Gene therapy has vast potential for treating and potentially curing a wide variety of disorders. However, gene delivery technologies require significant improvements in safety, efficiency, and expression stability before the majority of these diseases can be treated. Vectors based on adeno-associated virus (AAV) have proven themselves to be highly promising, both in the laboratory and the clinic, but they still suffer from several shortcomings. In particular, the majority of the human population has been exposed to AAV serotype 2, as well as other serotypes, and as a result the immune system is primed to neutralize AAV. Antibody neutralization of AAV vectors is an established problem, and cellular immune responses may also be a challenge. We will attempt to solve the former problem and will further investigate basic mechanisms involved in the latter. For the former, we have developed novel directed evolution technology to generate new mutants of AAV with new properties. Specifically, large libraries of virus with random point mutations in the capsid gene encoding the viral coat protein are generated, and variants with novel properties are selected using high throughput screens. We have utilized this approach to generate variants with altered receptor binding properties, as well as variants that escape neutralization by antibodies that greatly inhibit AAV gene delivery by the wild type capsid or coat proteins. We will study the potential of human antibody evading variants to mediate high efficiency gene delivery of the therapeutic gene erythropoietin to the muscle and liver of animals carrying anti-AAV antibodies. In addition, while AAV neutralization by antibodies is an established problem, much less is known about AAV interactions with other components of the immune system. Therefore, the basic mechanisms of immune neutralization of this virus by complement [and T cells] will be investigated to both in vitro and in vivo. In summary, viruses have naturally evolved for their own ends, which do not always meet the needs of a human therapeutic. The novel approaches developed in this work to re-evolve viruses into enhanced human therapeutics will therefore have broad and general impact on the molecular engineering of enhanced viral gene delivery vehicles, including alternate AAV serotypes as well as other vectors. Furthermore, it will yield insights into the responses of other immune system components to AAV.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
3R01HL081527-03S1
Application #
7851669
Study Section
Gene and Drug Delivery Systems Study Section (GDD)
Program Officer
Link, Rebecca P
Project Start
2007-06-08
Project End
2011-05-31
Budget Start
2009-06-01
Budget End
2010-05-31
Support Year
3
Fiscal Year
2009
Total Cost
$79,876
Indirect Cost
Name
University of California Berkeley
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Asokan, Aravind; Schaffer, David V; Samulski, R Jude (2012) The AAV vector toolkit: poised at the clinical crossroads. Mol Ther 20:699-708
Xu, Dan; Walker, Christopher M (2011) Continuous CD8ýýý T-cell priming by dendritic cell cross-presentation of persistent antigen following adeno-associated virus-mediated gene delivery. J Virol 85:12083-6
Yin, Lu; Greenberg, Kenneth; Hunter, Jennifer J et al. (2011) Intravitreal injection of AAV2 transduces macaque inner retina. Invest Ophthalmol Vis Sci 52:2775-83
Jang, Jae-Hyung; Schaffer, David V; Shea, Lonnie D (2011) Engineering biomaterial systems to enhance viral vector gene delivery. Mol Ther 19:1407-15
Kurpinski, Kyle; Lam, Hayley; Chu, Julia et al. (2010) Transforming growth factor-beta and notch signaling mediate stem cell differentiation into smooth muscle cells. Stem Cells 28:734-42
Jang, J-H; Koerber, J T; Gujraty, K et al. (2010) Surface immobilization of hexa-histidine-tagged adeno-associated viral vectors for localized gene delivery. Gene Ther 17:1384-9
Klimczak, Ryan R; Koerber, James T; Dalkara, Deniz et al. (2009) A novel adeno-associated viral variant for efficient and selective intravitreal transduction of rat Muller cells. PLoS One 4:e7467
Excoffon, Katherine J D A; Koerber, James T; Dickey, David D et al. (2009) Directed evolution of adeno-associated virus to an infectious respiratory virus. Proc Natl Acad Sci U S A 106:3865-70
Koerber, James T; Jang, Jae-Hyung; Schaffer, David V (2008) DNA shuffling of adeno-associated virus yields functionally diverse viral progeny. Mol Ther 16:1703-9
Schaffer, David V; Koerber, James T; Lim, Kwang-il (2008) Molecular engineering of viral gene delivery vehicles. Annu Rev Biomed Eng 10:169-94