Adeno-associated viral (rAAV) vectors can mediate safe gene transfer for the long-term correction of genetic diseases in animal models and efficiently deliver corrective genes for the treatment of human diseases. They are safe and persist in humans following delivery to lung, sinus, skeletal muscle, brain, and liver tissue. The ability to efficiently transduce different cell/tissue populations for corrective gene delivery has generated significant interest in understanding their basic biology. This includes their capsid structure, cellular tropism and interactions for entry, trafficking, uncoating, replication, DNA packaging, capsid assembly, and antibody neutralization. The long- range goal of this project is to obtain information on the AAV capsid transition dynamics required for efficient cell entry and intracellular trafficking to the nucleus for replication. Physical, biochemical, and genetic approaches will be used to identify sites on the capsid that are involved in structural changes that occur during cell entry and subsequent transport to the nucleus via the endocytic pathway. Four selected AAV serotypes (AAV1, AAV2, AAV5, and AAV8) which represent the spectrum of sequence and capsid structural diversity so far observed for the primate AAVs, will serve as our models for the proposed studies.
Specific aim 1 will utilize solution studies, employing limited proteolysis to coupled mass spectrometry to identify and measure dynamic protein regions.
Specific aim 2 will focus on crystallographic visualization of capsid transitions, providing a 3D platform onto which the data resulting from aim 1 can be annotated.
Specific aim 3 will validate the observations from specific aims 1 and 2 using biochemical and genetic approaches. It is anticipated that a better physical understanding of the AAVs, as is the main goal of this project, could give rise to a new generation of corrective viral gene delivery vectors with synergistic improvements in tissue tropism and transduction efficiencies.
Several Adeno-associated viral (rAAV) vectors can mediate safe gene transfer for the correction of genetic diseases and are in clinical trials. However, very little information is available on the physical transitions of the protein capsid necessary for permissive cellular infection and trafficking to the nucleus for replication. The availability of this information will be valuable for the development of next generation recombinant vectors with improved efficacy. This project aims to fill this dearth in our knowledge base of basic AAV biology.
|Llauró, Aida; Guerra, Pablo; Kant, Ravi et al. (2016) Decrease in pH destabilizes individual vault nanocages by weakening the inter-protein lateral interaction. Sci Rep 6:34143|
|Osting, Sue; Bennett, Antonette; Power, Shelby et al. (2014) Differential effects of two MRI contrast agents on the integrity and distribution of rAAV2 and rAAV5 in the rat striatum. Mol Ther Methods Clin Dev 1:4|
|Salganik, Maxim; Aydemir, Fikret; Nam, Hyun-Joo et al. (2014) Adeno-associated virus capsid proteins may play a role in transcription and second-strand synthesis of recombinant genomes. J Virol 88:1071-9|
|Rayaprolu, Vamseedhar; Kruse, Shannon; Kant, Ravi et al. (2014) Fluorometric Estimation of Viral Thermal Stability. Bio Protoc 4:|
|Maaty, Walid S; Lord, Connie I; Gripentrog, Jeannie M et al. (2013) Identification of C-terminal phosphorylation sites of N-formyl peptide receptor-1 (FPR1) in human blood neutrophils. J Biol Chem 288:27042-58|
|Rayaprolu, Vamseedhar; Kruse, Shannon; Kant, Ravi et al. (2013) Comparative analysis of adeno-associated virus capsid stability and dynamics. J Virol 87:13150-60|
|Tartaglia, Lawrence J; Bennett, Antonette; Plattner, Alexander S et al. (2013) Molecular cloning, overexpression, and an efficient one-step purification of ?5?1 integrin. Protein Expr Purif 92:21-8|
|Venkatakrishnan, Balasubramanian; Yarbrough, Joseph; Domsic, John et al. (2013) Structure and dynamics of adeno-associated virus serotype 1 VP1-unique N-terminal domain and its role in capsid trafficking. J Virol 87:4974-84|
|Salganik, Maxim; Venkatakrishnan, Balasubramanian; Bennett, Antonette et al. (2012) Evidence for pH-dependent protease activity in the adeno-associated virus capsid. J Virol 86:11877-85|
|Hilmer, Jonathan K; Bothner, Brian (2011) Physical signal modulation of time-of-flight mass analyzers increases precision and decreases noise. Rapid Commun Mass Spectrom 25:795-805|
Showing the most recent 10 out of 11 publications