scholarly journals Optimisation of a TALE nuclease targeting the HIV co-receptor CCR5 for clinical application

Gene Therapy ◽  
2021 ◽  
Author(s):  
Lea Isabell Schwarze ◽  
Dawid Głów ◽  
Tanja Sonntag ◽  
Almut Uhde ◽  
Boris Fehse
Keyword(s):  
Gene Editing ◽  
High Efficiency ◽  
The Novel ◽  
End Joining ◽  
Large Numbers ◽  
Novel Variant ◽  
Cleavage Domain ◽  
Mrna Electroporation ◽  
Receptor Ccr5

AbstractDisruption of the C-C-Chemokine-receptor-5 (CCR5) gene induces resistance towards CCR5-tropic HIV. Here we optimised our previously described CCR5-Uco-TALEN and its delivery by mRNA electroporation. The novel variant, CCR5-Uco-hetTALEN features an obligatory heterodimeric Fok1-cleavage domain, which resulted in complete abrogation of off-target activity at previously found homodimeric as well as 7/8 in silico predicted, potential heterodimeric off-target sites, the only exception being highly homologous CCR2. Prevailing 18- and 10-bp deletions at the on-target site revealed microhomology-mediated end-joining as a major repair pathway. Notably, the CCR5Δ55–60 protein resulting from the 18-bp deletion was almost completely retained in the cytosol. Simultaneous cutting at CCR5 and CCR2 induced rearrangements, mainly 15-kb deletions between the cut sites, in up to 2% of T cells underlining the necessity to restrict TALEN expression. We optimised in vitro mRNA production and showed that CCR5-on- and CCR2 off-target activities of CCR5-Uco-hetTALEN were limited to the first 72 and 24–48 h post-mRNA electroporation, respectively. Using single-cell HRMCA, we discovered high rates of TALEN-induced biallelic gene editing of CCR5, which translated in large numbers of CCR5-negative cells resistant to HIVenv-pseudotyped lentiviral vectors. We conclude that CCR5-Uco-hetTALEN transfected by mRNA electroporation facilitates specific, high-efficiency CCR5 gene-editing (30%–56%) and it is highly suited for clinical translation subject to further characterisation of off-target effects.

Novel somatic mutations in UBA1 as a cause of VEXAS syndrome

Blood ◽  
2021 ◽  
Author(s):  
James A Poulter ◽  
Jason Charles Collins ◽  
Catherine Cargo ◽  
Ruth M de Tute ◽  
Paul Evans ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Somatic Mutations ◽  
Clinical Phenotype ◽  
Splice Acceptor Site ◽  
Acceptor Site ◽  
The Novel ◽  
Disease Mechanism ◽  
Bone Marrow Biopsies ◽  
Novel Variant

Somatic mutations at methionine 41 (Met41) in UBA1, encoding the major E1 enzyme responsible for initiating ubiquitylation, were recently identified as the cause of a novel autoinflammatory disease, named VEXAS (Vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic). We sought to determine the prevalence of UBA1 mutations in a UK cohort of patients matching the VEXAS clinical phenotype. We identified 10 new patients with somatic mutations in UBA1, but only 8 had altered p.Met41. A novel variant, c.167C>T; p.Ser56Phe was identified, which was present in myeloid, and not lymphoid lineages and led to preferential loss of the catalytic activity of cytoplasmic UBA1. An additional novel variant, c.118-1G>C was identified at the splice acceptor site of exon 3 leading to altered splicing in vitro. Bone marrow biopsies from two patients with a Met41 substitution and the novel splice site variant were consistent with previously reported features of VEXAS. The bone marrow of the patient with the p.Ser56Phe variant was less similar, likely driven by a distinct but overlapping disease mechanism. Our study therefore confirms somatic p.Met41 substitutions in UBA1 as a major cause of VEXAS syndrome and identifies two new disease causing mutations.

scholarly journals Stress conditions promote the mating competency of Leishmania promastigotes in vitro marked by expression of the ancestral gamete fusogen HAP2

