Research Progress in Integration Sites for Cellular and Gene Therapy Products

doi:10.19803/j.1672-8629.20250151

Abstract

Abstract: Objective To elaborate the insertional integration mechanisms of retroviral and recombinant adeno-associated viral (rAAV) vectors, and review assessment Methods of integrated toxicity and analytic approaches to integration sites in order to call attention to the safety of cellular and gene therapy (CGT) products. Methods CNKI, PubMed and other databases were searched for literature related to integration sites of different vector types in general and toxicity mechanisms and investigational Methods in particular. Results Retroviral vectors were characterized by high-frequency random integration. The integrated toxicity was assessed using in vitro immortalization assay (IVIM), modified IVIM, or the transcript detection method. rAAV vectors were capable of low-frequency targeted integration, and the integrated toxicity was assessed using a neonatal mouse model of methylmalonic acidemia, where circulating cell-free nucleic acid was a more suitable characterization specimen. Besides, advantages and limitations of analytic techniques for integration sites, including WGS, LM-PCR, LAM-PCR, nr LAM-PCR, MGS-PCR, ITR-seq and AAV-seq, were compared. Conclusion Viral vectors are the primary gene delivery tool for the making of CGT products. The analysis of integration sites of vectors is crucial for ensuring product safety and preventing adverse drug reactions.

Key words: Cellular and Gene Therapy, Viral Vectors, Retrovirus, Lentivirus, Recombinant Adeno-Associated Virus, Integration Site, Assessment Model, Next-Generation Sequencing

CLC Number:

R954.5

QI Yujie, WANG Xin, ZHANG Jiahui, MA Enlong, GENG Xingchao. Research Progress in Integration Sites for Cellular and Gene Therapy Products[J]. Chinese Journal of Pharmacovigilance, 2025, 22(7): 721-727.

