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Nucleic Acids Research Jan 2018CRISPR systems have emerged as transformative tools for altering genomes in living cells with unprecedented ease, inspiring keen interest in increasing their specificity...
CRISPR systems have emerged as transformative tools for altering genomes in living cells with unprecedented ease, inspiring keen interest in increasing their specificity for perfectly matched targets. We have developed a novel approach for improving specificity by incorporating chemical modifications in guide RNAs (gRNAs) at specific sites in their DNA recognition sequence ('guide sequence') and systematically evaluating their on-target and off-target activities in biochemical DNA cleavage assays and cell-based assays. Our results show that a chemical modification (2'-O-methyl-3'-phosphonoacetate, or 'MP') incorporated at select sites in the ribose-phosphate backbone of gRNAs can dramatically reduce off-target cleavage activities while maintaining high on-target performance, as demonstrated in clinically relevant genes. These findings reveal a unique method for enhancing specificity by chemically modifying the guide sequence in gRNAs. Our approach introduces a versatile tool for augmenting the performance of CRISPR systems for research, industrial and therapeutic applications.
Topics: Base Sequence; Binding Sites; CRISPR-Cas Systems; DNA Cleavage; Gene Editing; Humans; K562 Cells; Phosphonoacetic Acid; RNA, Guide, CRISPR-Cas Systems
PubMed: 29216382
DOI: 10.1093/nar/gkx1199 -
Biochemistry Dec 2023CRISPR gene editing and control systems continue to emerge and inspire novel research and clinical applications. Advances in CRISPR performance such as optimizing the...
CRISPR gene editing and control systems continue to emerge and inspire novel research and clinical applications. Advances in CRISPR performance such as optimizing the duration of activity in cells, tissues, and organisms, as well as limiting off-target activities, have been extremely important for expanding the utility of CRISPR-based systems. By investigating the effects of various chemical modifications in guide RNAs (gRNAs) at defined positions and combinations, we find that 2'--methyl-3'-phosphonoacetate (MP) modifications can be substantially more effective than 2'--methyl-3'-phosphorothioate (MS) modifications at the 3' ends of single-guide RNAs (sgRNAs) to promote high editing yields, in some instances showing an order of magnitude higher editing yield in human cells. MP-modified 3' ends are especially effective at promoting the activity of guide RNAs cotransfected with Cas messenger RNA (mRNA), as the gRNA must persist in cells until the Cas protein is expressed. We demonstrate such an MP enhancement for sgRNAs cotransfected with a BE4 mRNA for cytidine base editing and also demonstrate that MP at the 3' ends of prime editing guide RNAs (pegRNAs) cotransfected with PE2 mRNA can promote maximal prime editing yields. In the presence of serum, sgRNAs with MP-modified 3' ends showed marked improvements in editing efficiency over sgRNAs with MS-modified 3' ends codelivered with Cas9 mRNA and showed more modest improvements at enhancing the activity of transfected ribonucleoprotein (RNP) complexes. Our results suggest that MP should be considered as a performance-enhancing modification for the 3' ends of synthetic gRNAs, especially in situations where the guide RNAs may be susceptible to exonuclease-mediated degradation.
Topics: Humans; CRISPR-Cas Systems; RNA, Guide, CRISPR-Cas Systems; Phosphonoacetic Acid; Gene Editing; RNA, Messenger
PubMed: 35436085
DOI: 10.1021/acs.biochem.1c00768 -
Journal of Medical Virology Dec 2022HSV-2 antiviral resistance mainly occurs in immunocompromised patients and especially in HIV-positive individuals receiving long-term antiviral treatment. Those...
