GENERAZIONE E CORREZIONE DI CELLULE STAMINALIPLURIPOTENTI INDOTTE NON VIRALI DA PAZIENTE SMA COMEMODELLO DI MALATTIA E SORGENTE PER LA TERAPIA CELLULARE

Spinal muscular atrophy (SMA) is among the most common genetic neurological diseases causing infant mortality. SMA is an autosomal recessive genetic disorder caused by mutations in the survival motor neuron 1 gene (SMN1), leading to the depletion of survival motor neuron (SMN) protein and resulting in the selective degeneration of spinal cord motor neurons. Patients with SMA exhibit muscle weakness and hypotonia. There is no cure for this disorder, which is devastating for patients and their families and a serious societal health problem. The human genome also harbors the SMN2 gene, which is almost identical to SMN1 except for a single nucleotide difference in SMN2. The splicing change resulting from this difference yields only 10% of the full-length protein and high levels of an unstable, truncated protein lacking exon 7 (SMNDelta7). Although worms, flies, and mice are useful for studying disease pathogenesis and drug screening, they have important limitations in recapitulating human diseases. One is that they lack SMN2, which can be introduced only with transgenic modifications. The possibility of reprogramming mature somatic cells to generate induced pluripotent stem cells (iPSCs) has enabled derivation of disease-specific pluripotent cells, offering unprecedented access to modeling human disease and for cell and gene therapy applications. Here, we successfully generated human SMA-iPSCs from a type 1 SMA patient and his unaffected father, using non-integrating episomal vectors and demonstrated their differentiation into motoneurons. Moreover, we employed single-stranded oligonucleotides to correct defective SMN1, the SMA gene, using SMN2. Corrected cell lines contained no exogenous sequences and appeared indistinguishable from healthy iPSCs. Non-viral SMA-iPSC-derived motor neurons reproduced disease-specific features while corrected SMA-specific-iPSCs gave rise to phenotypically rescued motor neurons in vitro and in vivo. Our next goal was to determine whether MNs derived from iPSCs survive and engraft appropriately within the SMA spinal cord, the effect of disease environment on grafted cells and vice versa, and whether transplantation can ameliorate the disease phenotype in SMA transgenic mice. iPSC-purified motoneurons were used for transplantation into the spinal cords of 1-day-old SMA mice. Transplantation of wild-type and corrected SMA motor neurons extended lifespan and ameliorated the phenotype of SMA mice. These results offer proof-of-concept that generating patient-specific iPSCs and motor neurons free of exogenous elements may be possible, with potential for research and clinical applications.