Add to citation manager EndNote|Ris|BibTeX

URL: https://zgywjj.magtechjournal.com/EN/10.19803/j.1672-8629.20250151

https://zgywjj.magtechjournal.com/EN/Y2025/V22/I7/721

References

[1] WANG L, LIU G, ZHENG L, et al.A New Era of Gene and Cell Therapy for Cancer: a Narrative Review[J]. Ann Transl Med, 2023, 11(9): 321.
[2] ANUROGO D, YULI PRASETYO BUDI N, THI NGO MH, et al. Cell and Gene Therapy for Anemia: Hematopoietic Stem Cells and Gene Editing[J]. Int J Mol Sci, 2021, 22(12): 6275.
[3] SHAIMARDANOVA AA, CHULPANOVA DS, MULLAGULOVA AI, et al.Gene and Cell Therapy for Epilepsy: a Mini Review[J]. Front Mol Neurosci, 2022, 15: 868531.
[4] BATTU R, RATRA D, GOPAL L.Newer Therapeutic Options for Inherited Retinal Diseases: Gene and Cell Replacement Therapy[J]. Indian J Ophthalmol, 2022, 70(7): 2316-2325.
[5] RHEE J, SHIH K C.Use of Gene Therapy in Retinal Ganglion Cell Neuroprotection: Current Concepts and Future Directions[J]. Biomolecules, 2021, 11(4): 581.
[6] WILLS CA, DRAGO D, PIETRUSKO RG.Clinical Holds for Cell and Gene Therapy Trials: Risks, Impact, and Lessons Learned[J]. Mol Ther Methods Clin Dev, 2023, 31: 101125.
[7] CETIN B, ERENDOR F, EKSI YE, et al.Gene and Cell Therapy of Human Genetic Diseases: Recent Advances and Future Directions[J]. J Cell Mol Med, 2024, 28(17): e70056.
[8] CHANCELLOR D, BARRETT D, NGUYEN-JATKOE L, et al.The State of Cell and Gene Therapy in 2023[J]. Mol Ther, 2023, 31(12): 3376-3388.
[9] WANG Y, QIU T, LIANG S, et al.An Overview of Cell and Gene Therapy Development in China[J]. Hum Gene Ther, 2022, 33(1-2): 14-24.
[10] GILL KP, DENHAM M.Optimized Transgene Delivery Using Third-Generation Lentiviruses[J]. Curr Protoc Mol Biol, 2020, 133(1): e125.
[11] SABATINO DE, BUSHMAN FD, CHANDLER RJ, et al.Evaluating the State of the Science for Adeno-Associated Virus Integration: an Integrated Perspective[J]. Mol Ther, 2022, 30(8): 2646-2663.
[12] MODLICH U, BOHNE J, SCHMIDT M, et al.Cell-Culture Assays Reveal the Importance of Retroviral Vector Design for Insertional Genotoxicity[J]. Blood, 2006, 108(8): 2545-2553.
[13] CHANDLER RJ, LAFAVE MC, VARSHNEY GK, et al.Vector Design Influences Hepatic Genotoxicity after Adeno-Associated Virus Gene Therapy[J]. J Clin Invest, 2015, 125(2): 870-880.
[14] CDE, NMPA. Guidelines for Non-Clinical Safety Evaluation of Gene Therapy Products (Trail)[EB/OL]. (2021-11-03)[2025-03-10]. https://www.cde.org.cn/zdyz/domesticinfopage?zdyzIdCODE=3c3eef7964f7950ca9a18b9ce095088c.
[15] CDE, NMPA. Guidelines for Long Term Follow-up Clinical Trail of Gene Therapy Products (Trail)[EB/OL]. (2021-12-03)[2025-03-10]. https://www.cde.org.cn/zdyz/domesticinfopage?zdyzIdCODE=948d4385437338fd7fd4919fd75f5f1a.
[16] CDE. Guidelines for Non-Clinical Safety Evaluation of Genetically Modified Cell Therapy Products (Trail)[EB/OL]. (2021-12-03)[2025-03-10]. https://www.cde.org.cn/zdyz/domesticinfopage?zdyzIdCODE=b7dfbba537d5ecc30659d715f5045acb.
[17] FDA. Preclinical Assessment of Investigational Cellular and Gene Therapy Products[EB/OL].[2025-03-10]. https://www.fda.gov/media/87564/download.
[18] FDA. Long Term Follow-up After Administration of Human Gene Therapy Products[EB/OL].[2025-03-10].https://www.fda.gov/media/113768/download.
[19] EMA. Guideline on Quality, Non-Clinical and Clinical Aspects of Medicinal Products Containing Genetically Modified Cells[EB/OL]. (2021-06-01)[2025-03-10].https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-quality-non-clinical-clinical-requirements-investigational-advanced-therapy-medicinal-products-clinical-trials_en.pdf.Guideline on Genetically Modified revised final-clean-adopted.
[20] BUSHMAN FD.Retroviral Insertional Mutagenesis in Humans: Evidence for Four Genetic Mechanisms Promoting Expansion of Cell Clones[J]. Mol Ther, 2020, 28(2): 352-356.
[21] WANG D, TAI, TAI PWL, GAO G.Adeno-Associated Virus Vector as A Platform for Gene Therapy Delivery[J]. Nat Rev Drug Discov, 2019, 18(5): 358-378.
[22] KREBS AS, MENDONCA LM, ZHANG P.Structural Analysis of Retrovirus Assembly and Maturation[J]. Viruses, 2021, 14(1): 54.
[23] PUNWANI D, KAWAHARA M, YU J, et al.Lentivirus Mediated Correction of Artemis-Deficient Severe Combined Immunodeficiency[J]. Hum Gene Ther, 2017, 28(1): 112-124.
[24] HUANG J, KHAN A, AU BC, et al.Lentivector Iterations and Pre-clinical Scale-up/Toxicity Testing: Targeting Mobilized CD34+ Cells for Correction of Fabry Disease[J]. Mol Ther Methods Clin Dev, 2017, 5: 241-258.
[25] SCHWARZER A, TALBOT SR, SELICH A, et al.Predicting Genotoxicity of Viral Vectors for Stem Cell Gene Therapy Using Gene Expression-Based Machine Learning[J]. Mol Ther, 2021, 29(12): 3383-3397.
[26] HOLMES R, ZÚÑIGA-PFLÜCKER JC. The OP9-DL1 System: Generation of T-Lymphocytes from Embryonic or Hematopoietic Stem Cells in vitro[J]. Cold Spring Harb Protoc, 2009(2): pdb. prot5156.
