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CRISPR-Cas9/gRNA exhibits therapeutic efficacy against latent human immunodeficiency virus (HIV) genome but the delivery of this therapeutic cargo to the brain remains as a challenge. In this research, for the first time, we demonstrated magnetically guided non-invasive delivery of a nano-formulation (NF), composed of Cas9/gRNA bound with magneto-electric nanoparticles (MENPs), across the blood-brain barrier (BBB) to inhibit latent HIV-1 infection in microglial (hμglia)/HIV (HC69) cells. An optimized ac-magnetic field of 60 Oe was applied on NF to release Cas9/gRNA from MENPs surface and to facilitate NF cell uptake resulting in intracellular release and inhibition of HIV. The outcomes suggested that developed NF reduced HIV-LTR expression significantly in comparison to unbound Cas9/gRNA in HIV latent hμglia/HIV (HC69) cells. These findings were also validated qualitatively using fluorescence microscopy to assess NF efficacy against latent HIV in the microglia cells. We believe that CNS delivery of NF (CRISPR/Cas9-gRNA-MENPs) across the BBB certainly will have clinical utility as future personalized nanomedicine to manage neuroHIV/AIDS.

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. Removal of integrated HIV DNA remains a roadblock for HIV cure. Here, Mancuso et al . show that intravenous administration of an adeno-associated virus-based CRISPR/Cas9 gene editing construct to SIV-infected macaques results in excision of integrated proviral DNA from infected blood cells and tissues known to be viral reservoirs.

AIDS remains incurable due to the permanent integration of HIV-1 into the host genome, imparting risk of viral reactivation even after antiretroviral therapy. New strategies are needed to ablate the viral genome from latently infected cells, because current methods are too inefficient and prone to adverse off-target effects. To eliminate the integrated HIV-1 genome, we used the Cas9/guide RNA (gRNA) system, in single and multiplex configurations. We identified highly specific targets within the HIV-1 LTR U3 region that were efficiently edited by Cas9/gRNA, inactivating viral gene expression and replication in latently infected microglial, promonocytic, and T cells. Cas9/gRNAs caused neither genotoxicity nor off-target editing to the host cells, and completely excised a 9,709-bp fragment of integrated proviral DNA that spanned from its 5′ to 3′ LTRs. Furthermore, the presence of multiplex gRNAs within Cas9-expressing cells prevented HIV-1 infection. Our results suggest that Cas9/gRNA can be engineered to provide a specific, efficacious prophylactic and therapeutic approach against AIDS.

CRISPR-associated protein 9 (Cas9)-mediated genome editing provides a promising cure for HIV-1/AIDS; however, gene delivery efficiency in vivo remains an obstacle to overcome. Here, we demonstrate the feasibility and efficiency of excising the HIV-1 provirus in three different animal models using an all-in-one adeno-associated virus (AAV) vector to deliver multiplex single-guide RNAs (sgRNAs) plus Staphylococcus aureus Cas9 (saCas9). The quadruplex sgRNAs/saCas9 vector outperformed the duplex vector in excising the integrated HIV-1 genome in cultured neural stem/progenitor cells from HIV-1 Tg26 transgenic mice. Intravenously injected quadruplex sgRNAs/saCas9 AAV-DJ/8 excised HIV-1 proviral DNA and significantly reduced viral RNA expression in several organs/tissues of Tg26 mice. In EcoHIV acutely infected mice, intravenously injected quadruplex sgRNAs/saCas9 AAV-DJ/8 reduced systemic EcoHIV infection, as determined by live bioluminescence imaging. Additionally, this quadruplex vector induced efficient proviral excision, as determined by PCR genotyping in the liver, lungs, brain, and spleen. Finally, in humanized bone marrow/liver/thymus (BLT) mice with chronic HIV-1 infection, successful proviral excision was detected by PCR genotyping in the spleen, lungs, heart, colon, and brain after a single intravenous injection of quadruplex sgRNAs/saCas9 AAV-DJ/8. In conclusion, in vivo excision of HIV-1 proviral DNA by sgRNAs/saCas9 in solid tissues/organs can be achieved via AAV delivery, a significant step toward human clinical trials. [Display omitted] Yin et al. use multiplex CRISPR/Cas9 genome editing technology to excise the HIV-1 provirus in a precise manner in three different HIV-1 animal models via in vivo AAV gene delivery. The feasibility of HIV excision in infected cells in vivo paves the way toward human clinical trials to cure HIV-1 infection.

Aging is the result of various compounding stresses that gradually overcome the homeostatic regulation of the cell, resulting in irreversible damage. This manifests as many acute and chronic conditions, the most common of which are neurodegeneration and dementia. Epidemiological studies have shown significant, strong correlations between viral infection and neurodegenerative diseases. This review overlays the characteristics of viral pathogenesis with the hallmarks of aging to discuss how active and latent viruses contribute to aging. Through our contextualization of myriad basic science papers, we offer explanations for premature aging via viral induction of common stress response pathways. Viruses induce many stresses: dysregulated homeostasis by exogenous viral proteins and overwhelmed protein quality control mechanisms, DNA damage through direct integration and epigenetic manipulation, immune-mediated oxidative stress and immune exhaustion, and general energy theft that is amplified in an aging system. Overall, this highlights the long-term importance of vaccines and antivirals in addition to their acute benefits.

