Brian Wigdahl

Latent human immunodeficiency virus type 1 (HIV-1) reservoirs in resting CD4+ T cells and myeloid-lineage cells such as macrophages and microglia remain the principal barrier to a cure, as antiretroviral therapy suppresses replication without eliminating integrated proviruses. These integrated proviruses are the main barrier to a cure. Current CRISPR-based therapeutic strategies as a cure for HIV-1 largely rely on endpoint measurements that cannot capture the temporal dynamics of viral activation and genome-editing activity over time. This thesis developed HIV-SCRIBE, a CRISPR-based molecular recorder in which a self-targeting guide RNA (stgRNA) locus is placed under a Tat-responsive minimal HIV-1 5'LTR promoter, coupling Cas9-mediated cleavage and error-prone repair to Tat-driven transcriptional activation to generate a durable molecular record of HIV-1 reactivation and, more broadly, of cell-type-specific CRISPR editing dynamics. The main objective of this thesis was to evaluate this molecular recorder system's ability to record Tat-dependent HIV-1 reactivation in HEK293T cells. Edits to the recorder, along with Cas9 protein and stgRNA production, were assessed at 48-hour, 72-hour, and one-week time points. Cas9 protein and stgRNA were detected at all time points examined; however, sequence analysis of recorder edits showed minimal editing across all time points, including one low-frequency variant, an insertion near the PAM site, that could itself limit further editing. RT-PCR further showed that although Tat was produced at each time point, it did not induce stgRNA expression. Future directions will need to determine whether the minimal promoter is functional in order to utilize the molecular recorder system across multiple cell types as originally designed.

Over a decade of advances in Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) and CRISPR-associated protein 9 (Cas9)-based technologies have culminated in the first-ever FDA-approved CRISPR/Cas-based therapy. Aside from this approved therapy for sickle cell anemia, several CRISPR/Cas-based therapies are currently under development or testing for a range of chronic diseases, including viral diseases like human immunodeficiency virus type 1 (HIV-1) infection, genetic diseases like familial hypercholesterolemia, and cancer. The success of these therapies hinges on the effective delivery of CRISPR/Cas9 components to target regions, efficient Cas endonuclease editing, repair profiles generated, and their resulting outcomes. Here, we discuss the factors that influence the generation of CRISPR/Cas9-generated repair edits, the overall profiles, and outcome prediction(s), as well as the analytical tools that have been developed to date. Finally, how this technology has been used towards a functional HIV-1 cure is discussed.

The dynamic latent proviral reservoir remains the principal barrier to a broad-spectrum cure for human immunodeficiency virus type 1 (HIV-1). Once an acute threat, HIV-1 is now a chronic disease for people living with HIV-1 (PLWH) who adhere to antiretroviral therapy (ART), though the latent reservoir endures, contributing to chronic disease syndromes, neuroinflammation, and HIV-1-associated neurocognitive disorders (HAND). Curative strategies for HIV-1 must target viral reservoirs across compartmental barriers, while navigating extensive inter- and intra-patient quasispecies diversity without causing additional harm to those undergoing treatment. Clustered Regularly Interspersed Short Palindromic repeats (CRISPR) gene editing has been proposed as a curative strategy, and the Staphylococcus aureus Cas9 (SaCas9) ortholog has emerged as the leading endonuclease for in vivo cure therapies. However, the two main components of CRISPR - the endonuclease and the guide RNA (gRNA) - remain poorly characterized in a comprehensive, Cas-ortholog and HIV-1-specific manner. Herein, the Cas:gRNA pairing for an HIV-1 cure strategy are evaluated computationally and experimentally. Computational interrogation of protospacer adjacent motif (PAM) availability identified the potential for safe, broad and effective (SBE) gRNA yield across HIV-1 quasispecies through the Nominate, Diversify, Narrow and Filter (NDNF) pipeline and characterized the trade-off between PAM breadth and off-target potential across a wide range of natural and engineered Cas orthologs. Experimentally, the optimal SaCas9:gRNA pair was interrogated through delivery of a 6,000 HIV-1-specific gRNA library to J-Lat 10.6 T lymphocytes followed by PMA/ionomycin stimulation, fluorescence-activated cell sorting (FACS), and next generation sequencing. Weighted comparisons between time points, reactivated, and silenced cell populations produced a ranked annotation of all 6,000 protospacers. Top SaCas9 gRNAs were distributed across multiple loci throughout the proviral genome, with the leading gRNA targeting vif. Critically, this analysis revealed a nuanced positional and nucleotide-specific mismatch preference for SaCas9 gRNAs that diverges from current design frameworks. Site-specific sequence logos derived from this analysis provide a practical framework for optimal SaCas9 gRNA design. Together these findings characterize the targeted components of CRISPR for an HIV-1 cure strategy and represent a significant advance toward a durable, safe and universal cure for HIV-1.

