Granted Patent
B2
US 12,234,472 · App. 18/047,341 · Granted Feb 25, 2025
Eukaryotic cells comprising adenovirus-associated virus polynucleotides
Inventors:
Michael Goren (Tarrytown, NY); Yu Zhao (Willison Park, NY); Alexandros Strikoudis (New York, NY); Darya Burakov (Tarrytown, NY); Gang Chen (Yorktown Heights, NY)
Assignee:
Regeneron Pharmaceuticals, Inc.
View Patent ↗
Loading inventors, assignments & file history…
Abstract
The present inventions provide eukaryotic cells, such as mammalian cells, that comprise adeno-associated virus (AAV) polynucleotides, including AAV capsid proteins (Cap), and are capable of expressing the polypeptides encoded by the AAV polynucleotides, and thereby are capable of producing AAV, including recombinant AAV. The eukaryotic cells also may comprise adenovirus (Ad) polynucleotides. The present inventions also provide methods of expressing AAV polynucleotides, as well as Ad polynucleotides, in eukaryotic cells, such as CHO cells, HEK 293 and BHK cells. The present inventions further provides other products and methods described herein.
Claims (31)
1. A eukaryotic cell from a eukaryotic cell line, wherein the eukaryotic cell comprises a polynucleotide comprising in order (i) a promoter, (ii) an intron, (iii) a first internal ribosome entry site, (iv) a first AAV Cap gene, (v) a second internal ribosome entry site, (vi) a second AAV Cap gene, and (vii) a polyadenylation site, wherein (i) to (vii) are operably linked.
2. The eukaryotic cell according to claim 1 , wherein the polynucleotide is integrated into a cell genome.
3. The eukaryotic cell according to claim 2 , wherein the eukaryotic cell is a CHO cell or a BHK cell.
4. The eukaryotic cell according to claim 2 , wherein the eukaryotic cell is a HEK 293 cell or a human amniotic cell.
5. The eukaryotic cell according to claim 1 , further comprising an operator.
6. The eukaryotic cell according to claim 5 , wherein the promoter is a CMV promoter and the operator is a Tet operator.
7. The eukaryotic cell according to claim 1 , further comprising:
a polynucleotide encoding AAV Rep;
a polynucleotide encoding Ad E1A;
a polynucleotide encoding Ad E1B;
a polynucleotide encoding Ad E2A or E2A orf;
a polynucleotide encoding Ad E4 or E4 orf 6;
a polynucleotide encoding VA RNA; and
a polynucleotide encoding AAV ITRs and a protein of interest.
8. A method of producing adeno-associated virus (AAV) Cap protein in cell culture, wherein the method comprises the steps of:
providing the cell culture that comprises eukaryotic cells, wherein a eukaryotic cell of the cell culture comprises a polynucleotide comprising in order (i) a promoter, (ii) an intron, (iii) a first internal ribosome entry site, (iv) a first AAV Cap gene, (v) a second internal ribosome entry site, (vi) a second AAV Cap gene, and (vii) a polyadenylation site, wherein (i) to (vii) are operably linked; and
culturing the eukaryotic cells in a culture medium to allow the eukaryotic cells to produce AAV VP1, VP2 and VP3 protein.
9. The method according to claim 8 , wherein the polynucleotide is integrated into a cell genome.
10. The method according to claim 9 , wherein the eukaryotic cell is a CHO cell or a BHK cell.
11. The method according to claim 9 , wherein the eukaryotic cell is a HEK 293 cell or a human amniotic cell.
12. The method according to claim 8 , wherein the eukaryotic cell further comprises an operator.
13. The method according to claim 12 , wherein the promoter is a CMV promoter and the operator is a Tet operator.
14. The method according to claim 8 , wherein the eukaryotic cell further comprises:
a polynucleotide encoding AAV Rep;
a polynucleotide encoding Ad E1A;
a polynucleotide encoding Ad E1B;
a polynucleotide encoding Ad E2A or E2A orf;
a polynucleotide encoding E4 or E4 orf 6;
a polynucleotide encoding VA RNA; and
a polynucleotide encoding AAV ITRs and a protein of interest, wherein the eukaryotic cell can produce recombinant AAV.
15. The method according to claim 8 , wherein the eukaryotic cells in the cell culture can express VP1, VP2 and VP3 proteins in a ratio of 1:2:9.3 of VP1 to VP2 to VP3.
Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Mar 21, 2024
From: GOREN, MICHAEL; ZHAO, YU; STRIKOUDIS, ALEXANDROS; BURAKOV, DARYA; CHEN, GANG
To: REGENERON PHARMACEUTICALS, INC.
Reel/Frame 066858/0113 →
Continuity (2)
Provisional Application
63256730
· Oct 18, 2021
References Cited (171)
US 5464758A
· Gossen et al.
· 1995
[cited by applicant]
US 5658785A
· Johnson
· 1997
[cited by applicant]
US 5981225A
· Kochanek et al.
· 1999
[cited by applicant]
US 5985846A
· Kochanek et al.
· 1999
[cited by applicant]
US 5989910A
· Mermod et al.
· 1999
[cited by applicant]
US 6423544B1
· Hardy
· 2002
[cited by applicant]
US 6558948B1
· Kochanek et al.
· 2003
[cited by applicant]
US 7232899B2
· Von Seggern et al.
· 2007
[cited by applicant]
US 7771997B2
· Chen et al.
· 2010
[cited by applicant]
US 8734809B2
· Gao
· 2014
[cited by examiner]
US 8852926B2
· Mo et al.
· 2014
[cited by applicant]
US 9315773B2
· Schiedner et al.
· 2016
[cited by applicant]
US 9371512B2
· Schiedner et al.
· 2016
[cited by applicant]
US 9469856B2
· Dou et al.
· 2016
[cited by applicant]
US 9534233B2
· Kochanek et al.
· 2017
[cited by applicant]
US 9777291B2
· Chatterjee
· 2017
[cited by applicant]
US 9783825B2
· Chatterjee
· 2017
[cited by applicant]
US 9803218B2
· Chatterjee
· 2017
[cited by applicant]
US 9816110B2
· Shen et al.
· 2017
[cited by applicant]
US 9834789B2
· Chatterjee et al.
· 2017
[cited by applicant]
US 20030192066A1
· Zhang et al.
· 2003
[cited by applicant]
US 20090191597A1
· Samulski
· 2009
[cited by examiner]
US 20130023033A1
· Wilson et al.
· 2013
[cited by applicant]
US 20180030480A1
· Shen et al.
· 2018
[cited by applicant]
US 20180267516A1
· Fister et al.
· 2018
[cited by applicant]
US 20190078099A1
· Zhou et al.
· 2019
[cited by applicant]
US 20190171188A1
· Fister
· 2019
[cited by applicant]
US 20200157567A1
· Cawood et al.
· 2020
[cited by applicant]
US 20220154215A1
· Greene
· 2022
[cited by applicant]
US 20220177854A1
· Chanas et al.
· 2022
[cited by applicant]
US 20220259572A1
· Gu et al.
· 2022
[cited by applicant]
US 20220307052A1
· Pechan et al.
· 2022
[cited by applicant]
US 20230048994A1
· Smith et al.
· 2023
[cited by applicant]
US 20230076955A1
· Cawood et al.
· 2023
[cited by applicant]
US 20230257770A1
· Cawood et al.
· 2023
[cited by applicant]
US 20230257831A1
· Cawood et al.
· 2023
[cited by applicant]
US 20230279427A1
· Hu et al.
· 2023
[cited by applicant]
US 20230287460A1
· Goren et al.
· 2023
[cited by applicant]
US 20230304062A1
· Zhao et al.
· 2023
[cited by applicant]
US 20230313228A1
· Cawood
· 2023
[cited by applicant]
US 20230357794A1
· Cawood et al.
