Lentivector-mediated SMN replacement in a mouse model of spinal muscular atrophy

Azzouz, Mimoun; Le, Thanh; Ralph, G. Scott; Walmsley, Lucy E.; Monani, Umrao R.; Lee, Debbie C P; Wilkes, Fraser; Mitrophanous, Kyriacos; Kingsman, Susan M.; Burghes, Arthur; Mazarakis, Nicholas D.

doi:10.1172/jci200422922

Cited by 41 publications

(57 citation statements)

References 24 publications

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“…This virus is an ideal candidate for gene therapy as it is replication deficient and certain AAV serotypes have been shown to have high tropism for both muscles and neurons and can use retrograde transport to travel from muscle to neurons in vivo. Previous studies have successfully used the retrograde transport ability of a pseudotyped lentivirus to deliver SMN cDNA (Azzouz et al, 2004). Although these data have direct implications for SMA therapy, they could also be transitioned to be applicable to many disease states in which it would be desirable to skip an exon, such as in muscular dystrophy, where skipping an exon or set of exons may return the proper reading frame.…”

Section: Discussionmentioning

confidence: 99%

A Negatively Acting Bifunctional RNA Increases Survival Motor Neuron Both In Vitro and In Vivo

Dickson

Osman²,

Lorson³

2008

Human Gene Therapy

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Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder and is the leading genetic cause of infant mortality. SMA is caused by the loss of survival motor neuron-1 (SMN1). In humans, a nearly identical copy gene is present called SMN2, but this gene cannot compensate for the loss of SMN1 because of a single silent nucleotide difference in SMN2 exon 7. This single-nucleotide difference attenuates an exonic splice enhancer, resulting in the production of an alternatively spliced isoform lacking exon 7, which is essential for protein function. SMN2, however, is a critical disease modifier and is an outstanding target for therapeutic intervention because all SMA patients retain SMN2 and SMN2 maintains the same coding sequence as SMN1. Therefore, compounds or molecules that increase SMN2 exon 7 inclusion hold great promise for SMA therapeutics. Bifunctional RNAs have been previously used to increase SMN protein levels and derive their name from the presence of two domains: an antisense RNA sequence specific to the target RNA and an untethered RNA segment that serves as a binding platform for splicing factors. This study was designed to develop negatively acting bifunctional RNAs that recruit hnRNPA1 to exon 8 and block the general splicing machinery from the exon 8. By blocking the downstream splice site, this could competitively favor the inclusion of SMN exon 7 and therefore increase full-length SMN production. Here we identify a bifunctional RNA that stimulated full-length SMN expression in a variety of cell-based assays including SMA patient fibroblasts. Importantly, this molecule was also able to induce SMN expression in a previously described mouse model of SMA and demonstrates a novel therapeutic approach for SMA as well as a variety of diseases caused by a defect in splicing.

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Section: Discussionmentioning

confidence: 99%

A Negatively Acting Bifunctional RNA Increases Survival Motor Neuron Both In Vitro and In Vivo

Dickson

Osman²,

Lorson³

2008

Human Gene Therapy

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show abstract

“…The blot was then stripped and re-probed against actin as a loading control motor neuron death occurs early in prenatal development and, in turn, this SMN-deWciency eVectively predetermines the fate of motor neurons. This model could account for why recent avenues into SMA therapies show elevated SMN protein levels, but result in a less robust eVect regarding overall survival and correction of the disease phenotype (Azzouz et al 2004;Haddad et al 2003). Regardless of the therapeutic mechanism, it will be critical to address when SMN levels must be elevated and what the speciWc role of SMN is in aVected tissues.…”

Section: Discussionmentioning

confidence: 99%

Novel aminoglycosides increase SMN levels in spinal muscular atrophy fibroblasts

et al. 2006

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Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality. SMA is caused by the homozygous absence of survival motor neuron-1 (SMN1). SMN2, a nearly identical copy gene, is retained in all SMA patients and encodes an identical protein as SMN1; however, SMN1 and SMN2 differ by a silent C to T transition which results in the production of an alternatively spliced isoform (SMNDelta7), which encodes a defective protein, demonstrating that the absence of the short peptide encoded by SMN exon 7 is critical in SMA development. Previously, we have shown that for some functions heterologous sequences can compensate for the exon 7 peptide, suggesting that the SMN C-terminus functions non-specifically. Consistent with this hypothesis, we now identify novel aminoglycosides that can induce SMN protein levels in patient fibroblasts. This hypothesis was supported, in part, by a novel fluorescent SMN read-through assay. Interestingly, however, through the development of a SMN exon 7-specific antibody, results suggested that levels of normal full-length SMN might also be elevated by aminoglycoside treatment. These results demonstrate that the compounds that promote read-through may provide an alternative platform for the discovery of compounds that induce SMN protein levels.

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“…Potentially the developmental signal that will eventually trigger motor neuron death occurs early in prenatal development and, in turn, this SMN deficiency effectively predetermines the fate of motor neurons. This model could account for why recent avenues into SMA therapies show elevated SMN protein levels but result in a less robust effect regarding overall survival and correction of the disease phenotype [34,35]. While the experiments described in this report have immediate implications for the development of an SMA therapy, the results could be used as a model for a broad range of genetic disorders in which correcting a splicing defect would restore functionality to a disease-causing gene by effectively being able to promote the inclusion of specific exons that are misprocessed in the disease state.…”

Section: Discussionmentioning

confidence: 99%

Stimulating Full-Length SMN2 Expression by Delivering Bifunctional RNAs via a Viral Vector

et al. 2006

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Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder that is the leading genetic cause of infant mortality. SMA is caused by the loss of survival motor neuron-1 (SMN1). In humans, a nearly identical copy gene is present, called SMN2. SMN2 is retained in all SMA patients and encodes an identical protein compared to SMN1. However, a single silent nucleotide difference in SMN2 exon 7 results in the production of a spliced isoform (called SMNDelta7) that encodes a nonfunctional protein. The presence of SMN2 represents a unique therapeutic target since SMN2 has the capacity to encode a fully functional protein. Here we describe an in vivo delivery system for short bifunctional RNAs that modulate SMN2 splicing. Bifunctional RNAs derive their name from the presence of two domains: an antisense RNA sequence specific to a target RNA and an untethered RNA segment that serves as a binding platform for splicing factors. Plasmid-based and recombinant adeno-associated virus vectors were developed that expressed bifunctional RNAs that stimulated SMN2 exon 7 inclusion and full-length SMN protein in patient fibroblasts. These experiments provide a mechanism to modulate splicing from a variety of genetic contexts and demonstrate directly a novel therapeutic approach for SMA.

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Lentivector-mediated SMN replacement in a mouse model of spinal muscular atrophy

Cited by 41 publications

References 24 publications

A Negatively Acting Bifunctional RNA Increases Survival Motor Neuron Both In Vitro and In Vivo

A Negatively Acting Bifunctional RNA Increases Survival Motor Neuron Both In Vitro and In Vivo

Novel aminoglycosides increase SMN levels in spinal muscular atrophy fibroblasts

Stimulating Full-Length SMN2 Expression by Delivering Bifunctional RNAs via a Viral Vector

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