Discovering human cell-compatible gene therapy virus variants via optimized screening in mouse models

Abstract

In gene therapy, intravenous injection of viral vectors reigns as the primary administration route. These vectors include adeno-associated viruses, adenoviruses, herpes viruses, rhabdoviruses and others. However, these naturally occurring viruses lack inherent tissue or organ tropism for tailored disease treatment. To address this, we devised an optimized process involving directed viral capsid evolution, organ-specific humanized mouse models and in vitro-in vivo virus screening. Our approach allows for the rapid generation specifically modified adeno-associated virus variants, surpassing the time required for natural evolution, which spans millions of years. Notably, these variants exhibit robust targeting of the liver, favouring chimeric human liver cells over murine hepatocytes. Furthermore, certain variants achieve augmented targeting with reduced off-target organ infection, thereby mitigating dosage requirements and enhancing safety in gene therapy.

References

[1] Verma IM, Somia N. Gene therapy – promises, problems and prospects. Nature. 1997;389:239-242.

[2] Hoy SM. Onasemnogene abeparvovec: first global approval. Drugs. 2019;79:1255-1262.

[3] Mendell JR, Al-Zaidy SA, Rodino-Klapac LR, et al. Current clinical applications of in vivo gene therapy with AAVs. Mol Ther. 2021;29:464-488.

[4] Frangoul H, Altshuler D, Cappellini MD, et al. CRISPR-Cas9 gene editing for sickle cell disease and beta-thalassemia. N Engl J Med. 2021;384:252-260.

[5] Liu XS, Wu H, Ji X, et al. Editing DNA methylation in the mammalian genome. Cell. 2016;167:233-247.e17.

[6] Montana CL, Kolesnikov AV, Shen SQ, Myers CA, Kefalov VJ, Corbo JC. Reprogramming of adult rod photoreceptors prevents retinal degeneration. Proc Natl Acad Sci U S A. 2013;110:1732-1737.

[7] Yao K, Qiu S, Wang YV, et al. Restoration of vision after de novo genesis of rod photoreceptors in mammalian retinas. Nature. 2018;560:484-488.

[8] Yu W, Mookherjee S, Chaitankar V, et al. Nrl knockdown by AAV-delivered CRISPR/Cas9 prevents retinal degeneration in mice. Nat Commun. 2017;8:14716.

[9] Zhu J, Ming C, Fu X, et al. Gene and mutation independent therapy via CRISPR-Cas9 mediated cellular reprogramming in rod photoreceptors. Cell Res. 2017;27:830-833.

[10] Browder KC, Reddy P, Yamamoto M, et al. In vivo partial reprogramming alters age-associated molecular changes during physiological aging in mice. Nat Aging. 2022;2:243-253.

[11] Kurita M, Araoka T, Hishida T, et al. In vivo reprogramming of wound-resident cells generates skin epithelial tissue. Nature. 2018;561:243-247.

[12] Lu Y, Brommer B, Tian X, et al. Reprogramming to recover youthful epigenetic information and restore vision. Nature. 2020;588:124-129.

[13] Rodriguez-Matellan A, Alcazar N, Hernandez F, Serrano M, Avila J. In vivo reprogramming ameliorates aging features in dentate gyrus cells and improves memory in mice. Stem Cell Reports. 2020;15:1056-1066.

[14] Ocampo A, Reddy P, Martinez-Redondo P, et al. In vivo amelioration of age-associated hallmarks by partial reprogramming. Cell. 2016;167:1719-1733.e12.

[15] Ribeiro R, Macedo JC, Costa M, et al. In vivo cyclic induction of the FOXM1 transcription factor delays natural and progeroid aging phenotypes and extends healthspan. Nat Aging. 2022;2:397-411.

[16] Yang JH, Hayano M, Griffin PT, et al. Loss of epigenetic information as a cause of mammalian aging. Cell. 2023;186:305-326.e327.

[17] Manno CS, Pierce GF, Arruda VR, et al. Successful transduction of liver in hemophilia by AAV-factor IX and limitations imposed by the host immune response. Nat Med. 2006;12:342-347.

[18] Nathwani AC, Tuddenham EGD, Rangarajan S, et al. Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N Engl J Med. 2011;365:2357-2365.

[19] Li C, Samulski RJ. Engineering adeno-associated virus vectors for gene therapy. Nat Rev Genet. 2020;21:255-272.

[20] Wang D, Tai PWL, Gao G. Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov. 2019;18:358-378.

[21] Gao G, Vandenberghe LH, Alvira MR, et al. Clades of adeno-associated viruses are widely disseminated in human tissues. J Virol. 2004;78:6381-6388.

[22] Miesbach W, Meijer K, Coppens M, et al. Gene therapy with adeno-associated virus vector 5-human factor IX in adults with hemophilia B. Blood. 2018;131:1022-1031.

[23] Pasi KJ, Rangarajan S, Mitchell N, et al. Multiyear follow-up of AAV5-hFVIII-SQ gene therapy for hemophilia a. N Engl J Med. 2020;382:29-40.

[24] Wilson JM, Flotte TR. Moving forward after two deaths in a gene therapy trial of myotubular myopathy. Hum Gene Ther. 2020;31:695-696.

