Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in the mouse

Abstract Background Hemophilia A, a bleeding disorder resulting from F8 mutations, can only be cured by gene therapy. A promising strategy is CRISPR-Cas9-mediated precise insertion of F8 in hepatocytes at highly expressed gene loci, such as albumin (Alb). Unfortunately, the precise in vivo integration efficiency of a long insert is very low (~ 0.1%). Results We report that the use of a double-cut donor leads to a 10- to 20-fold increase in liver editing efficiency, thereby completely reconstituting serum F8 activity in a mouse model of hemophilia A after hydrodynamic injection of Cas9-sgAlb and B domain-deleted (BDD) F8 donor plasmids. We find that the integration of a double-cut donor at the Alb locus in mouse liver is mainly through non-homologous end joining (NHEJ)-mediated knock-in. We then target BDDF8 to multiple sites on introns 11 and 13 and find that NHEJ-mediated insertion of BDDF8 restores hemostasis. Finally, using 3 AAV8 vectors to deliver genome editing components, including Cas9, sgRNA, and BDDF8 donor, we observe the same therapeutic effects. A follow-up of 100 mice over 1 year shows no adverse effects. Conclusions These findings lay the foundation for curing hemophilia A by NHEJ knock-in of BDDF8 at Alb introns after AAV-mediated delivery of editing components.

Download Full-text

Structural basis for proficient oxidized ribonucleotide insertion in double strand break repair

Nature Communications ◽

10.1038/s41467-021-24486-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Joonas A. Jamsen ◽

Akira Sassa ◽

Lalith Perera ◽

David D. Shock ◽

William A. Beard ◽

...

Keyword(s):

Mammalian Cells ◽

Time Lapse ◽

Strand Break ◽

Structural Basis ◽

End Joining ◽

Strand Breaks ◽

Nucleotide Pools ◽

Nucleotide Insertion ◽

Non Homologous End Joining

AbstractReactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs). Non-homologous end-joining (NHEJ) attaches broken chromosomal ends together in mammalian cells. Ribonucleotide insertion by DNA polymerase (pol) μ prepares breaks for end-joining and this is required for successful NHEJ in vivo. We previously showed that pol μ lacks discrimination against oxidized dGTP (8-oxo-dGTP), that can lead to mutagenesis, cancer, aging and human disease. Here we reveal the structural basis for proficient oxidized ribonucleotide (8-oxo-rGTP) incorporation during DSB repair by pol μ. Time-lapse crystallography snapshots of structural intermediates during nucleotide insertion along with computational simulations reveal substrate, metal and side chain dynamics, that allow oxidized ribonucleotides to escape polymerase discrimination checkpoints. Abundant nucleotide pools, combined with inefficient sanitization and repair, implicate pol μ mediated oxidized ribonucleotide insertion as an emerging source of widespread persistent mutagenesis and genomic instability.

Download Full-text

Non-homologous end joining pathway of the human pathogen Cryptococcus neoformans influences homologous integration efficiency but not virulence

Mycoscience ◽

10.1007/s10267-010-0038-7 ◽

2010 ◽

Vol 51 (4) ◽

pp. 272-280 ◽

Cited By ~ 8

Author(s):

Hao-Man Li ◽

Eric V. Virtudazo ◽

Akio Toh-e ◽

Masashi Yamaguchi ◽

Susumu Kawamoto ◽

...

Keyword(s):

Cryptococcus Neoformans ◽

Human Pathogen ◽

End Joining ◽

Homologous Integration ◽

Non Homologous End Joining ◽

Integration Efficiency

Download Full-text

Assessment of choriocapillary blood flow changes in response to half-dose PDT in chronic central serous chorioretinopathy using OCTA

10.21203/rs.2.18778/v5 ◽

2020 ◽

Author(s):

Juejun Liu ◽

Changzheng Chen ◽

Lu Li ◽

Yishuang Xu ◽

Zuohuizi Yi ◽

...

