Stem Cells Correct Mutation Causing Inherited Liver Disease

Jacquelyn K. Beals, PhD

October 12, 2011

October 12, 2011 — A new study provides evidence that human induced pluripotential stem cells (iPSCs) can be genetically corrected to generate cells that could be useful in clinical cell-based therapies. The study, published online October 12 in Nature, combined stem cell technologies to correct a point mutation in both alleles of the α1-1 antitrypsin (A1AT) gene (also known as SERPINA1; serine protease inhibitor).

The A1AT mutation dealt with in this study substitutes lysine for glutamic acid and causes abnormal α1-antitrypsin to build up in the liver cells (hepatocytes) that produce it. This abnormal protein polymerizes in the hepatocyte endoplasmic reticulum, impairing secretion and reducing plasma levels by as much as 85%; function of the mutant protein is also impaired. In patients with A1AT deficiency (A1ATD), the liver cell inclusions cause cirrhosis, which can only be treated by a liver transplant.

The present study, an international collaboration led by researchers at Cambridge University, United Kingdom, was announced at a Nature press briefing on October 11. In his introductory remarks, Allan Bradley, PhD, director emeritus, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, explained the challenges they faced.

"In this particular case, we needed to correct both copies of a gene, and we needed to do so in a circumstance where the genome of the cells was not damaged in any other way," he said. "We had to go in...and remove and change just 1 nucleotide out of 3 billion base pairs from the mother and 3 billion base pairs from the father."

Previous methods for targeting specific genes and selecting clones of interest left small genetic sites that could interfere with regulation and transcription of nearby genes. The alternative approach uses piggyBac, a mobile element (transposon) from moth DNA that can move around in mammalian DNA, including human embryonic stem cells. Gene segments "flanked by piggyBac inverted repeats" are excised without leaving residual sequences (as 1 manufacturer puts it, piggyBac "will remove the transposons from the genome, footprint-free").

Before using piggyBac to correct the A1AT point mutation, the investigators ran preliminary studies in mice. One study confirmed correction of an albino mutation in mouse iPSCs and its "seamless excision" by sequence analysis, and also observed normal function of the corrected allele (ie, pigment production) after injection into mouse blastocysts. This exercise showed that the piggyBac transposon could modify a mammalian genome at the level of a single base pair.

The next step paired piggyBac technology with zinc finger nuclease (ZFN) technology. ZFNs are a group of DNA-binding proteins that facilitate genome editing by producing double-strand breaks in DNA at locations that can be specified by the user.

"Basically, [ZFN] is a protein (2 proteins, actually) that are introduced into the cell, and they precisely cut the DNA at the point of the mutation. That cut then allows us to replace the bad copy with a good copy. And that has to go on both the copy from the mother and the copy from the father at the same time," Dr. Bradley explained in the press briefing. "So that was the challenge, and that is what we were able to do."

These technologies were combined and applied to A1AT-deficient iPSC lines from 3 patients. Among iPSC lines in which the A1AT mutation was corrected on both alleles, the correction was stable for more than 20 passages. In addition, these cell lines were still able to differentiate into cells of all 3 germ layers; modifying the genome did not reduce the cells' pluripotency.

The study also investigated whether the DNA cleavage and manipulation introduced any new mutations. Comparing a parental and corrected cell line identified 29 mutations, 25 of which were in the primary iPSC line, 3 of which arose during piggyBac excision, and 1 of which happened during the targeting. However, these final 4 mutations were inconsistent with the sites typically affected by ZFN or piggyBac integration. The authors concluded that using ZFNs with piggyBac can achieve "rapid and clean correction of a point mutation in human iPSCs without affecting their basic characteristics."

Further in vitro testing found that the corrected cell lines could differentiate normally and were capable of glycogen storage, uptake of low-density lipoprotein cholesterol, cytochrome P450 activity, and albumin secretion. Not only was mutant A1AT absent from the cells, but enzymatic activity of the secreted A1AT was comparable with A1AT of normal adult hepatocytes.

Finally, when transplanted into the livers of mice, the human "corrected iPSC-derived hepatocyte-like cells" colonized the liver lobes, produced human albumin, and did not lead to tumor formation. "Collectively these analyses demonstrate that genetic correction...resulted in functional restoration of A1AT in patient-derived cells," the study reports.

"These cells could repopulate the native liver, a term called therapeutic repopulation," commented Anil Dhawan, MD, from the Paediatric Liver Centre, King's College Hospital, London, United Kingdom, to Medscape Medical News via email. "The autologous cells (...IPs) will be much better, as hopefully they wouldn't trigger an immune response."

Asked about other disorders for which human iPSCs might be genetically corrected to generate cells for autologous cell-based therapies, Dr. Dhawan suggested "single gene defects like Crigler-Najjar syndrome, glycogenosis, Wilson's disease, [and] hyperlipidemias, to name a few."

In fact, A1ATD is among the more difficult diseases to treat, as both copies of the gene must be corrected. "These particular patients have 2 bad copies of the gene, and so correcting 1 isn't going to be sufficient for them," said Dr. Bradley in a telephone interview with Medscape Medical News. "You have to actually correct both, and that's because the bad protein accumulates in the cells, rather than getting out and doing its job, and that's not a great thing for the liver cells. Even if you correct 1, it's not ideal.

"Many human conditions would be restored by putting 1 copy back to normal, so in principle, there are easier examples than the one we chose," added Dr. Bradley. "There are many possible diseases that can be tried," he said. "So, yes, it could be applied elsewhere."

The study and study authors were supported by Wellcome Trust, the MRC Senior nonclinical fellowship, the Cambridge Hospitals National Institute for Health Research Biomedical Research Center, the Medical Research Council, Papworth National Health Services Trust, the Bill and Melinda Gates Foundation, Inserm, Institut Pasteur, and Japan Science and Technology Agency. One author is supported by a postdoctoral fellowship from the Japan Society for the Promotion of Science, 2 authors by Wellcome Trust Clinical Training Fellows, and another author by a fellowship from the International Human Frontiers Science Program Organization. Dr. Bradley and Dr. Dhawan have disclosed no relevant financial relationships.

Nature. Published online October 12, 2011.

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