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

> **NIH NIH R35** · UNIVERSITY OF MISSOURI-COLUMBIA · 2020 · $740,965

## Abstract

Project Description
Adeno-associated virus (AAV) is a leading gene therapy vector for delivery of DNA to correct genetic errors or
predispositions to disease. Our objective is an understanding of the virus-host interactions that mediate cell
entry. This foundation will support the widely sought goal of manipulating cell specificity, efficiently transducing
desired cells and reducing off-target side effects. This goal has motivated extensive genetic and phenotypic
characterization, which, with structure, provide an exceptional paradigm for general insights into viral entry.
Our binding and siRNA studies cast doubt on published co-receptors, motivating a gene trap screen in a
haploid cell line. The gene most frequently hit in AAV-resistant mutant cells, encoded a protein that we named
AAVR which binds with nM affinity, inhibits transduction, and is essential for all AAV serotypes and cell types
tested. We have expressed the AAV-binding domains, and will determine a structure of the complex by cryo-
electron microscopy (EM) before further characterizing the interactions through mutagenesis.
Other genes were implicated by the screen. We will determine which encoded proteins interact with AAV or
AAVR through photo-induced cross-linking, pull-down, and mass spectrometric identification of interacting
domains. These will be expressed for binding analysis, and hybrid x-ray/EM structure. The gene trap indicates
potential partners, not just at the cell surface, but during AAV's trafficking to the peri-nuclear trans Golgi
network, and which will illuminate how AAV's phospholipase A2 domain is released for endosomal escape.
EM methods, applicable to AAV, will be developed for general use. For refinement of hybrid structures, our
map-fitting optimization will incorporate parsimony restraints on model flexibility to avoid the common problem
of over-fitting at intermediate resolution. Difference map analysis will be improved, through model-based
calibration, to enhance the sensitivity with which small ligands and subtle conformational changes can be
analyzed by high resolution EM. This will be applicable to studies of AAV's attachment to extracellular glycans,
or to the binding of ligands to ion channels, ribosomes or enzyme complexes in other laboratories.
Within the MIRA framework, contributions to collaborative studies of protein dynamics will continue. These are
integrating crystal structure with NMR relaxation dispersion and residual dipolar couplings to characterize rate-
limiting milli-/micro-second protein motions in arginine kinase as it turns over. Our model system is illuminating
poorly understood general principles of intrinsic motions and conformational selection. These fundamental
questions are the primary goal, but the research also feeds back into methods development. The NMR
relaxation exchange data allows validation tests that our parsimonious model parameterization captures real
conformational changes. In summary, we will continue to work on ...

## Key facts

- **NIH application ID:** 9985934
- **Project number:** 5R35GM122564-04
- **Recipient organization:** UNIVERSITY OF MISSOURI-COLUMBIA
- **Principal Investigator:** MICHAEL S. CHAPMAN
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $740,965
- **Award type:** 5
- **Project period:** 2017-08-01 → 2022-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9985934

## Citation

> US National Institutes of Health, RePORTER application 9985934, Adeno-Associated Virus Gene Therapy Vectors: Molecular Interactions on Cell Entry (5R35GM122564-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9985934. Licensed CC0.

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