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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 -
Kidney360 Jul 2021
Topics: Allografts; Foscarnet; Kidney; Transplantation, Homologous
PubMed: 35368358
DOI: 10.34067/KID.0001062021 -
ACS Biomaterials Science & Engineering Apr 2022[ZrO][(FCN)(OH)] and Gd[FCN] inorganic-organic hybrid nanoparticles (IOH-NPs) are novel saline antiviral nanocarriers with foscarnet (FCN) as a drug anion. FCN as a...
[ZrO][(FCN)(OH)] and Gd[FCN] inorganic-organic hybrid nanoparticles (IOH-NPs) are novel saline antiviral nanocarriers with foscarnet (FCN) as a drug anion. FCN as a pyrophosphate analogue serves as a prototype of a viral DNA polymerase inhibitor. FCN is used for the treatment of herpesvirus infections, including the drug-resistant cytomegalovirus (CMV) and herpes simplex viruses, HSV-1 and HSV-2. The novel [ZrO][(FCN)(OH)] and Gd[FCN] IOH-NPs are characterized by aqueous synthesis, small size (20-30 nm), low material complexity, high biocompatibility, and high drug load (up to 44 wt % FCN per nanoparticle). The antiviral activity of the FCN-type IOH-NPs is probed for the human cytomegalovirus (HCMV). Moreover, the uptake of FCN-type IOH-NPs into vesicles, cytoplasm, and nuclei of nonphagocytic lung epithelial cells is evaluated. As a result, a promising antiviral activity of the FCN-type IOH-NPs that significantly outperforms freely dissolved FCN at the level of clinical formulations is observed, encouraging a future use of FCN-type IOH-NPs for the delivery of antivirals against respiratory viruses.
Topics: Antiviral Agents; Cytomegalovirus; Foscarnet; Herpesvirus 1, Human; Humans; Nanoparticles
PubMed: 35344659
DOI: 10.1021/acsbiomaterials.2c00074 -
Biomedicines Mar 2022Despite the eradication of smallpox four decades ago, poxviruses continue to be a threat to humans and animals. The arsenal of anti-poxvirus agents is very limited and...
Despite the eradication of smallpox four decades ago, poxviruses continue to be a threat to humans and animals. The arsenal of anti-poxvirus agents is very limited and understanding mechanisms of resistance to agents targeting viral DNA polymerases is fundamental for the development of antiviral therapies. We describe here the phenotypic and genotypic characterization of poxvirus DNA polymerase mutants isolated under selective pressure with different acyclic nucleoside phosphonates, including HPMPC (cidofovir), cHPMPC, HPMPA, cHPMPA, HPMPDAP, HPMPO-DAPy, and PMEO-DAPy, and the pyrophosphate analogue phosphonoacetic acid. Vaccinia virus (VACV) and cowpox virus drug-resistant viral clones emerging under drug pressure were characterized phenotypically (drug-susceptibility profile) and genotypically (DNA polymerase sequencing). Different amino acid changes in the polymerase domain and in the 3'-5' exonuclease domain were linked to drug resistance. Changes in the 3'-5' domain emerged earlier than in the polymerase domain when viruses acquired a combination of mutations. Our study highlights the importance of poxvirus DNA polymerase residues 314, 613, 684, 688, and 851, previously linked to drug resistance, and identified several novel mutations in the 3'-5' exonuclease domain (M313I, F354L, D480Y) and in the DNA polymerase domain (A632T, T831I, E856K, L924F) associated with different drug-susceptibility profiles. Furthermore, a combination of mutations resulted in complex patterns of cross-resistance. Modeling of the VACV DNA polymerase bearing the newly described mutations was performed to understand the effects of these mutations on the structure of the viral enzyme. We demonstrated the emergence of drug-resistant DNA polymerase mutations in complex patterns to be considered in case such mutations should eventually arise in the clinic.
