Single-cell and spatial transcriptomic analyses of gene therapy-associated retinal inflammation in non-human primates

Adeno-associated viral (AAV) vectors are rapidly advancing as gene therapies for retinal diseases, but gene therapy-associated uveitis (GTAU) limits their broader application. We assessed the ocular immune response to subretinal AAV gene therapy in two non-human primates (NHPs): NHP1 received bilateral AAV2-CAG-hRPE65 (voretigene neparvovec) at clinical dose; NHP2 received AAV8-GRK1-hRPGRco alongside an analogous mScarlet reporter vector in separate blebs. Longitudinal assessments over three months included multimodal imaging, electroretinography, cytokine profiling, followed by immunohistological, single-cell, and spatial transcriptomic analyses of retinal punches. Both therapies were well-tolerated with preservation of retinal structure and function. Single-cell RNA sequencing revealed that the AAV8 vector transduced 80% of cones/rods in treated areas, while AAV2 targeted 30% of RPE/rods. Transgene expression did not correlate with apoptotic markers. At three months, a persistent type 1 cell-mediated response was detected in the retina dominated by myeloid and T cells. Adjunctive intravitreal anti-TNF-α (adalimumab) did not mitigate this chronic anti-viral response. Spatial transcriptomic analysis and immunohistochemistry localized monocytic phagocytes to the subretinal space, consistent with upregulated cytokines (MCP-1/CCL2, IP-10/CXCL10, IL-8/CXCL8, and IL-6), implicating these cells in driving local inflammation. These findings elucidate the mechanism of GTAU and identify potential therapeutic targets to prevent immune-mediated complications in retinal gene therapy.