CRISPR + stem cells = gene therapy

CRISPR, the gene editing tool borrowed from bacteria, has been used to prevent muscular dystrophy in mouse embryos and treat liver disease in adult mice. Now it has also been used to treat stem cells from human muscular dystrophy patients. And this might be the key to making CRISPR gene therapy a viable treatment for human disease.

CRISPR is currently injected into animals. This is easy to do in embryos – especially at the single cell stage. However, it is much harder in adult animals. The first key problem is targeting the CRISPR ingredients to the right organ. The second is makeing sure that CRISPR doesn’t cause off target mutations in parts of the genome other than the disease-causing gene. Using CRISPR to treat stem cells instead of organs can avoid both these problems.

A team of scientists from Kyoto University recently took skin fibroblast cells from a patient with muscular dystrophy and turned them into induced pluripotent stem cells (iPSCs). Here’s a cool video on iPSCs:

The iPSCs from the patient all had a mutation in the dystrophin gene. The scientists used CRISPR or TALEN (Transcription activator-like effector nuclease) gene editing tools to correct the dystrophin mutation in the iPSCs.

They then tested the treated cells to see whether the gene editing treatment caused mutations in genes other than dystrophin. They did see evidence of slightly more mutations in the treated cells, compared with untreated cells. So they chose the cells with the least damage and turned these into skeletal muscle cells. They then sequenced the DNA in these cells to see whether they had corrected the dystrophin mutation.

They also stained the cells with a dystrophin antibody to see where the dystrophin was in the treated cells and did a western blot to detect how much normal dystrophin was in treated and untreated cells. There was no normal dystrophin DNA or protein in untreated cells. There was normal dystrophin DNA and normal full length protein or nearly full length protein in 50-90% of treated cells. And importantly the dystrophin was in the right place in the cell. They found that both gene editing technologies, CRISPR and TALEN, were equally effective in correcting the dystrophin mutation.

The most valuable part of using CRISPR on stem cells is that the cells can be easily screened to make sure that the disease-causing mutation has been corrected, and that no off target mutations have been caused, before the cells are used to treat the patient.

But iPSCs have still not been injected back into human patients for fear of causing cancer. They have been injected into monkeys and didn’t cause cancer if they were first differentiated into a particular type of cell, rather than left as stem cells that could become any cell (are still pluripotent). So hopefully gene editing stem cells works in human patients.

The Scientific Papers (all free and open access!):

Li et al. Precise Correction of the Dystrophin Gene in Duchenne Muscular Dystrophy
Patient Induced Pluripotent Stem Cells by TALEN and CRISPR-Cas9. Stem Cell Reports. 2015.

Takahashi et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007

Tanaka et al. Efficient and reproducible myogenic differentiation from human iPS cells: prospects for modeling Miyoshi Myopathy in vitro. PLoS ONE 2014.

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