· 2023
[cited by applicant]
EP 1230354A2
· 2002
[cited by applicant]
EP 1362096A2
· 2003
[cited by applicant]
EP 1743041A1
· 2007
[cited by applicant]
EP 2606128A1
· 2013
[cited by applicant]
EP 3456822A1
· 2019
[cited by applicant]
EP 3649239A1
· 2020
[cited by applicant]
WO 199953085A2
· 1999
[cited by applicant]
WO 2001034940A2
· 2001
[cited by applicant]
WO 2002066620A2
· 2002
[cited by applicant]
WO 2003101189A1
· 2003
[cited by applicant]
WO 2005106046A1
· 2005
[cited by applicant]
WO 2007133797A2
· 2007
[cited by applicant]
WO 2012041311A1
· 2012
[cited by applicant]
WO 2013190032A1
· 2013
[cited by applicant]
WO 2015092440A1
· 2015
[cited by applicant]
WO 2017140406A1
· 2017
[cited by applicant]
WO 2017221031A1
· 2017
[cited by applicant]
WO 2018150269A1
· 2018
[cited by applicant]
WO 2018150271A1
· 2018
[cited by applicant]
WO 2018177758A1
· 2018
[cited by applicant]
WO 2018189535A1
· 2018
[cited by applicant]
WO 2019020992A1
· 2019
[cited by applicant]
WO 2019030069A2
· 2019
[cited by applicant]
WO 2019057691A1
· 2019
[cited by applicant]
WO 2019073059A1
· 2019
[cited by applicant]
WO 2019126634A2
· 2019
[cited by applicant]
WO 2019141993A1
· 2019
[cited by applicant]
WO 2019155016A1
· 2019
[cited by applicant]
WO 2019157239A1
· 2019
[cited by applicant]
WO 2019175600A1
· 2019
[cited by applicant]
WO 202016148A1
· 2020
[cited by applicant]
WO 2020043869A2
· 2020
[cited by applicant]
WO 2020072480A1
· 2020
[cited by applicant]
WO 2020077411A1
· 2020
[cited by applicant]
WO 2020084034A1
· 2020
[cited by applicant]
WO 2020132165A1
· 2020
[cited by applicant]
WO 2020165603A1
· 2020
[cited by applicant]
WO 2020183133A1
· 2020
[cited by applicant]
WO 2020232366A2
· 2020
[cited by applicant]
WO 2021127432A1
· 2021
[cited by applicant]
WO 2021188892A1
· 2021
[cited by applicant]
WO 2022020712A1
· 2022
[cited by applicant]
WO 2022038369A1
· 2022
[cited by applicant]
WO 2022112218A1
· 2022
[cited by applicant]
WO 2022223954A1
· 2022
[cited by applicant]
WO 2023173105A2
· 2023
[cited by applicant]
Haifeng Chen, “Intron Splicing-mediated Expression of AAV Rep and Cap Genes and Production of AAV Vectors in Insect Cells”,(The American Society of Gene Therapy), Published Mar. 18, 2008, vol. 6 (Year: 2008).
[cited by examiner]
Renaud-Gabardos et al., “Internal Ribosome entry site-based vectors for combined gene therapy”, (World Journal of Experimental Medicine), Published Feb. 20, 2015, vol. 5 (Year: 2015).
[cited by examiner]
Chengwen et al, Engineering Adeno-associated Virus Vectors for Gene Therapy, (Nature Reviews Genetics), Published Feb. 10, 2020, vol. 21 (Year: 2020).
[cited by examiner]
Brown et al., Synthetic promoters for CHO cell engineering. Biotechnol Bioeng. Aug. 2014;111(8):1638-47.
[cited by applicant]
Ede et al., Quantitative Analyses of Core Promoters Enable Precise Engineering of Regulated Gene Expression in Mammalian Cells. ACS Synth Biol. May 20, 2016;5(5):395-404.
[cited by applicant]
Lagrange et al., New core promoter element in RNA polymerase II-dependent transcription: sequence-specific DNA binding by transcription factor IIB. Genes Dev. Jan. 1, 1998;12(1):34-44.
[cited by applicant]
Liu et al., Rapid establishment of a HEK 293 cell line expressing FVIII-BDD using AAV site-specific integration plasmids. BMC Res Notes. Sep. 10, 2014;7:626, 6 pages.
[cited by applicant]
Morita et al., Attenuated protein expression vectors for use in siRNA rescue experiments. Biotechniques. Aug. 2012;0(0):1-5.
[cited by applicant]
Ramos et al., The TetR family of transcriptional repressors. Microbiol Mol Biol Rev. Jun. 2005;69(2):326-56.
[cited by applicant]
Russell et al., Phage Bxb1 integrase mediates highly efficient site-specific recombination in mammalian cells. Biotechniques. Apr. 2006,40(4):462-464.
[cited by applicant]
Saxena et al., Design of Synthetic Promoters for Gene Circuits in Mammalian Cells. Methods Mol Biol. 2017;1651:263-273.
[cited by applicant]
Wissmann et al., Tn10 tet operator mutations affecting Tet repressor recognition. Nucleic Acids Res. May 27, 1986;14(10):4253-66.
[cited by applicant]
Luo et al., AAVS1-Targeted Plasmid Integration in AAV Producer Cell Lines. Hum Gene Ther Methods. Jun. 2017;28(3):124-138.
[cited by applicant]
White et al., Use of a negative selectable marker for rapid selection of recombinant vaccinia virus. Biotechniques. May 2011;50(5):303-9.
