The ISSCR Issues Statement on Human Germ Line Genome Modification

The International Society for Stem Cell Research has released a statement calling for a moratorium on attempts to apply nuclear genome editing of the human germ line in clinical practice

Sunday, 22 March 2015

In a statement released on Thursday, the International Society for Stem Cell Research called for a moratorium on attempts at clinical application of nuclear genome editing of the human germ line to enable more extensive scientific analysis of the potential risks of genome editing and broader public discussion of the societal and ethical implications.

Technologies used to introduce changes into the DNA sequence of cells have advanced rapidly, making genome editing increasingly simple. Genome editing is feasible, not just in the somatic cells of an adult organism, but also in early embryos, as well as the gametes (sperm and egg) that carry the inheritable, germ line DNA. Research involving germ line nuclear genome editing has been performed to date in many organisms, including mice and monkeys, and applications to human embryos are possible.

The ISSCR statement raises significant ethical, societal and safety considerations related to the application of nuclear genome editing to the human germ line in clinical practice. Current genome editing technologies carry risks of unintended genome damage, in addition to unknown consequences. Moreover, consensus is lacking on what, if any, therapeutic applications of germ line genome modification might be permissible.

The statement calls for a moratorium on attempts to apply nuclear genome editing of the human germ line in clinical practice, as scientists currently lack an adequate understanding of the safety and potential long term risks of germ line genome modification. Moreover, the ISSCR asserts that a deeper and more rigorous deliberation on the ethical, legal and societal implications of any attempts at modifying the human germ line is essential if its clinical practice is ever to be sanctioned.

In calling for the above moratorium, the ISSCR is not taking a position on the clinical testing of mitochondrial replacement therapy, a form of germ line modification that entails replacing the mitochondria (found outside the nucleus) in the eggs of women at risk of transmitting certain devastating diseases to their children.

Source: International Society for Stem Cell Research

Contact: Michelle Quivey

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Newer Genome Editing Tool Shows Promise in Engineering Human Stem Cells

Johns Hopkins study could advance use of stem cells for treatment and disease research

Tuesday, 06 January 2015

A powerful “genome editing” technology known as CRISPR has been used by researchers since 2012 to trim, disrupt, replace or add to sequences of an organism’s DNA. Now, scientists at Johns Hopkins Medicine have shown that the system also precisely and efficiently alters human stem cells.

In a recent online report on the work in Molecular Therapy, the Johns Hopkins team says the findings could streamline and speed efforts to modify and tailor human-induced pluripotent stem cells (iPSCs) for use as treatments or in the development of model systems to study diseases and test drugs.

“Stem cell technology is quickly advancing, and we think that the days when we can use iPSCs for human therapy aren’t that far away,” says Zhaohui Ye, Ph.D., an instructor of medicine at the Johns Hopkins University School of Medicine.

“This is one of the first studies to detail the use of CRISPR in human iPSCs, showcasing its potential in these cells.”

CRISPR originated from a microbial immune system that contains DNA segments known as clustered regularly interspaced short palindromic repeats. The engineered editing system makes use of an enzyme that nicks together DNA with a piece of small RNA that guides the tool to where researchers want to introduce cuts or other changes in the genome.

Previous research has shown that CRISPR can generate genomic changes or mutations through these interventions far more efficiently than other gene editing techniques, such as TALEN, short for transcription activator-like effector nuclease.

Despite CRISPR’s advantages, a recent study suggested that it might also produce a large number of “off-target” effects in human cancer cell lines, specifically modification of genes that researchers didn’t mean to change.

To see if this unwanted effect occurred in other human cell types, Ye, Linzhao Cheng, Ph.D., a professor of medicine and oncology in the Johns Hopkins University School of Medicine; and their colleagues pitted CRISPR against TALEN in human iPSCs, adult cells reprogrammed to act like embryonic stem cells. Human iPSCs have already shown enormous promise for treating and studying disease.

The researchers compared the ability of both genome editing systems to either cut out pieces of known genes in iPSCs or cut out a piece of these genes and replace it with another. As model genes, the researchers used JAK2, a gene that when mutated causes a bone marrow disorder known as polycythemia vera; SERPINA1, a gene that when mutated causes alpha1-antitrypsin deficiency, an inherited disorder that may cause lung and liver disease; and AAVS1, a gene that’s been recently discovered to be a “safe harbour” in the human genome for inserting foreign genes.

Their comparison found that when simply cutting out portions of genes, the CRISPR system was significantly more efficient than TALEN in all three gene systems, inducing up to 100 times more cuts. However, when using these genome editing tools for replacing portions of the genes, such as the disease-causing mutations in JAK2 and SERPINA1 genes, CRISPR and TALEN showed about the same efficiency in patient-derived iPSCs, the researchers report.

Contrary to results of the human cancer cell line study, both CRISPR and TALEN had the same targeting specificity in human iPSCs, hitting only the genes they were designed to affect, the team says. The researchers also found that the CRISPR system has an advantage over TALEN: It can be designed to target only the mutation-containing gene without affecting the healthy gene in patients, where only one copy of a gene is affected.

The findings, together with a related study that was published earlier in a leading journal of stem cell research (Cell Stem Cell), offer reassurance that CRISPR will be a useful tool for editing the genes of human iPSCs with little risk of off-target effects, say Ye and Cheng.

