Comparing molecular and computational approaches for detecting viral integration of AAV gene therapy constructs

• Published in: Molecular therapy. Methods & clinical development 2023-06, Vol.29, p.395-405
• Main Authors: Oziolor, Elias M., Kumpf, Steven W., Qian, Jessie, Gosink, Mark, Sheehan, Mark, Rubitski, David M., Newman, Leah, Whiteley, Laurence O., Lanz, Thomas A.
• Format: Article
• Language: English
• Subjects: AAV; EPTS; gene therapy; Original; targeted enrichment sequencing; TES; viral integration
• ISSN: 2329-0501; 2329-0501
• DOI: 10.1016/j.omtm.2023.04.009

Many current gene therapy targets use recombinant adeno-associated virus (AAV). The majority of delivered AAV therapeutics persist as episomes, separate from host DNA, yet some viral DNA can integrate into host DNA in different proportions and at genomic locations. The potential for viral integration leading to oncogenic transformation has led regulatory agencies to require investigation into AAV integration events following gene therapy in preclinical species. In the present study, tissues were collected from cynomolgus monkeys and mice 6 and 8 weeks, respectively, following administration of an AAV vector delivering transgene cargo. We compared three different next-generation sequencing approaches (shearing extension primer tag selection ligation-mediated PCR, targeted enrichment sequencing [TES], and whole-genome sequencing) to contrast the specificity, scope, and frequency of integration detected by each method. All three methods detected dose-dependent insertions with a limited number of hotspots and expanded clones. While the functional outcome was similar for all three methods, TES was the most cost-effective and comprehensive method of detecting viral integration. Our findings aim to inform the direction of molecular efforts to ensure a thorough hazard assessment of AAV viral integration in our preclinical gene therapy studies. [Display omitted] DNA was isolated from mouse and cynomolgus monkey following recombinant AAV treatment. Three methods for measuring viral integration were compared: shearing extension primer tag selection ligation-mediated PCR, targeted enrichment sequencing (TES), and whole-genome sequencing. All methods showed similar patterns of integration, but TES was the most cost-effective and comprehensive.