2021 ◽  
Author(s):  
Isabelle Louradour ◽  
Tiago Rodrigues Ferreira ◽  
Emma Duge ◽  
Nadira Karunaweera ◽  
Andrea Paun ◽  
...  
Keyword(s):  
High Efficiency ◽  
Wide Spectrum ◽  
Sexual Cycle ◽  
Sand Fly ◽  
Large Numbers ◽  
Transcriptomic Signature ◽  
Total Dna ◽  
Low Efficiency ◽  
Transformative Approach

Leishmania are protozoan parasites transmitted by the bite of sand fly vectors producing a wide spectrum of diseases in their mammalian hosts. These diverse clinical outcomes are directly associated with parasite strain and species diversity. Although Leishmania reproduction is mainly clonal, a cryptic sexual cycle capable of producing hybrid genotypes has been inferred from population genetic studies, and directly demonstrated by laboratory crosses. Experimentally, mating competency has been largely confined to promastigotes developing in the sand fly midgut. The ability to hybridize culture promastigotes in vitro has been limited so far to low efficiency mating between two L. tropica strains, L747 and MA37, that mate with high efficiency in flies. Here, we show that exposure of promastigote cultures to DNA damage stress produces a remarkably enhanced efficiency of in vitro hybridization of the L. tropica strains, and extends to other species, including L. donovani, L. infantum, and L. braziliensis, a capacity to generate intra- and interspecific hybrids. Whole genome sequencing and total DNA content analyses indicate that the hybrids are in each case full genome, polyploid hybrids. Single-cell RNA sequencing of the L747 and MA37 parental lines highlights the transcriptome heterogeneity of culture promastigotes and reveals discrete clusters that emerge post-irradiation in which genes potentially involved in genetic exchange are expressed, including the ancestral gamete fusogen HAP2. By generating reporter constructs for HAP2, we could select for mating-competent and mating-incompetent promastigotes. Overall, this work reveals that there are specific populations involved in Leishmania mating associated with a discernible transcriptomic signature, and that stress facilitated in vitro hybridization can be a transformative approach to generate large numbers of hybrid genotypes between diverse species and strains.

scholarly journals Understanding the diversity of genetic outcomes from CRISPR-Cas generated homology-directed repair

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Brett M. Sansbury ◽  
Amanda M. Hewes ◽  
Eric B. Kmiec
Keyword(s):  
Dna Repair ◽  
Enzymatic Activity ◽  
Broad Spectrum ◽  
Gene Editing ◽  
Cell Free Extract ◽  
End Joining ◽  
Homology Directed Repair ◽  
Editing Activity ◽  
Non Homologous End Joining

AbstractAs CRISPR-Cas systems advance toward clinical application, it is essential to identify all the outcomes of gene-editing activity in human cells. Reports highlighting the remarkable success of homology-directed repair (HDR) in the treatment of inherited diseases may inadvertently underreport the collateral activity of this remarkable technology. We are utilizing an in vitro gene-editing system in which a CRISPR-Cas complex provides the double-stranded cleavage and a mammalian cell-free extract provides the enzymatic activity to promote non-homologous end joining, micro-homology mediated end joining, and homology-directed repair. Here, we detail the broad spectrum of gene-editing reaction outcomes utilizing Cas9 and Cas12a in combination with single-stranded donor templates of the sense and nonsense polarity. This system offers the opportunity to see the range of outcomes of gene-editing reactions in an unbiased fashion, detailing the distribution of DNA repair outcomes as a function of a set of genetic tools.

scholarly journals Ku-Dependent Nonhomologous DNA End Joining inXenopus Egg Extracts

1999 ◽  
Vol 19 (4) ◽  
pp. 2585-2593 ◽  
Author(s):  
Paul Labhart
Keyword(s):  
Mammalian Cells ◽  
High Efficiency ◽  
End Joining ◽  
Free System ◽  
Egg Extract ◽  
Egg Extracts ◽  
Dependent Protein ◽  
Immunological Assays ◽  
Dna Ends

