceptor of the excisable element.
  9. 9 . The AAV production system of any one of claims 1-8 , wherein the exon of the excisable element comprises a stop codon in all coding frames.
  10. 10 . The AAV production system of claim 9 , wherein the exon of the excisable element comprises a nucleic acid sequence having at least 80% identity with the nucleic acid sequence of any one of SEQ ID NOs: 130-131.
  11. 11 . The AAV production system of any one of claims 1-10 , wherein the nucleic acid sequence encoding for Rep52 or Rep40 and/or the nucleic acid sequence encoding for Rep78 or Rep68 comprises the nucleic acid sequence of the excisable element.
  12. 12 . The AAV production system of claim 11 , wherein the AAV production component comprises the nucleic acid sequence having at least 80% identity with the nucleic acid sequence of any one of SEQ ID NOs: 137-139.
  13. 13 . The AAV production system of any one of claims 1-12 , wherein the nucleic acid sequence encoding for VP1, VP2, and/or VP3 comprises the nucleic acid sequence of the excisable element.
  14. 14 . The AAV production system of any one of claims 1-13 , wherein the nucleic acid sequence encoding for E2A and/or the nucleic acid sequence encoding for E4Orf6 comprises the nucleic acid sequence of the excisable element.
  15. 15 . The AAV production system of claim 14 , wherein the AAV production component comprises the nucleic acid sequence having at least 80% identity with the nucleic acid sequence of any one of SEQ ID NOs: 134-136.
  16. 16 . The AAV production system of any one of claims 1-15 , further comprising a helper component, wherein the helper component comprises one or more polynucleic acid molecules collectively encoding for: UL5, UL8, UL29, UL30, UL42, UL52, UL12, ICP10, ICP4, and ICP22.
  17. 17 . The AAV production system of any one of claims 1-16 , wherein the recombinase is a PhiC31 recombinase, a Cre recombinase, a VCre recombinase, a Flp recombinase, a Bxb1 recombinase, or a TP901 recombinase.
  18. 18 . The AAV production system of any one of claims 1-17 , 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.
  19. 19 . An engineered cell comprising an AAV production system according to any one of claims 1-18 .
  20. 20 . The engineered cell of claim 19 , wherein one or more polynucleic acid molecules of the AAV production component are stably integrated into the genome of the engineered cell.

Description

RELATED APPLICATION This application claims the benefit under 35 U.S.C. § 119 of U.S. provisional application Ser. No. 63/379,158, filed Oct. 12, 2022, the entire contents of which are incorporated by reference herein. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (A121070009WO00-SEQ-JRV.xml; Size: 344,150 bytes; and Date of Creation: Oct. 11, 2023) is herein 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 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 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; E4Orf6; VARNA; VP1; VP2; VP3; and AAP; at least one of which comprises a nucleic acid sequence of an excisable element, wherein the excisable element comprises, from 5′ to 3′: (i) a first intron splice donor; (ii) a first recombinase site; (iii) a first intron splice acceptor; (iv) an exon comprising a stop codon; (v) a second intron splice donor; (vi) a second recombinase site; (vii) a second intron splice acceptor; wherein the first recombinase site and the second recombinase site correspond to the recombinase of (a). 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 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 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. 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. In some embodiments, 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. In some embodiments, the first intron splice acceptor of the excisable element is stronger than the second intron splice acceptor of the excisable element. In some embodiments, the exon of the excisable element comprises a stop codon in all coding frames. In some embodiments, the exon of the excisable element comprises a nucleic acid sequence having at least 80% identity with the nucleic acid sequence of any one of SEQ ID NOs: 130-131. In some embodiments, the nucleic acid sequence encoding for Rep52 or Rep40 and/or the nucleic acid sequence encoding for Rep78 or Rep68 comprises the nucleic acid sequence of the excisable element. In some embodiments, the AAV production component comprises the nucleic acid sequence having at least 80% identity with the nucleic acid sequence of any one of SEQ ID NOs: 137-139. In some embodiments, the nucleic acid sequence encoding for VP1, VP2, and/or VP3 comprises the nucleic acid sequence of the excisable element. In some embodiments, the nucleic acid sequence encoding for E2A and/or the nucleic acid sequence encoding for E4Orf6 comprises the nucleic acid sequence of the excisable element. In some embodiments, the AAV production component comprises th