Keyword(s):

Blood Flow ◽

Central Serous Chorioretinopathy ◽

Early Stage ◽

Early Response ◽

Therapeutic Effects ◽

Chronic Central Serous Chorioretinopathy ◽

Half Dose ◽

Flow Changes

Abstract Background: Optical coherence tomography angiography (OCTA) is a principally new imaging technique that provide quantitative method to analyze choriocapillaris (CC) flow changes, while assessment of CC in vivo could be valuable in understanding the pathological mechanism of chronic central serous chorioretinopathy (CCSC) and the therapeutic effects of photodynamic therapy (PDT). In this study, we sought to quantify blood flow changes in CC of CCSC patients receiving half-dose PDT using OCTA.Methods: A total of 28 affected eyes and 24 unaffected eyes of 26 CCSC patients receiving half-dose PDT, and 40 eyes of 20 healthy gender- and age-matched subjects were retrospectively enrolled. The proportion of total areas of flow signal voids (FSV, %) in CC level of OCTA was assessed in both eyes of the CCSC patients at baseline and repeated in multiple sections at 1-week, 1-month, 3-month and 6-month intervals after PDT. In addition, CC patterns in response to PDT at early stage and their subsequent morphologic changes were qualitatively documented using OCTA. Results: For affected eyes, significant decrease in FSV was found at 6-m follow-up when compared with that at 1-m follow-up (p=0.036). When compared to normal control eyes, FSV in affected eyes was significantly higher at 1-m, 3-m and 6-m follow-up (p<0.05 for all), while FSV in unaffected eyes was significantly higher at baseline, 1-w, 1-m and 3-m follow-up (p<0.05 for all). Three CC patterns of early response to PDT were identified, including signs of recovery with more even flow signals, transient appearance of worse ischemia and secondary neovascularization within CC level.Conclusion: Abnormal CC flow attenuation remains in completely resolved eyes of CCSC patients treated with half-dose PDT.

Download Full-text

The anti-von Willebrand factor aptamer ARC1779 increases von Willebrand factor levels and platelet counts in patients with type 2B von Willebrand disease

Thrombosis and Haemostasis ◽

10.1160/th11-12-0889 ◽

2012 ◽

Vol 108 (08) ◽

pp. 284-290 ◽

Cited By ~ 40

Author(s):

Petra Jilma-Stohlawetz ◽

Paul Knöbl ◽

James C. Gilbert ◽

Bernd Jilma

Keyword(s):

Von Willebrand Factor ◽

Plasma Concentrations ◽

Fold Increase ◽

Von Willebrand Disease ◽

Platelet Counts ◽

Von Willebrand ◽

Willebrand Factor ◽

Ristocetin Cofactor

SummaryBlockade of hyperactive von Willebrand factor (VWF) by ARC1779 blunted the platelet drop induced by desmopressin in patients with type 2B von Willebrand disease (VWD). Thus, we hypothesised that ARC1779 may increase VWF levels and correct thrombocytopenia. Three thrombocytopenic patients suffering from type 2B VWD received a loading dose of 0.23 mg/kg ARC1779 followed by 4 μg/kg/min intravenously for 72 hours in a prospective clinical trial. ARC1779 was well tolerated and safe. Plasma concentrations of ARC1779 increased to 76 μg/ml (59–130) leading to an immediate decrease of free VWF A1 domains. VWF/FVIII levels increased as early as 12 h after start of infusion, peaked near the end of infusion, and returned to baseline at follow-up. VWF ristocetin cofactor activity (VWF:RCo) showed a median 10-fold increase 8 hours after end of infusion, while the median VWF-antigen and FVIII increase was less (5-fold and 4-fold, respectively). Most importantly inhibition of hyperactive VWF rapidly increased platelet counts from 40x109/l (38–58 x109/l) to a maximum of 146 x109/l (107–248 x109/l). In conclusion, ARC1779 markedly increases VWF/FVIII levels and most importantly improves or even corrects thrombocytopenia in VWD type 2B patients. This underscores the in vivo potency of ARC1779.

Download Full-text

Restoration of RPGR expression in vivo using CRISPR/Cas9 gene editing

Gene Therapy ◽

10.1038/s41434-021-00258-6 ◽

2021 ◽

Author(s):

Jessica D. Gumerson ◽

Amal Alsufyani ◽

Wenhan Yu ◽

Jingqi Lei ◽

Xun Sun ◽

...

