While the infection of activated CD4+ cells by HIV-1 in vivo normally results in a productive viral infection leading to cell death, a small number of infected T cells re-enter the resting state and this can lead to a latent HIV-1 infection. Latent HIV-1 infections result from integrated HIV-1 proviruses that are entirely transcriptionally silent yet can be reactivated from latency by stimulation by an appropriate antigen or reagent. While highly active antiretroviral therapy (HAART) can prevent HIV-1 spread and dramatically reduce viral loads, due to the progressive death of productively HIV-1-infected cells, a small population of latently HIV-1-infected T cells remains intact. As a result, when HAART is discontinued, HIV-1 can rapidly reappear and reconstitute a high level infection. Because the latently infected T-cell pool is extremely long lived, it has proven impossible to cure HIV-1 infected patients using HAART alone in the sense that these patients become entirely virus free and are able to discontinue HAART without virus rebound. Because latently HIV-1 infected T cells are, almost by definition, impossible to distinguish from other resting T cells, efforts to deplete the latent pool have previously focused on attempts to activate the proviruses present in latently infected cells using, for example, HDAC inhibitors, in the presence of HAART. However, it has become increasingly clear that this approach is unable to effectively activate the majority of latent proviruses. An alternative strategy, proposed here, is to target and destroy latent HIV-1 proviruses using vector-delivered RNA-guided endonucleases belonging to the CRISPR/Cas family of bacterial antiviral RNA- guided DNA endonucleases. In this application, we propose several approaches to effectively and specifically destroy the latent HIV-1 proviral reservoir using lentiviral vector delivery of Ca9 proteins and small guide RNAs that target one or more highly conserved regions of the HIV-1 genome. We will first optimize proviral targeting and expression of Cas9 and single guide RNAs (sgRNAs) and maximize lentiviral vector titer. Secondly, we will test various transient expression modalities for Cas9/sgRNA combinations and analyze their ability to destroy latent HIV-1 proviruses in culture, and finally, we will determine the ability of lentiviral vector- delivered Cas9/sgRNA complexes to ablate the latent HVI-1 proviral reservoir in vivo, using a humanized mouse model of HIV-1 infection.
While HIV-1 can be controlled by antiretroviral drugs, a cure has so far been impossible to achieve due to residual, latently HIV-1 infected cells. This grant seeks to develop targeted DNA endonucleases that could destroy HIV-1 DNA molecules in latently infected cells and achieve the complete clearance of HIV-1 from the body. DUMC 3025 - Durham, NC 27710 - tel (919) 684-3369 - fax (919) 681-8979 - firstname.lastname@example.org
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