Correction to: Rescue of premature aging defects in Cockayne syndrome stem cells by CRISPR/Cas9-mediated gene correction

Cockayne Syndrome (CS) is an autosomal recessive neurodegenerative premature aging disorder associated with defects in nucleotide excision repair (NER). Cells from CS patients, with mutations in CSA or CSB genes, present elevated levels of reactive oxygen species (ROS) and are defective in the repair of a variety of oxidatively generated DNA lesions. In this study, six purine lesions were ascertained in wild type (wt) CSA, defective CSA, wtCSB and defective CSB-transformed fibroblasts under different oxygen tensions (hyperoxic 21%, physioxic 5% and hypoxic 1%). In particular, the four 5′,8-cyclopurine (cPu) and the two 8-oxo-purine (8-oxo-Pu) lesions were accurately quantified by LC-MS/MS analysis using isotopomeric internal standards after an enzymatic digestion procedure. cPu levels were found comparable to 8-oxo-Pu in all cases (3–6 lesions/106 nucleotides), slightly increasing on going from hyperoxia to physioxia to hypoxia. Moreover, higher levels of four cPu were observed under hypoxia in both CSA and CSB-defective cells as compared to normal counterparts, along with a significant enhancement of 8-oxo-Pu. These findings revealed that exposure to different oxygen tensions induced oxidative DNA damage in CS cells, repairable by NER or base excision repair (BER) pathways. In NER-defective CS patients, these results support the hypothesis that the clinical neurological features might be connected to the accumulation of cPu. Moreover, the elimination of dysfunctional mitochondria in CS cells is associated with a reduction in the oxidative DNA damage.

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Hematopoietic stem cell dysfunction underlies the progressive lymphocytopenia in XLF/Cernunnos deficiency

Blood ◽

10.1182/blood-2014-05-574863 ◽

2014 ◽

Vol 124 (10) ◽

pp. 1622-1625 ◽

Cited By ~ 11

Author(s):

Serine Avagyan ◽

Michael Churchill ◽

Kenta Yamamoto ◽

Jennifer L. Crowe ◽

Chen Li ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem Cell ◽

Premature Aging ◽

Hematopoietic Stem ◽

Cell Dysfunction ◽

Key Points ◽

Deficient Mice

Key Points XLF-deficient mice recapitulate the lymphocytopenia of XLF-deficient patients. Premature aging of hematopoietic stem cells underlies the severe and progressive lymphocytopenia in XLF-deficient mice.

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p38a protects hematopoietic stem cells from physiological and premature aging stresses

Experimental Hematology ◽

10.1016/j.exphem.2017.06.116 ◽

2017 ◽

Vol 53 ◽

pp. S64

Author(s):

Keiyo Takubo ◽

Yukako Ootomo ◽

Daiki Karigane

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Premature Aging ◽

Hematopoietic Stem

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Detection and Isolation of Gene-Corrected Cells in Gaucher Disease Via a Fluorescence-Activated Cell Sorter Assay for Lysosomal Glucocerebrosidase Activity

Blood ◽

10.1182/blood.v89.9.3412 ◽

1997 ◽

Vol 89 (9) ◽

pp. 3412-3420 ◽

Cited By ~ 24

Author(s):

Matthew Lorincz ◽

Leonard A. Herzenberg ◽

Zhenjun Diwu ◽

John A. Barranger ◽

William G. Kerr

Keyword(s):

Stem Cells ◽

Gene Transfer ◽

Gaucher Disease ◽

Mononuclear Cells ◽

Disease Type ◽

Gene Correction ◽

Cell Sorter ◽

Hematopoietic Stem ◽

Gaucher Disease Type 1

Abstract Gaucher disease type 1 results from the accumulation of glucocerebroside in macrophages of the reticuloendothelial system, as a consequence of a deficiency in glucocerebrosidase (GC) activity. Recent improvements in the methodologies for introducing foreign genes into bone marrow stem cells have prompted several groups to test the efficacy of gene transfer therapy as a curative treatment for Gaucher disease. Limitations of this approach include the potential for insufficient engraftment of gene-corrected cells and incomplete transduction of hematopoietic stem cells using retroviral gene transfer. Overcoming these obstacles may be critical in the case of treatment for Gaucher disease type 1, because GC transduced cells have not been shown to have a growth advantage over noncorrected cells. Here, we describe the development and application of a novel, fluorescence-activated cell sorter based assay that directly quantitates GC activity at the single cell level. In a test of this application, fibroblasts from a Gaucher patient were transduced, and high expressing cells sorted based on GC activity. Reanalysis of cultured sorted fibroblasts reveals that these cells maintain high levels of enzymatic activity, compared with the heterogeneous population from which they were sorted. The assay is sufficiently sensitive to distinguish GC activity found in Gaucher patient monocytes from that in normal controls. Furthermore, preliminary results indicate that increased GC activity can be detected in transduced, CD34+ enriched peripheral blood mononuclear cells isolated from a Gaucher patient. This method should be a useful addition to current gene therapy protocols as a means to quantitatively assess gene correction of relevant cell populations and potentially purify transduced cells for transplantation.