[cited by applicant]
Wang et al., Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov. May 2019;18(5):358-378.
[cited by applicant]
Wentz et al., Influence of lactate, ammonia, and osmotic stress on adherent and suspension BHK cells. Enzyme Microb Technol. Jan. 1992;14(1):68-75.
[cited by applicant]
Worner et al., Adeno-associated virus capsid assembly is divergent and stochastic. Nat Commun. Mar. 12, 2021;12(1):1642, 9 pages.
[cited by applicant]
Weitzman et al., Adeno-associated virus biology. Methods Mol Biol. 2011;807:1-23.
[cited by applicant]
Gaidukov et al., A multi-landing pad DNA integration platform for mammalian cell engineering. Nucleic Acids Res. May 4, 2018;46(8):4072-4086.
[cited by applicant]
Hamaker et al., Site-specific Integration Ushers in a New Era of Precise CHO Cell Line Engineering. Curr Opin Chem Eng. Dec. 2018;22:152-160.
[cited by applicant]
Hein et al., Establishment of a Scalable Production Process using Stable Helper-Virus Free AAV Producer Cell Lines based on CEVEC's CAP Suspension Cell line. CEVEC Pharmaceuticals GmbH. Poster presentation, 1 page, (202…
[cited by applicant]
Hilliard et al., Systematic identification of safe harbor regions in the CHO genome through a comprehensive epigenome analysis. Biotechnol Bioeng. Feb. 2021;118(2):659-675.
[cited by applicant]
Kolling et al., State-change decisions and dorsomedial prefrontal cortex: the importance of time. Curr Opin Behav Sci. Aug. 2018;22:152-160.
[cited by applicant]
Lee et al., Construction of an rAAV Producer Cell Line through Synthetic Biology. ACS Synth Biol. Oct. 21, 2022;11(10):3285-3295. Pre-publication edition.
[cited by applicant]
Lee et al., Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway. Sci Rep. Feb. 25, 2015;5:8572, 11 pages.
[cited by applicant]
Papapetrou et al., Gene Insertion Into Genomic Safe Harbors for Human Gene Therapy. Mol Ther. Apr. 2016;24(4):678-84.
[cited by applicant]
Pellenz et al., New Human Chromosomal Sites with “Safe Harbor” Potential for Targeted Transgene Insertion. Hum Gene Ther. Jul. 2019;30(7):814-828.
[cited by applicant]
Ramachandra et al., Efficient recombinase-mediated cassette exchange at the AAVS1 locus in human embryonic stem cells using baculoviral vectors. Nucleic Acids Res. Sep. 1, 2011;39(16):e107, 13 pages.
[cited by applicant]
Sajgo et al., Dre—Cre sequential recombination provides new tools for retinal ganglion cell labeling and manipulation in mice. PLoS One. Mar. 7, 2014;9(3):e91435, 15 pages.
[cited by applicant]
Suzuki et al., VCre/VIoxP and SCre/SIoxP: new site-specific recombination systems for genome engineering. Nucleic Acids Res. Apr. 2011;39(8):e49, 11 pages.
[cited by applicant]
Tripp et al., Vector engineering of pRep-Cap and pHelper enhanced AAV productivity by triple transfection in suspension HEK293 cells. Lonza, ASGC Virtual. 17 pages, May 11-14, 2021.
[cited by applicant]
Wissing, Elevecta®—Helper virus-free AAV production with stable CAP® and HEK293 producer cells. Digital Week, Cell & Gene Therapy Manufacturing & Commercialization. 34 pages, Feb. 19, 2021.
[cited by applicant]
Ayuso et al., Production, purification and characterization of adeno-associated vectors. Curr Gene Ther. Dec. 2010;10(6):423-36.
[cited by applicant]
Brent et al., A eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor. Cell. Dec. 1985;43(3 Pt 2):729-36.
[cited by applicant]
Douin et al., Use and comparison of different internal ribosomal entry sites (IRES) in tricistronic retroviral vectors. BMC Biotechnol. Jul. 27, 2004;4:16, 12 pages.
[cited by applicant]
Gossen et al., Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A. Jun. 15, 1992;89(12):5547-51.
[cited by applicant]
Gossen et al., Transcriptional activation by tetracyclines in mammalian cells. Science. Jun. 23, 1995;268(5218):1766-9.