“CRISPR-mediated genome editing opens the door to many genetic applications in biologically relevant cells that can lead to better understanding of and potential cures for human diseases,” says Cheng.

Source: Johns Hopkins Medicine

Contact: Marin Hedin

Reference:

Efficient and Allele-Specific Genome Editing of Disease Loci in Human iPSCs

Cory Smith, Leire Abalde-Atristain, Chaoxia He, Brett R Brodsky, Evan M Braunstein, Pooja Chaudhari, Yoon-Young Jang, Linzhao Cheng and Zhaohui Ye

Molecular Therapy, December 16, 2014; doi:10.1038/mt.2014.226

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ZenMaster

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iPS Cells Used to Correct Genetic Mutations that Cause Muscular Dystrophy

iPS Cells Used to Correct Genetic Mutations that Cause Muscular Dystrophy

Thursday, 27 November 2014

This image shows immunofluorescence staining

of skeletal cells differentiated from DMD-iPS

cells. Untreated DMD skeletal cells do not

express dystrophin (green) due to the deletion of

exon 44. However, after any of the three

correction strategies are applied to iPS cells,

differentiation into skeletal cells results in normal

dystrophin expression. Scale bar, 50 μm.

Credit: Dr. Akitsu Hotta, Kyoto University.

Researchers at the Center for iPS Cell Research and Application (CiRA), Kyoto University, show that induced pluripotent stem (iPS) cells can be used to correct genetic mutations that cause Duchenne muscular dystrophy (DMD). The research, published in Stem Cell Reports, demonstrates how engineered nucleases, such as TALEN and CRISPR, can be used to edit the genome of iPS cells generated from the skin cells of a DMD patient. The cells were then differentiated into skeletal muscles, in which the mutation responsible for DMD had disappeared.

DMD is a severe muscular degenerative disease caused by a loss-of-function mutation in the dystrophin gene. It inflicts 1 in 3500 boys and normally leads to death by early adulthood. Currently, very little is available in terms of treatment for patients outside palliative care. One option gaining interest is genomic editing by TALEN and CRISPR, which have quickly become invaluable tools in molecular biology. These enzymes allow scientists to cleave genes at specific locations and then modify the remnants to produce a genomic sequence to their liking. However, programmable nucleases are not pristine and often mistakenly edit similar sequences that vary a few base pairs from the target sequence, making them unreliable for clinical use because of the potential for undesired mutations.

For this reason, induced pluripotent stem cells (iPS cells) are ideal models, because they provide researchers an abundance of patient cells on which to test the programmable nucleases and find optimal conditions that minimize off-target modifications. CiRA scientists took advantage of this feature by generating iPS cells from a DMD patient. They used several different TALEN and CRISPR to modify the genome of the iPS cells, which were then differentiated into skeletal muscle cells. In all cases, dystrophin protein expression was convalesced, and in some cases, the dystrophin gene was fully corrected.

One key to the success was the development of a computational protocol that minimized the risk of off-target editing. The team built a database that all possible permutations of sequences up to 16 base pairs long. Among these, they extracted those that only appear once in the human genome, i.e. unique sequences. DMD can be caused by several different mutations; in the case of the patient used in this study, it was the result of the deletion of exon 44. The researchers therefore built a histogram of unique sequences that appeared in a genomic region that contained this exon. They found a stack of unique sequences in exon 45.

to Akitsu Hotta, who headed the project and holds joint positions at CiRA and the Institute for Integrated Cell-Materials Sciences at Kyoto University:

"Nearly half the human genome consists of repeated sequences. So even if we found one unique sequence, a change of one or two base pairs may result in these other repeated sequences, which risks the TALEN or CRISPR editing an incorrect region. To avoid this problem, we sought a region that hit high in the histogram".

With this target, the team considered three strategies to modify the frame-shift mutation of the dystrophin gene: exon skipping by connecting exons 43 and 46 to restore the reading frame, frame shifting by incorporating insertion or deletion (indel) mutations, and exon knock-in by inserting exon 44 before exon 45. All three strategies effectively increased dystrophin synthesis in differentiated skeletal cells, but only the exon knock-in approach recovered the gene to its natural state. Importantly, editing showed very high specificity, suggesting that their computational approach can be used to minimize off-target editing by the programming nucleases.

Moreover, the paper provides a proof-of-principle for using iPS cell technology to treat DMD in combination with TALEN or CRISPR. The group now aims to expand this protocol to other diseases.

First author Lisa Li explains: "We show that TALEN and CRISPR can be used to correct the mutation of the DMD gene. I want to apply the nucleases to correct mutations for other genetic-based diseases like point mutations".

Source: Center for iPS Cell Research and Application - Kyoto University

Contact: Akemi Nakamura

Reference:

Precise correction of the DYSTROPHIN gene in Duchenne Muscular Dystrophy patient-derived iPS cells by TALEN and CRISPR-Cas9

Hongmei Lisa Li, Naoko Fujimoto, Noriko Sasakawa, Saya Shirai, Tokiko Ohkame, tetsushi Sakuma, Michihiro tanaka, Naoki Amano, Akira Watanabe, Hidetoshi Sakurai, Takashi Yamamoto, Shinya Yamanaka, and Akitsu Hotta

Stem Cell Reports, November 26, 2014, DOI: http://dx.doi.org/10.1016/j.stemcr.2014.10.013

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ZenMaster

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