Keyword(s):

Expression Vectors ◽

End Joining ◽

Reading Frame ◽

Guide Rna ◽

Domain Specific ◽

Somatic Gene ◽

Non Homologous End Joining ◽

Cone Opsins ◽

Inherited Retinal Degeneration

AbstractMutations in the gene for Retinitis Pigmentosa GTPase Regulator (RPGR) cause the X-linked form of inherited retinal degeneration, and the majority are frameshift mutations in a highly repetitive, purine-rich region of RPGR known as the OFR15 exon. Truncation of the reading frame in this terminal exon ablates the functionally important C-terminal domain. We hypothesized that targeted excision in ORF15 by CRISPR/Cas9 and the ensuing repair by non-homologous end joining could restore RPGR reading frame in a portion of mutant photoreceptors thereby correcting gene function in vivo. We tested this hypothesis in the rd9 mouse, a naturally occurring mutant line that carries a frameshift mutation in RPGRORF15, through a combination of germline and somatic gene therapy approaches. In germline gene-edited rd9 mice, probing with RPGR domain-specific antibodies demonstrated expression of full length RPGRORF15 protein. Hallmark features of RPGR mutation-associated early disease phenotypes, such as mislocalization of cone opsins, were no longer present. Subretinal injections of the same guide RNA (sgRNA) carried in AAV sgRNA and SpCas9 expression vectors restored reading frame of RPGRORF15 in a subpopulation of cells with broad distribution throughout the retina, confirming successful correction of the mutation. These data suggest that a simplified form of genome editing mediated by CRISPR, as described here, could be further developed to repair RPGRORF15 mutations in vivo.

Download Full-text

Autophosphorylation and Self-Activation of DNA-Dependent Protein Kinase

Genes ◽

10.3390/genes12071091 ◽

2021 ◽

Vol 12 (7) ◽

pp. 1091

Author(s):

Aya Kurosawa

Keyword(s):

Protein Kinase ◽

Conformational Changes ◽

Strand Break ◽

In Vivo Studies ◽

End Joining ◽

Dependent Protein Kinase ◽

Dependent Protein ◽

Non Homologous End Joining

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a member of the phosphatidylinositol 3-kinase-related kinase family, phosphorylates serine and threonine residues of substrate proteins in the presence of the Ku complex and double-stranded DNA. Although it has been established that DNA-PKcs is involved in non-homologous end-joining, a DNA double-strand break repair pathway, the mechanisms underlying DNA-PKcs activation are not fully understood. Nevertheless, the findings of numerous in vitro and in vivo studies have indicated that DNA-PKcs contains two autophosphorylation clusters, PQR and ABCDE, as well as several autophosphorylation sites and conformational changes associated with autophosphorylation of DNA-PKcs are important for self-activation. Consistent with these features, an analysis of transgenic mice has shown that the phenotypes of DNA-PKcs autophosphorylation mutations are significantly different from those of DNA-PKcs kinase-dead mutations, thereby indicating the importance of DNA-PKcs autophosphorylation in differentiation and development. Furthermore, there has been notable progress in the high-resolution analysis of the conformation of DNA-PKcs, which has enabled us to gain a visual insight into the steps leading to DNA-PKcs activation. This review summarizes the current progress in the activation of DNA-PKcs, focusing in particular on autophosphorylation of this kinase.

Download Full-text

Mono-homologous linear DNA recombination by the non-homologous end-joining pathway as a novel and simple gene inactivation method: a proof of concept study inDietziasp. DQ12-45-1b

10.1101/300723 ◽

2018 ◽

Author(s):

Shelian Lu ◽

Yong Nie ◽

Meng Wang ◽

Hong-Xiu Xu ◽

Dong-Ling Ma ◽

...

