Graduation Date

Spring 5-6-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Pharmaceutical Sciences

First Advisor

Howard E. Gendelman

Abstract

The persistence of integrated human immunodeficiency virus type 1 (HIV-1) proviral DNA despite combination antiretroviral therapy (ART) is the principal challenge to eliminating HIV-1 infection. This is made ever more complex based on the high viral mutation rates, diversity and resistance profiles. Taken together, these factors present principal obstacles towards any means to eliminate the virus from its infective human host. To address these factors, we have developed and validated a TatDE rLNP RNA delivery system. The system utilizes Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) guide RNAs (gRNAs) to target multiple exons of an integrated proviral HIV-1 (tat1-2/rev1-2/gp41). The stability and distribution of TatDE rLNP was first validated by cryo-transmission electron microscopy. Laboratory studies were employed next and demonstrated TatDE abilities to excise diverse HIV-1 strains from latently infected CD4+ T cells and mononuclear phagocytes. Work was performed in HIV-1 infected humanized mice, and conducted during ART support CRISPR-mediated excision of proviral DNA. Efficacy was determined by polymerase chain reaction (PCR), Sanger sequencing, and next-generation sequencing. Highly sensitive droplet digital PCR assay showed a significant difference in treated and untreated samples. Together, our data provide proof-of-concept for further development of CRISPR TatDE rLNP therapies as a potential means to eliminate integrated proviral HIV-1 DNA.

Comments

2023 Copyright, the authors

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