Preclinical Safety and Tolerability of an AAV Vector Expressing TLR9 in Mice and Hamsters

Toll-like receptor 9 (TLR9) is an innate DNA sensor that activates pro-inflammatory pathways and contributes to the maturation of antigen presenting cells. TLR9 deficiency has been implicated in impaired tumor immunosurveillance and attenuated responsiveness to immune checkpoint blockade. We developed an adeno-associated virus (AAV) vector encoding TLR9 to restore receptor expression in individuals with TLR9 deficiency and augment antitumor immunity. We first evaluated transgene biodistribution in mice 21 days after intraperitoneal administration of 1x10 ¹¹ vector genomes (vg) of AAV6.2FF-TLR9 using RNA in-situ hybridization. TLR9 was detected in liver, spleen, pancreas, ovary, heart, and lymph node, indicating systemic transduction and transcription following intraperitoneal delivery. To assess safety, mice and hamsters received low (1.5x10 ¹³ vg/kg) or high (4.5x10 ¹³ vg/kg) dose of AAV6.2FF-TLR9 intraperitoneally and were evaluated at 7, 28, and 56 days post-administration. Across both species and dosages, no adverse vector-related changes were observed in leukocyte counts, plasma biomarkers of hepatic or renal function, pro-inflammatory cytokines, or upon histopathological examination. The broad tissue expression and favorable safety profile of intraperitoneally delivered AAV6.2FF-TLR9 support progression of this vector toward clinical development as a gene replacement strategy to potentially enhance to efficacy of checkpoint blockade in patients with metastatic malignancies and impaired TLR9 signaling.