Keyword(s):

Genetic Manipulation ◽

Dna Recombination ◽

Proof Of Concept ◽

End Joining ◽

Circular Dna ◽

Linear Dna ◽

Genes Encoding ◽

Non Homologous End Joining ◽

Nhej Activity

ABSTRACTNon-homologous end-joining (NHEJ) is critical for genome stability because of its roles in double-strand break repair. Ku and ligase D (LigD) are the crucial proteins in this process, and strains expressing Ku and LigD can cyclize linear DNAin vivo.Herein, we established a proof-of-concept mono-homologous linear DNA recombination for gene inactivation or genome editing by which cyclization of linear DNAin vivoby NHEJ could be used to generate non-replicable circular DNA and could allow allelic exchanges between the circular DNA and the chromosome. We achieved this approach inDietziasp. DQ12-45-1b, which expresses Ku and LigD homologs and presents NHEJ activity. By transforming the strain with a linear DNA mono homolog to the sequence in chromosome, we mutated the genome. This method did not require the screening of suitable plasmids and was easy and time-effective. Bioinformatic analysis showed that more than 20% prokaryotic organisms contain Ku and LigD, suggesting the wide distribution of NHEJ activities. Moreover, theEscherichia colistrain also showed NHEJ activity when the Ku and LigD ofDietziasp. DQ12-45-1b were introduced and expressed in it. Therefore, this method may be a widely applicable genome editing tool for diverse prokaryotic organisms, especially for non-model microorganisms.IMPORTANCEThe non-model gram-positive bacteria lack efficient genetic manipulation systems, but they express genes encoding Ku and LigD. The NHEJ pathway inDietziasp. DQ12-45-1b was evaluated and was used to successfully knockout eleven genes in the genome. Since bioinformatic studies revealed that the putative genes encoding Ku and LigD ubiquitously exist in phylogenetically diverse bacteria and archaea, the mono-homologous linear DNA recombination by the NHEJ pathway could be a potentially applicable genetic manipulation method for diverse non-model prokaryotic organisms.

Download Full-text

Therapeutic effects of topically-administered guar gum nanoparticles in oxazolone-induced atopic dermatitis in mice

Biomedical Research and Therapy ◽

10.15419/bmrat.v5i5.444 ◽

2018 ◽

Vol 5 (5) ◽

pp. 2305-2325 ◽

Cited By ~ 1

Author(s):

Nandita Ghosh ◽

Shinjini Mitra ◽

Ena Ray Banerjee

Keyword(s):

Atopic Dermatitis ◽

Guar Gum ◽

Fold Increase ◽

Therapeutic Effects ◽

Cyamopsis Tetragonoloba ◽

Epidermal Thickness ◽

In Vivo Experiments ◽

Macrophage Populations

Introduction: Atopic dermatitis (AD) is a chronic disease of the skin, involving itchy, reddish and scaly lesions. It mainly affects children and has a high prevalence in developing countries. AD may occur due to environmental or genetic factors. Currently, all therapeutic strategies involve methods to simply alleviate the symptoms, and include lotions and corticosteroids, which have adverse effects. Use of phytochemicals and natural products has not yet been exploited fully. The particle used in this study is derived from Cyamopsis tetragonoloba, an edible polysaccharide with a galactomannan component. The mannose component mainly increases its specificity towards cellular uptake by mannose receptors, highly expressed by macrophages. The aim of this study was to determine the therapeutic effect of guar gum nanoparticles (GN) in vitro and in vivo in AD. Methods: To assess the wound healing capacity of GN, we first treated adherent fibroblast cells, with a scratch injury, with GN. GN successfully healed the wound caused by the scratch. In the in vivo experiments, Balb/c mice ears were treated topically with oxazolone (Oxa) to induce AD, and then were topically treated with GN. The ear thickness increased significantly until day 28 upon treatment with Oxa. Results: Application of GN showed a significant decrease in ear thickness as assessed on day 28. The total cell count of skin cells that showed a fold increase, when treated with Oxa, was again decreased after topical application of GN on the affected skin. The eosinophil count, as assessed by Giemsa staining, was also increased when treated with Oxa, while GN application led to a significant decrease. Serum IgE levels were restored by GN. T helper cell and macrophage populations, when examined by flow cytometry, showed an increase in percentage when treated with Oxa; the percentage was reduced after application of GN. Hematoxylin & Eosin (H&E) staining of the ear tissue showed an increase in epidermal thickness in Oxa-treated mice, while GN application showed reduced cellular infiltration and epidermal thickness. Conclusion: Overall, our results showed that GN, when administered topically, was successful in alleviating dermatitis caused by Oxa.

