Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells

Abstract

Human induced pluripotent stem cells (hIPSCs) represent a unique opportunity for regenerative medicine since they offer the prospect of generating unlimited quantities of cells for autologous transplantation as a novel treatment for a broad range of disorders1,2,3,4. However, the use of hIPSCs in the context of genetically inherited human disease will require correction of disease-causing mutations in a manner that is fully compatible with clinical applications3,5. The methods currently available, such as homologous recombination, lack the necessary efficiency and also leave residual sequences in the targeted genome6. Therefore, the development of new approaches to edit the mammalian genome is a prerequisite to delivering the clinical promise of hIPSCs. Here, we show that a combination of zinc finger nucleases (ZFNs)7 and piggyBac8,9 technology in hIPSCs can achieve bi-allelic correction of a point mutation (Glu342Lys) in the α1-antitrypsin (A1AT, also called SERPINA1) gene that is responsible for α1-antitrypsin deficiency (A1ATD). Genetic correction of hIPSCs restored the structure and function of A1AT in subsequently derived liver cells in vitro and in vivo. This approach is significantly more efficient than any other gene targeting technology that is currently available and crucially prevents contamination of the host genome with residual non-human sequences. Our results provide the first proof of principle for the potential of combining hIPSCs with genetic correction to generate clinically relevant cells for autologous cell-based therapies.

Human induced pluripotent stem cells derived hepatocytes: rising promise for disease modeling, drug development and cell therapy

“…Utilizing a combined technology of zinc finger nucleases (ZFNs) and piggyBac, the authors efficiently corrected the pathogenic point mutation in patient iPSC. The normal metabolic function was restored in hepatocytes derived from corrected iPSC, which demonstrated in vivo functionality (Yusa et al, 2011). This study proved a concept that inherited liver metabolic disorder can be cured through gene correction.…”

“…Yusa et al (2011) reported the targeted gene correction of α1-antitrypsin deficiency in human iPSC. Utilizing a combined technology of zinc finger nucleases (ZFNs) and piggyBac, the authors efficiently corrected the pathogenic point mutation in patient iPSC.…”

Promise and challenges of human iPSC-based hematologic disease modeling and treatment

“…Besides the improved culture conditions, particularly the discovery of ROCK inhibitor-mediated clonal cell growth, there have been some major breakthroughs in our efforts to enhance the recombination rates. The development of zinc finger nuclease (ZFN) technology and, more recently, the transcription activator-like effector nuclease (TALEN) technology have allowed significant enhancement of gene targeting efficiency in iPSCs [28,29,[54][55][56][57][58]. These exciting new developments will no doubt have impact on the realization of iPSC potentials.…”

Cut and Paste: restoring cellular function by gene correction

“…Moreover, even though the generated iPSCs and their derivatives bear certain mutations acquired during reprogramming, gene targeting and/or culture conditions, the mutations observed did not hamper the phenotypic correction in vivo, neither led to tumor formation. This work as well as several other reports published earlier this year highlight the need for exhaustive testing of iPSCs and their derivatives in the context of specific applications rather than absolute number of mutations acquired, i.e., iPSCs bearing mutations in genes compromising the function of differentiated cells should be discarded in favor of lines whose alterations do not hamper normal cell function upon differentiation and transplantation [13]. With these criteria in mind, one would rather prime functionality in a particular context instead of the absolute number of mutations observed.…”

“…More recently, Yusa et al described the development of a novel gene targeting approach by combining the use of ZFN and piggyBAC transposon technology [13]. In their approach the authors were able to excise back the targeting construct from the host genome in the absence of the typical genomic scars induced by other commonly employed excisable methods such as the Cre-LoxP system.…”