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

Department of Ophthalmology, Boston University, Boston, Massachusetts, United States
Molecular Therapy (Impact Factor: 6.23). 09/2007; 15(8):1471-8. DOI: 10.1038/
Source: PubMed


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.

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    • "Modulation of SMN levels in SMA tissue has been achieved by a variety of methodologies including and for example adenoassociated viral transduction [11] [27], antisense RNA technologies [7] [8] and histone deacetylase (HDAC) inhibitors [2] [4] [21]. One such HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA) significantly increases the level of SMN protein in SMA affected skeletal muscle [16] [18] and improves survival [18]. "
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    ABSTRACT: Spinal Muscular Atrophy (SMA), a primarily childhood form of motor neuron disease, is caused by reduced levels of a single, ubiquitously expressed protein: the Survival Motor Neuron (SMN) protein. Low levels of SMN cause motor neuron degeneration but recent reports describe effects of low SMN levels in multiple tissues and organs, including the vasculature. Previously we have reported a significant defect in the vascular beds of SMA affected skeletal muscle. Here we examine the effects of ubiquitously increasing SMN levels, via treatment with the histone deacetylase inhibitor SAHA, on this vascular defect in the Taiwanese: FVB.Cg-Tg(SMN2)2Hung Smn1(tm1Hung)/J mouse model of severe SMA. SAHA treatment resulted in an increase in the weight of SMA mice compared to untreated SMA mice and almost completely restored motor function at P10, the late symptomatic time point analysed. Vascular density in skeletal muscle was then assessed by morphological quantitation of immunofluorescence staining of vessels and quantitative fluorescent western blotting for a key endothelial protein PECAM-1. At P10 a severe vascular defect was present with a significant (P<0.01) ∼82% reduction in vascular density in SMA mice when compared to control littermates. SAHA significantly increases SMN levels and also increased vascular density in SMA mice (P<0.05), suggesting that the vascular defect in SMA mice is amendable to SAHA treatment.
    No preview · Article · Apr 2013 · Neuroscience Letters
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    • "Another example of successful use of trans-splicing in neurons in vivo is SMN exon 7 inclusion in the SMN2 transcript in a mouse model of spinal muscular atrophy (SMA) (18–20). In this paradigm, production of a functional SMN protein after trans-splicing has been inferred through changes in total SMN protein (18–20), but not demonstrated directly. "
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    ABSTRACT: Abnormal metabolism of the tau protein is central to the pathogenesis of a number of dementias, including Alzheimer's disease. Aberrant alternative splicing of exon 10 in the tau pre-mRNA resulting in an imbalance of tau isoforms is one of the molecular causes of the inherited tauopathy, FTDP-17. We showed previously in heterologous systems that exon 10 inclusion in tau mRNA could be modulated by spliceosome-mediated RNA trans-splicing (SMaRT). Here, we evaluated the potential of trans-splicing RNA reprogramming to correct tau mis-splicing in differentiated neurons in a mouse model of tau mis-splicing, the htau transgenic mouse line, expressing the human MAPT gene in a null mouse Mapt background.Trans-splicing molecules designed to increase exon 10 inclusion were delivered to neurons using lentiviral vectors. We demonstrate reprogramming of tau transcripts at the RNA level after transduction of cultured neurons or after direct delivery and long-term expression of viral vectors into the brain of htau mice in vivo. Tau RNA trans-splicing resulted in an increase in exon 10 inclusion in the mature tau mRNA. Importantly, we also show that the trans-spliced product is translated into a full-length chimeric tau protein. These results validate the potential of SMaRT to correct tau mis-splicing and provide a framework for its therapeutic application to neurodegenerative conditions linked to aberrant RNA processing.
    Full-text · Article · Mar 2013 · Human Molecular Genetics
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    • "Trans-splicing is a process by which two separately transcribed mRNAs are involved in a splicing event wherein a composite transcript is produced (reviewed in Wood et al., 2007). A trans-splicing approach promotes the inclusion of exon 7 in SMN2 mRNA transcripts in vitro and, to a lesser extent, increases SMN protein expression and function in SMA fibroblasts (Coady et al., 2007). By adding to the SMN2 trans-splicing vector a cassette that produces a small RNA that is complementary to the intron 7:exon 8 splice junction (the trans-splicing:antisense combination pMU3 vector), treatment with pMU3 resulted in a more marked increase in FL-SMN protein expression and small nuclear ribonucleoprotein (snRNP) assembly (an assayable function of SMN) than from the trans-splicing vector (Coady et al., 2008). "

    Preview · Article · Feb 2011 · Human gene therapy
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