AAV is a single stranded DNA virus which has shown great promise as a gene therapy vector. Recombinant AAV vectors delivering RPE65 gene have been demonstrated to cure Leber's congenital amaurosis (LCA) disease and patients are able to regain normal vision after receiving the vectors. In hemophilia B clinical trial, patient receiving AAV vectors were able to express therapeutic level of factor IX gene expression. Systemic delivery of rAAV into human subjects would require a lot more vectors. We anticipate the demand for high quality rAAV vector for human clinical trials will exceed the current capability in our field. To face this challenge, we propose to improve rAAV vector production utilizing a new concept to eliminate wild type replication rcAAV particles and engineer scalable manufacturing processes. We would also investigate the complimentary helper function of adenovirus over cytoplasmic carrier helper in facilitating rAAV replication and packaging. In order to further improve the quality of recombinant AAV production for human gene therapy, we hypothesize that unwanted replication competent AAV particle formation could be eliminated by sequestering the helper function and vector sequences into different cellular compartments. The production and process system will eliminate plasmid transfection and other procedures which are not feasible for large scale production. Hence, our three specific aims are: 1. To study enhancement of AAV encapsidation by adenovirus in a cytoplasmic helper packaging system; 2. To develop a rAAV production system utilizing a cytoplasmic rep/cap carrier; 3. To engineer a protocol for scalable production of rAAV vectors free of contaminants. Completion of these specific aims will greatly enhance the way of rAAV vectors to be used in the field of human gene therapy.

Public Health Relevance

The completion of this project will improve AAV production technology and allow a high quality rAAV vector to be produced economically in large scale. It has an immediate impact on human gene therapy and has the potential to improve the quality of life of patients with genetic diseases, especially for hemophilia which requires systemic delivery of vectors.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL114152-02
Application #
8424975
Study Section
Gene and Drug Delivery Systems Study Section (GDD)
Program Officer
Link, Rebecca P
Project Start
2012-02-15
Project End
2017-01-31
Budget Start
2013-02-01
Budget End
2014-01-31
Support Year
2
Fiscal Year
2013
Total Cost
$429,023
Indirect Cost
$148,616
Name
Temple University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
057123192
City
Philadelphia
State
PA
Country
United States
Zip Code
19122
Wang, Qizhao; Dong, Biao; Firrman, Jenni et al. (2014) Efficient production of dual recombinant adeno-associated viral vectors for factor VIII delivery. Hum Gene Ther Methods 25:261-8
St Laurent, Georges; Shtokalo, Dmitry; Dong, Biao et al. (2013) VlincRNAs controlled by retroviral elements are a hallmark of pluripotency and cancer. Genome Biol 14:R73