MANAN CHOPRA

PI: Karl J. Wahlin, Ph.D., UCSD Health, Department of Ophthalmology

Alternative Splicing and DNA Methylation Analysis of Age-Related Macular Degeneration (AMD) Human Retinal Tissue

Retinal degenerative diseases such as glaucoma, age-related macular degeneration (AMD), and retinitis pigmentosa affect 285 million people worldwide, and can all result in poor vision/vision loss that has varying etiologies and genetic factors involved. Most share a common neurodegenerative phenotype reflecting malfunction or total loss of retinal neurons. Oxford Nanopore Technologies long read sequencing has been increasingly used to investigate DNA methylation and gene expression patterns. These large reads that span hundreds of kilobases can capture entire transcripts in one read, enabling discovery of differentially expressed isoforms of genes of interest. I am taking advantage of this technology to investigate how AMD effects the methylation patterns of certain regions of the genome and the expression of certain genes/isoforms in contrast with healthy tissue. With the information, I hope to elucidate important genes in AMD processes for further research into the mechanisms and treatment of this disease.

Allison Christian

PI: Derek Welsbie, M.D., Ph.D., UCSD Health, Department of Ophthalmology

Cloning of S. marcescens Endonuclease for AAV Purification

Adeno-associated virus (AAV) is the leading viral vector for gene therapies, primarily for its ability to efficiently deliver transgenes with relatively low toxicity and risk of host-genome integration. In the production of AAV, endonucleases are often required to digest producer cell genomic DNA. This reduces viral lysate viscosity and degrades contaminating nucleic acids—both of which decrease DNA fouling and maximize AAV column binding during chromatography purification. While benzonase is most commonly used for viral production, its prohibitive cost makes AAV purification broadly expensive and inaccessible. Here, we outline chromatography purification of the S. marcescens endonuclease as an efficacious alternative for producer cell genomic DNA digestion. This method lowers the overall cost and increases the yield of endonuclease, which can make way for higher throughput, larger scale AAV production imperative for the future of gene therapies.