ABS 030: Cloning of Cell-Type-Specific cis-Regulatory Modules (CRMs) in the AAV vectors

Menglin Li ¹ ² ³, Thiri Wai Wai ¹ ² ³, Marina Nimnual ¹ ² ³, Yi-Rong Peng ¹ ² ³

¹ Jules Stein Eye Institute, Department of Ophthalmology
² David Geffen School of Medicine
³ David Geffen School of Medicine, University of California, Los Angeles, CA, USA.

Van Wickle (2025) Volume 1, ABS030

Introduction: Neurodegenerative retinal diseases have impacted over 300 million people worldwide, causing irreversible blindness. This calls for a high demand to find therapies to treat these diseases. The retinal diseases can be classified into age-related macular degeneration, diabetic retinopathy, and glaucoma.

Due to various mutation possibilities, extensive genetic heterogeneity is a big obstacle in developing gene therapy. For this research, I will be focusing on Adeno-associated virus (AAVs) as the gene therapy. AAVs are very useful in delivering elements to specific cell types in a treatment way. Previous studies have shown that, CRMs, cis-regulatory modules, such as promoters and enhancers that are specific to cell types, are able to guide the specific delivery of AAVs.
Citation: Yan, W., Peng, Y.-R., van Zyl, T., Regev, A., Shekhar, K., Juric, D., & Sanes, J. R. (2020). Cell Atlas of The Human Fovea and Peripheral Retina. Scientific Reports, 10(1), 9802.

Methods: We identified the candidate CRMs for each cell type. Then we cloned these CRMs into AAV vectors. First we amplify CRMs through genomic PCR, extract the CRMs products through gel extraction, perform in-fusion of CRM and AAV vector to get plasmid, transform the plasmid into bacterial cells, and extract the plasmid through mini-prep.

Discussion: This project will contribute to creating a toolbox for targeting specific cells, with potential applications for future therapeutic treatments.