Interrogating the roles of truncated/mutated inverted terminal repeat sequences in adeno-associated virus vectors in transgene stability through episome formation and host cell genome integration

Gene therapies based on adeno-associated virus (AAV) vectors have been a revolutionary medical advancement in treating human genetic diseases. With a single dose, AAV-mediated gene therapies can confer long-term correction or abatement of disease for the lifetime of the patient. However, recent findings have found that vector DNA integration into the host-cell genome occurs at much higher frequencies than previously thought. Furthermore, integrated transgenes seem to substantially contribute to the long-term stable expression of the therapy. These reports have drastically shifted the safety status of AAVs. The only viral elements retained in AAV vectors used in gene therapy are the inverted terminal repeat (ITR) sequences found at both the ends of the vector genome. The ITRs are essential for the stability for the AAV vector genome after entry into the host cell. We recently revealed that vectors manufactured for gene therapy can harbor truncated or mutated (t/m)ITRs with fairly high frequencies. Interestingly, these types of imperfect ITRs match the truncated ITRs observed in integration sites. I, therefore, hypothesize that truncated/mutated ITRs in AAV viruses and in packaged vectors can influence the frequency of integration. This project proposal is divided into three main aims to define if and how truncated/mutated ITRs can alter the outcomes of AAV genomes, with paradigm-shifting implications for AAV-based gene therapy applications. • Aim 1: To examine ITR structures among wildtype AAVs. Examination of wildtype ITRs, which are proven to also integrate into the host cell infected with AAV, will help to inform on how ITR integrity in gene therapy vectors influences integration. We will perform advanced NGS-based identification of ITRs among natural proviral AAV genomes found in non-human primate tissues and test the ability of wtAAVs to spontaneously form t/mITRs. • Aim 2: To track the post-entry outcomes of vectors that bear a high percentage of t/mITRs. We will test vectors that harbor t/mITRs or wildtype (wt)ITRs for their ability to integrate into the host cell genome. We will develop novel cell culture and NGS-based workflows to interrogate t/mITR-mediated integration in cultured human hepatocytes and in humanized liver mouse models. • Aim 3: To find correlation between integration frequencies in treated non-human primate tissues with the prevalence of t/mITRs in test vectors. Examination of tissues from human patients receiving gene therapy is hard to justify. Non-human primate models are the best proxy to track the kinetics of integration. We will, therefore, examine tissues treated with AAV vectors sourced from previous studies to determine the rate of integration and the structure of ITRs in multiple treated tissues of NHPs. Project Number: 1R01GM155648-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Phillip Tai | Institution: UNIV OF MASSACHUSETTS MED SCH WORCESTER, WORCESTER, MA | Award Amount: $1,675,000 | Activity Code: R01 | Study Section: Special Emphasis Panel[ZRG1 BBBT-X (81)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11133188