AAV2.7m8 transduction of stage 2 human retinal organoids induces highly variable responses in innate and inflammatory gene expression and cytokine secretion.

The preclinical evaluation of recombinant AAV (rAAV) gene therapy vectors should consider host innate and inflammatory immune responses which can cause harmful ocular inflammation and decrease the efficiency of transgene delivery. In this study we determined which cell types expressed the transgene following transduction of 75-day-old human pluripotent stem cell (hPSC)-derived retinal organoids (RO) with the gene delivery vector AAV2.7m8-CAG-EGFP (AAVGFP). We then examined the changes in RO inflammatory gene expression and cytokine secretion following AAVGFP transduction. Our results indicated that RO cell types including photoreceptors (PR), retinal progenitor cells (RPCs), retinal ganglion cells (RGC), horizontal cells, and amacrine cells were transduced by AAVGFP. PCR array analysis following AAVGFP RO transduction detected up regulation of innate and adaptive immune response genes including chemokine (CC) genes, cluster of differentiation (CD) genes, interleukin (IL) genes, and transcription factors. The changes in gene expression following RO transduction varied drastically depending on the manufacturer and the production lot of AAVGFP, and were also not consistent between RO batches transduced with the same lot of AAVGFP. Analysis of RO supernatants indicated that several pro- and anti-inflammatory cytokines including sCD40L, IL-4, IL-6, IL-10, IL-13, IL-17A and MCP-1 were secreted following AAVGFP transduction, but the secretion pattern of these cytokines varied between transductions. These data indicate that intermediate-stage differentiating human ROs derived from the same hPSC line have highly variable responses to rAAV gene delivery vectors.