Eradication of the latent HIV viral reservoir: Targeted disruption of the integrated HIV provirus using engineered meganucleases and quantitation of the latent HIV reservoir using multiplex ddPCR

Thesis (Ph.D.)--University of Washington, 2016-09The major barrier to HIV cure is the establishment of a long-lived latent viral reservoir that is not affected by combination ant-retroviral therapy and is not cleared by the immune system. When HIV positive patients on combination anti-retroviral therapy with undetectable viremia interrupt therapy, viremic rebound occurs within two weeks of discontinuing cART. This viral rebound is due to stochastic reactivation of HIV transcription and replication in the long-lived viral reservoir. HIV cure necessitates eradication of this long-lived reservoir. To eradicate the HIV proviral reservoir, we are using HIV-specific engineered meganucleases. Mutations in essential HIV genes can disrupt the integrated provirus and could prevent reactivation from latency. I demonstrate that engineered meganucleases can introduce mutations in an integrated HIV provirus. The HIV-specific engineered meganucleases also cleave and introduce mutations in genomic off-target sites. We show that a second generation of an HIV-specific meganuclease developed using structure guided protein engineering, has an improved off-target toxicity profile and retains activity at the HIV target site. I further demonstrate that expression of the three prime repair exonuclease-2 (Trex2) in combination with either the meganuclease or fusion megaTALs increases the frequency of mutations at the HIV target site. The compact size of meganucleases and the increasing ease with which they can be redesigned to recognize new DNA sequences makes meganucleases an attractive tool for HIV cure applications. HIV cure also requires precise quantitation of the viral reservoir. Current assays used to measure the HIV viral reservoir are imprecise and tend to either over or underestimate the size of the functional reservoir. In the setting of both structured and analytical treatment interruptions it is critical that the reservoir is measured accurately so that patients do not discontinue cART in the setting of large functional reservoirs. I describe here a multiplex ddPCR assay that can simultaneously detect up to six viral genes in the same reaction. The multiplex ddPCR assay gives an estimate of the completeness of the integrated provirus, which can be used to measure levels of functional integrated HIV provirus. I hypothesize that this multiplex ddPCR assay gives a truer estimate of the size of the functional HIV reservoir