HIV-SCRIBE is developed, a CRISPR-based molecular recorder in which a self-targeting guide RNA 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.
Abstract
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.
The combination knockout of CCR5, MOGS, and viral sequences profoundly reduces HIV-1 replication in an ex vivo cellular model, that is, HIV-1-infected peripheral blood mononuclear human cells, thus offering a pathway to launch further preclinical studies.
Z. Safaei, Anna Bellizzi, Hong Liu et al.· Human Gene Therapy· 0 citations
CRISPR technology is emerging as a promising therapeutic approach for eliminating chronic viral infections, such as herpesviruses and HIV. Here, for the first time, we demonstrate in vitro that CRISPR can be used to excise the HTLV-1 genome and reduce proviral loads in PBMCs from HAM/TSP (HTLV-1-associated myelopathy/tropical spastic paraparesis) patients. Single treatment with CRISPR-RNP (ribonucleoprotein) complexes composed of two gRNAs targeting the HTLV-1 env gene and 3’LTR sequences resulted in excision of a 2613 bp segment of the proviral genome, spanning tax and HBZ genes, without detectable off-target activity. Furthermore, CRISPR treatment led to over 50% reduction in proviral loads 5 days post-electroporation. Our data indicate that CRISPR-Cas9 gene editing can be used as a therapeutic strategy to eliminate HTLV-1 DNA from infected cells and may serve as a platform for curing HAM/TSP.
Samuel Brancazio, K. Khalili, Steven Jacobson et al.· Journal of Neurovirology· 0 citations
ABSTRACT The persistent HIV-1 reservoir includes a subset of cells harboring transcriptionally repressed latent HIV-1 that contributes to rebound upon antiretroviral treatment (ART) interruption. However, the majority of the reservoir consists of defective proviral genomes with mutations that prevent production of HIV-1 virions. People with HIV (PWH), even with suppression of viremia, demonstrate comorbidities of the central nervous system, heart, gut, and general aging-associated inflammation. Previously, we identified a transcriptionally active element within the envelope gene (env) of HIV-1, which mediates the expression of aberrant HIV-1 RNAs. We hypothesize that spurious expression of defective proviruses contributes to the general inflammation that drives these comorbidities. We observed correlations between levels of inflammatory cytokines in serum of PWH and levels of HIV-1 transcripts from this intragenic promoter. To investigate the impact of defective proviruses, we employed CRISPR-Cas9 to render the 5′ long terminal repeat (LTR), which acts as the enhancer and promoter for proviral transcription, non-functional. HIV-1-infected cells harboring this deletion produce significantly higher levels of IP-10 and IL-8 in vitro in both monocytic cell lines and primary monocyte-derived macrophages. Transcripts generated from the env promoter include a 5′ cap and polyA tail, and the induction of IP-10 expression was dependent on the cytosolic innate immune sensing pathway components MDA5 and MAVS and not cGAS and RIG-I. We propose that defective HIV proviruses contribute to chronic inflammation in PWH through an MDA5-dependent induction of type I interferon pathways. IMPORTANCE People with HIV-1 are at higher risk of developing age-associated comorbidities and immune exhaustion even when receiving antiviral treatments and having no detectable viremia. Transcription and translation have been documented from latent and defective proviruses, but their impact on inflammation associated with chronic HIV-1 infection remains poorly understood. The significance of this work is in identifying a role for defective HIV-1 proviruses and correlating their transcription in triggering a type I interferon response. These results highlight the importance of the persistent defective HIV-1 proviruses and understanding their impact on driving chronic inflammation to inform future strategies to assure people with HIV-1 healthy living and aging. People with HIV-1 are at higher risk of developing age-associated comorbidities and immune exhaustion even when receiving antiviral treatments and having no detectable viremia. Transcription and translation have been documented from latent and defective proviruses, but their impact on inflammation associated with chronic HIV-1 infection remains poorly understood. The significance of this work is in identifying a role for defective HIV-1 proviruses and correlating their transcription in triggering a type I interferon response. These results highlight the importance of the persistent defective HIV-1 proviruses and understanding their impact on driving chronic inflammation to inform future strategies to assure people with HIV-1 healthy living and aging.
Jonathan Kilroy, Aparna Deokar, Samantha Patalano et al.· Journal of Virology· 0 citations
Despite its efficiency to prevent viral multiplication, antiretroviral therapy does not affect HIV-1 latently-infected cells. These cells do not produce significant amounts of viruses and constitute HIV-1 reservoir. To purge this long-lived viral reservoir, the "shock and kill" strategy relies on the use of latency reversing agents (LRAs) to induce activation of latent cells. All LRAs developed until now target cellular proteins and are therefore not specific for HIV-infected cells. Here we present a new LRA that binds and activates HIV-1 Tat which is the key regulator for viral transcription and latency reversal. This molecule termed D10 was designed to bind to the major groove of the Tat protein, and found to activate Tat transcriptional activity by stabilizing the HIV transcription complex. This LRA induces strong HIV production by latent cell lines and latent cells from people living with HIV-1. On latent cells from PBMCs, D10 is active at ∼50 nM, the concentration required to stabilize HIV transcription complex. D10 is the first Tat activator available and the first LRA that targets an HIV protein.
