Decoupling Efficacy from Toxicity: Engineering Spatial Control in AAV-Mediated Gene Therapy

Abstract

Hepatotoxicity poses a critical safety challenge for AAV-mediated gene therapy. To mitigate this, we evaluated strategies to minimize off-target hepatic transduction using an antibody expression model. We compared (i) muscle-restricted wild-type AAV9 expression and (ii) a novel myotropic capsid variant, AAV.eM. In humanized B-NDG mice bearing Raji-Luc lymphomas, intravenous administration of AAV9-MHCK7 encoding an CD19CD3 bispecific T-cell engager failed to reduce tumor burden. Conversely AAV.eM-MHCK7-CD19CD3 substantially alleviated tumor burden and achieved lymphoma clearance. By leveraging tissue-specific microRNAs, precise restriction of AAV.eM-mediated transgene expression to skeletal or cardiac muscle was achieved. Incorporating a heart-specific miR-208a binding site into the transgenes 3UTR did not compromise therapeutic efficacy when delivered via AAV.eM-MHCK7. Intramuscular delivery of AAV9-MHCK7-CD19CD3 or AAV.eM-MHCK7-CD19CD3 both cleared Raji-Luc tumors at a dose of 5 x 1012 vg/kg, underscoring the advantage of localized and targeted rAAV delivery over systemic administration. Notably, only AAV.eM-MHCK7-CD19CD3 achieved tumor eradication at a tenfold lower intramuscular dose (5 x 1011 vg/kg), reducing manufacturing costs and risks of dose-dependent immunogenicity and toxicity. Our findings demonstrate that combining tissue-specific targeting--via engineered capsids or tissue-selective promoters--with local delivery robustly reduces off-target hepatic expression, providing a strategic framework for enhancing the safety of AAV-based gene therapies.