Zinc finger nucleases (ZFN)-mediated CCR5 disruption has shown to be a promising strategy for HIV gene therapy, but the latent viral reservoir still remains the major obstacle towards sterilizing or even a functional cure for HIV-1 infection. Resting CD4+ memory T cells are thought to be the major source of latency, and these also constitute a particularly stable reservoir of virus. ZFN targeting the conserved sequences in the 3' and the 5' LTR was reported to precisely excise the entire provirus in J-Lat cells, but the difficulty in delivering ZFNs to resting primary CD4+ T cells limited the potential f this approach for latency reduction. Recently, we have developed a method by using HIV env pseudotyping non-integrating lentivirus, successfully delivered CCR5-ZFN to resting CD4+ T cells and showed effectiveness in vivo in Hu-PBL mice. We have also found in preliminary studies that using Sindbis virus glycoprotein engineered to express ZZ-domain (Fc binding domain from protein A) provides another easy way to target resting CD4+ T cells. Here the pseudotyped lentivirus is treated with CD4 antibody (the ZZ domain containing lentivirus can bind any antibody) before infecting T cells. We could demonstrate targeting in >70% of resting CD4+ T cells. Thus we propose to target resting CD4+ T cells with non-integrating lentivirus carrying LTR and/or CCR5 ZFNs via HIV env-pseudotyped or Sindbis virus env-ZZ-domain, by which it would enable easy gene editing. We will use this method to edit CCR5 and/or HIV-LTR. Therefore, CCR5 disruption in bystander CD4+ T cells would protect uninfected cells and LTR/ZFN would excise integrated provirus in infected primary CD4+ T cells. The combination of these two strategies would potentially provide complete HIV protection. The long-term goal of this proposed research is to develop an alternative effective and safe HIV therapy that might offer a functional cure with simplified regimen. We propose to: 1) Generate and optimize reagents for Zinc Finger Nuclease (ZFN)- based gene editing in resting CD4+ T cells; 2) Evaluate whether ex vivo ZFN-modified PBMCs from ART suppressed individuals are protected from reactivation of endogenous virus after reconstitution in Hu/PBL mice

Public Health Relevance

Zinc finger nucleases (ZFN)-mediated CCR5 disruption has shown to be a promising strategy for HIV gene therapy, but the latent viral reservoir still remains the major obstacle towards sterilizing or even a functional cure for HIV-1 infection. Resting CD4+ memory T cells are thought to be the major source of HIV latency, and we have developed a method to deliver ZFN components to resting CD4+ T cells. Therefore, in this proposed research we will deliver both ZFNs targeting CCR5 and HIV provirus to potentially reach a complete HIV protection.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Small Research Grants (R03)
Project #
5R03AI120696-02
Application #
9098594
Study Section
AIDS Discovery and Development of Therapeutics Study Section (ADDT)
Program Officer
Poon, Betty
Project Start
2015-06-26
Project End
2017-05-31
Budget Start
2016-06-01
Budget End
2017-05-31
Support Year
2
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Texas Tech University
Department
Type
Graduate Schools
DUNS #
City
Lubbock
State
TX
Country
United States
Zip Code
79430