This proposal seeks to develop potent and broadly active transcriptional blockades of HIV-1 and novel approaches to modify the genome to abrogate CCR5 expression and deliver therapeutic genes specifically and safely to this site. This submission is a competing renewal for GM065059. In the first funding period of this grant we created novel zinc finger transcription factors, ZF-TFs that target conserved sites in the HIV-1 genome and inhibit viral replication in T cell lines and primary human cells and significantly advanced methods for the development of ZF-TFs. Here we will extend these studies to create a potent cocktail of ZF-TFs that, when delivered, will pose significant challenges to viral escape. We also demonstrated that we could engineer and evolve zinc finger-recombinase fusion proteins (RecZFs) capable of excising a target gene from human cells as well as targeting integration into human cells. Here we aim to further develop this approach to allow us to phenocopy the natural protective 32 mutation in CCR5 through targeted deletion of this gene in primary cells. Furthermore we will advance our studies of RecZFs to enable us to site-specifically integrate our HIV-1 gene blockade expression cassettes into the CCR5 locus, disrupting CCR5 expression. We will address the potential of these approaches to alter the course of HIV-1 infection in T cell lines, primary T cells, and monocytes derived from CD34+ cells. The net result of this study will be an increased understanding of HIV-1 gene regulation, methods to control HIV-1 replication, novel tools to modify the human genome, and a new approach to safely and specifically deliver therapeutic genes. These results will have significant implications for HIV-1 gene therapy and gene therapy in general. Relevance: While highly active anti-retroviral therapy (HAART) has changed the face of HIV-1 disease, viral resistance, toxicity, side-effects of the drug cocktail, cost issues, and patient compliance are problematic for a significant percentage of the affected population We propose to develop a powerful new and long lasting approach to HIV-1 gene therapy that addresses these problems and we hope that its application will significantly improve the treatment of HIV-1 infected individuals. The new approach we develop will be applicable to other diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
AIDS Discovery and Development of Therapeutics Study Section (ADDT)
Program Officer
Tompkins, Laurie
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Scripps Research Institute
La Jolla
United States
Zip Code
Sakkhachornphop, Supachai; Barbas 3rd, Carlos F; Keawvichit, Rassamee et al. (2012) Zinc finger protein designed to target 2-long terminal repeat junctions interferes with human immunodeficiency virus integration. Hum Gene Ther 23:932-42
da Silva, Frederico Aires; Li, Min; Rato, Sylvie et al. (2012) Recombinant rabbit single-chain antibodies bind to the catalytic and C-terminal domains of HIV-1 integrase protein and strongly inhibit HIV-1 replication. Biotechnol Appl Biochem 59:353-66
Schwimmer, L J; Gonzalez, B; Barbas 3rd, C F (2012) Benzoate X receptor zinc-finger gene switches for drug-inducible regulation of transcription. Gene Ther 19:458-62
Gaj, Thomas; Guo, Jing; Kato, Yoshio et al. (2012) Targeted gene knockout by direct delivery of zinc-finger nuclease proteins. Nat Methods 9:805-7
Gaj, Thomas; Mercer, Andrew C; Gersbach, Charles A et al. (2011) Structure-guided reprogramming of serine recombinase DNA sequence specificity. Proc Natl Acad Sci U S A 108:498-503
Gersbach, Charles A; Gaj, Thomas; Gordley, Russell M et al. (2011) Targeted plasmid integration into the human genome by an engineered zinc-finger recombinase. Nucleic Acids Res 39:7868-78
Guo, Jing; Gaj, Thomas; Barbas 3rd, Carlos F (2010) Directed evolution of an enhanced and highly efficient FokI cleavage domain for zinc finger nucleases. J Mol Biol 400:96-107
Gonzalez, Beatriz; Schwimmer, Lauren J; Fuller, Roberta P et al. (2010) Modular system for the construction of zinc-finger libraries and proteins. Nat Protoc 5:791-810
Gersbach, Charles A; Gaj, Thomas; Gordley, Russell M et al. (2010) Directed evolution of recombinase specificity by split gene reassembly. Nucleic Acids Res 38:4198-206
Tschulena, Ulrich; Peterson, Kenneth R; Gonzalez, Beatriz et al. (2009) Positive selection of DNA-protein interactions in mammalian cells through phenotypic coupling with retrovirus production. Nat Struct Mol Biol 16:1195-9

Showing the most recent 10 out of 20 publications