Interdisciplinary Training

Esther Mintzer

"Design and evolution of homing endonucleases targeting essential HIV genes"

The most significant barrier to the eradication of HIV is its ability to establish life-long latent infection. Current attempts to purge the reservoir of latently infected T-cells have been unsuccessful thus far.  In comparison, little effort has been focused on the targeted inactivation of the integrated HIV genome. The engineering of homing endonucleases to target and cleave essential HIV sequences is a promising solution to the problem of latency. Homing endonucleases (HEs) are a class of enzymes that cleave DNA with exquisite specificity, having extended target sites of 14-40 base pairs. One side-effect of DNA cleavage by HEs is DNA repair by non-homologous end-joining (NHEJ), which often results in the introduction of mutations, usually deletions, at the site of cleavage. Thus, NHEJ can actually be exploited to inactivate the targeted gene. My hypothesis is that the HIV provirus can be cleaved by homing endonucleases designed to target sequences in HIV, leading to deletion of essential sequences and inactivation of the latent virus. Through a combination of computational design and directed evolution, homing endonucleases that target conserved sequences in essential HIV genes will be generated and tested in an in vitro model of HIV latency.