PROJECT SUMMARY/ABSTRACT The molecular tools that allow for targeting specific cell types in mammalian retina are essential for understanding how this complex tissue works and responds to disease. Adeno-associated viruses (AAVs) provide safe and long-lasting expression and offer a powerful way to target retinal cells in a rapid, cost- effective, and efficient manner. The challenge is to develop AAV-based tools that allow for cell-type- or subtype-specific labeling and manipulation so that only the desired retinal cell types will be targeted. One of the major strategies for developing cell-type-specific AAVs is to integrate mini-promoters that can drive cell-type-specific gene expression into AAVs. To design such mini-promoters, Cis-regulatory modules (CRMs) in the genome, e.g., enhancers, which are considered as the primary determinants of cell-type-specific gene expression, are often combined with minimal basal promoters. Multiple methods have been developed to nominate cell-type-specific CRMs for AAVs. However, the efficiency has not been high, especially for non- abundant cell types or sub-types in the retina. We have pioneered the study of cell-type-specific CRMs in the retina and explored strategies that can increase the efficiency of identifying them. Putative CRMs are often nominated based on chromatin accessibility due to the binding of transcription factors (TFs) and consequent low nucleosome occupancy. However, many of the chromatin accessible putative CRMs are not active and fail to drive cell-type-specific gene expression in vivo. We found that pre-screening of chromatin accessible putative CRMs based on the density of cell-type-specific TF binding sites (TFBSs) can significantly increase the efficiency of identifying active cell-type-specific CRMs. Based on this result, we hypothesize that cell-type-specific CRMs can be efficiently designed based on cell-type-specific chromatin accessibility and by enriching for cell-type-specific TFBSs, and integrating these CRMs into AAVs can render specificity. The proposed studies will test this hypothesis in two aims. In Aim 1, we will determine whether cell-type- specific CRMs can be efficiently nominated based on cell-type-specific chromatin accessibility and TFBSs enrichment. In Aim 2, we will determine whether assembling cell-type-specific TFBSs into synthetic CRMs can provide cell-type-specificity in AAVs. In summary, the proposed studies will develop novel strategies for designing cell-type-specific CRMs and generate valuable AAV tools to advance retinal studies. As AAV-based gene therapy was approved by FDA for treating inherited retinal dystrophy (e.g., Luxturna), this study could also benefit future gene therapy.