PubMed: 35327382
DOI: 10.3390/biomedicines10030580 -
Frontiers in Microbiology 2022The appearance of drug-resistant mutations in UL54 DNA polymerase and UL97 kinase genes is problematic for the treatment of human cytomegalovirus (HCMV) diseases. During...
The appearance of drug-resistant mutations in UL54 DNA polymerase and UL97 kinase genes is problematic for the treatment of human cytomegalovirus (HCMV) diseases. During treatment of HCMV infection in a pediatric hematopoietic cell transplant recipient, H600L and T700A mutations and E576G mutation were independently found in the UL54 gene. Foscarnet (FOS; phosphonoformic acid) resistance by T700A mutation is reported. Here, we investigated the role of novel mutations in drug resistance by producing recombinant viruses and a model polymerase structure. The H600L mutant virus showed an increase in resistance to ganciclovir (GCV) by 11-fold and to FOS and cidofovir (CDV) by 5-fold, compared to the wild type, while the E756G mutant virus showed an increase in resistance to FOS by 9-fold and modestly to CDV by 2-fold. With the FOS-resistant T700A mutation, only H600L produced increased FOS resistance up to 37-fold, indicating an additive effect of these mutations on FOS resistance. To gain insight into drug resistance mechanisms, a model structure for UL54 polymerase was constructed using the yeast DNA polymerase as a template. In this model, HCMV DNA polymerase contains a long palm loop domain of which H600 and T700 are located on each end and T700 interacts with the FOS binding pocket. Our results demonstrate that H600L and E756G mutations in UL54 polymerase are novel drug-resistant mutations and that the acquisition of both H600L and T700A mutations in the DNA-binding loop confers increased resistance to FOS treatment, providing novel insights for the mechanism acquiring foscarnet resistance.
PubMed: 35185843
DOI: 10.3389/fmicb.2022.771978 -
Medicine Feb 2022Cytomegalovirus (CMV) disease is relatively uncommon in nontransplant hematological patients. Moreover, cutaneous manifestations of CMV diseases have scarcely been...
RATIONALE
Cytomegalovirus (CMV) disease is relatively uncommon in nontransplant hematological patients. Moreover, cutaneous manifestations of CMV diseases have scarcely been reported and are probably under-recognized.
PATIENT CONCERNS
We describe a patient with large B-cell lymphoma who developed a band-form, erythematous lesion over his left abdomen soon after the second course of rituximab, cyclophosphamide, doxorubicin, vincristine, prednisolone chemotherapy.
DIAGNOSES
The lesion was initially mistaken for bacterial cellulitis or herpes zoster and was histologically confirmed as cutaneous CMV infection. Subsequent work-up also detected CMV viremia and the presence of CMV meningoencephalitis.
INTERVENTIONS
The patient was treated with ganciclovir plus CMV immune globulin followed by foscarnet.
OUTCOMES
Although the patient's cutaneous lesion resolved, his cognitive impairment did not recover, and he developed a fatal multi-organ failure 1 month later.
LESSONS
Cutaneous CMV disease can herald multisystem involvement and an unfavorable prognosis in immunocompromised hosts. It should be ruled out with biopsy in patients with hematological malignancy who have cutaneous lesions refractory to antibacterial therapy.
Topics: Antiviral Agents; Cytomegalovirus Infections; Fatal Outcome; Foscarnet; Ganciclovir; Hematologic Neoplasms; Humans; Immunocompromised Host; Male; Skin Diseases
PubMed: 35119018
DOI: 10.1097/MD.0000000000028721 -
Transplant Infectious Disease : An... Apr 2022Foscarnet is an important drug for the treatment of cytomegalovirus infection in patients undergoing hematopoietic stem cell transplantation (HSCT). Foscarnet is often... (Observational Study)
Observational Study
Long-term use of foscarnet is associated with an increased incidence of acute kidney injury in hematopoietic stem cell transplant patients: A retrospective observational study.
