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In vitro and in vivo modeling of gene therapy vector stability when challenged by natural virus infection in human hepatocytes

Award Number: R21AI183080
ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 11/13/2024
PERIOD OF PERFORMANCE END DATE: 10/31/2026

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In vitro and in vivo modeling of gene therapy vector stability when challenged by natural virus infection in human hepatocytes - PROJECT SUMMARY Gene therapies based on adeno-associated virus (AAV) vectors have been a revolutionary medical advancement in treating human genetic diseases. With a single dose, AAV-mediated gene therapies can confer long-term correction or abatement of disease for the lifetime of the patient. Recombinant (r)AAVs that are used for the transfer of therapeutic genes are considered safe, partly because these vectors are removed of all viral genes. The only viral elements that are retained are the inverted terminal repeat (ITR) sequences that are at both the ends of the vector genome. However, this feature may make them vulnerable to natural viral infections from wildtype (wt)AAV and helper viruses, such as adenoviruses (AdV), herpesviruses, or papillomaviruses. It is speculated that these challenges may destabilize the therapeutic vector genome or may amplify them in a process called “mobilization”. Unfortunately, models for these events have yet to be explored, and the extent to which these processes can alter the abundance or structure of the vector genomes are unknown. Understanding the stability of rAAV genomes in treated tissues under these naturally occurring and unavoidable circumstances is critically important for the gene therapy field. This project proposal aims to define if and how natural virus infections that can act to alter the composition of AAV-based gene therapy in human hepatocytes, a prevalent gene therapy target for rAAV-based treatments. The objectives are to develop novel methods to characterize the structure of vector genomes as they transition from linear species, to extrachromosomal episomes, and to vector genomes that have integrated into the host cell genome. The bases of this work leverages expertise in long-read sequencing technologies, bioinformatics, and vectorology to address these decades-long unanswered questions. The grant proposal is divided into two main aims: • Aim 1. Track changes in vector genome abundance and episomal configurations within transduced primary human hepatocytes following wtAAV and wtAdV infection. A relevant in vitro model will be developed to track the kinetics of transgene expression and to assess the stability of rAAV genomes in transduced human hepatocytes following wtAAV and wtAdV infection over time. • Aim 2. Assessing the ability for wtAAV and wtAdV infection to destabilize rAAV episomes in humanized mouse livers. Whether wtAAV and wtAdV infection will act to destabilize or mobilize rAAV genomes in human hepatocytes, or whether they will act to drive integration of vectors into the host cell genome will be tested in the Fah/Rag2/Il2rgc-deficient (FRG)-humanized liver mouse model.


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 = $167,500 )
2026	2026	UNIVERSITY OF MASSACHUSETTS MEDICAL SCHOOL	55 LAKE AVE N	WORCESTER	MA	01655	WORCESTER	USA	Allergy and Infectious Diseases Research	000	2	11/28/2025	NON-COMPETING CONTINUATION	$167,500
														Subtotal = $167,500

Issue Date FY: 2025 ( Subtotal = $284,331 )
2025	2025	UNIVERSITY OF MASSACHUSETTS MEDICAL SCHOOL	55 LAKE AVE N	WORCESTER	MA	01655	WORCESTER	USA	Allergy and Infectious Diseases Research	000	1	11/13/2024	NEW	$284,331
														Subtotal = $284,331

Grand Total All Awards = $451,831

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In vitro and in vivo modeling of gene therapy vector stability when challenged by natural virus infection in human hepatocytes

Award Number: R21AI183080

ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

PERIOD OF PERFORMANCE START DATE: 11/13/2024

PERIOD OF PERFORMANCE END DATE: 10/31/2026

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