Stem cell-based therapy for α1-antitrypsin deficiency

Human induced pluripotent stem cells offer the possibility of generating unlimited quantities of cells for autologous transplantation. By correcting the genetic defect underlying Z-allele α1-antitrypsin deficiency, we recently provided the first proof of principle for application of human induced pluripotent stem cells in the treatment of inherited genetic disorders. Several important safety concerns will need to be addressed before this can be translated into clinical practice.

is due to the scarce availability of large numbers of highquality hepato cytes. An additional problem, common to whole-organ transplantation, is the continued requirement for lifelong harmful immunosuppression. Seminal work by Takahashi and Yamanaka raised the exciting possibility that induced pluripotent stem cells (iPSCs) could be used to generate large quantities of high-quality cells for autolo gous transplantation [8].
We explored the prospect of iPSC-based cell therapy by deriving fi broblasts from skin biopsies of individuals who are homozygous for Z-allele α 1 -antitrypsin defi ciency (PiZ). Th ese cells were reprogrammed with retroviral constructs that overexpressed key pluripotency-asso ciated transcription factors to produce patient-specifi c iPSCs. Th e stem cells were then diff erentiated using a novel inhouse protocol to produce hepatocytes that recapitulated many of the features of the clinical phenotype [9]. Specifi cally the hepatocyte-like cells from PiZ homozygotes formed polymers that were retained within the endoplasmic reticulum [9]. Th is potentially limitless supply of cells provided us with a useful new model since, unlike previous cell models, the mutant α 1 -antitrypsin is under the control of the endogenous promoter.
Th e use of iPSCs within the context of treating individuals with PiZ would require correction of the underlying genetic abnormality in a manner fully compatible with clinical applications. Th e genetic defect responsible for PiZ (Glu342Lys) was therefore targeted using a combination of engineered zinc fi nger nucleases and a piggyBac donor vector in patient-specifi c human iPSCs [10]. Th rough this approach we successfully demonstrated for the fi rst time an effi cient gene-editing technique capable of restoring normal structure, function and secretion of α 1 -antitrypsin in subsequently derived liver cells. Th is genetic correction did not leave residual exogenous sequences in the targeted iPSC genome. Th e drawback of this approach is that retroviral reprogramming vectors remain within the genome. We therefore derived human iPSC lines using a nonintegrating RNA (Sendai) virus and produced corrected hepatocytes with stable karyotypes in almost completely chemically defi ned culture conditions. Given the concern surrounding the safety of stem cell products, however, more eff ort was required to understand the genetic stability of human iPSC lines. Lines were characterised not only at the Abstract Human induced pluripotent stem cells off er the possibility of generating unlimited quantities of cells for autologous transplantation. By correcting the genetic defect underlying Z-allele α 1 -antitrypsin defi ciency, we recently provided the fi rst proof of principle for application of human induced pluripotent stem cells in the treatment of inherited genetic disorders. Several important safety concerns will need to be addressed before this can be translated into clinical practice. chromosome number level (by G-banding), but also for copy number variation (using array-based comparative genomic hybridisation) and single base pair resolution (using whole exome sequencing). A large proportion of Sendai virus-reprogrammed human iPSCs carried the correct numbers of chromosomes but had signifi cant copy number variation detected by array-based com parative genomic hybridisation. Lines genetically stable by array-based comparative genomic hybridisation could be identifi ed and these were subjected to whole exome sequencing. Th is analysis revealed that whilst the gene correction technique did not perturb the genome, the initial derivation of nonretroviral human iPSC lines induced 29 exomic point mutations.
Th e biological relevance of the point mutations in iPSC-derived hepatocytes is unclear as none of the new mutations occurred in genes known to predispose to cancer. Moreover, to the best of our knowledge such an in-depth analysis of human cell lines has not previously been performed. We therefore investigated the behaviour of the cells in vivo by injecting genetically corrected iPSC-derived liver cells into a mouse model of liver injury. Th is assay confi rmed the functional capacity of our cells and importantly demonstrated that the point mutations did not cause catastrophic carcinogenic sequelae since none of the mice developed tumours. In total, our results provided proof of principle for the potential of combining human iPSCs with gene therapy techniques to generate cells for autologous cell-based treatment of individuals with α 1 -antitrypsin defi ciency.
Several challenges remain before this technology can be applied within the context of a clinical trial. First, the cell type produced in vitro remains of a foetal nature in terms of its functionality, and great eff orts must now be made to improve protocols of diff erentiation to realise the fi nal step of maturation in order to achieve truly adult-like cells. Th e fi nal step of maturation, however, may perhaps only be achieved within an extracellular niche of the human liver. If this was to be the case then, as long as the iPSC-derived hepatocytes can be shown to be safe, a move towards the clinic seems rational. Defi ning safety will require considerable discussion amongst scientists and clinicians alike and will need more comprehensive animal data than we had the time to perform in our study. In the interim, our work opens the possibility that, with the correct level of screening, iPSCderived products may soon realise their much anticipated use for the treatment of human disorders.

Competing interests
The authors declare that they have no competing interests.