P. Tong, Laetitia Marty, Nawel Chekrit et al.· bioRxiv· 0 citations
Despite the success of antiretroviral therapy in controlling HIV-1 replication, achieving a cure remains a significant challenge. Blocking viral replication has proven particularly challenging due to the highly efficient nature of cell-to-cell transmission (C-CT), as antibodies often fail to access the virological synapse. Here, we studied the effects of immune-based strategies, including ADCC and CAR NK/T cells, on HIV-1 transmission between cells.
We developed a highly sensitive assay for precise quantification of cell lysis using luciferase activity and built CAR NK/T based on the VRC01 and PGT121 bnAbs. We then utilized the assay to assess cellular cytotoxicity mediated by antibodies and CAR NK/T against target cells expressing HIV-1 envelope glycoproteins (Envs). We have previously developed an ultrasensitive HIV-1 C-CT assay, using a replication-competent HIV-1 vector and an activated nanoluciferase reporter system to quantify viral transmission in primary T cells. Using our nanoluciferase reporter assay, we studied the effects of a) PBMCs and soluble IgGs, and b) CAR NK/T on HIV-1 C-CT.
We detected cytotoxic activity of T and NK cells engineered to express VRC01 and PGT121 CARs against CEM.NKR cells that express luciferase and HIV-1 Envs as targets. HIV-1 C-CT of the NL43(AD8) molecular clone, which contains all HIV-1 genes, exhibits significant resistance to bnAbs but could be efficiently blocked by ADCC. Moreover, CAR PBMCs exhibited increased specific killing across a range of envelope-expressing target cells at various E: T cell ratios. Among the CARs tested, the PGT121 bnAb-based CAR consistently outperformed others in mediating cellular cytotoxicity.
These results provide proof of concept that targeting HIV-1 C-CT through ADCC and CAR NK/T cell therapies can overcome the limitations of bnAbs. This study offers the first evidence to test these approaches to effectively target HIV-1 C-CT, highlighting their potential in studying HIV-1 treatment strategies.
NIH U01 Award, 2022 - 2027
Vaccines and Immunotherapy (VAC)
ABSTRACT Human immunodeficiency virus type 1 (HIV-1) persists in latent reservoirs, mainly within CD4+ T cells, which are refractory to antiretroviral therapy, and can lead to rapid viral rebound upon treatment interruption. The “shock and kill” strategy aims to eliminate latent reservoirs by inducing viral transcription through latency-reversing agents (LRAs), thereby exposing infected cells to immune-mediated clearance. We developed and validated a simple, low-cost, and highly reproducible in vitro screening protocol to evaluate the efficacy and safety of LRAs, using a two-color flow cytometry assay on ACH2 cells, a well-characterized model of HIV-1 latency. Using this method, we identified PEP005 and CUDC-907 as the most potent LRAs across multiple experimental settings. Their reactivation capacity was further confirmed through transcriptomic analysis, which revealed a significant upregulation of viral RNA copies following stimulation. In addition, we collaborated with Dompé for using the Exscalate platform, an innovative computer-aided drug discovery approach, together with the experimental validation, which led to the identification of Tandutinib, a tyrosine kinase and mTOR inhibitor, as a novel LRA candidate with appreciable latency-reversing activity in the ACH2 model. While the ACH2 cell line does not fully recapitulate the complexity of HIV latency in vivo, it offers a robust and scalable system for early-stage screening and prioritization of candidate LRAs. Importantly, the future application of these LRAs in ex vivo samples derived from people living with HIV, with a particular focus on pediatric samples, will be crucial to deepen our understanding of latency reactivation in clinically relevant settings and age-specific immune environments. IMPORTANCE This study addresses a major obstacle to curing human immunodeficiency virus type 1 (HIV-1) infection: the persistence of latent viral reservoirs that are not eliminated by current therapies. We developed a simple and reproducible assay to identify compounds capable of reactivating latent virus, a key step in cure strategies. Using this approach, we identified effective latency-reversing compounds and, through collaboration with Dompé using the EXSCALATE platform, we also identified Tandutinib as a promising new candidate for further investigation. This study addresses a major obstacle to curing human immunodeficiency virus type 1 (HIV-1) infection: the persistence of latent viral reservoirs that are not eliminated by current therapies. We developed a simple and reproducible assay to identify compounds capable of reactivating latent virus, a key step in cure strategies. Using this approach, we identified effective latency-reversing compounds and, through collaboration with Dompé using the EXSCALATE platform, we also identified Tandutinib as a promising new candidate for further investigation.
A. Neri, Arianna Rotili, Elena Morrocchi et al.· Microbiology spectrum· 0 citations