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Effective Mutant Gene Knockdown in Disease Specific iPSCs

“Rapid Generation of Functional Dopaminergic Neurons From Human Induced Pluripotent Stem Cells Through a Single-Step Procedure Using Cell Lineage Transcription Factors”

From Stem Cells Translational Medicine

A study recently published in Stem Cells TM from the group of Tobias Cantz at the Hannover Medical School, Germany has united two exciting technologies; the use of third generation lentiviral vectors (LVs), recently shown to have amazing potency in ex vivo gene therapy, and that of the ever evolving field of patient specific induced pluripotent stem cells (iPSCs).   Excitingly, they have demonstrated the efficacy of LVs in gene silencing using the expression of a short hairpin RNA (shRNA) directed against mutant mRNA in a disease specific iPSC line (Eggenschwiler et al).

Mouse iPSCs generated contain a mutated human PiZ isoform of α-1-antitrypsin (A1AT) (Warlich E et al), whose accumulation leads to neonatal hepatitis, liver cirrhosis, and hepatocarcinoma (Sveger, and Eriksson et al). As PiZ A1AT heterozygous individuals do not show disease phenotype (Hultcrantz  and Mengarelli, and Bals), this suggests that a reduction in levels could be a relevant therapeutic approach for homozygous carriers. To establish if this is in fact true, a CAG promoter was used to express a short hairpin RNA (shRNA) directed against PiZ A1AT and introduced into the PiZ iPSCs.   Excitingly, expression of the shRNA did not affect pluripotency and during differentiation of PiZ iPSCs a 90% reduction in human PiZ A1AT was observed while other hepatic markers were not affected.   Next, PiZ-shRNA and scrambled-shRNA control iPSC lines were injected into blastocysts for chimera formation, leading to mice with high levels of transgenes in all tissues at E13.5 and analysis of CD45- cells (haematopoietic marker) from the liver, which contain the hepatoblasts, found a ∼90% knockdown of PiZ hA1AT similar to that seen in vitro, whereas other liver markers, such as Afp, Ttr, and Ck18, were not affected.   Analysis of PiZ A1AT protein in whole liver lysates at E15.5 found levels of chimerism of 25%, 40% and 50% and even controlling for this, PiZ A1AT was significantly reduced.

Next, fibroblasts from a human female patient with severe A1AT deficiency (PiZZ homozygosity) suffering from acute liver disease were transduced with a polycistronic lentiviral reprogramming vector.   Resultant iPSCs were then transduced with lentiviral PiZ-shRNA or scr-shRNA and differentiated towards the hepatic lineage using a cytokine-mediated strategy.   Excitingly, this led to a 90% reduction in PiZ hA1AT mRNA levels at day 18, while most hepatic markers were not altered.   A reduction in the protein level of PiZ A1AT of around 66% was also noted in the shRNA hepatic cells.   Furthermore, the mature hepatic cells expressing the shRNA at the end of the hepatic differentiation protocol also had reduced PiZ A1AT levels, and also secreted albumin into the medium and showed cytochrome P450 1A1 activity, typical of normal hepatic cells.

Together, the authors demonstrate success in the knockdown of a disease-causing mutant DNA using a lentiviral vector expressing a shRNA in patient-specific iPSCs and the successful differentiation to mature hepatic cells.   This technology has the potential to be used for multiple other disease-specific cells to generate clonal cell lines with well-defined properties that could allow for safe use in future therapy.   The authors do not that this type of manipulation will be useful for in vitro studies, but to be clinically relevant other strategies must be explored, such as ZFN- or TALEN-based gene editing, even though they are more laborious strategies.


  • Bals R (2010) Alpha-1-antitrypsin deficiency. Best Pract Res Clin Gastroenterol 24:629–633
  • Eriksson S et al (1986) Risk of cirrhosis and primary liver cancer in alpha 1-antitrypsin deficiency. N Engl J Med 314:736–739
  • Hultcrantz R, Mengarelli S (1984) Ultrastructural liver pathology in patients with minimal liver disease and alpha 1-antitrypsin deficiency: A comparison between heterozygous and homozygous patients. Hepatology 4:937–945
  • Sveger T (1976) Liver disease in alpha1-antitrypsin deficiency detected by screening of 200,000 infants. N Engl J Med 294:1316–1321
  • Warlich E et al (2011) Lentiviral vector design and imaging approaches to visualize the early stages of cellular reprogramming. Mol Ther 19:782–789

From Stem Cells Translational Medicine

Stem Cell Correspondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.