Disruption of the Survival Motor Neuron ( SMN) gene in pigs using ssDNA

Spinal Muscular Atrophy (SMA) is an autosomal recessive neurodegenerative disease that is a result of a deletion or mutation of the SMN1 ( Survival Motor Neuron ) gene. A duplicated and nearly identical copy, SMN2 , serves as a disease modifier as increasing SMN2 copy number decreases the severity of the disease. Currently many therapeutic approaches for SMA are being developed. Therapeutic strategies aim to modulate splicing of SMN2 -derived transcripts, increase SMN2 gene expression, increase neuro-protection of motor neurons, stabilize the SMN protein, replace the SMN1 gene and reconstitute the motor neuron population. It is our goal to develop a pig animal model of SMA for the development and testing of therapeutics and evaluation of toxicology. In the development of a SMA pig model, it was important to demonstrate that the human SMN2 gene would splice appropriately as the model would be based on the presence of the human SMN2 transgene. In this manuscript, we show splicing of the human SMN1 and SMN2 mini-genes in porcine cells is consistent with splicing in human cells, and we report the first genetic knockout of a gene responsible for a neurodegenerative disease in a large animal model using gene targeting with single-stranded DNA and somatic cell nuclear transfer.