Genetic conversion of an SMN2 gene to SMN1: a novel approach to the treatment of spinal muscular atrophy.

Spinal muscular atrophy (SMA), a recessive, neuromuscular disease, is caused by a mutation or deletion in the SMN1 gene. The SMN2 gene is present in the same region of chromosome 5 and is similar in DNA sequence to SMN1 except for a T at position +6 of exon 7 that is likely the predominant functional difference between the two genes. This change alters RNA splicing which results in the removal of exon 7 from the mature mRNA; only 10% full-length transcripts are produced from the SMN2 gene. Our lab has shown that single-stranded oligonucleotides (ODN) can be used to repair genes with single base mutations within the context of the native chromosome. Here, we used SMN2-sequence-specific ODNs to direct the exchange of a T to a C in an SMA skin fibroblast cell line from a type 1 patient. The cells were transfected with ODNs of either 47 or 75 bases in length and designed to hybridize to either the transcribed or non-transcribed DNA strand of the SMN2 gene. We analyzed the genotype of these cells using a well-established Taqman probe-based PCR assay, restriction enzyme digestion, and cycle sequencing. Conversion of the SMN2 genotype to SMN1 was detected when the specific ODN was added. As a result, we observed an increase in production of full-length SMN mRNA, measured by qRT-PCR, and SMN protein, measured by western blotting. Finally, properly localized SMN protein was detected by the accretion of gemini of coiled bodies (gems) only in targeted cells. This is the first report of the use of ODNs to direct genetic conversion of SMN2 to SMN1 in human cells from SMA patients.