Seamless gene correction of beta-thalassemia mutations in patient-specific cells

(Press-News.org) August 5, 2014 – A major hurdle in gene therapy is the efficient integration of a corrected gene into a patient's genome without mutating off-target sites. In a paper published today in Genome Research, scientists have used CRISPR/Cas genome editing technology to seamlessly and efficiently correct disease-causing mutations in cells from patients with β-thalassemia.

β-thalassemia results from inherited DNA mutations in the hemoglobin beta (HBB) gene, resulting in reduced HBB expression in red blood cells and, in the most severe forms, anemia. The only established curative treatment is hematopoietic stem cell transplantation; however, this treatment requires a matched donor. Gene therapy, which delivers a corrected copy of a gene into patient cells, could bypass the need for a donor. Previous attempts using a virus to randomly insert a normal gene into the genome has been successful in one β-thalassemia patient, but the long-term effect of viral insertion is not yet known.

To correct HBB mutations directly in a patient's genome, researchers first generated induced pluripotent stem cells, or iPSCs, from skin cells of patients. The real breakthrough came when they applied CRISPR/Cas9 to precisely engineer a double strand DNA break at the HBB locus in these cells, allowing a donor plasmid with the corrected sites to be efficiently integrated, thus replacing the mutated sites. The donor plasmid also contained selectable markers to identify cells with corrected copies of the gene. These selectable markers were subsequently removed with transposase and a second round of selection, generating a seamless, corrected version of HBB in the patient's genome.

Importantly, the researchers could differentiate the corrected iPSCs into mature blood cells, and these blood cells showed restored expression of hemoglobin. However, much work is needed before these cells could be transplanted back into a patient for treating β-thalassemia. "Although we and others are able to differentiate iPSCs into blood cell progenitors as well as mature blood cells, the transplantation of the progenitors into mouse models to test them has so far proven very difficult," said senior author Yuet Wai Kan from the University of California, San Francisco. "I believe it will take quite a few more years before we can apply it in a clinical setting."

INFORMATION: Scientists from the University of California, San Francisco and the Blood Systems Research Institute contributed to this study.

This study was funded by the National Institutes of Health.

Media contacts: The authors are available for more information by contacting: Kristen Bole, University Relations, University of California, San Francisco (kristen.bole@ucsf.edu, +1-415-476-2743)

Interested reporters may obtain copies of the manuscript via email from Peggy Calicchia, Administrative Assistant, Genome Research (calicchi@cshl.edu, +1-516-422-4012).

About the article: The manuscript will be published online ahead of print on 5 August 2014. Its full citation is as follows: Xie F, Ye L, Chang JC, Beyer AI, Wang J, Muench MO, Kan YW. 2014. Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac. Genome Res doi: 10.1101/gr.173427.114

About Genome Research: Launched in 1995, Genome Research is an international, continuously published, peer-reviewed journal that focuses on research that provides novel insights into the genome biology of all organisms, including advances in genomic medicine. Among the topics considered by the journal are genome structure and function, comparative genomics, molecular evolution, genome-scale quantitative and population genetics, proteomics, epigenomics, and systems biology. The journal also features exciting gene discoveries and reports of cutting-edge computational biology and high-throughput methodologies.

About Cold Spring Harbor Laboratory Press: Cold Spring Harbor Laboratory Press is an internationally renowned publisher of books, journals, and electronic media, located on Long Island, New York. Since 1933, it has furthered the advance and spread of scientific knowledge in all areas of genetics and molecular biology, including cancer biology, plant science, bioinformatics, and neurobiology. The Press is a division of Cold Spring Harbor Laboratory, an innovator in life science research and the education of scientists, students, and the public. For more information, visit our website at http://cshlpress.org/

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