ABSTRACT An extract from activated Xenopus eggs joins both matching and nonmatching ends of exogenous linear DNA substrates with high efficiency and fidelity (P. Pfeiffer and W. Vielmetter, Nucleic Acids Res. 16:907–924, 1988). In mammalian cells, such nonhomologous end joining (NHEJ) is known to require the Ku heterodimer, a component of DNA-dependent protein kinase. Here I investigated whether Ku is also required for the in vitro reaction in the egg extract. Immunological assays indicate that Ku is very abundant in the extract. I found that all NHEJ was inhibited by autoantibodies against Ku and that NHEJ between certain combinations of DNA ends was also decreased after immunodepletion of Ku from the extract. The formation of a joint between a DNA end with a 5′-protruding single strand (PSS) and an end with a 3′-PSS, between two ends with 3′-PSS, and between two blunt ends was most Ku dependent. On the other hand, NHEJ between two DNA ends bearing 5′-PSS was Ku independent. These results show that theXenopus cell-free system will be useful to biochemically dissect the role of Ku in eukaryotic NHEJ.

scholarly journals Stress conditions promote Leishmania hybridization in vitro marked by expression of the ancestral gamete fusogen HAP2 as revealed by single-cell RNAseq

eLife ◽  
2022 ◽  
Vol 11 ◽  
Author(s):  
Isabelle Louradour ◽  
Tiago Rodrigues Ferreira ◽  
Emma Duge ◽  
Nadira D Karunaweeera ◽  
Andrea Paun ◽  
...  
Keyword(s):  
Single Cell ◽  
High Efficiency ◽  
Wide Spectrum ◽  
Sexual Cycle ◽  
Sand Fly ◽  
Large Numbers ◽  
Transcriptomic Signature ◽  
Low Efficiency ◽  
Transformative Approach

Leishmania are protozoan parasites transmitted by the bite of sand fly vectors producing a wide spectrum of diseases in their mammalian hosts. These diverse clinical outcomes are directly associated with parasite strain and species diversity. Although Leishmania reproduction is mainly clonal, a cryptic sexual cycle capable of producing hybrid genotypes has been inferred from population genetic studies, and directly demonstrated by laboratory crosses. Experimentally, mating competence has been largely confined to promastigotes developing in the sand fly midgut. The ability to hybridize culture promastigotes in vitro has been limited so far to low efficiency crosses between two L. tropica strains, L747 and MA37, that mate with high efficiency in flies. Here, we show that exposure of promastigote cultures to DNA damage stress produces a remarkably enhanced efficiency of in vitro hybridization of the L. tropica strains, and extends to other species, including L. donovani, L. infantum, and L. braziliensis, a capacity to generate intra- and interspecific hybrids. Whole genome sequencing and total DNA content analyses indicate that the hybrids are in each case full genome, mostly tetraploid hybrids. Single-cell RNA sequencing of the L747 and MA37 parental lines highlights the transcriptome heterogeneity of culture promastigotes and reveals discrete clusters that emerge post-irradiation in which genes potentially involved in genetic exchange are expressed, including the ancestral gamete fusogen HAP2. By generating reporter constructs for HAP2, we could select for promastigotes that could either hybridize or not in vitro. Overall, this work reveals that there are specific populations involved in Leishmania hybridization associated with a discernible transcriptomic signature, and that stress facilitated in vitro hybridization can be a transformative approach to generate large numbers of hybrid genotypes between diverse species and strains.

scholarly journals Clinical and Functional Characterization of Novel INSR Variants in Two Families With Severe Insulin Resistance Syndrome

2021 ◽  
Vol 12 ◽  
Author(s):  
Qiaoli Zhou ◽  
Jing Yu ◽  
Xuewen Yuan ◽  
Chunli Wang ◽  
Ziyang Zhu ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Insulin Resistance Syndrome ◽  
Type A ◽  
Chinese Children ◽  
The Novel ◽  
Severe Insulin Resistance ◽  
Functional Assays ◽  
Novel Variants ◽  
Novel Variant

