Application and optimization of a gene targeting strategy called Small Fragment Homologous Replacement for the treatment of the Spinal Muscolar Atrophy

The majority of patients affected by spinal muscular atrophy (SMA) have deletion of the survival of motor neuron 1 (SMN1) gene, but they retain a “nonfunctional” copy of the duplicate gene (SMN2) in their genome. SMN2 produces defective SMN protein because of a C>T transition in exon 7, which causes the skipping of exon 7 during SMN mRNA maturation. Many attempts have been made to correct altered SMN gene expression and to increase the level of normal SMN protein, but to date an effective treatment for this disease has not been established. Small Fragment Homologous Replacement (SFHR) is a site-specific gene modification approach that has the potential to maintain the genomic organization necessary for expression. The target modification in the genome is mediated by small DNA fragments (SDFs) 400–800 bp in length. In this study we used SFHR to induce a T > C transition at codon 280 in exon 7 of the SMN2 gene in order to produce an increase in functional SMN protein. SDFs were transfected in vitro into cells obtained from five human fetal chorionic villi of embryos, homozygous for the SMN1 deletion, by either electroporation or microinjection. Transfected SMA cells showed an increase of up to 53% in full-length SMN mRNA compared with untransfected controls, as detected by real-time polymerase chain reaction. Consistent with the RNA data, immunocytochemistry and immunoblotting revealed a significant 2-fold increase in wild-type SMN protein. Furthermore, genotype and phenotype of transfected cells remained stable after several in vitro passage