723. AAV Mediated Cancer Targeting: Systemic Trafficking to Tumor Is More Important Than Vector Tumor Cell Interaction

Abstract

Adding tumor specific ligands to enhance vector tumor cell interaction is the conventional concept to generate tumor targeting adeno-associated viral vector (AAV). However, it remains poorly proved whether high AAV tumor cell interaction contributes to high tumor localization in vivo following systemic delivery. Here, we conducted directed evolution selections on patient derived xenograft models using a complex AAV capsid library. Uniquely, we compared the pressure for AAV tumor cell interaction alone (intratumoral library injection) and multi-layer pressure including traveling to the tumor and infection (intravenous library injection). Distinct patterns of AAV capsid motifs were identified after intratumoral and intravenous screenings. Motifs isolated from intratumoral screenings were named tumor specific motifs and those isolated from intravenous screenings were named systematic trafficking motifs. AAVs with tumor specific motifs but not systemic trafficking motifs showed significantly increased tumor cell transduction in vitro, indicating enhanced vector target cell interaction after intratumoral-based selections. Interestingly, following systemic delivery, AAVs with systemic trafficking motifs mediated hundreds of folds higher transgene expression than those with tumor specific motifs and wild type AAV in vivo. The combination of both motifs further increases the tumor tropism but not the transduction efficiency in vivo. When analyzing the AAV genome biodistribution by quantifying the genome copy number, the systemic trafficking motifs greatly reduced the native AAV tropism, which potentiated higher accessibility of AAV to the tumor. In contrast, AAVs only bearing tumor specific motifs maintained native AAV tropism and failed to mediate increased genome localization in tumor. Furthermore, in two independent patient derived xenograft models and two different tumor types, our novel AAV vector armed with combined motifs all showed hundreds of folds increase in transduction efficiency with no detectable off-targeting expression. In conclusion, contradictory to the prevailing theory, our study demonstrated that the AAV tumor cell interaction did not contribute to increased tumor localization but just specificity in vivo. Therefore, targeting is not only depending on AAV tumor cell interaction but also, and more importantly, depending on the accessibility of AAV to the tumor cells following systemic delivery. Future cancer directed AAV vector design should take into account the complex processes during systemic delivery as well as the vector tumor cell interaction.