ObjectiveDefects in the insulin receptor (INSR) gene cause various severe insulin resistance conditions, including Donohue syndrome (DS), Rabson-Mendenhall syndrome (RMS) and type A insulin resistance (type A-IR). This study aimed to investigate the clinical characterization and molecular defects in three Chinese children with INSR-related insulin resistance syndrome.MethodsWe reviewed the clinical data of three Chinese children with INSR-related insulin resistance syndrome from two unrelated kindreds. Genetic analysis was performed using whole-exome sequencing and the effects of the novel variants were further assessed by in vitro functional assays.ResultsThe proband with type A-IR presented with acanthosis nigricans, hypertrichosis, and euglycemia with mild insulin resistance in early childhood. His sister presented with features typical of type A-IR and was diagnosed with diabetes mellitus with severe insulin resistance at the age of 9.8 years. The proband with DS showed typical dysmorphic characteristics, severe intrauterine growth retardation, extreme insulin resistance, fasting hypoglycemia and postprandial hyperglycemia from birth. The heterozygote variants c.[3670G>A]; c.[3614C>T] were identified in both siblings with type A-IR; and c.[749_751del]; c.[3355C>T] in the patient with DS. In vitro studies showed that the novel variant c.749_751del [p.(Thr250del)] in the α-subunit, reduced expression of the mature INSR protein and severely impaired INSR function. In contrast, the novel variant c.3670G>A [p.(Val1224Met)] in the β-subunit had no effect on total protein expression and phosphorylation of INSR and Akt, suggesting that the variant p.Val1224Met appeared to be tolerated and was not responsible for the severe insulin resistance.ConclusionOur study detailed the clinical features of three patients with type A-IR and DS, and identified two novel variants in the INSR gene. Functional assays indicated the novel variant p.Thr250del was pathogenic. In contrast, the novel variant p.Val1224Met was suggested to be tolerated by our experimental data, even though bioinformatics analyses predicted the variant as deleterious.

scholarly journals Diminished neutralization responses towards SARS-CoV-2 Omicron VoC after mRNA or vector-based COVID-19 vaccinations

2021 ◽  
Author(s):  
Henning Jacobsen ◽  
Monika Strengert ◽  
Henrike Maass ◽  
Mario Alberto Ynga Durand ◽  
Barbora Kessel ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Immune Escape ◽  
Significant Risk ◽  
Neutralization Assay ◽  
The Novel ◽  
Wild Type ◽  
Neutralization Activity ◽  
Novel Variant

Background: SARS-CoV-2 variants accumulating immune escape mutations provide a significant risk to vaccine-induced protection. The novel variant of concern Omicron (B.1.1.529) has to date the largest number of amino acid alterations in its Spike protein. Thus, it may efficiently escape recognition by neutralizing antibodies, allowing breakthrough infections in convalescent and vaccinated individuals. Aims: We analysed neutralization activity after vaccination with all mRNA-, vector- or heterologous immunization schemes currently available in Europe at peak response and in a longitudinal follow-up with BNT162b2 vaccinees to define immune escape potential of the Omicron VoC. Methods: We tested sera by in vitro neutralization assay towards SARS-CoV-2 B.1, Omicron, Beta and Delta pseudotypes Results: All vaccines apart from Ad26.CoV2.S showed high levels of responder rates (93.3-100%) towards SARS-CoV-2 wild-type, but some reductions in neutralizing Beta and Delta VoC pseudotypes. The novel Omicron variant had the biggest impact, both in terms of response rates and neutralization titres among responders. Only mRNA-1273 showed a 100% response rate to Omicron and induced the highest titres of neutralizing antibodies, followed by heterologous prime-boost approaches. Homologous BNT162b2 vaccination or vector-based formulations with AZD1222 or Ad26.CoV2.S performed less well with peak responder rates of 33%, 50% and 9%, respectively. However, Omicron responder rates in BNT162b2 recipients were maintained in our six month longitudinal follow-up and even slightly increased to 47%, indicating cross-protection against Omicron is maintained over time. Conclusions: Overall, our data strongly argues for urgent booster doses in individuals who were previously vaccinated with BNT162b2, or a vector-based immunization scheme.