HSV-2 antiviral resistance mainly occurs in immunocompromised patients and especially in HIV-positive individuals receiving long-term antiviral treatment. Those situations can be challenging as few alternatives are available for HSV infection management. To describe clinical and virological significance of two novel potential HSV-2 resistance mutations after treating an obese patient with a pseudotumoral genital HSV-related lesion. Consecutive different antiviral treatments were used: valacyclovir (VACV) then foscarnet (FOS) then topical cidofovir (CDV) and finally imiquimod. Under VACV, genotypic resistance testing revealed a novel mutation within viral thymidine kinase (TK, gene UL23) not previously reported but probably accounting for antiviral resistance: W89G, similar to W88R mutation reported in HSV-1 TK, known to be associated with ACV resistance for HSV-1. Under FOS, while initial mutations were still present, a second genotypic resistance testing performed on persisting lesions showed a novel mutation within viral DNA polymerase (DNA pol, gene UL30): C625R. All three antivirals used in this case are small molecules and pharmacokinetics of VACV, FOS, and CDV have not been evaluated in animals and there are very few studies in human. As small molecules are poorly bound to proteins and distribution volume is increased in obese patients, there is risk of underdosage. This mechanism is suspected to be involved in emergence of resistance mutation and further data is needed to adapt, closely to patient profile, antiviral dosage. This report describes a chronic HSV-2 genital lesion, with resistance to current antivirals and novel mutations within viral TK and DNA pol which may confer antiviral resistance.
Topics: Acyclovir; Antiviral Agents; Cidofovir; DNA-Directed DNA Polymerase; Drug Resistance, Viral; Foscarnet; Genitalia; Herpes Simplex; Herpesvirus 2, Human; Humans; Imiquimod; Mutation; Obesity; Thymidine Kinase; Valacyclovir
PubMed: 35973907
DOI: 10.1002/jmv.28070 -
BMJ Case Reports May 2021We present a 24-year-old man with a 2-year history of progressive right-sided monocular vision loss with no other symptoms. An MRI showed a meningioma compressing the...
We present a 24-year-old man with a 2-year history of progressive right-sided monocular vision loss with no other symptoms. An MRI showed a meningioma compressing the right optic nerve and the cavernous sinus. The tumour was partially resected. Eight days after discharge the patient was admitted with fever, a severe stabbing headache, insomnia, nausea and vomiting. A FilmArray panel and a cerebral biopsy were performed which were positive for herpes simplex virus 1 (HSV-1). An MRI of the brain showed asymmetric bilateral lesions in the frontobasal region with predominance of the right side. Acyclovir was started and continued until completing 21 days. A month after discharge, he started experiencing insomnia, trichotillomania, limb tremor, persistence of abulia, apathy and emotional lability. An HSV-1 encephalitis relapse was suspected, acyclovir and foscarnet were started. Due to the poor response to antiviral therapy CSF was tested, which was positive for anti-NMDA receptor encephalitis. A treatment course of intravenous immunoglobulin was started with a favourable outcome.
Topics: Acyclovir; Adult; Antiviral Agents; Encephalitis, Herpes Simplex; Foscarnet; Humans; Male; Neoplasm Recurrence, Local; Young Adult
PubMed: 34039543
DOI: 10.1136/bcr-2020-241136 -
Transplantation and Cellular Therapy Jan 2021The high incidence of human herpesvirus-6 (HHV-6) reactivation, potentially interfering with engraftment after umbilical cord blood (UCB) hematopoietic cell...
