Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells

Nature Communications, Journal Year: 2015, Volume and Issue: 6(1)

Published: March 10, 2015

To combat hostile viruses, bacteria and archaea have evolved a unique antiviral defense system composed of clustered regularly interspaced short palindromic repeats (CRISPRs), together with CRISPR-associated genes (Cas). The CRISPR/Cas9 develops an adaptive immune resistance to foreign plasmids viruses by creating site-specific DNA double-stranded breaks (DSBs). Here we adapt the human cells for intracellular against viruses. Using HIV-1 infection as model, our results demonstrate that disrupts latently integrated viral genome provides long-term new infection, expression replication in cells. We show engineered human-induced pluripotent stem stably expressing HIV-targeted can be efficiently differentiated into HIV reservoir cell types maintain their challenge. These unveil potential therapeutic strategy infections.

Language: Английский

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Microglial Cells: The Main HIV-1 Reservoir in the Brain

Frontiers in Cellular and Infection Microbiology, Journal Year: 2019, Volume and Issue: 9

Published: Oct. 24, 2019

Despite efficient combination of the antiretroviral therapy (cART) which has significantly decreased mortality and morbidity HIV-1 infection, a definitive HIV cure not been achieved. Hidden in cellular anatomic reservoirs is major hurdle toward functional cure. Microglial cells, CNS resident macrophages, are one latent HIV-1. These cells believed to be involved both emergence drugs resistance reseeding peripheral tissues. Moreover, these long-life also development HIV-1-associated neurocognitive diseases (HAND). Clearing infected from brain therefore crucial achieve However, many characteristics microglial central nervous system might preclude eradication reservoirs. Better understandings specific molecular mechanisms latency should help design new molecules strategies preventing HAND achieving an needed circumvent limitations associated anatomical sanctuaries with barriers such as blood barrier (BBB) that reduce access drugs.

Language: Английский

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An In-Depth Comparison of Latency-Reversing Agent Combinations in Various In Vitro and Ex Vivo HIV-1 Latency Models Identified Bryostatin-1+JQ1 and Ingenol-B+JQ1 to Potently Reactivate Viral Gene Expression

PLoS Pathogens, Journal Year: 2015, Volume and Issue: 11(7), P. e1005063 - e1005063

Published: July 30, 2015

The persistence of latently infected cells in patients under combinatory antiretroviral therapy (cART) is a major hurdle to HIV-1 eradication. Strategies purge these reservoirs are needed and activation viral gene expression one promising strategy. Bromodomain Extraterminal (BET) bromodomain inhibitors (BETi) compounds able reactivate latent proviruses positive transcription elongation factor b (P-TEFb)-dependent manner. In this study, we tested the reactivation potential protein kinase C (PKC) agonists (prostratin, bryostatin-1 ingenol-B), which known activate NF-κB signaling pathway as well P-TEFb, used alone or combination with P-TEFb-releasing agents (HMBA BETi (JQ1, I-BET, I-BET151)). Using vitro post-integration latency model cell lines T-lymphoid myeloid lineages, demonstrated that PKC acted potent latency-reversing (LRAs) their combinations led synergistic at mRNA levels. Mechanistically, combined treatments higher activations P-TEFb than corresponding individual drug treatments. Importantly, observed ex vivo cultures CD8+-depleted PBMCs from 35 cART-treated HIV-1+ aviremic percentage reactivated following bryostatin-1+JQ1 treatment was identical anti-CD3+anti-CD28 antibodies control stimulation. Remarkably, resting CD4+ T isolated 15 patients, ingenol-B+JQ1 released infectious viruses levels similar obtained effects two were already detected 24 hours post-stimulation. These results constitute first demonstration LRA exhibiting such effect represent proof-of-concept for co-administration different types LRAs strategy reduce size reservoirs.