Cytoplasmic Granules in Lymphocytes from Rats Dosed with SK&F 14336-D

1971 ◽  
Vol 29 ◽  
pp. 556-557
Author(s):  
T. G. Merrill ◽  
B. J. Payne ◽  
A. J. Tousimis
Keyword(s):  
Lipid Storage ◽  
Pokeweed Mitogen ◽  
Electron Microscopic ◽  
Cytoplasmic Granules ◽  
Storage Diseases ◽  
Tay Sachs Disease ◽  
Large Numbers ◽  
Microscopic Studies ◽  
Neurovisceral Lipidosis

Rats given SK&F 14336-D (9-[3-Dimethylamino propyl]-2-chloroacridane), a tranquilizing drug, developed an increased number of vacuolated lymphocytes as observed by light microscopy. Vacuoles in peripheral blood of rats and humans apparently are rare and are not usually reported in differential counts. Transforming agents such as phytohemagglutinin and pokeweed mitogen induce similar vacuoles in in vitro cultures of lymphocytes. These vacuoles have also been reported in some of the lipid-storage diseases of humans such as amaurotic familial idiocy, familial neurovisceral lipidosis, lipomucopolysaccharidosis and sphingomyelinosis. Electron microscopic studies of Tay-Sachs' disease and of chloroquine treated swine have demonstrated large numbers of “membranous cytoplasmic granules” in the cytoplasm of neurons, in addition to lymphocytes. The present study was undertaken with the purpose of characterizing the membranous inclusions and developing an experimental animal model which may be used for the study of lipid storage diseases.

The Antiviral and Antimalarial Drug Repurposing in Quest of Chemotherapeutics to Combat COVID-19 Utilizing Structure-Based Molecular Docking

2020 ◽  
Vol 23 ◽  
Author(s):  
Sisir Nandi ◽  
Mohit Kumar ◽  
Mridula Saxena ◽  
Anil Kumar Saxena
Keyword(s):  
Molecular Docking ◽  
In Silico ◽  
Drug Repurposing ◽  
Clinical Settings ◽  
The Novel ◽  
In Silico Docking ◽  
Biochemical Mechanisms ◽  
Novel Coronavirus ◽  
In Silico Molecular Docking

Background: The novel coronavirus disease (COVID-19) is caused by a new strain (SARS-CoV-2) erupted in 2019. Nowadays, it is a great threat that claims uncountable lives worldwide. There is no specific chemotherapeutics developed yet to combat COVID-19. Therefore, scientists have been devoted in the quest of the medicine that can cure COVID- 19. Objective: Existing antivirals such as ASC09/ritonavir, lopinavir/ritonavir with or without umifenovir in combination with antimalarial chloroquine or hydroxychloroquine have been repurposed to fight the current coronavirus epidemic. But exact biochemical mechanisms of these drugs towards COVID-19 have not been discovered to date. Method: In-silico molecular docking can predict the mode of binding to sort out the existing chemotherapeutics having a potential affinity towards inhibition of the COVID-19 target. An attempt has been made in the present work to carry out docking analyses of 34 drugs including antivirals and antimalarials to explain explicitly the mode of interactions of these ligands towards the COVID-19protease target. Results: 13 compounds having good binding affinity have been predicted towards protease binding inhibition of COVID-19. Conclusion: Our in silico docking results have been confirmed by current reports from clinical settings through the citation of suitable experimental in vitro data available in the published literature.

Synthetic and semi-synthetic drugs as promising therapeutic option for the treatment of COVID-19

2020 ◽  
Vol 20 ◽  
Author(s):  
Ekta Shirbhate ◽  
Preeti Patel ◽  
Vijay K Patel ◽  
Ravichandran Veerasamy ◽  
Prabodh C Sharma ◽  
...  
Keyword(s):  
Therapeutic Option ◽  
Antiviral Treatment ◽  
The Novel ◽  
Clinical Experiences ◽  
Synthetic Compounds ◽  
Synthetic Drugs ◽  
Global Pandemic ◽  
The World ◽  
Novel Coronavirus

: The novel coronavirus disease-19 (COVID-19), a global pandemic that emerged from Wuhan, China has today travelled all around the world, so far 216 countries or territories with 21,732,472 people infected and 770,866 deaths globally (as per WHO COVID-19 update dated August 18, 2020). Continuous efforts are being made to repurpose the existing drugs and develop vaccines for combating this infection. Despite, to date, no certified antiviral treatment or vaccine prevails. Although, few candidates have displayed their efficacy in in vitro studies and are being repurposed for COVID-19 treatment. This article summarizes synthetic and semi-synthetic compounds displaying potent activity in their clinical experiences or studies against COVID-19 and also focuses on mode of action of drugs being repositioned against COVID-19.

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