ORCID ID

0000-0003-1084-6838

Graduation Date

Fall 12-18-2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Immunology, Pathology & Infectious Disease

First Advisor

Howard E. Gendelman, M.D.

Second Advisor

Bhavesh Kevadiya, Ph.D.

Third Advisor

Larisa Y. Poluektova, M.D., Ph.D.

Fourth Advisor

Geoffrey M. Thiele, Ph.D.

Abstract

A critical barrier to achieving a functional cure for infection by human immunodeficiency virus type one (HIV-1) rests in the presence of latent proviral DNA integrated in the nuclei of host CD4+ T cells and mononuclear phagocytes. Accordingly, HIV-1-infected patients must adhere to lifelong regimens of antiretroviral therapy (ART) to prevent viral rebound, CD4+ T cell decline, and progression to acquired immunodeficiency syndrome (AIDS). Gene editing using clustered regularly interspersed short palindromic repeat (CRISPR)-Cas9 technology stands as one means to inactivate integrated proviral DNA. We devised a mosaic gRNA CRISPR-Cas9 system- TatDE- that targets viral transcriptional regulator genes tat / rev, while also accounting for clinical HIV-1 sequence diversity. TatDE effectively reduced viral replication in all 7 HIV-1 strains tested by an average of 82%, and attenuated latency reversal in HIV-1-infected ACH2 T cells by 94%. In establishing the TatDE system, we identify a positive association between CRISPR target conservation and antiviral efficacy. Moreover, TatDE’s simultaneous cleavage of 5 HIV-1 exons (tat1-2 / rev1-2 / and gp41) suggest that exonic disruption may be a guiding principal in designing CRISPR for HIV-1 elimination. Limitations to utilizing an alternate vector, recombinant adeno-associated virus 9 (rAAV9), for HIV-1 removal are experimentally addressed. Preliminary data exploring the design of virus-like particles and nanoparticles for CRISPR-Cas9 delivery are presented. To guide the development of curative nanoparticles for HIV-1 infection, europium sulfide (EuS) theranostic nanoprobes were devised. EuS aided in the identification of dynamin-mediated endocytosis as a mechanism for particle uptake in CD4+ T cells and macrophages. Additionally, EuS facilitated the non-invasive characterization of nanoformulation biodistribution using magnetic resonance imaging (MRI) and single photon emission computed tomography (SPECT-CT). Thus, advances in the arenas of molecular biology, theranostics, and material science coalesce in providing a pathway towards HIV-1 elimination.

Comments

Dr. Channabasavaiah Gurumurthy, Ph.D. also served as an advisor in this work.

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