Thursday, July 3, 2025 7/3/2025

Preserving Genome Integrity In AAV-Mediated Gene Therapy

Award Number: R01HL160748
ORGANIZATION: NATIONAL HEART, LUNG, & BLOOD INSTITUTE
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 02/01/2022
PERIOD OF PERFORMANCE END DATE: 01/31/2026

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Preserving Genome Integrity In AAV-Mediated Gene Therapy - ABSTRACT Adeno-associated virus (AAV) vectors are in clinical development for delivery of genes to treat multiple genetic diseases including hemophilia. While progress has been made to optimize gene delivery, in some studies the required AAV vector doses were high, leading to toxicity and even fatal outcomes in one study. These findings highlight the need for novel approaches to reduce the AAV vector dose to minimize liver toxicity, anti-AAV immune responses, and genotoxicity. Our recent studies and work from others have identified an underappreciated limitation to efficient gene correction with AAV vectors. In a long term study of AAV gene delivery of FVIII in hemophilia A dogs, we found that most of the AAV vector genomes were highly rearranged in transduced liver tissues. These rearrangements typically disrupted the transgene, and so would compromise expression of the transgene product—unexpectedly, our data indicated that most of the AAV vector genomes present did not produce functional protein after transduction. These rearranged AAV genomes were present in integrated forms but also in AAV concatemers that may be episomal forms. It is unclear whether these rearrangements occurred during vector production or after transduction of the target cells, though data is accumulating that at least some of the rearrangements originate in vector producer cells. Our hemophilia A dog study also identified integration events in the canine genome within genes linked to cell growth and cancer that were associated with clonal expansions. Validation of integrated AAV DNA in these expanded clones by sequence analysis showed that in all cases integrated vectors were highly rearranged, with only one of five encoding an intact transgene. An extensive literature documents interactions of AAV with host DNA repair pathways in both vector producer and target cells, though the influence of host factors in AAV DNA rearrangements is mostly unstudied. We hypothesize that modulation of host cell pathways can suppress AAV DNA rearrangements, thereby allowing improved transgene expression per vector DNA copy. In this proposal, we will 1) implement a deep sequencing method to quantify rearrangement frequency in a statistically rigorous fashion, 2) identify cellular pathways that can be modulated with small molecules, siRNAs, or microRNAs that suppress vector rearrangements, and 3) devise novel delivery strategies that support efficient pathway modulation, suppress vector rearrangement, and boost transgene output per vector copy. These methods will be assessed during AAV vector production (Specific Aim 1) and after AAV delivery in the transduced target cells (Specific Aim 2). Our deliverables at the end of the project will be a greatly enhanced understanding of the interaction of AAV with host cell DNA handling pathways, and methods for modulating these pathways to allow safe and effective gene delivery at lower vector doses.


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: 2025 ( Subtotal = $580,716 )
2025	2025	THE CHILDREN'S HOSPITAL OF PHILADELPHIA	3401 CIVIC CENTER BLVD	PHILADELPHIA	PA	19104	PHILADELPHIA	USA	Blood Diseases and Resources Research	000	4	2/28/2025	NON-COMPETING CONTINUATION	$580,716
														Subtotal = $580,716

Issue Date FY: 2024 ( Subtotal = $632,334 )
2024	2024	THE CHILDREN'S HOSPITAL OF PHILADELPHIA	3401 CIVIC CENTER BLVD	PHILADELPHIA	PA	19104	PHILADELPHIA	USA	Blood Diseases and Resources Research	000	3	1/31/2024	NON-COMPETING CONTINUATION	$580,716
2024	2024	THE CHILDREN'S HOSPITAL OF PHILADELPHIA	3401 CIVIC CENTER BLVD	PHILADELPHIA	PA	19104	PHILADELPHIA	USA	Blood Diseases and Resources Research	001	3	6/21/2024	NON-COMPETING CONTINUATION	$51,618
														Subtotal = $632,334

Issue Date FY: 2023 ( Subtotal = $645,239 )
2023	2023	CHILDREN'S HOSPITAL OF PHILADELPHIA, THE	3401 CIVIC CENTER BLVD	PHILADELPHIA	PA	19104	PHILADELPHIA	USA	Blood Diseases and Resources Research	000	2	2/6/2023	NON-COMPETING CONTINUATION	$645,239
														Subtotal = $645,239

Issue Date FY: 2022 ( Subtotal = $631,452 )
2022	2022	CHILDREN'S HOSPITAL OF PHILADELPHIA, THE	3401 CIVIC CENTER BLVD	PHILADELPHIA	PA	19104	PHILADELPHIA	USA	Blood Diseases and Resources Research	000	1	1/31/2022	NEW	$631,452
2022	2022	CHILDREN'S HOSPITAL OF PHILADELPHIA, THE	3401 CIVIC CENTER BLVD	PHILADELPHIA	PA	19104	PHILADELPHIA	USA	Blood Diseases and Resources Research	001	1	3/11/2022	NEW	$0
														Subtotal = $631,452

Grand Total All Awards = $2,489,741

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Preserving Genome Integrity In AAV-Mediated Gene Therapy

Award Number: R01HL160748

ORGANIZATION: NATIONAL HEART, LUNG, & BLOOD INSTITUTE

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

PERIOD OF PERFORMANCE START DATE: 02/01/2022

PERIOD OF PERFORMANCE END DATE: 01/31/2026

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