[27] SCHMITT TM, ZÚÑIGA-PFLÜCKER JC. Induction of T Cell Development from Hematopoietic Progenitor Cells by Delta-like-1 in vitro[J]. Immunity, 2022, 17(6): 749-756.
[28] BASTONE AL, DZIADEK V, JOHN-NEEK P, et al.Development of an in vitro Genotoxicity Assay to Detect Retroviral Vector-Induced Lymphoid Insertional Mutants[J]. Mol Ther Methods Clin Dev, 2023, 30: 515-533.
[29] LIDONNICI MR, PALEARI Y, TIBONI F, et al.Multiple Integrated Non-Clinical Studies Predict the Safety of Lentivirus-Mediated Gene Therapy for β-thalassemia[J]. Mol Ther Methods Clin Dev, 2018, 11: 9-28.
[30] GONG X, GAO H, WANG W, et al.Intramuscular Injection of rAAV2-Retro for Low Motor Neuron Transduction: Evaluating Five Promoters[J]. Int J Med Sci, 2025, 22(4): 775-789.
[31] SCHNEPP BC, JENSEN RL, CHEN CL, et al.Characterization of Adeno-Associated Virus Genomes Isolated from Human Tissues[J]. J VIROL, 2005, 79(23): 14793-14803.
[32] NIEMEYER GP, HERZOG RW, MOUONT J, et al.Long-Term Correction of Inhibitor-Prone Hemophilia B Gogs Treated with Liver-DirectedAAV2-Mediated Factor IX Gene Therapy[J]. Blood, 2009, 113(4): 797-806.
[33] MARTINS KM, BRETON C, ZHENG Q, et al.Prevalent and Disseminated Recombinant and Wild-Type Adeno-Associated Virus Integration in Macaques and Humans[J]. Hum Gene Ther, 2023, 34(21-22): 1081-1094.
[34] DONSANTE A, MILLER DG, LI Y, et al.AAV Vector Integration Sites in Mouse Hepatocellular Carcinoma[J]. Science, 2007, 317(5837): 477.
[35] SHEN W, LIU S, OU L. rAAV Immunogenicity, Toxicity, and Durability in 255 Clinical Trials: a Meta-Analysis[J]. Front Immunol, 2022, 13: 1001263.
[36] WANG JH, GESSLER DJ, ZHAN W, et al.Adeno-Associated Virus as a Delivery Vector for Gene Therapy of Human Diseases[J]. Signal Transduct Tar, 2024, 9(1): 78.
[37] CESANA D, CALABRIA A, RUDILOSSO L, et al.Retrieval of Vector Integration Sites from Cell-Free DNA[J]. Nat Med, 2021, 27(8): 1458-1470.
[38] MANDLIK JS, PATIL AS, SINGH S. Next-Generation Sequencing (NGS): Platforms and Applications[J]. J Pharm Bioallied Sci, 2024, 16(Suppl 1): 41-45.
[39] JOBANPUTRA V, WRZESZCZYNSKI KO, BUTTNER R, et al.Clinical Interpretation of Whole-Genome and Whole-Transcriptome Sequencing for Precision Oncology[J]. Semin Cancer Biol, 2022, 84: 23-31.
[40] WELLS DW, GUO S, SHAO W, et al.An Analytical Pipeline for Identifying and Mapping the Integration Sites of HIV and Other Retroviruses[J]. BMC Genomics., 2020, 21(1): 216.
[41] SCHMIDT M, SCHWARZWAELDER K, BARTHOLOMAE C, et al.High-Resolution Insertion-Site Analysis by Linear Amplification-Mediated PCR (LAM-PCR)[J]. Nat Methods, 2007, 4(12): 1051-1057.
[42] WANG W, BARTHOLOMAE CC, GABRIEL R, et al.The LAM-PCR Method to Sequence LV Integration Sites[J]. Methods Mol Biol, 2016, 1448: 107-120.
[43] BEARD BC, ADAIR JE, TROBRIDGE GD, et al.High-Throughput Genomic Mapping of Vector Integration Sites in Gene Therapy Studies[J]. Methods Mol Biol, 2014, 1185: 321-344.
[44] ZHOU X Y, JIA F Y, WU X L, et al.Applicability of Reference Cells in Lentiviral Vector Integration Site Detection with Different Methods[J]. Chin J Microbiol Immunol(中华微生物学和免疫学杂志), 2023, 43(10): 791-801.
[45] BRETON C, CLARK PM, WANG L, et al.ITR-Seq, a Next-Generation Sequencing Assay, Identifies Genome-Wide DNA Editing Sites in vivo Following Adeno-Associated Viral Vector-Mediated Genome Editing[J]. BMC Genomics., 2020, 21(1): 239.
[46] GREIG JA, MARTINS KM, BRETON C, et al.Integrated Vector Genomes May Contribute to Long-Term Expression in Primate Liver after AAV Administration[J]. Nat Biotechnol, 2024, 42(8): 1232-1242.
[47] HANLON KS, KLEINSTIVER BP, GARCIA SP, et al.High Levels of AAV Vector Integration into CRISPR-Induced DNA Breaks[J]. Nat Commun, 2019, 10(1): 4439.
[48] KARIMZADEH M, ARLIDGE C, ROSTAMI A, et al.Human Papill-omavirus Integration Transforms Chromatin to Drive Oncogenesis[J]. Genome Biol, 2023, 24(1): 142.
[49] KAMAL M, LAMEIRAS S, DELOGER M, et al.Human Papilloma Virus (HPV) Integration Signature in Cervical Cancer: Identification of MACROD2 Gene as HPV Hot Spot Integration Site[J]. Brit J Cancer, 2021, 124(4): 777-785.
[50] SPEEL EJ.HPV Integration in Head and Neck Squamous Cell Carcinomas: Cause and Consequence[J]. Recent Results Cancer Res, 2017, 206: 57-72.
[51] KOODAMVETTY A, THANGAVEL S.Advancing Precision Medicine: Recent Innovations in Gene Editing Technologies[J]. Adv Sci, 2025, 12(14): e2410237.
[52] DABIRI H, SAFARZADEH KP, HABIBI AM, et al.Site-Specific Transgene Integration in Chimeric Antigen Receptor(CAR) T Cell Therapies[J]. Biomark Res, 2023, 11(1): 67.
[53] PANDEY S, GAO XD, KRASNOW NA, et al.Efficient Site-Specific Integration of Large Genes in Mammalian Cells via Continuously Evolved Recombinases and Prime Editing[J]. Nat Biomed Eng, 2025, 9(1): 22-39.
[54] DURRANT MG, PERRY NT, PAI JJ, et al.Bridge RNAs Direct Programmable Recombination of Target and Donor DNA[J]. Nature, 2024, 630(8018): 984-993.