GENERAZIONE E CORREZIONE DI CELLULE STAMINALIPLURIPOTENTI INDOTTE NON VIRALI DA PAZIENTE SMA COMEMODELLO DI MALATTIA E SORGENTE PER LA TERAPIA CELLULARE
Tesi di Dottorato
Data di Pubblicazione:
2012
Citazione:
GENERAZIONE E CORREZIONE DI CELLULE STAMINALIPLURIPOTENTI INDOTTE NON VIRALI DA PAZIENTE SMA COMEMODELLO DI MALATTIA E SORGENTE PER LA TERAPIA CELLULARE / C. Simone ; direttore della scuola: M. Clerici ; tutore: G. Comi ; correlatore: S. Corti. Universita' degli Studi di Milano, 2012 Feb 08. 24. ciclo, Anno Accademico 2011. [10.13130/simone-chiara_phd2012-02-08].
Abstract:
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.
Tipologia IRIS:
Tesi di dottorato
Elenco autori:
C. Simone
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