Language: Английский

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Current Status of Latency Reversing Agents Facing the Heterogeneity of HIV-1 Cellular and Tissue Reservoirs

Frontiers in Microbiology, Journal Year: 2020, Volume and Issue: 10

Published: Jan. 24, 2020

One of the most explored therapeutic approach aiming at eradicating HIV-1 reservoirs is “shock and kill” strategy which based on reactivation in latently-infected cells (“shock” phase), while maintaining ART order to prevent spreading infection by neosynthesized virus. This kind would allow “kill” phase during then die from viral cytopathic effects or host cytolytic effector mechanisms following reactivation. Several latency reversing agents (LRAs) with distinct mechanistic classes have been characterized reactivate gene expression. Some LRAs tested terms their potential purge latent vivo clinical trials, showing that possible. However, alone failed reduce size reservoirs. Together inability immune system clear LRA-activated lack specificity these LRAs, heterogeneity largely contributes limited success trials using LRAs. Indeed, established numerous cell types are phenotypes metabolic properties, influenced patient history. Hence, silencing expression cellular tissue need be better understood rationally improve this cure hopefully reach success.

Language: Английский

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HIV DNA Is Frequently Present within Pathologic Tissues Evaluated at Autopsy from Combined Antiretroviral Therapy-Treated Patients with Undetectable Viral Loads

Journal of Virology, Journal Year: 2016, Volume and Issue: 90(20), P. 8968 - 8983

Published: July 28, 2016

HIV infection treatment strategies have historically defined effectiveness through measuring patient plasma RNA. While combined antiretroviral therapy (cART) can reduce viral load (pVL) to undetectable levels, the degree that is eliminated from other anatomical sites remains unclear. We investigated DNA levels in 229 varied autopsy tissues 20 HIV-positive (HIV(+)) cART-treated study participants with low or VL and cerebrospinal fluid (CSF) prior death who were enrolled National Neurological AIDS Bank (NNAB) longitudinal cohort. Extensive medical histories obtained for each participant. Autopsy specimens, including at least six brain nonbrain per participant, reviewed by pathologists. DNA, measured quantitative droplet digital PCR, was identified 48/87 82/142 >200 copies/million cell equivalents. No participant found be completely free of tissue HIV. Parallel sequencing studies some recovered intact Abnormal histological findings all participants, especially brain, spleen, lung, lymph node, liver, aorta, kidney. All demonstrated pathology. Ninety-five percent had atherosclerosis, 75% died cancer. This assists characterizing locations HIV, particular, macrophage-rich tissues, such as central nervous system (CNS) testis. Additional are needed determine if promotes pathogenesis inflammatory diseases, HIV-associated neurocognitive disorders, cancer, atherosclerosis.It well-known levels; however, cART cannot clear infection. An ongoing question is, "Where hiding?" A well-studied reservoir "resting" T cells, which isolated blood products succumb once activated. Less-studied reservoirs samples, unknown penetration, contain a comparably diverse spectrum potentially HIV-infected immune important since <2% body lymphocytes actually reside blood. examined specimens HIV(+) while on >50% infected. Additionally, we considerable pathology participants' substantiates tissue-associated present despite inform future into persistence.

Language: Английский

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Epigenetic control of HIV-1 post integration latency: implications for therapy

Clinical Epigenetics, Journal Year: 2015, Volume and Issue: 7(1)

Published: Sept. 24, 2015

With the development of effective combined anti-retroviral therapy (cART), there is significant reduction in deaths associated with human immunodeficiency virus type 1 (HIV-1) infection. However, complete cure HIV-1 infection difficult to achieve without elimination latent reservoirs which exist infected individuals even under cART regimen. These established during early have long life span, include resting CD4+ T cells, macrophages, central nervous system (CNS) resident macrophage/microglia, and gut-associated lymphoid tissue/macrophages, can actively produce upon interruption cART. Several epigenetic non-epigenetic mechanisms been implicated regulation viral latency. Epigenetic such as histone post translational modifications (e.g., acetylation methylation) DNA methylation proviral microRNAs are involved establishment The better understanding modulating latency could give clues for eradication these reservoirs. latency-reversing agents (LRA) found reactivating vitro, ex vivo, vivo. Some target elicit expression order kill latently cells through cytopathic effect or host immune response. therapeutic approaches aimed at achieving a sterilizing (elimination from body). In present review, we will discuss our current epigenomics how this information be moved laboratory bench patient's bedside.

