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Cellular perturbations to enhance precise therapeutic genome editing to treat FTD/ALS

Award Number: F32AG081085
ORGANIZATION: NATIONAL INSTITUTE ON AGING
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS
PERIOD OF PERFORMANCE START DATE: 04/01/2023
PERIOD OF PERFORMANCE END DATE: 03/31/2025

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Cellular perturbations to enhance precise therapeutic genome editing to treat FTD/ALS - PROJECT SUMMARY CRISPR holds great promise toward therapeutically editing pathological mutations in the gene fused in sarcoma (FUS), known to cause 5% of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) cases. The FUS gene is ideal for therapeutic editing, as it is a dominant-negative genetic mutation, allowing for one allele to produce enough of the FUS protein for cells to survive. However, there are several challenges that must be addressed before this promise can be realized, including the inefficient delivery of Cas9 and gRNA into cells and the lack of indel formation after Cas9 cleavage. Towards this, my lab has established a robust neuronal differentiation protocol via doxycycline-inducible transcription factors. With this protocol, I can derive phenotypic neurons from iPSCs that recapitulate disease phenotypes within 7 days13. We have additionally engineered virus-like particles (VLP), that harbor no genetic material, to deliver Cas9 protein and gRNA to neurons12. In this grant, I propose exploring small perturbations within neurons to gain further insights into how CRISPR therapies can be designed for the precise editing of neurodegenerative diseases. I have collected preliminary data with neurons and iPSCs, using the same CRISPR editing strategy, and found that iPSCs and neurons have divergent editing outcomes. Specifically, we used gRNA designed to cleave sequences in the NEFL and B2M genes. In both genes, we found that iPSCs had multiple editing outcomes not present in neurons, commonly large deletions. Editing in neurons frequently resulted in no indel being produced. The few indels that do occur in neurons appear to be almost exclusively small insertions (+1 indel) are produced via mutagenic NHEJ. These small indels are ideal to cause frameshifts and induce nonsense-mediated decay and represent an optimal strategy for therapeutic editing in neurons. I propose studying how iPSC-neurons can be perturbed to increase the prevalence of our desired editing outcome. Using our directed differentiation protocol as well as our VLP delivery system, I will explore how indel frequency and identity are affected by changes in the transcription of the edited gene and the expression and activity of DNA damage proteins. In Aim 1, I will alter the transcription of genes to explore how interactions with RNA polymerase change editing frequency and indel formation. In Aim 2, I will investigate how changing the expression or activity of DNA repair enzymes affects editing outcomes. The insight gained from the work described in this grant can be used to precisely edit neurons to silence pathological genetic mutations, like FUS mutations. Future studies would also apply this editing strategy to other mutated genes in neurons and potentially other post-mitotic cells. The superior training environment provided at the Gladstone Institutes will allow me the opportunity to explore these aims with expert research mentorship, support from core facilities, and beneficial collaborations.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2026 ( Subtotal = $0 )
2026	2024	J.DAVID GLADSTONE INSTITUTES	1650 OWENS ST	SAN FRANCISCO	CA	94158	SAN FRANCISCO	USA	Aging Research	000	2	2/24/2026	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2024 ( Subtotal = $74,284 )
2024	2024	J.DAVID GLADSTONE INSTITUTES	1650 OWENS ST	SAN FRANCISCO	CA	94158	SAN FRANCISCO	USA	Aging Research	000	2	5/15/2024	NON-COMPETING CONTINUATION	$74,284
														Subtotal = $74,284

Issue Date FY: 2023 ( Subtotal = $69,080 )
2023	2023	J.DAVID GLADSTONE INSTITUTES	1650 OWENS ST	SAN FRANCISCO	CA	94158	SAN FRANCISCO	USA	Aging Research	001	1	3/29/2023	NEW	$0
2023	2023	J.DAVID GLADSTONE INSTITUTES	1650 OWENS ST	SAN FRANCISCO	CA	94158	SAN FRANCISCO	USA	Aging Research	000	1	3/29/2023	NEW	$69,080
														Subtotal = $69,080

Grand Total All Awards = $143,364

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Cellular perturbations to enhance precise therapeutic genome editing to treat FTD/ALS

Award Number: F32AG081085

ORGANIZATION: NATIONAL INSTITUTE ON AGING

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS

PERIOD OF PERFORMANCE START DATE: 04/01/2023

PERIOD OF PERFORMANCE END DATE: 03/31/2025

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