Since their discovery in 1971, the polyomaviruses JC (JCPyV) and BK (BKPyV), isolated from patients with progressive multifocal leukoencephalopathy and polyomavirus-associated nephropathy, respectively, remained for decades as the only known members of the Polyomaviridae family of viruses of human origin. Over the past five years, the application of new genomic amplification technologies has facilitated the discovery of several novel human polyomaviruses (HPyVs), bringing the present number to 10. These HPyVs share many fundamental features in common such as genome size and organization. Infection by all HPyVs is widespread in the human population, but they show important differences in their tissue tropism and association with disease. Much remains unknown about these new viruses. In this review, we discuss the problems associated with studying HPyVs, such as the lack of culture systems for the new viruses and the gaps in our basic understanding of their biology. We summarize what is known so far about their distribution, life cycle, tissue tropism, their associated pathologies (if any), and future research directions in the field.

Current antiretroviral therapy does not eliminate the integrated and transcriptionally silent HIV-1 provirus in latently infected cells. Recently, a “shock and kill” strategy has been extensively explored to eradicate the HIV-1 latent reservoirs for a permanent cure of AIDS. The therapeutic efficacy of currently used agents remains disappointing because of low efficiency, non-specificity and cellular toxicity. Here we present a novel catalytically-deficient Cas9-synergistic activation mediator (dCas9-SAM) technology to selectively, potently and persistently reactivate the HIV-1 latent reservoirs. By screening 16 MS2-mediated single guide RNAs, we identified long terminal repeat (LTR)-L and O that surround the enhancer region (-165/-145 for L and -92/-112 for O) and induce robust reactivation of HIV-1 provirus in HIV-1 latent TZM-bI epithelial, Jurkat T lymphocytic and CHME5 microglial cells. This compulsory reactivation induced cellular suicide via toxic buildup of viral proteins within HIV-1 latent Jurkat T and CHME5 microglial cells. These results suggest that this highly effective and target-specific dCas9-SAM system can serve as a novel HIV-latency-reversing therapeutic tool for the permanent elimination of HIV-1 latent reservoirs.

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the central nervous system (CNS) caused by reactivation of the human polyomavirus JCV gene expression and its replication in oligodendrocytes, the myelin producing cells in the brain. Once a rare disease seen in patients with lymphotproliferative and myeloproliferative disorders, PML has been seen more frequently in HIV-1 positive/AIDS patients as well as patients undergoing immunomodulatory therapy due for autoimmune disorders including multiple sclerosis, rheumatoid arthritis, and others. As of now there is no cure for PML and in most cases disease progression leads to death within two years. Similar to other polyomaviruses, the JCV genome is small circular double stranded DNA that includes coding sequences for the viral early protein, T-antigen, which is critical for directing viral reactivation and lytic infection. Here, we employ a newly developed gene editing strategy, CRISPR/Cas9, to introduce mutations in the viral genome and, by inactivating the gene encoding T-antigen, inhibit viral replication. We first used bioinformatics screening and identified several potential targets within the JCV T-antigen gene that can serve as sites for the creation of guide RNAs (gRNAs) for positioning the Cas9 nuclease on the designated area of the viral genome for editing. Results from a series of integrated genetic and functional studies showed that transient or conditional expression of Cas9 and gRNAs specifically targets the DNA sequences corresponding to the N-terminal region of T-antigen, and by introducing mutation, interferes with expression and function of of the viral protein, hence suppressing viral replication in permissive cells. Results from SURVEYOR assay revealed no off-target effects of the JCV-specific CRISPR/Cas9 editing apparatus. These observations provide the first evidence for the employment of a gene editing strategy as a promising tool for the elimination of the JCV genome and a potential cure for PML.

Least component-based delivery of drug-tagged-nanocarriers across blood-brain-barriers (BBB) will allow site-specific and on-demand release of therapeutics to prevent CNS diseases. We developed a non-invasive magnetically guided delivery of magneto-electric nanocarriers (MENCs), ~20 nm, 10 mg/kg, across BBB in C57Bl/J mice. Delivered MENCs were uniformly distributed inside the brain and were non-toxic to brain and other major organs, such as kidney, lung, liver and spleen and did not affect hepatic, kidney and neurobehavioral functioning.

JC Virus (JCPyV), a member of the Polyomaviridiæ family, is a human neurotropic virus with world-wide distribution. JCPyV is the established opportunistic infectious agent of progressive multifocal leukoencephalopathy, a fatal demyelinating disease, which results from the cytolytic infection of oligodendrocytes. Mutations in the regulatory region of JCPyV determine the different viral strains. Mad-1 the strain associated with PML contains two 98 base pair repeats, whereas the archetype strain (CY), which is the transmissible form of JCPyV, contains only one 98 tandem with two insertions of 62 and 23 base pairs respectively. The oncogenicity of JCPyV has been suspected since direct inoculation into the brain of rodents and primates resulted in the development of brain tumors and has been attributed to the viral protein, T-Antigen. To further understand the oncogenicity of JCPyV, a transgenic mouse colony containing the early region of the archetype strain (CY), under the regulation of its own promoter was generated. These transgenic animals developed tumors of neural crest origin, including: primitive neuroectodermal tumors, medulloblastomas, adrenal neuroblastomas, pituitary tumors, malignant peripheral nerve sheath tumors, and glioblastomas. Neoplastic cells from all different phenotypes express T-Antigen. The close parallels between the tumors developed by these transgenic animals and human CNS tumors make this animal model an excellent tool for the study of viral oncogenesis.