SURVIVAL OF THE FITTEST: CHALLENGING TRANSDUCED CELLS WITH HIV-1 REPLICATION

Harvard Medical School, Boston MA

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A variety of genetic strategies, including shRNA or viral entry inhibitors, have been documented to inhibit HIV-1 replication in vitro. Translation of these effective in vitro strategies to clinically efficacious treatments has been limited in large part due to the low levels of genetically modified cells that can be achieved in vivo. However, in clinical scenarios where expression of the therapeutic gene may protect transduced cell from pathogenic effects, the transduced cells may have a selective proliferative advantage and repopulate the cellular compartment. To assess the potential of various HIV gene therapy strategies to protect cells expressing genetic inhibitors from virus-induced cytopathicity and to provide for a selective advantage in vitro, the CD4-positive cell line CEMx174 was transduced with lentiviral vectors expressing GFP along with either a HIV-1 tat/rev-specific small hairpin (sh)RNA or the viral entry inhibitor M87o. After infection with HIV-1, the percentage of GFP-positive cells in the CEMx174-M87o population increased from 30 to greater than 95 percent, but was unchanged in uninfected cells, and in infected CEMx174-GFP cells and CEMx174-sh(tat/rev) cells. To assess the lower limit for selective outgrowth, viral replication and GFP expression were examined in mixtures of CEMx174-M87o and untransduced cells ranging from 1 to 100 percent following HIV-1 NL4-3 infection. After 21 days, the percentage of GFP-positive cells in the CEMx174-M87o mixture increased from 1 to great than 95 percent. Intracellular expression of HIV-1 p24 Gag increased in transduced cells through day 9, but then receded by day 21. These results demonstrate very potent inhibition of HIV-1 viral replication with the viral entry inhibitor M87o and, importantly, a survival advantage of transduced cells in vitro.