The high incidence of human herpesvirus-6 (HHV-6) reactivation, potentially interfering with engraftment after umbilical cord blood (UCB) hematopoietic cell transplantation (HCT), remains a major challenge. To potentially address this problem, we evaluated the effect of prophylactic foscarnet administered twice daily beginning on day +7 and continuing through engraftment in 25 patients. To determine the impact of foscarnet on HHV-6, engraftment, and other transplantation outcomes, we compared results in 61 identically treated patients with hematologic malignancies. Treatment and control groups underwent reduced-intensity conditioning UCB HCT with a conditioning regimen of fludarabine, cyclophosphamide, and total body irradiation 200 cGy with or without antithymocyte globulin (ATG), using sirolimus plus mycophenolate mofetil immune suppression. The treatment and control groups were similar in terms of age, disease risk, use of ATG, Hematopoietic Cell Transplantation Comorbidity Index, and graft CD34 cell dose; however, foscarnet-treated patients were less likely to receive a double UCB graft and to be treated more recently (2016 to 2018). The cumulative incidence of HHV-6 reactivation by day +100 was 63% for all patients (95% confidence interval [CI], 51% to 75%) and was not significantly different between the 2 groups. HHV-6 reactivation occurred at a median of 34 days in the foscarnet group and 25.5 days in the control group. The incidence of neutrophil engraftment at day 42 was higher in the foscarnet group compared with the control group (96%; [95% CI, 83% to 100%] versus 75% [95% CI, 64% to 85%]; P< .01). The cumulative incidence of platelet engraftment by 6 months was 92% (95% CI, 69% to 100%) for the foscarnet group versus 75% (95% CI, 60% to 90%) for the control group (P= .08), and multivariate analysis identified the use of foscarnet as an independent predictor of better platelet engraftment. No patients died as a result of graft failure in recipients of foscarnet, whereas 5 patients died from graft failure in the control group. Six-month overall survival (OS) and nonrelapse mortality (NRM) were better in the foscarnet group (96% versus 72% [P= .02] and 4% versus 18% [P= .07], respectively). Even though foscarnet prophylaxis did not prevent HHV-6 viremia, we observed a delay in time to HHV-6 reactivation, a trend toward differences in engraftment, NRM, and OS compared with historical controls.
Topics: Cord Blood Stem Cell Transplantation; Foscarnet; Graft vs Host Disease; Hematopoietic Stem Cell Transplantation; Herpesvirus 6, Human; Humans
PubMed: 33053448
DOI: 10.1016/j.bbmt.2020.10.008 -
Medicine Dec 2021Intraocular infection of Epstein-Barr virus (EBV) may cause severe visual loss. However, it is relatively rare, and there is no consensus on its treatment.
RATIONALE
Intraocular infection of Epstein-Barr virus (EBV) may cause severe visual loss. However, it is relatively rare, and there is no consensus on its treatment.
PATIENT CONCERNS
A 44-year-old woman complained of a right-eye floater and exhibited a unilateral exudative change along the retinal veins at the Department of Ophthalmology, St. Luke's International Hospital.
DIAGNOSIS
EBV retinitis was diagnosed based on EBV-positive (9.09 × 103 copies/μl) and cytomegalovirus-negative results in the aqueous humor.
INTERVENTIONS
Oral prescription of valaciclovir hydrochloride, and an intravitreal injection of foscarnet sodium hydrate was administered. However, the retinal infiltration progressed, and vitreous opacity with cellular infiltration appeared. Intravitreal methotrexate (MTX) injection effectively suppressed retinal and vitreous infiltration. However, she developed optic-nerve papillitis, and central retinal vein occlusion related to the severe swelling of the optic-nerve, and began steroid pulse therapy. Considering the increase in intraocular EBV levels to 6.4 × 104 copies/ml, we restarted intravitreal foscarnet injections replacing MTX. This in turn rapidly reduced the EBV levels to 3.27 × 104 copies/ml, followed by papillitis alleviation.
OUTCOMES
The intraocular MTX administration reduced the inflammatory vitreous and retinal infiltration, but not the EBV load, while foscarnet reduced the EBV load and papillitis, but not vitreous infiltration.
LESSONS
The retinal infiltration may have involved EBV infection to the retinal neurons but also EBV-free reactive inflammatory cells. EBV infection to the neurons may have been, at least partially, treated by intravitreal foscarnet treatment, and the reactive inflammatory cells by intravitreal MTX. Further observations are warranted to reach a consensus on treating intraocular EBV infection.
Topics: Adult; Epstein-Barr Virus Infections; Female; Foscarnet; Herpesvirus 4, Human; Humans; Methotrexate; Papilledema; Pulse Therapy, Drug; Retinitis; Steroids; Treatment Outcome
PubMed: 35049237
DOI: 10.1097/MD.0000000000028101 -
Journal of Virology Jun 2019In order to identify host cellular DNA metabolic enzymes that are involved in the biosynthesis of hepatitis B virus (HBV) covalently closed circular (ccc) DNA, we...
