Engineering robust AAV vectors: Addressing ITR instability for effective cancer gene therapy

Introduction: Cancer remains one of the most challenging diseases to treat due to the complexity of tumor biology and barriers to effective drug delivery. Gene therapy using adeno-associated viruses (AAVs) offers a promising approach for oncology, particularly with the development of engineered capsids such as AAV-DJ, which demonstrate enhanced tissue penetration and targeted gene expression in tumor microenvironments. Importantly, AAV-based strategies are also being explored in neuro-oncology, where capsid variants capable of crossing the blood-brain barrier provide new opportunities for treating aggressive brain tumors such as glioblastoma. The AAV genome consists of a therapeutic transgene flanked by 145 bp inverted terminal repeats (ITRs), which are prone to mutations and deletions but also essential for viral packaging. While capsid engineering has improved tumor targeting and systemic delivery, ITR instability remains a critical challenge for consistent therapeutic outcomes. Methods: In this study, we evaluated the impact of five common ITR mutations on vector integrity and performance. HEK293T cells were co-transfected with transfer, Rep/Cap (AAV-DJ), and helper plasmids for viral packaging. ITR mutations were confirmed via Sanger sequencing, and purified vectors were assessed for viral titers using qPCR and transduction efficiency via GFP fluorescence microscopy. Mutant ITRs were subsequently repaired to wild-type sequences to compare recovery of function. To model therapeutic relevance, AAV vectors were tested in tumor-bearing mice using immunofluorescence markers to assess transgene expression within cancerous tissues. Results: Even minor ITR alterations, such as single base pair deletions, demonstrated significantly reduced viral titers and transduction efficiency, with more pronounced effects in constructs carrying large therapeutic cassettes. Although mutant vectors retained some tumor-targeting capability, wild-type ITR constructs achieved the highest efficiency. Conclusion: This work underscores the critical role of ITR integrity in AAV-based oncology applications, including neuro-oncology. Even small sequence disruptions can compromise vector potency, highlighting the need for rigorous quality control during vector design and production. These findings inform the development of robust AAV platforms for cancer gene therapy, ensuring optimal delivery and therapeutic impact.