iPSC - Projects

Title: Identification of AAV capsids for potent transduction of somatic mammalian cells
Summary: To find an efficient AAV capsid to deliver reprogramming factors to murine embryonic fibroblasts (MEF, an ideal target cell for reprogramming), we screened our large collection of AAV wildtypes and mutants using a YFP reporter. This included AAV-DJ that we had previously derived by DNA family shuffling (Grimm et al., J. Virol 2008). Impressively, we succeeded in identifying 6 AAV variants which transduce MEF close to 100%, with best results obtained with AAV-DJ (dark blue bars).
People: Elena Sénis, Eike Kienle

Title: Generation of recombinant AAV vectors expressing iPS reprogramming factors
Summary: We next cloned the cDNAs for the four classical reprogramming factors Oct4, Sox2, Klf4 and c-Myc into conventional single-stranded or into our proprietary (Grimm et al., Nature 2006) self-complemenary AAV vector genomes. In line with our previous findings, the latter then yielded much more potent gene expression in MEF than the standard single-stranded AAVs (both packaged into AAV-DJ), that even surpassed the levels achievable with a widely used lentiviral vectors system.
People: Elena Sénis, Stefan Mockenhaupt

Title: Use of optimized and highly potent AAV vectors for somatic reprogramming
Summary: Using the most potent capsids identified in project #1 and the optimized vector genomes from project #2, we are currently comprehensively testing numerous conditions to identify ideal settings for MEF reprogramming. Interestingly, thus far, we have consistently noted a striking and unanticipated cytotoxic effect particularly with the Sox2-expressing vector after multiple successive infection rounds, whose molecular mechanism we are presently investigating in more detail.
People: Elena Sénis in collaboration with J. Utikal & A. Schambach

Title: Screening for AAV capsids that potently infect mammalian stem cells
Summary: Towards our long-term goal, to genetically modify iPSC with therapeutic sequences prior to differentiation into new somatic cells, we screened our AAV vector collection of various human and murine stem cells, including iPSC. To date, our best results include close to 100% transduction efficiencies with our AAV-DJ capsid as well as with selected peptide display mutants. These capsids and data hence provide excellent starting points for the upcoming creation of vectors for iPSC engineering.
People: Elena Sénis, Eike Kienle

Title: Implementation of an AAV-based marker/tracking system to label and identify iPSC
Summary: Based on the specific down-regulation of the let7 miRNA in iPSC cells as compared to parental somatic cells, we created AAV vectors expressing a GFP reporter tagged with multiple let7 binding sites. As hoped for, we found that this vector can potently and specifically identify fully reprogrammed iPSC, as evidenced by the co-localization (white arrows) of GFP (green) with Oct4 staining (red). As expected, the GFP/Oct4 correlation is best with AAVs containing 4x let7 binding sites.
People: Elena Sénis, Stefan Mockenhaupt

Title: Stable engineering of human iPSC to achieve protection against viral infections
Summary: Our ultimate goal towards which we are working is to use our iPSC-specific AAV vectors (see above, project #4) to stably integrate shRNAs directed against the Hepatitis C virus (HCV) genome into the endogenous miR-122 locus, so that they will be expressed under a cellular and presumably safe promoter once the iPSC have been differentiated to hepatocytes. The latter should then be protected against HCV in case they are used for transplantation into patients with liver damage.
People: Elena Sénis, Stefan Mockenhaupt, in collaboration with T. Cantz (MPI Münster) and H. Willenbring (USCF, CA, USA)

Contact: E-Mail (Last update: 26/02/2012)