In order to identify host cellular DNA metabolic enzymes that are involved in the biosynthesis of hepatitis B virus (HBV) covalently closed circular (ccc) DNA, we developed a cell-based assay supporting synchronized and rapid cccDNA synthesis from intracellular progeny nucleocapsid DNA. This was achieved by arresting HBV DNA replication in HepAD38 cells with phosphonoformic acid (PFA), a reversible HBV DNA polymerase inhibitor, at the stage of single-stranded DNA and was followed by removal of PFA to allow the synchronized synthesis of relaxed circular DNA (rcDNA) and subsequent conversion into cccDNA within 12 to 24 h. This cccDNA formation assay allows systematic screening of the effects of small molecular inhibitors of DNA metabolic enzymes on cccDNA synthesis but avoids cytotoxic effects upon long-term treatment. Using this assay, we found that all the tested topoisomerase I and II (TOP1 and TOP2, respectively) poisons as well as topoisomerase II DNA binding and ATPase inhibitors significantly reduced the levels of cccDNA. It was further demonstrated that these inhibitors also disrupted cccDNA synthesis during HBV infection of HepG2 cells expressing sodium taurocholate cotransporting polypeptide (NTCP). Mechanistic analyses indicate that whereas TOP1 inhibitor treatment prevented the production of covalently closed negative-strand rcDNA, TOP2 inhibitors reduced the production of this cccDNA synthesis intermediate to a lesser extent. Moreover, small interfering RNA (siRNA) knockdown of topoisomerase II significantly reduced cccDNA amplification. Taking these observations together, our study demonstrates that topoisomerase I and II may catalyze distinct steps of HBV cccDNA synthesis and that pharmacologic targeting of these cellular enzymes may facilitate the cure of chronic hepatitis B. Persistent HBV infection relies on stable maintenance and proper functioning of a nuclear episomal form of the viral genome called cccDNA, the most stable HBV replication intermediate. One of the major reasons for the failure of currently available antiviral therapeutics to cure chronic HBV infection is their inability to eradicate or inactivate cccDNA. We report here a chemical genetics approach to identify host cellular factors essential for the biosynthesis and maintenance of cccDNA and reveal that cellular DNA topoisomerases are required for both synthesis and intracellular amplification of cccDNA. This approach is suitable for systematic screening of compounds targeting cellular DNA metabolic enzymes and chromatin remodelers for their ability to disrupt cccDNA biosynthesis and function. Identification of key host factors required for cccDNA metabolism and function will reveal molecular targets for developing curative therapeutics of chronic HBV infection.
Topics: Antiviral Agents; DNA Topoisomerases; DNA, Circular; DNA, Viral; Foscarnet; Genome, Viral; Hep G2 Cells; Hepatitis B; Hepatitis B virus; Hepatocytes; Humans; RNA, Small Interfering; Virion; Virus Replication
PubMed: 30867306
DOI: 10.1128/JVI.02230-18 -
Chemical Science Feb 2015Fosfazinomycin A is a phosphonate natural product in which the C-terminal carboxylate of a Val-Arg dipeptide is connected to methyl 2-hydroxy-2-phosphono-acetate...
Fosfazinomycin A is a phosphonate natural product in which the C-terminal carboxylate of a Val-Arg dipeptide is connected to methyl 2-hydroxy-2-phosphono-acetate (Me-HPnA) via a unique hydrazide linkage. We report here that Me-HPnA is generated from phosphonoacetaldehyde (PnAA) in three biosynthetic steps through the combined action of an -methyltransferase (FzmB) and an α-ketoglutarate (α-KG) dependent non-heme iron dioxygenase (FzmG). Unexpectedly, the latter enzyme is involved in two different steps, oxidation of the PnAA to phosphonoacetic acid as well as hydroxylation of methyl 2-phosphonoacetate. The -methyltransferase (FzmH) was able to methylate Arg-NHNH () to give Arg-NHNHMe (), constituting the second segment of the fosfazinomycin molecule. Methylation of other putative intermediates such as desmethyl fosfazinomycin B was not observed. Collectively, our current data support a convergent biosynthetic pathway to fosfazinomycin.
