Gene Corrected Spinal Muscular Atrophy-Induced Pluripotent Stem Cells And Motoneuron As A Model And Cell Source For Transplantation

Objective: To describe the correction of human spinal muscular atrophy (SMA)-induced pluripotent stem cells (iPSCs) and motoneurons using targeted gene correction with single-stranded oligonucleotides. Background Spinal muscular atrophy (SMA) is among the most common genetic neurological diseases causing infant mortality. Reprogramming adult human cells to induced pluripotent stem cells (iPSCs) allows obtaining patient-specific cells possible. However, the use of SMA iPSCs will require their genetic correction in a manner that is compatible with clinical applications. Design/Methods: We generated iPSCs from fibroblasts from a patient with SMA and his father using a non-viral method. Cells were nucleofected with oriP/EBNA1 vectors encoding six reprogramming factors. We used SMN2 sequence-specific ODNs to direct the exchange of a T to C at position +6 of exon 7, thus converting SMN2 into SMN1 in the SMA-iPSCs. The iPSCs were fully characterized and differentiated using a protocol to promote motoneuron commitment. The phenotype of cells was tested by morphological, gene expression, and protein analysis. iPSC-purified motoneurons were transplanted into the spinal cords of SMA mice. Histochemical and neuropathological analyses were conducted. Survival and neuromuscular function were investigated. Results: SMA corrected cell lines contained no exogenous sequences and appeared indistinguishable from healthy iPSCs. Non-viral SMA-iPSC-derived motoneurons reproduced disease-specific features (reductions in cell number, cell size, and axon length) while corrected SMA-specific-iPSCs gave rise to phenotypically rescued motoneurons in vitro and in vivo after transplantation in SMA spinal cord. Different splicing profiles, detected in vitro by microarray analysis in SMA motoneurons compared to wild-type, were normalized after the genetic correction. Transplantation of wild-type and corrected SMA motoneurons extended lifespan (>50%) and ameliorated the phenotype of SMA mice significantly more than SMA motoneurons and untreated animals. Conclusions: These results offer proof-of-concept that generating patient-specific corrected iPSCs and motoneurons free of exogenous elements may be possible, with potential for research and clinical applications. Disclosure: Dr. Corti has nothing to disclose. Dr. Nizzardo has nothing to disclose. Dr. Simone has nothing to disclose. Dr. Falcone has nothing to disclose. Dr. Nardini has nothing to disclose. Dr. Ronchi has nothing to disclose. Dr. Donadoni has nothing to disclose. Dr. Salani has nothing to disclose. Dr. Riboldi has nothing to disclose. Dr. Menozzi has nothing to disclose. Dr. Bonaglia has nothing to disclose. Dr. Magri has nothing to disclose. Dr. Bresolin has nothing to disclose. Dr. Comi has received research support from Telethon Italy and SMA Europe.