Human Molecular Genetics Advance Access published online on February 19, 2009
Human Molecular Genetics, doi:10.1093/hmg/ddp076
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Delivery of bifunctional RNAs that target an intronic repressor and increase SMN levels in an animal model of spinal muscular atrophy
1 Department of Molecular Microbiology and Immunology 2 Department of Veterinary Pathobiology, Bond Life Sciences Center, University of Missouri, Columbia, MO, 65211
* Corresponding author: Christian L. Lorson, Department of Veterinary Pathobiology, Bond Life Sciences Center, 1201 Rollins Road, Room 471G, University of Missouri, Columbia, MO 65211-7310, Tel: (573) 884-2219, Fax: (573) 884-9395, Email: lorsonc{at}missouri.edu
Received January 12, 2009; Revised February 9, 2009; Accepted February 12, 2009
Spinal muscular atrophy (SMA) is a motor neuron disease caused by the loss of survival motor neuron-1 (SMN1). A nearly identical copy gene, SMN2, is present in all SMA patients, which produces low levels of functional protein. Although the SMN2 coding sequence has the potential to produce normal, full-length SMN, approximately 90% of SMN2-derived transcripts are alternatively spliced and encode a truncated protein lacking the final coding exon (exon 7). SMN2, however, is an excellent therapeutic target. Previously, we developed bifunctional RNAs that bound SMN exon 7 and modulated SMN2 splicing. To optimize the efficiency of the bifunctional RNAs, a different anti-sense target was required. To this end, we genetically verified the identity of a putative intronic repressor and developed bi-functional RNAs that target this sequence. Consequently, there is a two-fold mechanism of SMN induction: inhibition of the intronic repressor and recruitment of SR proteins via the SR-recruitment sequence of the bifunctional RNA. The bifunctional RNAs effectively increased SMN in human primary SMA fibroblasts. Lead candidates were synthesized as 2'-O-methyl RNAs and were directly injected in the central nervous system of SMA mice. Single RNA injections were able to illicit a robust induction of SMN protein in the brain and throughout the spinal column of neonatal SMA mice. In a severe model of SMA, mean life span was extended following delivery of bifunctional RNAs. This technology has direct implications for the development of a SMA therapy, but also lends itself to a multitude of diseases caused by aberrant pre-mRNA splicing.
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