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.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
Project #
Application #
Study Section
Gene and Drug Delivery Systems Study Section (GDD)
Program Officer
Link, Rebecca P
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Temple University
Internal Medicine/Medicine
Schools of Medicine
United States
Zip Code
Wang, Q; Dong, B; Firrman, J et al. (2016) Evaluation of the biological differences of canine and human factor VIII in gene delivery: implications in human hemophilia treatment. Gene Ther 23:597-605
Moore, Andrea R; Dong, Biao; Chen, Lingxia et al. (2015) Vaccinia virus as a subhelper for AAV replication and packaging. Mol Ther Methods Clin Dev 2:15044
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
Dong, Biao; Duan, Xunbao; Chow, Hoi Yee et al. (2014) Proteomics analysis of co-purifying cellular proteins associated with rAAV vectors. PLoS One 9:e86453
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
Wang, Feng; Cui, Xiuling; Wang, Mingxi et al. (2013) A reliable and feasible qPCR strategy for titrating AAV vectors. Med Sci Monit Basic Res 19:187-93
Dong, Biao; Moore, Andrea R; Dai, Jihong et al. (2013) A concept of eliminating nonhomologous recombination for scalable and safe AAV vector generation for human gene therapy. Nucleic Acids Res 41:6609-17
Lu, Hui; Qu, Guang; Yang, Xiao et al. (2011) Systemic elimination of de novo capsid protein synthesis from replication-competent AAV contamination in the liver. Hum Gene Ther 22:625-32