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Naïve Induced Pluripotent Stem Cells Generated From β-Thalassemia Fibroblasts Allow Efficient Gene Correction With CRISPR/Cas9

Stem Cells Translational Medicine ◽

10.5966/sctm.2015-0157 ◽

2015 ◽

Vol 5 (1) ◽

pp. 8-19 ◽

Cited By ~ 32

Author(s):

Yuanyuan Yang ◽

Xiaobai Zhang ◽

Li Yi ◽

Zhenzhen Hou ◽

Jiayu Chen ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Gene Correction ◽

Efficient Gene ◽

Induced Pluripotent

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The ATPase Domain but Not the Acidic Region of Cockayne Syndrome Group B Gene Product Is Essential for DNA Repair

Molecular Biology of the Cell ◽

10.1091/mbc.10.11.3583 ◽

1999 ◽

Vol 10 (11) ◽

pp. 3583-3594 ◽

Cited By ~ 30

Author(s):

Robert M. Brosh ◽

Adayabalam S. Balajee ◽

Rebecca R. Selzer ◽

Morten Sunesen ◽

Luca Proietti De Santis ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Excision Repair ◽

Uv Light ◽

Genetic Disorder ◽

Cockayne Syndrome ◽

Premature Aging ◽

Atpase Domain ◽

Acidic Region

Cockayne syndrome (CS) is a human genetic disorder characterized by UV sensitivity, developmental abnormalities, and premature aging. Two of the genes involved, CSA andCSB, are required for transcription-coupled repair (TCR), a subpathway of nucleotide excision repair that removes certain lesions rapidly and efficiently from the transcribed strand of active genes. CS proteins have also been implicated in the recovery of transcription after certain types of DNA damage such as those lesions induced by UV light. In this study, site-directed mutations have been introduced to the human CSB gene to investigate the functional significance of the conserved ATPase domain and of a highly acidic region of the protein. The CSB mutant alleles were tested for genetic complementation of UV-sensitive phenotypes in the human CS-B homologue of hamster UV61. In addition, theCSB mutant alleles were tested for their ability to complement the sensitivity of UV61 cells to the carcinogen 4-nitroquinoline-1-oxide (4-NQO), which introduces bulky DNA adducts repaired by global genome repair. Point mutation of a highly conserved glutamic acid residue in ATPase motif II abolished the ability of CSB protein to complement the UV-sensitive phenotypes of survival, RNA synthesis recovery, and gene-specific repair. These data indicate that the integrity of the ATPase domain is critical for CSB function in vivo. Likewise, the CSB ATPase point mutant failed to confer cellular resistance to 4-NQO, suggesting that ATP hydrolysis is required for CSB function in a TCR-independent pathway. On the contrary, a large deletion of the acidic region of CSB protein did not impair the genetic function in the processing of either UV- or 4-NQO-induced DNA damage. Thus the acidic region of CSB is likely to be dispensable for DNA repair, whereas the ATPase domain is essential for CSB function in both TCR-dependent and -independent pathways.

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Long targeting arms do not increase the efficiency of homologous recombination in the β-globin locus of murine embryonic stem cells

Blood ◽

10.1182/blood-2003-03-0708 ◽

2003 ◽

Vol 102 (4) ◽

pp. 1531-1533 ◽

Cited By ~ 12

Author(s):

Zhi Hong Lu ◽

Jason T. Books ◽

Richard M. Kaufman ◽

Timothy J. Ley

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Homologous Recombination ◽

Globin Gene ◽

Embryonic Stem ◽

Regulatory Sequences ◽

Globin Genes ◽

Gene Correction ◽

Murine Embryonic Stem Cells ◽

Alternative Approaches

Abstract The correction of mutant β-globin genes has long been a therapeutic goal for patients with β-thalassemia or hemoglobinopathies. The use of homologous recombination (HR) to achieve this goal is an attractive approach because it eliminates the need to include regulatory sequences in the therapeutic construct, and it eliminates mutagenesis induced by random integration. However, HR is a very inefficient process for gene correction, and its efficiency is probably locus dependent. The length of targeting arms is thought to be a determinant of targeting efficiency, so we compared the ability of standard (8-kb) versus very long (16-, 24-, and 110-kb) regions of homology to correct a mutant murine β-globin gene in embryonic stem cells. Increasing the length of the targeting sequences did not increase the efficiency of HR in this locus, suggesting that alternative approaches will be required to improve the efficiency of this approach for globin gene correction.

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