[cited by applicant]
Iida et al., Inducible gene expression by retrovirus-mediated transfer of a modified tetracycline-regulated system. J Virol. Sep. 1996;70(9):6054-9.
[cited by applicant]
Jaubert et al., Tetracycline-regulated transactivators driven by the involucrin promoter to achieve epidermal conditional gene expression. J Invest Dermatol. Aug. 2004;123(2):313-8.
[cited by applicant]
Labow et al., Conversion of the lac repressor into an allosterically regulated transcriptional activator for mammalian cells. Mol Cell Biol. Jul. 1990;10(7):3343-56.
[cited by applicant]
Louvion et al., Fusion of GAL4-VP16 to a steroid-binding domain provides a tool for gratuitous induction of galactose-responsive genes in yeast. Gene. Sep. 6, 1993;131(1):129-34.
[cited by applicant]
Mattioni et al., Regulation of protein activities by fusion to steroid binding domains. Methods Cell Biol. 1994;43 Pt A:335-52.
[cited by applicant]
Murphy et al., Estrogen regulation of protein expression and signaling pathways in the heart. Biol Sex Differ. Mar. 10, 2014;5(1):6, 7 pages.
[cited by applicant]
No et al., Ecdysone-inducible gene expression in mammalian cells and transgenic mice. Proc Natl Acad Sci U S A. Apr. 16, 1996;93(8):3346-51.
[cited by applicant]
Ortiz et al., Tetracycline-inducible gene expression in Trichomonas vaginalis. Molecular and Biochemical Parasitology. Apr. 25, 2003;128(1):43-49.
[cited by applicant]
Pedone et al., A tunable dual-input system for on-demand dynamic gene expression regulation. Nat Commun. Oct. 2, 2019;10(1):4481, 13 pages.
[cited by applicant]
Pinto et al., Precise integration of inducible transcriptional elements (PrIITE) enables absolute control of gene expression. Nucleic Acids Res. Jul. 27, 2017;45(13):15 pages.
[cited by applicant]
Sadowski et al., GAL4-VP16 is an unusually potent transcriptional activator. Nature. Oct. 6, 1988;335(6190):563-4.
[cited by applicant]
Sato et al., Generation of mouse iPS cells using an inducible expression of transgenes via the cumate gene-switch. Anal Biochem. Jun. 15, 2020;599:113748, 7 pages.
[cited by applicant]
Schmidt et al., Adeno-associated virus type 2 Rep78 induces apoptosis through caspase activation independently of p53. J Virol. Oct. 2000;74(20):9441-50.
[cited by applicant]
Sergeeva et al., Multicopy Targeted Integration for Accelerated Development of High-Producing Chinese Hamster Ovary Cells. ACS Synth Biol. Sep. 18, 2020;9(9):2546-2561.
[cited by applicant]
Shin et al., Streamlined Human Cell-Based Recombinase-Mediated Cassette Exchange Platform Enables Multigene Expression for the Production of Therapeutic Proteins. ACS Synth Biol. Jul. 16, 2021;10(7):1715-1727.
[cited by applicant]
Sisson et al., Expression of the reverse tetracycline-transactivator gene causes emphysema-like changes in mice. Am J Respir Cell Mol Biol. May 2006;34(5):552-60.
[cited by applicant]
Song et al., Investigation of arc repressor DNA-binding specificity by comparative molecular dynamics simulations. J Biomol Struct Dyn. 2015;33(10):2083-93.
[cited by applicant]
International Search Report and Written Opinion for Application No. PCT/US2022/078266, dated Feb. 17, 2023, 24 pages.
[cited by applicant]
International Search Report and Written Opinion for Application No. PCT/US2022/078271, dated Feb. 6, 2023, 22 pages.
[cited by applicant]
Invitation to Pay Additional Fees for Application No. PCT/US2022/078275, dated Feb. 17, 2023, 14 pages.
[cited by applicant]
Bae et al., Design and Testing of Vector-Producing HEK293T Cells Bearing a Genomic Deletion of the SV40 T Antigen Coding Region. Mol Ther Methods Clin Dev. Jul. 9, 2020;18:631-638.
[cited by applicant]
Su et al., Self-attenuating adenovirus enables production of recombinant adeno-associated virus for high manufacturing yield without contamination. Nat Commun. Mar. 7, 2022;13(1):1182, with supplementary information, 32…
[cited by applicant]
Sun et al. Expression of heme oxygenase-1 and GFP gene mediated by recombinant adeno-associated-virus in transplanted liver in rats.
[cited by applicant]