RNA-seq analysis of trans-differentiated ARPE-19 cells transduced by AAV9-AIPL1 vectors

Inherited retinal disorders (IRD) have become a primary focus of gene therapy research since the success of AAV-based therapeutics (voretigene neparvovec-rzyl) for Leber congenital amaurosis type 2 (LCA2). Dozens of monogenic IRDs could be potentially treated with a similar approach using adeno-associated virus (AAV) to transfer a functional gene into the retina. Here, we present the results of design, production and in vitro testing of the AAV serotype 9 (AAV9) vector carrying the codon-optimized (co) copy of aryl hydrocarbon receptor interacting protein like-1 (AIPL1) as a possible treatment for LCA4. The pAAV-AIPL1co was able to successfully transduce retinal pigment epithelium cells (ARPE-19) and initiate expression of human AIPL1. Intriguingly, cells transduced with AAV9-AIPL1co showed much less antiviral response than AAV9-AIPL1wt (wild type AIPL1 ) transduced. RNA-sequencing (RNA-seq) analysis of trans-differentiated ARPE-19 cells transduced with AAV9-AIPL1co demonstrated the significant differences in expression of genes involved in innate immune response. In contrast, AAV9-AIPL1wt induced prominent activation of multiple interferon-stimulated genes. The key part of possible regulatory molecular mechanism is activation of dsRNA-responsive antiviral oligoadenylate synthetases, and significant increase in level of histone coding genes’ transcripts overrepresented in RNA-seq data (i.e. H1, H2A, H2B, H3 and H4). The RNA-seq data suggests that AAV9-AIPL1co exhibiting less immunogenicity than AAV9-AIPL1wt can be used for potency testing using relevant animal models to develop future therapeutics for LCA4. ARPE-19 cells were transduced with AAV9-AIPL1wt, AAV9-AIPL1co or AAV9-GFP (control) vectors. RNA-sequencing of these samples and of non-transduced cells was performed, 3 replicates for each condition