Genetic Correction Of Human Induced Pluripotent Stem Cells From SMA Patient

Spinal muscular atrophy (SMA) is a class of neurodegenerative disease characterized by loss of motor neurons in the anterior horn of the spinal cord, resultant weakness and atrophy. SMA associated with mutations in the survival motor neurons (SMN1) gene, which leads to deletion or unfunctional motor neuron protein. There are no approved therapies for SMA in clinical. SMNlgene is located on chromosome5q13.■SMN2, a homologous of SMN1, which has only two base pair difference with SMN1, is retained in all SMA patients. In our research, we used pMN-Neo combined with transcription activator like effector nucleases (TALEN) for in situ mutagenesis of SMN2, to get into an SMN1-like gene, which can express functional SMN protein in induced pluripotent stem cells(iPSCs) of SMA patient origin, to achieve autologous gene therapy for SMA.Part I:Generate SMA patient-specific autologous iPSCs.Objective:Induced SMA patient autologous iPSCs from SMA patient urine cells.Methods:We tried to generate SMA patient-specific iPSCs from the urine cells of SMA patient by the retrovirus-mediated transfection of four transcription factors, namely Oct4, Sox2, c-Myc, and Klf4. And, valproic acid sodium (VPA) was added to the medium during subsequent culture. The iPSC clones were characterized by morphology, karyotype analysis, alkaline phosphatase stainining, immunofluorescence and in vivo differentiation experiment.Results:(1) The characterization of SMA-iPSCs by immunoflurescence staining of pluripotent cell markers is consistent with normal ESCs. And the cells were alkaline phosphatase staining positive.(2) Karyotype analysis of the P15generation of SMA-iPSCs showed no mutations in the induced cells at chromosome level, which is consistent with the karyotype analysis of patient peripheral blood.Conclusion:Based on the preliminary results, the SMA-iPSCs have been successfully generated from the urine cells of SMA patient.Part Ⅱ:Construction and targeting of nonvival SMA gene therapy vector.Objective:To construct SMN2gene targeting vector, pMN-Neo, for in sitro mutagenesis. Combined with TELAN technique to target pMN-Neo to the region of exon7and exon8of SMN2gene in the SMA-iPSCs genome. Then screen the SMA-iPSCs clones which stably express SMN proteins.Methods:(1) Construction of the vector pMN with molecular techniques, for instance, PCR amplification, restriction enzyme digestion, ligasion and so on. Then insert the open reading frame of Neo constitute to the SMA gene therapy vector, pMN-Neo.(2) Transfecting pMN-Neo into single SMA-iPSCs by nucleofection.Results:(1) Restriction enzyme analysis assay showed the inserted fragment to be the theoretical size, and sequencing analysis showed the theoretical sequence, indicating successful construction of the construct.(2)After transfection and screening for13days by G418, we get122clones.Conclusion:The construction of TALEN and the pMN-Neo vector are successful. We prelimarily gained SMA-iPS targeting cells for identification.