Recipient Organization
IOWA STATE UNIVERSITY
S. AND 16TH ELWOOD
AMES,IA 50011
Performing Department
VETERINARY MEDICINE
Non Technical Summary
Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality. Here, we will characterize a novel intronic element that plays a critical role in pathogenesis of SMA. In addition, we will use this element as a target for the antisense-mediated correction of SMA gene.
Animal Health Component
20%
Research Effort Categories
Basic
60%
Applied
20%
Developmental
20%
Goals / Objectives
Spinal muscular atrophy (SMA) is caused by loss of Survival of Motor Neuron 1 (SMN1) gene. A nearly identical copy of the gene, SMN2, fails to compensate for the loss SMN1 due to a C-to-T mutation at position 6 in exon 7 (C6U mutation in mRNA). The C6U mutation leads to exon 7 exclusion during pre-mRNA splicing, resulting in the synthesis of a truncated nonfunctional protein. It is generally believed that correction of SMN2 exon 7 splicing holds the promise for cure. For this we need to identify a gene-specific target. Towards this aim, we have discovered an intronic inhibitory element that we have named as Intronic Splicing Silencer N1 (abbreviated as ISS-N1). Blocking of ISS-N1 by an antisense oligonucleotide (or ASO) fully corrects SMN2 exon 7 splicing in patient cells. This grant proposal is aimed at validating ISS-N1 as a therapeutic target for ASO-mediated gene correction.
Project Methods
In aim 1, we will test ASOs that are active at low concentrations but are not toxic at higher concentrations. Based on mutational analyses, ISS-N1 is comprised of a fifteen-nucleotide-long sequence. To block this region, we have begun to test shorter ASOs from 10-to-15 nucleotides. In fact, we have already obtained a 15mer ASO that fully restores exon 7 inclusion when transfected (50 nM) with SMN2 minigene. A 12mer ASO was also able to substantially restore exon 7 inclusion. It is possible that shifting the annealing positions will help to increase the efficiency of shorter ASOs. The results of structure probing will be useful to find such annealing positions that fall within a loop. Although we will perform our initial experiments with 2'-O-methyl (2'-O-Me) phosphorothioate ASOs, there are other known modifications that could be substituted for certain improvements. Some of these modifications may render better annealing properties, higher specificity, higher nuclease
resistance and efficient nuclear transport. In aim 2, we will perform experiments in normal mice carrying human SMN2. These mice are ideally suited for checking the effect of compounds including ASOs on splicing of human SMN2. The splicing pattern of human SMN2 will be determined by RT-PCR. To compare the relative efficacy of delivery and antisense effect, up to 20 ASOs (selected from Aim 1) with different chemistry and/or length will be tested. Control experiments will be carried out to examine any illicit or toxic effects of ASOs on growth and development of mice. Final experiments will be done in SMA mice. We will administer selected ASOs in SMA mice and determine the changes in the level of full-length SMN in different tissues, including spinal cord and muscle. We will also monitor growth, development and progression of disease in the ASO-treated SMA mice. We will determine the synergistic effect by administering efficient ASOs in SMA mice treated with other drugs that have
previously shown stimulatory effect on exon 7 inclusion from SMN2.