Human immunodeficiency virus type 1 (HIV-1) is a retrovirus that integrates into the host cell’s DNA as a provirus. Transcription from the provirus is regulated in large part by cellular proteins and epigenetic factors. These may be repressive or permissive to productive infection. The host factors that regulate this balance are therefore attractive targets for HIV-1 therapeutics. Indeed, proviral chromatin is the focus of two of the current HIV-1 cure strategies. “Shock and Kill” uses latency reversal agents to open the provirus’s chromatin, promoting high levels of gene expression that induce the killing of infected cells. “Block and Lock” uses latency promoting agents to induce heterochromatin, blocking transcription and forcing HIV-1 into a state of deep latency. Here, the compounds investigated in both strategies are reviewed, including their chemical structures, mechanisms of action, and clinical results. Finally, the use of CRISPR-Cas therapeutics and the impact of chromatin architecture on its efficacy are discussed.

In the context of neurodegeneration, activated microglia facilitate inflammation via secretion of TNF-α, IL-1α, and C1q. Astrocytes exposed to this signaling array polarize to a reactive inflammatory phenotype, termed A1 or A1-like. Astrocytes are essential for neuronal survival, synaptic support, and blood–brain barrier (BBB) function, but A1-like astrocytes upregulate inflammatory gene expression, downregulate neurotrophic factors, and secrete neurotoxic signals. The consequences of A1-like polarization on BBB function are unknown but may have etiological implications for some diseases. Frequently identified by upregulation of complement component 3 (C3), A1-like astrocytes have been characterized in neurodegenerative disorders like Alzheimer’s disease, with polarization correlated with disease progression and severity. However, the role of A1-like astrocytes in neurodegeneration associated with chronic viral infections, like HIV-1-associated neurocognitive disorder (HAND), remains unclear. An in vitro system using primary human astrocytes, as well as a BBB model featuring primary human brain microvascular endothelial cells (BMECs) co-cultured with astrocytes, was used to elucidate cellular and molecular consequences of chronic astrocyte activation. As measured by whole transcriptome analysis and protein expression assays, repeated treatment with TNF-α, IL-1α, and C1q induced A1-like polarization of astrocytes both in monoculture and in a BBB model, resulting in increased secretion of pro-inflammatory signals. No substantial change to BBB permeability was observed. In contrast, exposure to HIV-1 viral protein Tat did not independently induce A1-like polarization. Ongoing investigations into the effect of astrocyte polarization on BBB integrity and treatment with pathogenic proteins may provide insights into the role of neurotoxic astrocytes in neurovirologic pathologies.

Herpes simplex virus type 1 (HSV-1) causes a lifelong infection due to latency established in the trigeminal ganglia, which is the source of recurrent outbreaks of cold sores. The lifelong persistence of HSV-1 is further facilitated by the lack of cure strategies, unsuccessful vaccine development, and the inability of the host immune system to clear HSV-1. Despite the inefficiencies of the immune system, the course of HSV-1 infection remains under strict immunological control. Specifically, HSV-1 is controlled by a CD8⁺ T cell response that is cytotoxic to HSV-1-infected cells, restricts acute infection, and uses noncytolytic mechanisms to suppress reactivation in the TG. When this CD8⁺ T cell response is disrupted, reactivation of latent HSV-1 occurs. With antiviral therapies unable to cure HSV-1 and prophylactic vaccine strategies failing to stimulate a protective response, we propose non-thermal plasma (NTP) as a potential therapy effective against recurrent HSV-1 infection. We have demonstrated that NTP, when applied directly to HSV-1-infected cells, has antiviral effects and stimulates cellular stress and immunomodulatory responses. We further propose that the direct effects of NTP will lead to long-lasting indirect effects such as reduced viral seeding into the TG and enhanced HSV-1-specific CD8⁺ T cell responses that exert greater immune control over HSV-1 infection.

Previous studies have shown that two CCAAT enhancer binding protein (C/EBP) sites, upstream of the transcriptional start site, are necessary for subtype B human immunodeficiency virus type 1 (HIV-1) gene expression in cells of the monocytemacrophage lineage. However, no studies have been reported concerning the C/EBP sites within the non-subtype B HIV-1 LTRs or downstream of the transcriptional start site within the subtype B LTR. Electrophoretic mobility shift (EMS) analyses demonstrated that one of three putative downstream C/EBP binding sites (downstream element three, DS3) within the subtype B LTR was able to form a DNA-protein complex containing C/EBP[beta]. By utilizing the DS3-9C variant, which exhibits low DNA binding affinity for C/EBP[beta], it has been shown that the DS3 binding site is important for HIV-1 basal transcription. The DS3-9C-containing LTR exhibits a lower basal transcription level and is able to be transactivated by C/EBP[beta]-2 to a lower extent compared to the parental HIV-1 subtype B LTR. However, upon activation with Tat alone or with C/EBP[beta]-2 together, the DS3-9C-containing LTRs exhibit a similar or even greater transcriptional activation than that of the parental LTR. HIV-1 molecular clone viruses containing the DS3-9C C/EBP binding site variant have revealed a decreased replication capacity and a delayed rate of replication. These results suggest that the DS3-9C configuration might be related to virual persistence and reactivation in cells of the monocyte-macrophage lineage. Furthermore, one C/EBP binding site has been identified within the subtype C LTR. Subtype B and C LTRs are able to be activated by C/EBP[beta]-2 to similar levels in the monocytic cell line, U-937. The interaction observed between C/EBP[beta]-2 and Tat with the subtype B and C LTRs suggest the functionally important transactivation ability of C/EBP[beta] among different subtypes that, under certain circumstances, may be cell type dependent.

