Targeted gene alteration in SMA patient cells: Genetic conversion of an SMN2 gene to SMN1 increases full-length SMN production.

摘要

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease that is characterized by the loss of alpha motor neurons leading to muscle atrophy and eventually death. SMA is caused by a mutation or deletion in the survival motor neuron (SMN) gene. This gene is present in two copies, SMN1 and SMN2, with each differing by 5 nucleotide substitutions, all of which are silent mutations. However, at position +6 in exon 7 of SMN2 there is a T in the place of a C, which causes an exonic splice enhancer, and the majority of transcripts produced from SMN2 are lacking exon 7. Patients with SMA rely on the number of copies of SMN2 and, for this reason; disease severity is inversely related to SMN2 copy number. Currently there is no cure or adequate treatment available but many studies are underway to develop therapies that facilitate an increase in full-length SMN. Some of these are histone deacetylase inhibitors (HDI) that work in increasing global transcription. They have been found to increase SMN transcript as well as protein but their effects are transient and short-lived. Another promising treatment for SMA comes in the form of gene repair with the use of single stranded oligonucleotides (ssODN). In fact, it was observed that in patient cells, the ssODN-mediated gene conversion was able to alter the base in SMN2 to become SMN1-like that was evaluated on the genomic level by several assays; quantitative PCR, restriction enzyme digestion, and cycle sequencing. The gene conversion led to an increase in full-length SMN mRNA and this effect was observed out to 7 days after oligonucleotide treatment. Most importantly, the cells treated with oligonucleotide were able to produce statistically significant increases in the amount of cellular gems, which is a universal measure for the SMN protein. Thus, the use of ssODN-mediated gene therapy in SMA patients' cells was successful and the ssODN led to a robust increase in full-length SMN mRNA and a significant increase in cellular gems.