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Pathogenic hotspots illuminate mechanism and therapeutic potential in arrhythmogenic cardiomyopathy

Award Number: R01HL168059
ORGANIZATION: NATIONAL HEART, LUNG, & BLOOD INSTITUTE
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 04/05/2023
PERIOD OF PERFORMANCE END DATE: 02/29/2028

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Pathogenic hotspots illuminate mechanism and therapeutic potential in arrhythmogenic cardiomyopathy - PROJECT SUMMARY Recent exponential advancement of genome engineering technology has revived enthusiasm for its implementation in genetic cardiomyopathies. This is especially promising for arrhythmogenic cardiomyopathy (ACM), a cause of sudden cardiac death and end stage heart failure. Most early genome engineering therapies have focused on gene replacement; however, a significant minority of ACM variants likely act via dominant negative disease mechanisms that will not respond to gene replacement therapy. RNA Binding Motif 20 (RBM20) and plakophilin 2 (PKP2) are genes associated with deadly forms of ACM in which there are both dominant negative and haploinsufficient pathogenic variants. Variants in these genes cause cardiomyopathy and arrhythmia by disrupting global cardiomyocyte transcriptional splicing and desmosomal structure, respectively. hat these variants are clustered in pathogenic hotspots that align to known and novel functional protein domains, indicating that focused study of these hotspots can illuminate differential disease mechanisms and potentially reduce the burden of therapeutic design. Our central hypothesis is that variants in pathogenic hotspots of RBM20 and PKP2 have differential downstream mechanisms that converge on ACM disease phenotypes, and that these pathogenic hotspots allow the design of a genome engineering strategy to edit many pathogenic variants with a single reagent. In Aim 1, we will identify haploinsufficient vs. dominant negative variants in RBM20. We then use high throughput genome engineering techniques to create a library of these variants in induced pluripotent stem cell cardiomyocytes. We will apply a combination of single cell library preparation and long read RNAseq to define the downstream consequences of each disease mechanism on splicing of known and novel RBM20 targets. In Aim 2, we focus on a novel dominant negative mechanism for C-terminal PKP2 truncating variants in which they lose their plasma membrane localization, sequestering critical desmosome components in the cytoplasm. We will use variant effect mapping to define downstream mechanisms of a library of pathogenic PKP2 truncating variants, and will define the role of a novel PKP2 interactor on PKP2 membrane localization. In Aim 3, we will extend our work showing the feasibility of single prime editing (PE) reagents for correction of multiple variants in a pathogenic hotspot in vitro: We will design engineered prime editing (epe)gRNAs with the newest high efficiency PEmax construct for the PKP2 C- terminus hotspot and dominant negative RBM20 RS domain hotspot in vitro. We will then use innovative methods to package PEmax in AAVMYO to correct two pathogenic murine Rbm20 RS domain variants in vivo using the same epegRNA. We will go on to measure the effect of this editing on deep ACM phenotypes. In summary, this project will capitalize on our identification of pathogenic hotspots in RBM20 and PKP2 to provide a comprehensive evaluation of variant-level disease mechanism in these genes, and demonstrate the potential of hotspot directed prime editing as a tractable genome engineering therapeutic.


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 = $722,530 )
2026	2026	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Cardiovascular Diseases Research	000	4	4/21/2026	NON-COMPETING CONTINUATION	$722,530
														Subtotal = $722,530

Issue Date FY: 2025 ( Subtotal = $750,316 )
2025	2025	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Cardiovascular Diseases Research	000	3	2/26/2025	NON-COMPETING CONTINUATION	$675,285
2025	2025	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Cardiovascular Diseases Research	001	3	8/19/2025	NON-COMPETING CONTINUATION	$75,031
														Subtotal = $750,316

Issue Date FY: 2024 ( Subtotal = $745,470 )
2024	2024	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Cardiovascular Diseases Research	000	2	2/29/2024	NON-COMPETING CONTINUATION	$684,616
2024	2024	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Cardiovascular Diseases Research	001	2	6/21/2024	NON-COMPETING CONTINUATION	$60,854
														Subtotal = $745,470

Issue Date FY: 2023 ( Subtotal = $771,818 )
2023	2023	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Cardiovascular Diseases Research	000	1	4/5/2023	NEW	$771,818
2023	2023	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Cardiovascular Diseases Research	001	1	6/5/2023	NEW	$0
														Subtotal = $771,818

Grand Total All Awards = $2,990,134

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Pathogenic hotspots illuminate mechanism and therapeutic potential in arrhythmogenic cardiomyopathy

Award Number: R01HL168059

ORGANIZATION: NATIONAL HEART, LUNG, & BLOOD INSTITUTE

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 04/05/2023

PERIOD OF PERFORMANCE END DATE: 02/29/2028

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