1347 Using Living Human Glioblastoma Samples to Develop Tumor-Targeted AAV Gene Therapy Vectors

INTRODUCTION: Glioblastoma multiforme is the most common primary intracranial malignant brain tumor in adults, known for its aggressive behavior and poor prognosis. Several therapeutic approaches have been described; unfortunately, no treatment alternatives have substantially impacted the disease course. Gene therapy promises the ability to deliver specific sequences into certain cells, but we still lack the ability to target specific tumoral cell types in the human brain. METHODS: To develop selective AAV gene therapy vectors, we have adapted an organotypic model that keeps alive resected human glioblastoma specimens under specialized culture conditions for more than two weeks. We apply panels of AAVs to the tissue, allowing time for transgene expression to occur. By immunostaining and single nuclei sequencing, we can define which cell types our AAVs are able to enter. RESULTS: A preliminary panel of 14 well-established AAV gene therapy vectors shows that the majority of existing capsids, including those used in clinical trials (AAV2 and AAV9), have limited transduction of glioblastoma tumor cells. Notably, promoter selection makes a significant difference, with all capsids’ transgene expression improved with the CAG promoter as compared with EF1a. Interestingly, AAV2-retro shows significantly better transduction regardless of the promoter used, with up to 60% of GFAP+ tumor cells expressing a GFP transgene. AAV2-retro differs from AAV2 by a short segment of 10 amino acids, offering a focused area for selective mutation to identify alterations that further improve the efficiency and specificity of transduction for tumor cells. CONCLUSIONS: Development of a highly selective and efficient AAV vector for glioblastoma would allow either convection-enhanced delivery into the infiltrated but still salvageable brain, or suggest opportunities for locally delivered intra-arterial viruses able to cross the disrupted blood-brain barrier.