[25] Kuzmin DA, Shutova MV, Johnston NR, et al. The clinical landscape for AAV gene therapies. Nat Rev Drug Discov. 2021;20:173-174.

[26] Rodriguez-Marquez E, Meumann N, Buning H. Adeno-associated virus (AAV) capsid engineering in liver-directed gene therapy. Expert Opin Biol Ther. 2021;21:749-766.

[27] Chiorini JA, Kim F, Yang L, Kotin RM. Cloning and characterization of adeno-associated virus type 5. J Virol. 1999;73:1309-1319.

[28] Farris KD, Pintel DJ. Improved splicing of adeno-associated viral (AAV) capsid protein-supplying pre-mRNAs leads to increased recombinant AAV vector production. Hum Gene Ther. 2008;19:1421-1427.

[29] Deverman BE, Pravdo PL, Simpson BP, et al. Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nat Biotechnol. 2016;34:204-209.

[30] Dandri M, Burda MR, Török E, et al. Repopulation of mouse liver with human hepatocytes and in vivo infection with hepatitis B virus. Hepatology. 2001;33:981-988.

[31] Tateno C, Kawase Y, Tobita Y, et al. Generation of novel chimeric mice with humanized livers by using Hemizygous cDNA-uPA/SCID mice. PLoS One. 2015;10:e0142145.

[32] Ravindra Kumar S, Miles TF, Chen X, et al. Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types. Nat Methods. 2020;17:541-550.

[33] DiMattia MA, Nam HJ, van Vliet K, et al. Structural insight into the unique properties of adeno-associated virus serotype 9. J Virol. 2012;86:6947-6958.

[34] Rose JA, Maizel JV, Inman JK, Shatkin AJ. Structural proteins of adenovirus-associated viruses. J Virol. 1971;8:766-770.

[35] Opie SR, Warrington KH, Agbandje-McKenna M, Zolotukhin S, Muzyczka N. Identification of amino acid residues in the capsid proteins of adeno-associated virus type 2 that contribute to heparan sulfate proteoglycan binding. J Virol. 2003;77:6995-7006.

[36] Govindasamy L, DiMattia MA, Gurda BL, et al. Structural insights into adeno-associated virus serotype 5. J Virol. 2013;87:11187-11199.

[37] Song X, Guo Y, Duo S, et al. A mouse model of inducible liver injury caused by tet-on regulated urokinase for studies of hepatocyte transplantation. Am J Pathol. 2009;175:1975-1983.

[38] Kay MA. Selecting the best AAV capsid for human studies. Mol Ther. 2015;23:1800-1801.

[39] Lisowski L, Dane AP, Chu K, et al. Selection and evaluation of clinically relevant AAV variants in a xenograft liver model. Nature. 2014;506:382-386.

[40] Vercauteren K, Hoffman BE, Zolotukhin I, et al. Superior in vivo transduction of human hepatocytes using engineered AAV3 capsid. Mol Ther. 2016;24:1042-1049.

[41] Wang L, Bell P, Somanathan S, et al. Comparative study of liver gene transfer with AAV vectors based on natural and engineered AAV capsids. Mol Ther. 2015;23:1877-1887.

[42] Lin R, Zhou Y, Yan T, et al. Directed evolution of adeno-associated virus for efficient gene delivery to microglia. Nat Methods. 2022;19:976-985.

[43] Tabebordbar M, Lagerborg KA, Stanton A, et al. Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species. Cell. 2021;184:4919-4938.e22.

[44] Kruepunga N, Hakvoort TBM, Hikspoors J, Kohler SE, Lamers WH. Anatomy of rodent and human livers: what are the differences? Biochim Biophys Acta Mol Basis Dis. 2019;1865:869-878.

[45] Martignoni M, Groothuis GM, de Kanter R. Species differences between mouse, rat, dog, monkey and human CYP-mediated drug metabolism, inhibition and induction. Expert Opin Drug Metab Toxicol. 2006;2:875-894.

[46] Yu Y, Ping J, Chen H, et al. A comparative analysis of liver transcriptome suggests divergent liver function among human, mouse and rat. Genomics. 2010;96:281-289.

[47] Nathwani AC, Reiss UM, Tuddenham EGD, et al. Long-term safety and efficacy of factor IX gene therapy in hemophilia B. N Engl J Med. 2014;371:1994-2004.

[48] Westhaus A, Cabanes-Creus M, Rybicki A, et al. High-throughput in vitro, ex vivo, and in vivo screen of adeno-associated virus vectors based on physical and functional transduction. Hum Gene Ther. 2020;31:575-589.

[49] Berry GE, Asokan A. Cellular transduction mechanisms of adeno-associated viral vectors. Curr Opin Virol. 2016;21:54-60.

[50] Ding W, Zhang L, Yan Z, Engelhardt JF. Intracellular trafficking of adeno-associated viral vectors. Gene Ther. 2005;12:873-880.

[51] Konkle BA, Walsh CE, Escobar MA, et al. BAX 335 hemophilia B gene therapy clinical trial results: potential impact of CpG sequences on gene expression. Blood. 2021;137:763-774.

[52] Grimm D, Lee JS, Wang L, et al. In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses. J Virol. 2008;82:5887-5911.

RIGHTS & PERMISSIONS

2023 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.