BACKGROUND
Foscarnet is an important drug for the treatment of cytomegalovirus infection in patients undergoing hematopoietic stem cell transplantation (HSCT). Foscarnet is often discontinued because of the development of acute kidney injury (AKI). Thus, the identification of factors leading to the development of AKI is beneficial. This study aimed to investigate the incidence of AKI and the factors influencing AKI development in HSCT patients treated with foscarnet.
METHODS
This was a retrospective observational study. Patients who underwent HSCT and received foscarnet at the Department of Hematology, Osaka City University Hospital, were identified from medical records. The patients were classified into AKI and non-AKI groups, and the risk factors associated with AKI were evaluated. For continuous variables, receiver-operating characteristic (ROC) curve analysis was used to calculate the optimal cutoff value.
RESULTS
Thirty-five patients (47 cases) were assigned to the AKI (51.1%, 24/47) and non-AKI groups (48.9%, 23/47). The AKI group had a significantly longer foscarnet administration period than the non-AKI group (p = 0.049). The appropriate cutoff value for the foscarnet administration period using the ROC curve was 27 days. The incidence of AKI was significantly higher in cases who received foscarnet for more than 27 days (11/14, 78.6%) compared to those who received less than 27 days (13/33, 39.4%) (odds ratio: 5.64, 95% confidence interval 1.32-24.2, p = 0.024).
CONCLUSION
The incidence of AKI was 51.1% in HSCT patients treated with foscarnet, and foscarnet administration for more than 27 days may be associated with the incidence of AKI.
Topics: Acute Kidney Injury; Foscarnet; Hematopoietic Stem Cell Transplantation; Humans; Incidence; Retrospective Studies; Risk Factors; Transplantation, Homologous
PubMed: 35114030
DOI: 10.1111/tid.13804 -
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 -
Anais Brasileiros de Dermatologia 2022
Topics: Acquired Immunodeficiency Syndrome; Acyclovir; Antiviral Agents; Foscarnet; Herpes Genitalis; Herpes Simplex; Humans; Imiquimod
PubMed: 35039209
DOI: 10.1016/j.abd.2020.10.019 -
Romanian Journal of Ophthalmology 2021To report a case of acute retinal necrosis (ARN) and to emphasize special aspects of the management. Factors that must be considered. We present the case of an...
To report a case of acute retinal necrosis (ARN) and to emphasize special aspects of the management. Factors that must be considered. We present the case of an 83-year-old woman examined for acute vision loss in her left eye (LE). Background: diabetes, pseudophakic in her LE; subluxated intraocular lens (IOL) and advanced pseudoexfoliative glaucoma in her right eye (RE). The visual acuity (VA) was hand movements in both eyes. Funduscopic examination revealed vitritis, temporal area of retinal necrosis with peripapillary choroiditis spots and macular haemorrhages in her LE and OCT showed a cystic macular edema. A positive polymerase chain reaction (PCR) test for Varicella Zoster Virus (VZV) in aqueous humor of her LE was found. She underwent intravenous Acyclovir 10 mg per kg every 8 hours. She received two doses of adjunctive intravitreal Foscarnet (2.4 mg/ 0.1 mL) in the first 3 days of treatment (2 days between doses). After 3 days of treatment, she started with intravenous prednisone 60 mg per day. The VA of her LE was 0,8 and the retinal necrosis activity was stationary. In fundoscopic examination, vitritis and retinal hemorrhages have disappeared. At that moment there were no foci of chorioretinitis or macular edema although retinal ischemia persisted at the inferior nasal level. The role of adjunctive intravitreal antiviral therapy in combination with systemic treatment revealed promising results. Corticosteroids can be used topically and orally to decrease the severe inflammatory response associated with ARN. Early treatment is crucial to optimize visual and anatomic outcomes.
Topics: Acyclovir; Aged, 80 and over; Eye Infections, Viral; Female; Foscarnet; Herpesvirus 3, Human; Humans; Retinal Necrosis Syndrome, Acute
PubMed: 35036649
DOI: 10.22336/rjo.2021.53