Download Full-text

Leaky severe combined immunodeficiency in mice lacking non-homologous end joining factors XLF and MRI

10.1101/2020.03.04.976829 ◽

2020 ◽

Author(s):

Sergio Castañeda-Zegarra ◽

Qindong Zhang ◽

Amin Alirezaylavasani ◽

Marion Fernandez-Berrocal ◽

Rouan Yao ◽

...

Keyword(s):

Severe Combined Immunodeficiency ◽

Embryonic Lethality ◽

Combined Immunodeficiency ◽

End Joining ◽

Mild Phenotype ◽

Reduced Body ◽

Non Homologous End Joining ◽

Mature Lymphocyte ◽

And Function

AbstractNon-homologous end-joining (NHEJ) is a DNA repair pathway required to detect, process, and ligate DNA double-stranded breaks (DSBs) throughout the cell cycle. The NHEJ pathway is necessary for V(D)J recombination in developing B and T lymphocytes. During NHEJ, Ku70 and Ku80 form a heterodimer that recognizes DSBs and promotes recruitment and function of downstream factors PAXX, MRI, DNA-PKcs, Artemis, XLF, XRCC4, and LIG4. Mutations in several known NHEJ genes result in severe combined immunodeficiency (SCID). Inactivation of Mri, Paxx or Xlf in mice results in normal or mild phenotype, while combined inactivation of Xlf/Mri, Xlf/Paxx, or Xlf/Dna-pkcs leads to late embryonic lethality. Here, we describe three new mouse models. We demonstrate that deletion of Trp53 rescues embryonic lethality in mice with combined deficiencies of Xlf and Mri. Furthermore, Xlf-/-Mri-/-Trp53+/- and Xlf-/-Paxx-/-Trp53+/- mice possess reduced body weight, severely reduced mature lymphocyte counts, and accumulation of progenitor B cells. We also report that combined inactivation of Mri/Paxx results in live-born mice with modest phenotype, and combined inactivation of Mri/Dna-pkcs results in embryonic lethality. Therefore, we conclude that XLF is functionally redundant with MRI and PAXX during lymphocyte development in vivo. Moreover, Mri genetically interacts with Dna-pkcs and Paxx.

Download Full-text

Linear-double-stranded DNA (ldsDNA) based AND logic computation in mammalian cells

10.1101/266056 ◽

2018 ◽

Author(s):

Weijun Su ◽

Chunze Zhang ◽

Shuai Li

Keyword(s):

Synthetic Biology ◽

Mammalian Cells ◽

Rapid Development ◽

Genetic Circuits ◽

End Joining ◽

Double Stranded Dna ◽

Logic Computation ◽

Key Enzyme ◽

Non Homologous End Joining

AbstractSynthetic biology employs engineering principles to redesign biological system for clinical or industrial purposes. The development and application of novel genetic devices for genetic circuits construction will facilitate the rapid development of synthetic biology. Here we demonstrate that mammalian cells could perform two- and three-input linear-double-stranded DNA (ldsDNA) based Boolean AND logic computation. Through hydrodynamic ldsDNA delivery, two-input ldsDNA-base AND-gate computation could be achieved in vivo. Inhibition of DNA-PKcs expression, a key enzyme in non-homologous end joining (NHEJ), could significantly downregulate the intensity of output signals from ldsDNA-based AND-gate. We further reveal that in mammalian cells ldsDNAs could undergo end processing and then perform AND-gate calculation to generate in-frame output proteins. Moreover, we show that ldsDNAs or plasmids with identical overlapping sequences could also serve as inputs of AND-gate computation. Our work establishes novel genetic devices and principles for genetic circuits construction, thus may open a new gate for the development of new disease targeting strategies and new protein genesis methodologies.

Download Full-text