Trans-splicing-mediated improvement in a severe mouse model of spinal muscular atrophy

Abstract

Spinal muscular atrophy is a leading genetic cause of infantile death and occurs in approximately 1/6000 live births. SMA is caused by the loss of Survival Motor Neuron-1 (SMN1), however, all patients retain at least one copy of a nearly identical gene called SMN2. While SMN2 and SMN1 are comprised of identical coding sequences, the majority of SMN2 transcripts are alternatively spliced, encoding a truncated protein that is unstable and nonfunctional. Considerable effort has focused upon modulating the SMN2 alternative splicing event since this would produce more wild-type protein. Recently we reported the development of an optimized trans-splicing system that involved the coexpression of a SMN2 trans-splicing RNA and an antisense RNA that blocks a downstream splice site in SMN2 pre-mRNA. Here, we demonstrate that in vivo delivery of the optimized trans-splicing vector increases an important SMN-dependent activity, snRNP assembly, in disease-relevant tissue in the SMA mouse model. A single injection of the vector into the intracerebral-ventricular space in SMA neonates also lessens the severity of the SMA phenotype in a severe SMA mouse model, extending survival approximately 70%. Collectively, these results provide the first in vivo demonstration that SMN2 trans-splicing leads to a lessening of the severity of the SMA phenotype and provide evidence for the power of this strategy for reprogramming genetic diseases at the pre-mRNA level.

Figure 1.

Trans-splicing detection in SMA tissues. Four representative severe SMA mice (Smn^−/−;SMN2^+/+) were assayed for trans-splicing using trans-splicing specific primers. The panel of gels shows RT-PCR results indicating the biodistribution of the trans-SMN RNA. Tissues were harvested 24 h posttransfection and mCycA was used as a positive control for loading and RT-PCR. Molecular weight markers are indicated on the left of each gel.

Figure 2.

Trans-splicing mediated increases in functional SMN levels in vivo. A, In vivo restoration of severe SMA neonatal UsnRNP assembly capacity is mediated by SMN trans-splicing RNA plasmid injections. U1 and U11 snRNAs were radiolabeled in vitro and incubated with the respective extracts. Y12 antibody was used to immunoprecipitate snRNP complexes from tissue extracts derived from treated SMA mice or unaffected heterozygote mice. U1^KO is a negative control RNA that lacks the Sm nucleation site. Mouse lumbar spinal cord sections were removed from the vertebrae and passed through a 25 gauge needle before the assembly process. The negative control SMA mouse was injected with pMU2-tsRNA^KO plasmid. HeLa cell extract served as a positive control. Representative results from three independent experiments are shown. B, Western blots of spinal cords from ASO-tsRNA-treated mice demonstrated increases in SMN protein. 10% SDS-PAGE gels are shown developed with an anti-SMN monoclonal antibody. Six separate SMA mice were used to generate statistical significance between treatment and control transfections. Equal amounts of protein were loaded in each well as determined by Lowry assay.

Figure 3.

Intracerebroventricular delivery of optimized trans-splicing vector extends average life span in a severe model of SMA. Neonatal pups were injected with a mixture of PEI/Glucose and plasmid vector via a single intracerebral-ventricular injection. The pMU3 intracerebroventricular injection produced an approximate ∼70% increase in SMA mouse survival. Kaplan–Meier curve depicts negative controls vehicle (Glucose/PEI) and tsRNA^KO dying at a maximum 7 d. n = 15 mice for each group. p < 0.001.