Search

Spinal muscular atrophy (SMA), an autosomal recessive neuromuscular disorder, is the leading genetic cause of infant mortality. SMA is caused by the homozygous loss of Survival Motor Neuron-1 (SMN1). In humans, a nearly identical copy gene is present, SMN2. SMN2 is retained in all SMA patients and encodes the same protein as SMN1. However, SMN1 and SMN2 differ by a silent C-to-T transition at the 5' end of exon 7, causing alternative splicing of SMN2 transcripts and low levels of full-length SMN. SMA is monogenic and therefore well suited for gene-replacement strategies. Recently, self-complementary adeno-associated virus (scAAV) vectors have been used to deliver the SMN cDNA to an animal model of disease, the SMNΔ7 mouse. In this study, we examine a severe model of SMA, Smn(-/-);SMN2(+/+), to determine whether gene replacement is viable in a model in which disease development begins in utero. Using two delivery paradigms, intracerebroventricular injections and intravenous injections, we delivered scAAV9-SMN and demonstrated a two to four fold increase in survival, in addition to improving many of the phenotypic parameters of the model. This represents the longest extension in survival for this severe model for any therapeutic intervention and suggests that postsymptomatic treatment of SMA may lead to significant improvement of disease severity.

Spinal muscular atrophy (SMA), a neurodegenerative disease, is the second most common genetic disorder and the leading genetic cause of infantile death. SMA arises from the loss of Survival Motor Neuron-1 (SMN1), leading to degeneration of lower motor neurons and, consequently, the atrophy of voluntary muscles. A duplicated copy gene called SMN2 exists in humans. SMN2 is unable to fully compensate for the loss of SMN1 because it produces very low levels of functional SMN protein due to an alternative splicing event. A C/T transition in SMN2 exon 7 results in a transcript lacking exon 7 and, therefore, creates a truncated SMN protein that cannot fully compensate for the loss of SMN1. However, SMN2 is an ideal target for therapeutic strategies that redirect this critical splicing event. Previously, we developed the first trans-splicing strategy to increase the full-length mRNA and functional SMN protein from the SMN2 gene. To improve the trans-splicing efficacy, we then developed a single-vector system that expressed a trans-splicing RNA (tsRNA) and an antisense blocking the downstream splice site. This single vector greatly enhanced trans-splicing of SMN2 transcripts in vitro and in vivo. In this report, we have added a neurotrophic factor [insulin-like growth factor (IGF)-1] to this single vector to determine whether neuroprotection and SMN induction provide greater protection in an SMA animal model. Intracerebroventricular injection of the trans-splicing/IGF vector significantly increased SMN protein in brain and spinal cord of SMAΔ7 mice and lessened the severity of disease in a more severe mouse model as evidenced by an extension of life span and increased body mass.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder and is the leading genetic cause of infant mortality. SMA is caused by the loss of survival motor neuron-1 (SMN1). In humans, a nearly identical copy gene is present called SMN2, but this gene cannot compensate for the loss of SMN1 because of a single silent nucleotide difference in SMN2 exon 7. This single-nucleotide difference attenuates an exonic splice enhancer, resulting in the production of an alternatively spliced isoform lacking exon 7, which is essential for protein function. SMN2, however, is a critical disease modifier and is an outstanding target for therapeutic intervention because all SMA patients retain SMN2 and SMN2 maintains the same coding sequence as SMN1. Therefore, compounds or molecules that increase SMN2 exon 7 inclusion hold great promise for SMA therapeutics. Bifunctional RNAs have been previously used to increase SMN protein levels and derive their name from the presence of two domains: an antisense RNA sequence specific to the target RNA and an untethered RNA segment that serves as a binding platform for splicing factors. This study was designed to develop negatively acting bifunctional RNAs that recruit hnRNPA1 to exon 8 and block the general splicing machinery from the exon 8. By blocking the downstream splice site, this could competitively favor the inclusion of SMN exon 7 and therefore increase full-length SMN production. Here we identify a bifunctional RNA that stimulated full-length SMN expression in a variety of cell-based assays including SMA patient fibroblasts. Importantly, this molecule was also able to induce SMN expression in a previously described mouse model of SMA and demonstrates a novel therapeutic approach for SMA as well as a variety of diseases caused by a defect in splicing.