Article

Restoration of SMN function: delivery of a trans-splicing RNA re-directs SMN2 pre-mRNA splicing.

Department of Veterinary Pathobiology, Bond Life Sciences Center, University of Missouri, Columbia, Missouri 65211-7310, USA.
Molecular Therapy (Impact Factor: 6.43). 09/2007; 15(8):1471-8. DOI: 10.1038/sj.mt.6300222
Source: PubMed

ABSTRACT Spinal muscular atrophy (SMA) is caused by loss of survival motor neuron-1 (SMN1). A nearly identical copy gene called SMN2 is present in all SMA patients; however SMN2 produces low levels of functional protein due to alternative splicing. Recently a therapeutic approach has been developed referred to as trans-splicing. Conceptually, this strategy relies upon pre-messenger RNA (pre-mRNA) splicing occurring between two separate molecules: (i) the endogenous target RNA and (ii) the therapeutic RNA that provides the correct RNA sequence via a trans-splicing event. SMN trans-splicing RNAs were initially examined and expressed from a plasmid-backbone and shown to re-direct splicing from a SMN2 mini-gene as well as from endogenous transcripts. Subsequently, recombinant adeno-associated viral vectors were developed that expressed and delivered trans-splicing RNAs to SMA patient fibroblasts. In the severe SMA patient fibroblasts, SMN2 splicing was redirected via trans-splicing to produce increased levels of full-length SMN mRNA and total SMN protein levels. Finally, small nuclear ribonucleoprotein (snRNP) assembly, a critical function of SMN, was restored to SMN-deficient SMA fibroblasts following treatment with the trans-splicing vector. Together these results demonstrate that the alternatively spliced SMN2 exon 7 is a tractable target for replacement by trans-splicing.

0 Bookmarks
 · 
100 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: GNE myopathy is a rare neuromuscular autosomal recessive disease, resulting from mutations in the gene UDP N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). The most frequent mutation is the single homozygous missense mutation, M712T-the Middle Eastern mutation-located ten amino acids before the end of the protein. We have used an adeno-associated virus (AAV)-based trans-splicing (TS) vector as a gene therapy tool to overcome this mutation by replacing the mutated last exon of GNE by the wild-type exon while preserving the natural endogenous regulatory machinery. We have designed relevant plasmids directed either to mouse or to human GNE. Following transfection of C2C12 murine muscle cells with the mouse TS vectors, we have been able to detect by nested RT-PCR trans-spliced molecules carrying the wild-type exon 12 of GNE. Similarly, transfection of HEK293 human cells with the human-directed TS vectors resulted in the generation of trans-spliced human GNE RNA molecules. Furthermore, infection of primary muscle cells from a GNE myopathy patient carrying the homozygous M712T mutation, with an AAV8-based viral vector carrying a human-directed TS construct, resulted in the generation of wild-type GNE transcripts in addition to the mutated ones. These studies provide a proof of concept that the TS approach could be used to partially correct the Middle Eastern mutation in GNE myopathy patients. These results provide the basis for in vivo research in animal models using the AAV platform with TS plasmids as a potential genetic therapy for GNE myopathy.
    Neuromolecular medicine 11/2013; 16(2). · 5.00 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Spinal muscular atrophy (SMA) is a debilitating neuromuscular disease caused by the loss of survival of motor neuron (SMN) protein. Previously we demonstrated that ISIS 396443, an antisense oligonucleotide (ASO) targeted to the SMN2 pre-mRNA, is a potent inducer of SMN2 exon 7 inclusion and SMN protein expression, and improves function and survival of mild and severe SMA mouse models. Here we demonstrate that ISIS 396443 is the most potent ASO in central nervous system (CNS) tissues of adult mice, compared to several other chemically modified ASOs. We evaluated methods of ISIS 396443 delivery to the CNS and characterized its pharmacokinetics and pharmacodynamics in rodents and non-human primates (NHPs). Intracerebroventricular (ICV) bolus injection is a more efficient method of delivering ISIS 396443 to the CNS of rodents, compared to ICV infusion. For both methods of delivery, the duration of ISIS-396443-mediated SMN2 splicing correction is long-lasting, with maximal effects still observed 6 months after treatment discontinuation. Administration of ISIS 396443 to the CNS of NHPs by a single intrathecal bolus injection results in widespread distribution throughout the spinal cord. Based upon these preclinical studies, we have advanced ISIS 396443 into clinical development.
    Journal of Pharmacology and Experimental Therapeutics 04/2014; · 3.89 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Spinal muscular atrophy (SMA) is an autosomal recessive genetic disease, which causes death of motor neurons in the anterior horn of spinal cord. Genetic cause of SMA is the deletion or mutation of SMN1 gene, which encodes the SMN protein. Although SMA patients include SMN2 gene, a duplicate of SMN1 gene, predominant production of exon 7 skipped isoform from SMN2 pre-mRNA fails to rescue SMA patients. Here we show that hnRNP M, a member of hnRNPs protein family, when knocked down, promote exon 7 skipping of both SMN2 and SMN1 pre-mRNA. By contrast, overexpression of hnRNP M promotes exon 7 inclusion of both SMN2 and SMN1 pre-mRNA. Significantly, hnRNP M promotes exon 7 inclusion in SMA patient cells. Thus, we conclude that hnRNP M promotes exon 7 inclusion of both SMN1 and SMN2 pre-mRNA. . we also demonstrate that hnRNP M contacts an enhancer on exon 7, which was previously shown to provide binding site for tra2β. We present evidence that hnRNP M and tra2β contact overlapped sequence on exon 7 but with slightly different RNA sequence requirements. In addition, hnRNP M promotes U2AF65 recruitment on the flanking intron of exon 7. We conclude that hnRNP M promotes exon 7 inclusion of SMN1 and SMN2 pre-mRNA through targeting an enhancer on exon 7 through recruiting U2AF65. Our results provide a clue that hnRNP M is a potential therapeutic target for SMA.
    Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 04/2014; · 5.44 Impact Factor

Full-text (2 Sources)

Download
44 Downloads
Available from
May 26, 2014