A heterozygous hexanucleotide (GGGGCC) repeat expansion in a single allele of the C9orf72 gene is the most frequent genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), two fatal and irreversible neurodegenerative diseases without effective treatment. Given that the cellular dysfunction caused by this mutation is multifactorial, targeting the gene itself by CRISPR/Cas9 gene editing is a potentially curative intervention. This work proposes novel applications of CRISPR gene editing technology to edit or silence the pathogenic C9orf72 disease gene in FTD/ALS patient derived iPSC. Three editing strategies will be evaluated for ability to correct the pathologic C9orf72 repeat expansion, including: A) excising exon 1a to silence transcription of the repeat expansion, B) excising the repeat expansion itself, C) allele-specific inactivation of the mutant allele (Aim 1a). In addition, the efficiency of gene editing, off target effects, and ability to correct cellular pathology will be investigated in C9FTD/ALS patient-derived neurons and microglia (Aim 1b). Finally, new single- molecule sequencing technology to accurately measure the repeat expansion size will be employed to size the repeat expansion in various patient iPSC lines and human tissue (Aim 2). This work combines novel CRISPR/Cas9 editing technologies, cellular disease modeling, molecular and genomic analysis, and bioinformatics to address these aims. My goal in seeking a K08 Mentored Clinical Scientist Research Career Development Award is to acquire the necessary knowledge and scientific training to launch my career as an independent R01 funded academic investigator. My long-term goal is to make major advances in the therapeutic approaches to neurodegenerative diseases, including dementias. My doctoral and clinical training provide me with the impetus to address clinically relevant and high impact hypothesis driven research questions. The exceptional research environment at the UCSF affiliated Gladstone Institutes combines leaders across many fields with cutting-edge technology and equipment. I have assembled a team of highly accomplished mentors and advisors to guide me on my path to becoming an independent investigator. My training plan is designed to advance my technical skill set in areas that are new to me (CRISPR gene editing, genomic analysis, bioinformatics, and advanced cellular disease modeling) as well as provide experience in areas critical to successfully running a lab (scientific communication and lab management). Completing the research and obtaining the skill set I propose here will prepare me well to obtain an R01 or equivalent funding to launch my career as an independent investigator.

Public Health Relevance

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are two fatal and incurable neurodegenerative diseases linked by a shared genetic cause ? a heterozygous hexanucleotide (GGGGCC) repeat expansion in a single allele of the C9orf72 gene. The goal of this work is to develop novel CRISPR based therapeutic gene editing technologies and test whether gene editing can reverse the cellular pathology caused by this repeat expansion in patient derived cells. The results of these studies will advance our use of CRISPR technologies for therapeutic editing in FTD/ALS, inform our understanding of the regulation of C9orf72 gene, and will be applicable to many other repeat expansion and single gene disorders.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Clinical Investigator Award (CIA) (K08)
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Neurological Sciences Training Initial Review Group (NST)
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Gubitz, Amelie
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University of California San Francisco
Schools of Medicine
San Francisco
United States
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