Spinal muscular atrophy and the therapeutic application of trans-splicing RNA

Abstract

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Development of RNA modalities to re-direct pathogenic pre-mRNA splicing events is the key to curing monogenic diseases. Improving the efficiency of these molecules in vivo is critical as they move towards clinical applications. Spinal muscular atrophy (SMA) is caused by loss of SMN1. A nearly identical copy gene called SMN2 produces low levels of functional protein due to alternative splicing. This research characterized critical exon splicing elements involved and was later utilized in the development of successful molecular therapy. Then a trans-splicing RNA (tsRNA) that re-directed SMN2 splicing was designed that contains elements of the first study. Next this study demonstrates reducing the competition between endogenous splices sites enhanced the efficiency of trans-splicing. A single vector system was developed that expressed the SMN tsRNA and a splice-site blocking antisense (ASO-tsRNA). The ASOtsRNA vector significantly elevated SMN levels in primary SMA patient fibroblasts, within the central nervous system of SMA mice and increased SMNdependent in vitro snRNP assembly. These results demonstrate that the ASOtsRNA strategy provides insight into the trans-splicing mechanism and a means of significantly enhancing SMA trans-splicing activity in vivo.