PubMed: 25621145
DOI: 10.1039/C4SC03095H -
Ophthalmic Surgery, Lasers & Imaging... Feb 2015A 60-year-old woman with a history of recurrent headaches and blurred vision presented with bilateral optic disc edema. Optic neuritis was suspected, and intravenous... (Review)
Review
A 60-year-old woman with a history of recurrent headaches and blurred vision presented with bilateral optic disc edema. Optic neuritis was suspected, and intravenous methylprednisonlone was administered. Her vision declined to hand motions in both eyes, and subsequent evaluation revealed bilateral acute retinal necrosis with bilateral central retinal artery occlusions (CRAO). Aqueous humor polymerase chain reaction analysis was positive for herpes simplex virus (HSV), establishing a diagnosis of HSV-associated bilateral acute retinal necrosis (ARN) and meningitis. CRAO has rarely been reported in association with ARN, and a fulminant course with bilateral CRAO in association with ARN has not been previously reported. This case emphasizes the importance of careful peripheral examination in patients with presumptive optic neuritis, judicious use of systemic corticosteroid in this context, and the retinal vaso-obliterative findings that may be observed in the pathogenesis of ARN.
Topics: Antibodies, Viral; Antiviral Agents; Aqueous Humor; DNA, Viral; Drug Therapy, Combination; Eye Infections, Viral; Female; Fluorescein Angiography; Foscarnet; Ganciclovir; Herpes Simplex; Herpesvirus 2, Human; Humans; Magnetic Resonance Imaging; Meningitis, Viral; Middle Aged; Polymerase Chain Reaction; Retinal Artery Occlusion; Retinal Necrosis Syndrome, Acute
PubMed: 25707059
DOI: 10.3928/23258160-20150213-24 -
Investigative Ophthalmology & Visual... Jun 2020Herpes simplex virus type I (HSV-1) infection of corneal epithelial cells activates ataxia telangiectasia mutated (ATM), an apical kinase in the host DNA damage response...
PURPOSE
Herpes simplex virus type I (HSV-1) infection of corneal epithelial cells activates ataxia telangiectasia mutated (ATM), an apical kinase in the host DNA damage response pathway, whose activity is necessary for the progression of lytic HSV-1 infection. The purpose of this study is to investigate the mechanism of ATM activation by HSV-1 in the corneal epithelium, as well as its functional significance.
METHODS
Mechanistic studies were performed in cultured human corneal epithelial cell lines (hTCEpi, HCE), as well as in esophageal (EPC2) and oral (OKF6) cell lines. Transfection-based experiments were performed in HEK293 cells. HSV-1 infection was carried out using the wild-type KOS strain, various mutant strains (tsB7, d120, 7134, i13, n208), and bacterial artificial chromosomes (fHSVΔpac, pM24). Inhibitors of ATM (KU-55933), protein synthesis (cycloheximide), and viral DNA replication (phosphonoacetic acid) were used. Outcomes of infection were assayed using Western blotting, qRT-PCR, immunofluorescence, and comet assay.
RESULTS
This study demonstrates that HSV-1-mediated ATM activation in corneal epithelial cells relies on the viral immediate early gene product ICP4 and requires the presence of the viral genome in the host nucleus. We show that ATM activation is independent of viral genome replication, the ICP0 protein, and the presence of DNA lesions. Interestingly, ATM activity appears to be necessary at the onset of infection, but dispensable at the later stages.
CONCLUSIONS
This study expands our understanding of HSV-1 virus-host interactions in the corneal epithelium and identifies potential areas of future investigation and therapeutic intervention in herpes keratitis.
Topics: Cells, Cultured; DNA Damage; DNA Replication; DNA, Viral; Epithelium, Corneal; Eye Infections, Viral; Herpesvirus 1, Human; Humans; Keratitis, Herpetic; Virus Replication
PubMed: 32543665
DOI: 10.1167/iovs.61.6.39