UK scientists took stem cells made from the skin cells of patients with an inherited liver disease called alpha1-antitrypsin deficiency, used “molecular scissors” to effect a “clean” repair of the gene mutation that causes the disease, and showed, both in test tubes and in mice, that the gene worked correctly when the stem cells made new cells that were almost like liver cells. Nature reports the study, led by researchers from the Wellcome Trust Sanger Institute and the University of Cambridge, in its 12 October online issue.

The study is significant because it uses a tidy method that leaves no remnants of the repair mechanism behind, which could otherwise introduce unacceptable risks in a clinical setting: thus it is a new way of making a “clean correction” to the defect gene.

Because of this, it brings closer the possibility of patient-specific stem cell therapies, whereby corrected stem cells are used to grow working liver cells inside the patient and thus avoid the need for expensive and often risky liver transplants.

Co-author Professor Allan Bradley, Director Emeritus of the Wellcome Trust Sanger Institute, told the press:

“We have developed new systems to target genes and integrated all the components to correct, efficiently, defects in patient cells.”

“Our systems leave behind no trace of the genetic manipulation, save for the gene correction,” he added.

Co-author Professor David Lomas, Professor of Respiratory Biology at the University of Cambridge and Consultant Physician at Addenbrooke’s and Papworth Hospitals, has spent the last 20 years working on the mechanism of alpha1-antitrypsin deficiency and cares for patients with the condition. He said:

“As there is currently no cure for this disease other than liver transplantation, and given the increasing strains being placed on the national liver transplant programme as a result of the sharp increase in the frequency of liver disease, alternative therapies for genetic and other liver diseases are urgently being sought.”

Bradley, Lomas and colleagues targeted a gene defect that causes cirrhotic liver disease and increases the risk of lung cancer and emphysema. The defect results in alpha1-antitrypsin deficiency (A1ATD), the most common known inherited disorder of the liver and lung, occurring in about one in 2000 people of North European origin.

The defect that causes the disease occurs in a single nucleotide change in the gene that codes for alpha1-antitrypsin (A1AT), an enzyme inhibitor that normally protects bodily tissues against excessive inflammation. People with the defective gene cannot release A1AT properly from the liver, where it stays trapped, and causes the damage that leads to liver cirrhosis and lung emphysema.

Currently, the only way to treat the cirrhotic liver is via transplant.

Bradley told Nature News that a genetic cure would require a complete replacement of the defective gene throughout the liver, because any remaining mutant protein would continue to accumulate and cause damage:

“You can’t just put in a normal copy because that’s not sufficient to change the disease,” said Bradley.

That’s why the researchers turned to stem cells, because they can be coaxed into becoming virtually any cell in the body, including new liver cells. The idea was if it were possible to repair the DNA in the stem cells, then perhaps they could regenerate new tissue that lacked cells with the gene defect.

There are two types of stem cell: embryonic and induced Pluripotent (iPS). Embryonic are the “gold standard”, they make the best stem cells, but there are lots of ethical problems with using them, not least because it requires the destruction of many embryos.

Then scientists found they could reprogram skin cells and blood cells to become pluripotent (able to make a range of other cells) like embryonic stem cells, and the idea of the iPS cell was born. But as time went on, they discovered lab-cultured iPS cells were perhaps not quite as easy and safe as first thought, for instance they can accumulate DNA mutations that then cause uncontrolled growth of tissue.

So the team faced a difficult challenge: if a problem with iPS cells is that they accumulate DNA defects, then they had better make sure when they went in to repair the single nucleotide change in the A1AT gene, they did not leave behind any remnants of the repair mechanism (such as foreign DNA) that might facilitate this.

And reading their report, it appears that they have done this, and checked they did it.

Building on previous work from Cambridge where they transformed skin cells into liver cells by reprogramming stem cells, the team successfully and accurately corrected an alpha1-antitrypsin gene in an established cell line containing the mutation.

They used “molecular scissors” in the form of an engineered molecule called a “zinc-finger nuclease” to find and cut the faulty A1AT gene in iPS cells made from skin cells of people with A1ATD.

Then they used a self-inserting DNA molecule called “piggyBac” to replace the defective portion. Afterwards, they removed the piggyBac sequences from the cells, so when they coaxed them to differentiate into liver cells, these did not show any trace of residual DNA damage at the site of correction.

The iPS cells formed cells that showed some of the properties of hepatocytes, the liver cells that are most affected by A1ATD.

14 days after the researchers transplanted the gene-corrected hepatocyte-like cells into mice, some of them had integrated into the liver and were producing human A1AT.

Thus the researchers proved that the accurate copy of the gene was now active in the hepatocyte-like cells by showing the presence of normal A1AT in both the test tube and mouse experiments.

Dr Ludovic Vallier, senior author of the study and an expert in human pluripotent stem cell biology, said:

“This study represents a first step toward personalised cell therapy for genetic disorders of the liver.”

“We still have major challenges to overcome before any clinical applications but we have now the tools necessary to advance toward this essential objective,” said Vallier, who is Medical Research Council (MRC) senior Fellow and Principal Investigator at the University of Cambridge’s MRC Centre for Stem Cell Biology and Regenerative Medicine and Department of Surgery.

When they have analyzed stem cells before, the team has discovered their genomes often contain mutations, although it is not clear what causes them. But in this study they were careful to employ the latest sequencing techniques to find cells with the smallest number of mutations, and then look at what happens to these when they differentiate into tissue cells.

They suggest screening of stem cells is going to be a very important part of making therapies that use these methods safe.

In a final step, the researchers took cells directly from a patient with A1ATD and corrected the gene defect exactly as they did with the cell line: they found the corrected cells produced normal A1AT.

Lomas said:

“It is a quite remarkable series of results, founded on strong research and the generous participation of our patients. One of the next steps will be exploring the use of this technique in human trials.”

Bradley agreed, “These are early steps but, if this technology can be taken into treatment, it will offer great possible benefits for patients.”

Written by Catharine Paddock PhD