Degree Name
PhD (Doctor of Philosophy)
Program
Biomedical Sciences
Date of Award
12-2022
Committee Chair or Co-Chairs
Zhi Q. Yao
Committee Members
Jonathan P. Moorman, Shunbin Ning, Antonio E. Rusinol, Krishna Singh
Abstract
Human Immunodeficiency Virus (HIV) infection initiates major metabolic and cell- survival complications. Anti-retroviral therapy (ART) is the current approach to suppress active HIV replication to a level of undetected viral load, but it is not a curative approach. Newer and sophisticated gene editing technologies could indeed be a potent antiviral therapy to achieve a clinical sterilization/cure of HIV infection. Chronic HIV patients, even under a successful ART regimen, exhibit a low-grade inflammation, immune senescence, premature aging, telomeric DNA attrition, T cell apoptosis, and cellular homeostasis. In this dissertation, we investigated CD4 T cell homeostasis, degree of T cell apoptosis, an associated telomeric DNA damage, DNA damage repair signaling, and the apoptotic pathways in CD4 T cells during HIV infection with or without ART treatment. Our data support a DNA damage accumulation, and impaired DNA damage repair in chromosome ends via recruitment of 53BP1 protein to the damaged foci. We found that a key player of DNA damage and repair enzyme, ATM, and its associated checkpoint proteins (CHK1, CKH2) are affected by HIV infection. HIV infection also altered another multifunctional master regulator protein AKT that is crucial in maintaining cellular homeostasis.
Curing HIV is the ultimate redemption against HIV-associated complications. To explore the possibility of a functional cure, we investigated the use of a transient and a non-viral CRISPR/Cas9-based gene editing technology targeting the latently incorporated HIV provirus.
After performing a nucleofection/electroporation using an in vitro formulated ribonucleoprotein (RNP) constituting a synthetic guide RNA (gRNA) and Cas9 nuclease protein, we demonstrated a significant (maximum 97%) reduction of HIV-mRNA and p24-capsid protein expression, upon stimulation (using PMA) and latency reactivation of latently HIV-infected CD4 T cells and latent-monocytes. Notably, the RNP treatment did not induce any cytotoxic effects, without affecting the abilility of cell proliferation. A sequence specific cleavage of HIV-provirus in two crucial gene locations (targeting vpr/tat genes) showed the most significant suppression of HIV reactivation or latency reversal. We have used DNA sequencing, and T7EI assay to confirm the target-site-specific cleavage of the HIV-proviral genome. Our data confirm the activation of non- homologous end joining (NHEJ) pathway to repair the double-stranded DNA break created by the CRISPR/Cas9 treatment. Taken together, this study provides a new gene therapeutic approach using synthetic gRNA/Cas9 targeting HIV genome, which warrant further in vivo animal and human studies.
Document Type
Dissertation - unrestricted
Recommended Citation
Khanal, Sushant, "Investigating the PI3K/AKT/ATM Pathway, Telomeric DNA Damage, T Cell Death, and CRISPR/Cas9-mediated Gene Editing During Acute and Chronic HIV Infection" (2022). Electronic Theses and Dissertations. Paper 4136. https://dc.etsu.edu/etd/4136
Copyright
Copyright by the authors.
Included in
Immune System Diseases Commons, Immunology of Infectious Disease Commons, Immunotherapy Commons, Medical Immunology Commons, Medical Molecular Biology Commons, Virology Commons, Virus Diseases Commons