AAV is a single stranded DNA virus which has shown great promise as a gene therapy vector. In clinical trial, rAAV vectors delivering RPE65 gene have been demonstrated to cure Leber's congenital amaurosis (LCA) diseases and patients are able to regain normal vision after receiving the vectors. However, its use in other genetic diseases such as hemophilia still face considerable challenge. Systemic delivery of rAAV into human subjects would require a lot more vectors than targeting the retina tissue. Prelimary studies have demonstrated more than 99.9% of rAAV vectors were wasted in the varous stages of rAAV transduction. In addition, rAAV genomes packaging preference remains an unsettling issues. In order to further develop recombinant AAV for human gene therapy, we hypothesize that it would eliminate unwanted replication competent AAV particle formation and improve rAAV production by sequetering the helper function and vector sequencing into different cellular compartment. Advanced genomics and proteomics will be introduced to study rAAV packaging preference and encapsidation mechanism. Finally, we identified that cytoplasm is major site for rAAV genome loss. An innovative assay will be established to track rAAV genomes in cytoplasm host and mechanism will be studied to reduce rAAV genomes in cytoplasm of the host cells. Hence, our three specific aims are 1. To develop and optimize the next generation of rAAV packaging system~ 2. To characterize rAAV genome integrity and rAAV packaging mechanisms~ 3. To study the molecular status of recombinant AAV genomes in the cytoplasm. Completion of specific aims in this application will markedly enhance the way of rAAV vectors to be used in the human gene therapy field.

Public Health Relevance

The completion of this project may provide a solution for producing high quality rAAV vector economically. Our work may help design optimized vector for human gene therapy and improve the quality of life of patients with genetic diseases.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL080789-08
Application #
8496093
Study Section
Special Emphasis Panel (ZRG1-BST-N (90))
Program Officer
Mcdonald, Cheryl
Project Start
2006-02-15
Project End
2015-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
8
Fiscal Year
2013
Total Cost
$327,769
Indirect Cost
$113,541
Name
Temple University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
057123192
City
Philadelphia
State
PA
Country
United States
Zip Code
19122
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