Safer gene editing approaches towards HIV-1 gene therapy

3^rd International Conference on HIV/AIDS, STDs & STIs

November 30 - December 02, 2015 Atlanta, USA

Manjunath N Swamy

Paul L Foster School of Medicine, USA

Keynote: J AIDS Clin Res

Abstract :

CCR5 gene disruption is a promising method for HIV-1 gene therapy and recent gene editing technologies such as ZFNs and CRISPR/Cas9 provide methods for such disruption. However, one major concern of using nucleases is off-target effects associated with their long-term expression. Thus, successful clinical translation of gene editing strategies necessitates the development of safe and effective methods for their transient expression in relevant cells. We have modified the ZFN and CRISPR/ Cas9 gene editing technologies to provide for transient expression of nucleases. We used non-integrating lentivirus (NILV) for transient expression of ZFNs and pseudotyped the virus with HIV-envelope for targeted delivery to CD4 T cells. Both activated and resting primary CD4 T cells transduced with CCR5-ZFNs NILV showed resistance to HIV-1 infection in vitro. Furthermore, NILV transduced resting CD4 T cells from HIV-1 seronegative individuals were resistant to HIV-1 challenge when reconstituted into NOD-scid IL2r�?³c null (NSG) mice. Likewise, endogenous virus replication was suppressed in NSG mice reconstituted with CCR5-ZFN-transduced resting CD4 T cells from treatment naive as well as ART-treated HIV-1 seropositive patients. Taken together, NILV pseudotyped with HIV envelope provides a simple and clinically viable strategy for HIV-1 gene therapy. Since the CRISPR/Cas9 system provides an easier way for gene editing, we also modified this system for transient expression of Cas9 protein. For this purpose, we pre-packaged Cas9 protein (Cas9P LV) in lentiviral particles and showed its effectiveness for gene disruption in cells, including primary T cells expressing specific sgRNAs. We then constructed an â�?�?all in oneâ�? lentivirus to express sgRNAs in association with prepackaged Cas9 protein (sgRNA/Cas9P LV). We successfully edited CCR5 in TZM-bl cells by this approach. Using a sgRNA targeting HIV LTR, we also were able to disrupt HIV provirus in the J-LAT model of viral latency. Moreover, we also found that pre-packaging Cas9 protein in LV particle reduced off-target editing of chromosome 4:-29134166 locus by CCR5 sgRNA, compared to continued expression from the vector. These results show that sgRNA/Cas9P LV can be used as a safer approach for human gene therapy.

Biography :

Manjunath N Swamy is currently Prof. Biomedical Sciences and Co-Director of the Center of Emphasis in Infectious Diseases. He obtained his MD degree from the All India Institute of Medical Sciences, New Delhi, India and received Post-doctoral training at the Tufts-New England Medical Center. He was as an investigator at the Immune Disease Institute, Harvard Medical School before joining the TTUHSC

Email: manjunath.swamy@ttuhsc.edu