Elimination of HIV-1 requires clearance and removal of integrated proviral DNA from infected cells and tissues. Here, sequential long-acting slow-effective release antiviral therapy (LASER ART) and CRISPR-Cas9 demonstrate viral clearance in latent infectious reservoirs in HIV-1 infected humanized mice. HIV-1 subgenomic DNA fragments, spanning the long terminal repeats and the Gag gene, are excised in vivo, resulting in elimination of integrated proviral DNA; virus is not detected in blood, lymphoid tissue, bone marrow and brain by nested and digital-droplet PCR as well as RNAscope tests. No CRISPR-Cas9 mediated off-target effects are detected. Adoptive transfer of human immunocytes from dual treated, virus-free animals to uninfected humanized mice fails to produce infectious progeny virus. In contrast, HIV-1 is readily detected following sole LASER ART or CRISPR-Cas9 treatment. These data provide proof-of-concept that permanent viral elimination is possible.
We used NOD/SCID mice, also known as NRG, to assess the ability of lentivirus-mediated intravenous delivery of CRISPR in editing the HIV-1 genome from the circulating PBMC engrafts, some of which homed within several animal solid tissues. Lentivirus-mediated delivery of a multiplex of guide RNAs accompanied by Cas9 endonuclease led to the excision of the targeted region of the viral genome positioned within the HIV-1 LTR from the in-vitro-infected human peripheral blood mononuclear cells (PBMCs) embedded in the spleens of NRG mice. Similarly, the treatment of NRG mice harboring PBMC engrafts derived from HIV-1-positive patients with the therapeutic lentivirus eliminated the presence of the viral DNA fragment in the blood, as well as in the spleen, lung, and liver, of the engrafted animals. Sanger sequence analysis of the viral DNA after treatment with the lentiviral vectors expressing Cas9 and gRNAs verified the editing and removal of the proviral DNA fragment from the viral genome at the predicted sites. This proof-of-concept study, for the first time, demonstrates successful excision of the HIV-1 proviral DNA from patient immune cell engrafts in humanized mice upon treatment with lentivirus-expressing CRISPR and causes a decline in the level of replication-competent virus.
Elimination of HIV DNA from infected individuals remains a challenge in medicine. Here, we demonstrate that intravenous inoculation of SIV-infected macaques, a well-accepted non-human primate model of HIV infection, with adeno-associated virus 9 (AAV9)-CRISPR/Cas9 gene editing construct designed for eliminating proviral SIV DNA, leads to broad distribution of editing molecules and precise cleavage and removal of fragments of the integrated proviral DNA from the genome of infected blood cells and tissues known to be viral reservoirs including lymph nodes, spleen, bone marrow, and brain among others. Accordingly, AAV9-CRISPR treatment results in a reduction in the percent of proviral DNA in blood and tissues. These proof-of-concept observations offer a promising step toward the elimination of HIV reservoirs in the clinic.
ObjectivePatients undergoing immune modulatory therapies for the treatment of autoimmune diseases such as multiple sclerosis, and individuals with an impaired-immune system, most notably AIDS patients, are in the high risk group of developing progressive multifocal leukoencephalopathy (PML), an often lethal disease of the brain characterized by lytic infection of oligodendrocytes in the central nervous system (CNS) with JC virus (JCV). The immune system plays an important regulatory role in controlling JCV reactivation from latent sites by limiting viral gene expression and replication. However, little is known regarding the molecular mechanisms responsible for this regulation.Methods and ResultsHere, we investigated the impact of soluble immune mediators secreted by activated PBMCs on viral replication and gene expression by cell culture models and molecular virology techniques. Our data revealed that viral gene expression and viral replication were suppressed by soluble immune mediators. Further studies demonstrated that soluble immune mediators secreted by activated PBMCs inhibit viral replication induced by T-antigen, the major viral regulatory protein, by suppressing its expression in glial cells. This unexpected suppression of T-antigen was mainly associated with the suppression of translational initiation. Cytokine/chemokine array studies using conditioned media from activated PBMCs revealed several candidate cytokines with possible roles in this regulation. Among them, only IFN-γ showed a robust inhibition of T-antigen expression. While potential roles for IFN-β, and to a lesser extent IFN-α have been described for JCV, IFN-γ has not been previously implicated. Further analysis of IFN-γ signaling pathway revealed a novel role of Jak1 signaling in control of viral T-antigen expression. Furthermore, IFN-γ suppressed JCV replication and viral propagation in primary human fetal glial cells, and showed a strong anti-JCV activity.ConclusionsOur results suggest a novel role for IFN-γ in the regulation of JCV gene expression via downregulation of the major viral regulatory protein, T-antigen, and provide a new avenue of research to understand molecular mechanisms for downregulation of viral reactivation that may lead to development of novel strategies for the treatment of PML.
Background Heavy and chronic ethanol (EtOH) exposure can cause significant structural and functional damage to the adult brain. The most devastating consequence of EtOH exposure is the neurotoxicity associated with the depletion of neurons. Regulation of splice variants in the brain can modulate protein functions, which may ultimately affect behaviors associated with alcohol dependence and EtOH-mediated neurotoxicity. Since alcohol consumption is associated with neurotoxicity, it is possible that altered splicing of survival and pro-survival factors during the development of alcoholism may contribute to the neurotoxicity. Methods Primary human neurons and a neuroblastoma cell line were exposed to different concentrations of EtOH for various time periods. Cell viability and neuronal marker expression were analyzed by MTT assay and immunoblotting, respectively. Effect of EtOH exposure on splicing regulatory protein expression and alternative splicing of candidate genes were analyzed by a biochemical approach. Transcriptional activity of SRSF1 gene was determined by reporter gene analysis. Results Our results suggest that EtOH exposure to neuronal cells at 25 mM and higher concentrations are detrimental. In addition, EtOH exposure caused a dramatic reduction in serine-arginine rich splicing factor 1 (SRSF1) expression levels. Furthermore, EtOH exposure led to pre-mRNA missplicing of Mcl-1, a pro-survival member of the Bcl-2 family, by downregulating the expression levels of serine/arginine rich splicing factor 1 (SRSF1). Moreover, ectopic expression of both SRSF1 and MCL-1L isoform was able to recover EtOH-mediated neurotoxicity. Conclusions Our results suggest that ethanol exposure can lead to pre-mRNA missplicing of Mcl-1 in neuronal cells. Our results indicate that ethanol exposure of neurons leads to a decrease in the ratio of Mcl-1L/Mcl-1S by favoring pro-apoptotic Mcl-1S splicing over anti-apoptotic Mcl-1L isoform suggesting that Mcl-1S may play a crucial role in neurotoxicity associated with alcohol consumption.
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