Project Summary/Abstract
Alternative splicing (AS) is the fundamental mechanism of generating isoform diversity in eukaryotic cells.
AS occurs at an especially high frequency in the retina and other nervous system tissues, contributing to many
important cellular and physiological functions within the cells, including tightly regulated and complex processes
like neuronal development. Moreover, dysregulation of AS can have a substantial impact on retinal survival and
function. Retinitis pigmentosa (RP) is a group of inherited retinal diseases that cause dysfunction and
degeneration of the light-sensitive photoreceptor layer of the eye, resulting in irreversible vision loss and
blindness in over 1.5 million people worldwide. Yet, therapeutic options for these patients remain limited. Many
mutations that cause mis-splicing of a gene cause RP. Furthermore, defects in the regulation of AS, including
mutations in spliceosome components and other associated splicing factors, also cause disease. It is unknown
why retinal cells are so particularly, and often exclusively, susceptible to aberrant splicing, despite these defects
often occurring in either ubiquitously expressed genes or from mutated splicing factors found in all tissues of the
body. By combining novel long-read RNA-sequencing technology with CRISPR engineering and 3D human stem
cell organoid models, we can acquire a detailed understanding of AS and learn how dysregulated AS contributes
to retina-specific disease. To further investigate the AS landscape in human retinal development and disease,
this proposal aims to 1) comprehensively examine the AS events in rod and cone photoreceptors that occur
during differentiation of human stem cell-derived retinal organoids, 2) investigate how splicing factor mutations
alter normal AS in the retina compared to other neurons, and 3) understand how dysregulated gene expression
that results from aberrant splicing causes retina-specific degeneration. During the mentored phase of this
proposal, I will take advantage of the many strengths of my multidisciplinary team of mentor/co-mentors,
advisors, and collaborators to perform and analyze the single cell long-read transcriptomic experiments with the
developing retinal organoids and acquire the training needed regarding brain organoid differentiation and
CRISPR methodologies to successfully transition myself to the independent research phase. In the independent
phase, I will use the splicing factor mutant cell lines generated in the Zack lab to further understand the
mechanism(s) of retinal-specific degeneration caused by dysregulated AS events. The experiments proposed
will not only provide the retinal field with a more complete understanding of AS in the retina, knowledge which
can possibly be harnessed to design new treatment options for RP, but the training plan we have developed will
also provide a robust pathway to establishing my successful and productive independent research career that
extends well beyond the aims of this grant.