Language: Английский

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Preclinical development and qualification of ZFN-mediated CCR5 disruption in human hematopoietic stem/progenitor cells

Molecular Therapy — Methods & Clinical Development, Journal Year: 2016, Volume and Issue: 3, P. 16067 - 16067

Published: Jan. 1, 2016

Gene therapy for HIV-1 infection is a promising alternative to lifelong combination antiviral drug treatment. Chemokine receptor 5 (CCR5) the coreceptor required R5-tropic of human cells. Deletion CCR5 renders cells resistant infection, and potential cure has been shown through allogeneic stem cell transplantation with naturally occurring homozygous deletion in donor hematopoietic stem/progenitor (HSPC). The requirement HLA-matched HSPC bearing deletions prohibits widespread application this approach. Thus, strategy disrupt genomic sequences using zinc finger nucleases was developed. Following discussions regulatory agencies, we conducted IND-enabling preclinical vitro vivo testing demonstrate feasibility (preclinical) safety nucleases-based disruption HSPC. We report here clinical-scale manufacturing process necessary deliver CCR5-specific mRNA electroporation data. Our results effective biallelic up 72.9% modified colony forming units from adult mobilized maintenance vivo. Tumorigenicity studies demonstrated initial product safety; further are ongoing subjects infected ([email protected]).

Language: Английский

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Barriers for HIV Cure: The Latent Reservoir

AIDS Research and Human Retroviruses, Journal Year: 2018, Volume and Issue: 34(9), P. 739 - 759

Published: July 28, 2018

Thirty-five years after the identification of HIV-1 as causative agent AIDS, we are still in search vaccines and treatments to eradicate this devastating infectious disease. Progress has been made understanding molecular pathogenesis infection, which crucial for development current therapy regimens. However, despite their efficacy at limiting active viral replication, these drugs unable purge latent reservoir: a pool cells that harbor transcriptionally inactive, but replication-competent proviruses, represent main barrier from affected individuals. In review, discuss advances field have allowed better latency, including diverse cell types constitute reservoir, factors influencing tools study well prospective therapeutic approaches target latently infected cells, so functional cure HIV/AIDS can become reality.

Language: Английский

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New Challenges of HIV-1 Infection: How HIV-1 Attacks and Resides in the Central Nervous System

Cells, Journal Year: 2019, Volume and Issue: 8(10), P. 1245 - 1245

Published: Oct. 13, 2019

Acquired immunodeficiency syndrome (AIDS) has become one of the most devastating pandemics in recorded history. The main causal agent AIDS is human virus (HIV), which infects various cell types immune system that express CD4 receptor on their surfaces. Today, combined antiretroviral therapy (cART) standard treatment for all people with HIV; although it improved quality life living HIV (PLWH), cannot eliminate latent reservoir virus. Therefore HIV/AIDS turned from a fatal disease to chronic requiring lifelong treatment. Despite significant viral load suppression, been observed at least half patients under cART present HIV-associated neurocognitive disorders (HAND), have related HIV-1 infection and replication central nervous (CNS). Several studies focused elucidating mechanism by can invade CNS how generate effects seen HAND. This review summarizes research its interaction an emphasis generation HAND, enters CNS, relationship between cells effect these cells.

Language: Английский

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A highly multiplexed droplet digital PCR assay to measure the intact HIV-1 proviral reservoir

Cell Reports Medicine, Journal Year: 2021, Volume and Issue: 2(4), P. 100243 - 100243

Published: April 1, 2021

Quantifying the replication-competent HIV reservoir is essential for evaluating curative strategies. Viral outgrowth assays (VOAs) underestimate because they fail to induce all proviruses. Single- or double-region DNA overestimate it exclude many defective We designed two triplex droplet digital PCR assays, each with 2 unique targets and 1 in common, normalize results PCR-based T cell counts. Both are specific, sensitive, reproducible. Together, estimate number of proviruses containing five primer-probe regions. Our 5-target on average 12.1-fold higher than correlate paired quantitative VOA (Spearman's ρ = 0.48) but a markedly smaller previous assays. In patients antiretroviral therapy, decay rates blood CD4+ cells faster intact proviruses, provirus frequencies similar mucosal circulating cells.

Language: Английский

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