Progressive retinal dystrophies, including retinitis pigmentosa and macular degeneration, are frequently caused by dominant negative mutations in which the disease could be cured by the silencing the mutant allele. Until recently, this task was impossible. However, the advent of CRISPR/Cas9 genome editing raises the exciting possibility of curing the disease by selectively inactivating the dominant disease-causing allele, while preserving the normal allele. As a proof-of-concept, we will focus on bestrophin, a protein encoded by the BEST1 gene that forms a calcium-activated chloride channel expressed in the retinal pigment epithelium (RPE). The most common BEST1-related disease is called ?Best disease? (BD), which is caused by >200 different dominant-negative protein coding mutations that result in defective chloride channel function, sub- retinal lipid accumulation, and macular atrophy. Induced pluripotent stem cells (iPSCs) from BD patients develop into RPE with disease phenotypes, such as abnormal chloride channel conductance and bestrophin mislocalization. DNA excision with dual cutting Cas9 is remarkably efficient, and by targeting Cas9 with guide RNAs (gRNA) to common polymorphisms on the same allele as (in cis with) the disease mutation, we propose to eliminate the disease protein. By targeting common polymorphisms, we hope to treat a majority of BD patients with just a few gRNA pairs. Although therapeutic editing for BD is promising, many daunting challenges remain. 1. How can we be confident that inactivation of the disease allele will cure the disease? 2. How do we efficiently identify polymorphisms in cis within a 20-30 kb genomic window that can be used for dual Cas9 excision of one allele, while leaving the other allele intact? (Fig. 5). 3. What are the ideal methods to introduce editing DNA/RNA/proteins into RPE for efficient and specific editing? 4. How do we assess the off- target editing for different SNPs and different methods of inserting Cas9 into cells? 5. Can we minimize off- target DNA damage using alternative forms of Cas9? We will systematically address each of these questions with a combination of bioinformatics, cell biology, and bioengineering with these aims:
Aim 1. Determine the efficacy of allele-specific editing and the rescue of BD-associated RPE phenotypes using fluorescent reporter iPSCs Aim 2. Test allele-specific gRNAs for inactivation of disease alleles in RPE from 10 BD patients Aim 3. Determine the fidelity of the most robust allele-specific editing BD is a fertile testing ground for therapeutic editing, since RPE can readily be derived from iPSCs for in vitro studies, and is already the target of cell and gene therapy trials. Our studies also have larger implications: our methods are applicable to any dominant negative genetic disease where the selective removal of a single allele could be therapeutic. For instance, dominant negative disease of photoreceptors (e.g., RHO), auditory cells, nervous system, and muscle, are potential targets in the future.

Public Health Relevance

We are focused on therapeutic editing to cure Best disease by inactivating the dominant negative mutant allele of BEST1. Our studies could lead to new treatments for Best disease as well as other disorders caused by dominant negative mutations.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Diseases and Pathophysiology of the Visual System Study Section (DPVS)
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Shen, Grace L
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J. David Gladstone Institutes
San Francisco
United States
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