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Adeno-Associated Virus Gene Therapy Vectors: Molecular Interactions on Cell Entry

Award Number: R35GM122564
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 08/01/2017
PERIOD OF PERFORMANCE END DATE: 07/31/2028

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Adeno-Associated Virus Gene Therapy Vectors: Molecular Interactions on Cell Entry - Project Summary / Abstract Adeno-associated virus (AAV) is a leading delivery vector for gene therapies of a wide range of genetic disease and predilections. The FDA has recently approved AAV-mediated treatments for spinal muscular atrophy (SMA) and RPE-associated retinal dystrophy, while 150 clinical trials are ongoing. However, inefficient transduction requires high doses with risk of immunotoxicity that has been evident in clinical trials. A genome-wide screen for host proteins, needed for AAV cell transduction, changed the understanding of AAV entry. Hitherto-uncharacterized AAVR was identified as a key receptor, while the downstream role of GPR108 is most likely in endosomal escape. AAVR cryo-EM structure will be put into biological context. Studies of GPR108 will start by pinpointing the entry step and cell location where AAV and GPR108 associate, and identification of the domains with which AAV interacts. The approach to structure will be holistic. Overall configuration will come from cryo-electron tomography (ET) of AAV complexed with native-like GPR108 nanodiscs. This will be integrated with high resolution cryo-electron microscopy (EM) of AAV complexed with expressed extracellular domains of GPR108. Triggers will be investigated of an AAV conformational transition that releases a virally-sequestered phospholipase A2 for endosomal escape, as AAV traffics to the nucleus. Antibody neutralization of AAV is widely considered mediated by interference with glycan-binding or of a post-entry step. This will be reevaluated after finding overlap in the binding of AAVR and several neutralizing monoclonal antibodies. Prevalent in vivo modes of binding will first be established through cryo-EM using polyclonal antibodies from pooled human serum. Mutations, directed towards escape, will be used to test whether receptor-binding and entry are, in fact, primary mechanisms of neutralization, and whether escape to rapid neutralization by pre-existing antibodies can be designed without harm to receptor-mediated cell entry. Computer methods, optimizing atomic structure vs. cryo-EM maps, prototyped during AAV studies, will be developed and disseminated. Modules will implement the RSRef algorithm, unique in matching to map values, a sum of 3D atomic profiles of refinable experimental resolution. This will be combined with open source force fields, opening an emerging application of atomic resolution cryo-EM in determining hydrogen bonding networks and protonation states. For resolutions below 3 Å, restraints eliminating unnecessary freedom will be introduced to limit the over-fitting that is an under-appreciated problem in EM refinement. The project’s main goal is a molecular understanding of AAV’s host interactions, providing structural and mechanistic foundations for engineering needed improvements in vector efficiency and specificity in development of safe gene therapies. Broader collateral impact will come from a model system for viral entry that will be put to rigorous practical test, and computer methods for improving the precision of EM structures.


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 = $0 )
2025	2024	UNIVERSITY OF MISSOURI SYSTEM	121 UNIVERSITY HALL	COLUMBIA	MO	65211	BOONE	USA	Biomedical Research and Research Training	000	7	11/6/2024	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2024 ( Subtotal = $765,461 )
2024	2024	UNIVERSITY OF MISSOURI SYSTEM	121 UNIVERSITY HALL	COLUMBIA	MO	65211	BOONE	USA	Biomedical Research and Research Training	002	7	8/16/2024	NON-COMPETING CONTINUATION	$765,461
2024	2023	UNIVERSITY OF MISSOURI SYSTEM	121 UNIVERSITY HALL	COLUMBIA	MO	65211	BOONE	USA	Biomedical Research and Research Training	001	6	6/3/2024	COMPETING CONTINUATION	$89,764
2024	2021	UNIVERSITY OF MISSOURI SYSTEM	121 UNIVERSITY HALL	COLUMBIA	MO	65211	BOONE	USA	Biomedical Research and Research Training	000	5	6/3/2024	NON-COMPETING CONTINUATION	-$89,764
														Subtotal = $765,461

Issue Date FY: 2023 ( Subtotal = $779,586 )
2023	2023	UNIVERSITY OF MISSOURI SYSTEM	121 UNIVERSITY HALL	COLUMBIA	MO	65211	BOONE	USA	Biomedical Research and Research Training	000	6	8/15/2023	COMPETING CONTINUATION	$779,586
														Subtotal = $779,586

Issue Date FY: 2021 ( Subtotal = $740,965 )
2021	2021	UNIVERSITY OF MISSOURI SYSTEM	316 UNIVERSITY HALL	COLUMBIA	MO	65211	BOONE	USA	Biomedical Research and Research Training	000	5	7/27/2021	NON-COMPETING CONTINUATION	$740,965
														Subtotal = $740,965

Issue Date FY: 2020 ( Subtotal = $740,965 )
2020	2020	UNIVERSITY OF MISSOURI SYSTEM	316 UNIVERSITY HALL	COLUMBIA	MO	65211	BOONE	USA	Biomedical Research and Research Training	000	4	7/27/2020	NON-COMPETING CONTINUATION	$740,965
														Subtotal = $740,965

Issue Date FY: 2019 ( Subtotal = $740,965 )
2019	2019	UNIVERSITY OF MISSOURI SYSTEM	316 UNIVERSITY HALL	COLUMBIA	MO	65211	BOONE	USA	Biomedical Research and Research Training	002	3	7/30/2019	NON-COMPETING CONTINUATION	$740,965
2019	2018	UNIVERSITY OF MISSOURI SYSTEM	316 UNIVERSITY HALL	COLUMBIA	MO	65211	BOONE	USA	Biomedical Research and Research Training	001	2	2/3/2019	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$120,224
2019	2017	OREGON HEALTH & SCIENCE UNIVERSITY	3181 SW SAM JACKSON PARK RD	PORTLAND	OR	97239	MULTNOMAH	USA	Biomedical Research and Research Training	000	1	2/3/2019	NEW	-$120,224
														Subtotal = $740,965

Issue Date FY: 2018 ( Subtotal = $705,187 )
2018	2018	UNIVERSITY OF MISSOURI SYSTEM	316 UNIVERSITY HALL	COLUMBIA	MO	65211	BOONE	USA	Biomedical Research and Research Training	000	2	9/19/2018	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$705,187
2018	2017	OREGON HEALTH & SCIENCE UNIVERSITY	3181 SW SAM JACKSON PARK RD	PORTLAND	OR	97239	MULTNOMAH	USA	Biomedical Research and Research Training	001	1	9/20/2018	NEW	$0
														Subtotal = $705,187

Issue Date FY: 2017 ( Subtotal = $713,417 )
2017	2017	OREGON HEALTH & SCIENCE UNIVERSITY	3181 SW SAM JACKSON PARK RD	PORTLAND	OR	97239	MULTNOMAH	USA	Biomedical Research and Research Training	000	1	8/1/2017	NEW	$713,417
														Subtotal = $713,417

Grand Total All Awards = $5,186,546

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Adeno-Associated Virus Gene Therapy Vectors: Molecular Interactions on Cell Entry

Award Number: R35GM122564

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

PERIOD OF PERFORMANCE START DATE: 08/01/2017

PERIOD OF PERFORMANCE END DATE: 07/31/2028

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