The Adeno-associated virus recombinant AAV (rAAV) gene delivery system is entering a crucial and exciting phase with the promise of more than 20 years of intense research now realized in a number of successful human clinical trials. However, two major challenges that must be overcome for full realization are transient or low transgene expression and the detrimental effect of circulating host antibodies. The primary goal of our research is to use structural biology approaches, combined with biochemistry, molecular biology, and in vivo animal studies to develop AAV viral vectors that overcome these challenges and have improved clinical therapeutic efficacy. A key component to success is to identify regions of the capsid that participate in cellular interactions leading to or altering infection, i particular cell receptor attachment and interaction with host immunity, especially neutralizing antibodies. In this project, we will dissect the determinants of tissue transduction and also determine if these overlap with antigenic regions of the capsid. To get a general understanding we will compare AAV1/6, AAV5, AAV8, and AAV9, which represent the range of sequence and structure diversity among the AAV serotypes and have shown promise for clinical applications. We will take a reductionist approach to tackle the problem. We will first use structural biology, namely X-ray crystallography and cryo-electron microscopy and image reconstruction, to define the most antigenically reactive regions of the AAV capsids and then determine their role in infection through molecular and biochemical methods. The goal will then be to identify and mutate the residues that do not affect infection, characterize resulting vectors with respect to their antigenic reactivity against human serum, including those from post-AAV clinical gene therapy patients, and use the data arising as the guiding principle for general antibody escape AAV vector design. These will be tested in vivo, including the use of a clinically relevant hemophilia B mouse model. The overall impact of this project will be an understanding of the AAV capsid determinants necessary for successful infection and the neutralization of this process by host antibodies, a fundamental goal of virology. This information is critical for the gene therapy community in order to engineer recombinant AAV vectors for successful gene delivery in the presence of a host immune response, a natural host defense mechanism that cannot be prevented but must be circumvented in gene delivery applications because it dramatically reduces or eliminates gene expression.

Public Health Relevance

The AAV gene delivery vector system has entered an exciting phase with a number of successful human clinical trials that report therapeutic efficacy. The full realization of the translational benefits of this system will require the development of novel vectors with the capacity to retain their natural tropisms along with the ability to evade the human antibody immune response to the capsid.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-IDM-U (02))
Program Officer
Marino, Pamela
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Florida
Schools of Medicine
United States
Zip Code
Bennett, Antonette; Patel, Saajan; Mietzsch, Mario et al. (2017) Thermal Stability as a Determinant of AAV Serotype Identity. Mol Ther Methods Clin Dev 6:171-182
Pittman, Nikéa; Misseldine, Adam; Geilen, Lorena et al. (2017) Atomic Resolution Structure of the Oncolytic Parvovirus LuIII by Electron Microscopy and 3D Image Reconstruction. Viruses 9:
Tse, Longping Victor; Klinc, Kelli A; Madigan, Victoria J et al. (2017) Structure-guided evolution of antigenically distinct adeno-associated virus variants for immune evasion. Proc Natl Acad Sci U S A 114:E4812-E4821
Smith, Richard H; Hallwirth, Claus V; Westerman, Michael et al. (2016) Germline viral ""fossils"" guide in silico reconstruction of a mid-Cenozoic era marsupial adeno-associated virus. Sci Rep 6:28965
Aydemir, Fikret; Salganik, Maxim; Resztak, Justyna et al. (2016) Mutants at the 2-Fold Interface of Adeno-associated Virus Type 2 (AAV2) Structural Proteins Suggest a Role in Viral Transcription for AAV Capsids. J Virol 90:7196-204
Huang, Lin-Ya; Patel, Ami; Ng, Robert et al. (2016) Characterization of the Adeno-Associated Virus 1 and 6 Sialic Acid Binding Site. J Virol 90:5219-30
Li, Chengwen; Wu, Shuqing; Albright, Blake et al. (2016) Development of Patient-specific AAV Vectors After Neutralizing Antibody Selection for Enhanced Muscle Gene Transfer. Mol Ther 24:53-65
Tseng, Yu-Shan; Vliet, Kim Van; Rao, Lavanya et al. (2016) Generation and characterization of anti-Adeno-associated virus serotype 8 (AAV8) and anti-AAV9 monoclonal antibodies. J Virol Methods 236:105-110
Li, Baozheng; Ma, Wenqin; Ling, Chen et al. (2015) Site-Directed Mutagenesis of Surface-Exposed Lysine Residues Leads to Improved Transduction by AAV2, But Not AAV8, Vectors in Murine Hepatocytes In Vivo. Hum Gene Ther Methods 26:211-20
Halder, Sujata; Van Vliet, Kim; Smith, J Kennon et al. (2015) Structure of neurotropic adeno-associated virus AAVrh.8. J Struct Biol 192:21-36

Showing the most recent 10 out of 56 publications