Induced pluripotent stem cells provide a fascinating tool to study disease-associated changes directly in the cell type affected in vivo. In such studies, iPS cells derived from healthy donors are typically used as reference point. However, considering the genetic variability between human individuals, an ideal control for studying monogenic diseases would be gene-corrected cells from the same patient. So far, classic methods of targeted genetic modification have proven inefficient in human pluripotent stem cells. Here we present the efficient gene correction of an expanded polyglutamin-containing allele in human neural stem cells (lt-NES cells; Koch et al., PNAS 106(9):3225-30, 2009) generated from iPSC derived from a patient with Machado Joseph Disease (MJD). In MJD, the monoallelic expansion of a polyglutamin-encoding CAG motif in exon 10 is causative for the formation of ATXN3-containing aggregates and neurodegeneration. To exchange the mutation-containing exon we took advantage of the ability of recombinant adeno-associated viral vectors (AAV) to efficiently target homologous chromosomal loci and introduce defined sequences with high fidelity. Using exon-flanking homology arms the expanded exon was exchanged by the non-expanded exon amplified from the healthy allele from the same patient. From a single viral transduction of 750.000 cells, an average of 220±40 clonal neural stem cell lines could be generated. Out of 60 clones analyzed by PCR and 22 clones analyzed by Western Blotting, all showed reversion to a non-expanded CAG-containing allele. Our data suggest that AAV-mediated gene correction could represent a fast and efficient approach to generate isogenic controls in iPSC-based disease modeling.
|Number of pages||1|
|Publication status||Published - 12 Nov 2011|
|Event||SfN Neuroscience 2011 - Washinton DC, United States|
Duration: 12 Nov 2011 → 16 Nov 2011
|Conference||SfN Neuroscience 2011|
|Period||12/11/11 → 16/11/11|