The human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) regulates viral gene expression. The viral transactivator protein Tat plays a pivotal role in transcriptional activation via interaction with the transactivation response element (TAR) of viral RNA. Studies in the pre-HAART era demonstrated HIV-1-infected peripheral blood (PB)-derived LTR variants containing a C-to-T change at position 3 of C/EBP site I (3T), or at position 5 of Sp site Ill (5T) were preferentially encountered in late stage disease. The 3T and 5T configurations exhibited low binding affinity for their cognate transcription factors. Our recent studies conducted during the HAART era have identified a number of HIV-1-infected patients within the DREXELM ED HIV/AIDS Genetic Analysis Cohort harboring LTR variants containing 5T Sp III. Transient expression analyses demonstrated that most LTRs containing either the 5T or co-selected 3T/5T configurations exhibited basal, stimulated, and Tat-mediated transcription activities similar to LTRs carrying the conB sequence at these LTR binding sites in Jurkat T cells and monocytic U-937 cells. However, the patient 19 clone 18 5T-containing LTR could not be transactivated by either the laboratory strain IIIB Tat or by Tat derived from the patient 19. When nucleotide +32 (A) of the TAR element of the defective 19-18 LTR was changed to conB (G), Tat-mediated LTR transactivation was only partially restored. However, this could be further enhanced by changing the sequence of the three Sp binding sites to the conB sequence. Results also indicate that selective pressures are likely placed on the virus at least during discrete phases of HIV disease that result in a viral LTR-Tat structural complex that may be more optimized for more effective viral transcription, possibly in specific cellular phenotypes. Furthermore, results are reported that suggest the HIV-1 Tat exon II may contain molecular determinants that play an important role in LTR-directed Tat-mediated transactivation at low concentrations of transactivator protein. The results suggest that genetic variation in Tat exon II may have more impact on Tat-mediated transactivation at low Tat concentrations that may be present during activation from latency, as compared to productive replication in CD4+ T cells.

Preclinical microbicide development efforts have focused on the development of the polyanionic compound poly(styrene-alt-maleic acid) (alt-PSMA). In vitro experiments demonstrated that alt-PSMA is a potent inhibitor of infection by R5 and X4 strains of HIV-1. alt-PSMA was shown to be an effective inhibitor of HIV-1 laboratory strains and clinical isolates, as well as both cell-free and cell-associated forms of infectivity. In keeping with the development of a safe inhibitor, alt-PSMA was also characterized as having low cytotoxicity. High levels of HIV-1 inhibition and low levels of cytotoxicity were confirmed in assays using primary human peripheral blood mononuclear cells (PBMCs). Furthermore, in compound washout experiments designed to mimic product leakage and the loss of active compounds following application, there were no adverse effects associated with the use of alt-PSMA. This was not true of other polyanionic HIV-1 inhibitors (including carrageenan, cellulose sulfate, and polystyrene sulfonate), which caused increases in HIV-1 infection subsequent to compound removal in experiments using cell lines and human PBMCs in conjunction with R5 and X4 viruses. The favorable attributes of alt-PSMA-potent inhibition of HIV-1, low cytotoxicity, no adverse effects in washout assays-suggested the continued development of alt-PSMA not only as a single agent but also as a component of a combination microbicide. The successes achieved using highly active antiretroviral therapy (HAART) have suggested that higher efficacy can be achieved by using compound combinations in microbicides. In investigations of potential compound combinations, the activity of alt-PSMA was assessed in combination with a fusion inhibitor (cyanovirin), an entry inhibitor (polystyrene sulfonate), or a reverse transcriptase inhibitor (efavirenz). Analyses performed using CalcuSyn and MacSynergy software indicated that the greatest degree of synergistic antiviral activity was achieved by the combination of alt-PSMA and cyanovirin. These results suggest that synergy with alt-PSMA is established in a combination that also includes a compound with a similar but non-overlapping mechanism of action. These combination studies, as well as experiments focused on the activity of alt-PSMA as a single compound, strongly indicate that alt-PSMA is a compound that deserves further assessment as a potential candidate microbicide and a partner in a combination microbicide.