Modulation of modifiers of Pre-mRNA splicing: A therapeutic strategy for Amenable Inherited Diseases

Modifier genes involved in pre-mRNA splicing may offer novel therapeutic targets for reducing the severity of some genetic diseases. Spinal muscular atrophy (SMA) is a devastating neurodegenerative disease most commonly caused by the homozygous loss of the survival motor neuron 1 (SMN1) gene. The absence of SMN is embryonic lethal, however humans have one or more copies of a nearly identical gene, SMN2, that provides low levels of full length SMN (FL-SMN). A single nucleotide change in SMN2 exon 7 creates a splice silencer recognition site that leads to the predominant production of non-functional transcripts missing exon 7. The splicing of SMN2 exon 7 is regulated by numerous positive and negative splicing factors that function collaboratively to influence exon recognition by the spliceosome. Splice switching antisense oligonucleotide (AO) manipulation of the expression and therefore intracellular concentrations of these splicing factors has potential as a means of restoring FL-SMN from the otherwise suboptimal SMN2 gene. In this study, three splicing factors involved in the processing of SMN2, Human transformer 2 Beta 1 (TRA2-β1), Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and Src associated in mitosis, of 68kDa (SAM68), were selected as targets for AO manipulation. TRA2-β1 is a positive splicing factor that promotes exon selection, with increased concentrations shown to restore FL-SMN levels produced from SMN2. AOs were designed to mediate upregulation of TRA2-β1 and thereby increase levels of SMN protein. Results from TRA2-β AO treated SMA patient cells showed a significant increase in the amount of functional SMN protein generated from the SMN2 gene. Therefore, AO mediated TRA2-β1 upregulation has the potential in the development of a combination therapy for SMA. HnRNP A1 and SAM68 are negative splicing factors that promote SMN2 exon 7 exclusion. AOs were designed to target hnRNP A1 and SAM68 frame-shifting exons for removal during pre-mRNA splicing in an attempt to knockdown respective protein expression and thereby facilitate increased FL-SMN and SMN protein production. AO vi mediated knockdown of hnRNP A1 and SAM68 was achieved resulting in enhanced functional SMN expression in SMA patient cells. In addition, the lead AOs targeting TRA2-β, hnRNP A1 and SAM68 were evaluated in combination with the Anti ISS-N1 AO that is the current antisense treatment available to SMA patients. Combination AO treatments in SMA cells showed an additive effect in increasing SMN protein expression. Whilst the focus of this study was to manipulate splicing factors involved in SMN2 splicing as a means to increase SMN protein and thereby reduce the severity of SMA, this approach can be extended to other diseases potentially amenable to amelioration by manipulation of the factors regulating alternative splicing. Therefore, this study also explores a similar strategy for reducing the severity of retinitis pigmentosa 11 (adRP11), a rare autosomal dominant disease caused by mutations in a splicing factor called pre-mRNA processing factor 31 (PRPF31). The protein produced from the normal PRPF31 allele is unable to meet retinal splicing demands required for the survival of photoreceptors and retinal pigment epithelia and thus patients experience progressive loss of vision. PRPF31 expression is negatively regulated by a transcription factor called CCR-NOT transcription factor subunit 3 (CNOT3). AOs were designed to knockdown CNOT3 transcript levels in an attempt to increase expression from the normal PRPF31 allele and thus improve the adRP11 phenotype. To date, several AOs targeting frame-shifting exons in CNOT3 have mediated CNOT3 exon skipping and increased PRPF31 levels. This study shows that AO-mediated manipulation of splicing factor concentrations has the potential to alter the splicing outcomes of disease-causing genes. However, global effects on the splicing of other genes needs to be considered and fully investigated in order to determine the therapeutic potential of this approach