Excision of reprogramming transgenes improves the differentiation potential of iPS cells generated with a single excisable vector.

The residual presence of integrated transgenes following the derivation of induced pluripotent stem (iPS) cells is highly undesirable. Here we demonstrate efficient derivation of iPS cells free of exogenous reprogramming transgenes using an excisable polycistronic lentiviral vector. A novel version of this vector containing a reporter fluorochrome allows direct visualization of vector excision in living iPS cells in real time. We find that removal of the reprogramming vector markedly improves the developmental potential of iPS cells and significantly augments their capacity to undergo directed differentiation in vitro. We further propose that methods to efficiently excise reprogramming transgenes with minimal culture passaging, such as those demonstrated here, are critical since we find that iPS cells may acquire chromosomal abnormalities, such as trisomy of chromosome 8, similar to embryonic stem cells after expansion in culture. Our findings illustrate an efficient method for the generation of transgene-free iPS cells and emphasize the potential beneficial effects that may result from elimination of integrated reprogramming factors. In addition, our results underscore the consequences of long-term culture that will need to be taken into account for the clinical application of iPS cells.

Gene therapy for Wiskott-Aldrich syndrome: rescue of T-cell signaling and amelioration of colitis upon transplantation of retrovirally transduced hematopoietic stem cells in mice.

The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency that is caused by mutations in the recently identified WASP gene. WASP plays an important role in T-cell receptor-mediated signaling to the actin cytoskeleton. In these studies we assessed the feasibility of using retroviral gene transfer into WASP-deficient hematopoietic stem cells (HSCs) to rescue the T-cell signaling defect that is characteristic of WAS. Upon transplantation of WASP-deficient (WKO) HSCs that have been transduced with WASP-expressing retroviruses, mature B and T cells developed in normal numbers. Most importantly, the defect in antigen receptor-induced proliferation was significantly improved in T cells. Moreover, the susceptibility of colitis by WKO HSCs was prevented or ameliorated in recipient bone marrow chimeras by retrovirus-mediated expression of WASP. A partial reversal of the T-cell signaling defect could also be achieved following transplantation of WASP-deficient HSCs expressing the WASP-homologous protein N-WASP. Furthermore, we have documented a selective advantage of WT over WKO cells in lymphoid tissue using competitive repopulation experiments and Southern blot analysis. Our results provide proof of principle that the WAS-associated T-cell signaling defects can be improved upon transplantation of retrovirally transduced HSCs without overt toxicity and may encourage clinical gene therapy trials.