253. AAV Delivery of a Trans-Splicing RNA Re-Directs SMN2 Splicing and Results in Increased Full-Length SMN
Authors: Tristan H. Coady, Monir Shababi, Christian L. Lorson
Journal: Molecular Therapy (2006)

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. SMN2 produces low levels of functional protein, while nearly 90% of SMN2-derived transcripts are alternatively spliced and encode a truncated protein that lacks the final coding exon (exon 7). While SMN is ubiquitously expressed, motor neurons are especially sensitive to the low levels of SMN and this heightened sensitivity leads to the development of SMA. Even though the majority of SMN2-derived transcripts are alternatively spliced and encode a protein that is truncated and unstable, the presence of SMN2 represents a unique therapeutic target because the SMN2 gene has the capacity to encode a fully functional protein. SMN1 and SMN2 differ by 10 nucleotides, however, none of these non-polymorphic differences alter the SMN coding sequence. Rather, due to a subtle disruption of a critical pre-mRNA processing regulatory element, SMN2 transcripts are alternatively spliced. Therefore, by increasing the inclusion of SMN2 exon 7 at the pre- mRNA splicing step, this would result in the production of increased levels of full-length SMN transcript, and in turn, fully functional SMN protein.In this study, the goal was to develop a trans-splicing system that results in an increase in full-length SMN expression from the SMN2 gene. Recently a therapeutic approach has been developed referred to as trans-splicing. Conceptually, this strategy relies upon pre- mRNA splicing occurring between two different molecules: 1) the endogenous mutant RNA and 2) the therapeutic RNA that provides the correct RNA sequence via a trans-splicing event. We have identified trans-splicing RNAs that efficiently increase SMN full- length expression and result in elevated levels of SMN protein. The trans-splicing RNAs were initially examined and expressed from a plasmid-backbone and shown to re-direct splicing from an 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 re-directed to produce increased levels of full-length SMN and total SMN protein levels were similarly elevated following the delivery of trans-splicing RNAs.These experiments shed light on potential therapeutic avenues for SMA, but provide a mechanist tool that could be used to address a number of genetic disorders that are due to an alternative splicing event. 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. SMN2 produces low levels of functional protein, while nearly 90% of SMN2-derived transcripts are alternatively spliced and encode a truncated protein that lacks the final coding exon (exon 7). While SMN is ubiquitously expressed, motor neurons are especially sensitive to the low levels of SMN and this heightened sensitivity leads to the development of SMA. Even though the majority of SMN2-derived transcripts are alternatively spliced and encode a protein that is truncated and unstable, the presence of SMN2 represents a unique therapeutic target because the SMN2 gene has the capacity to encode a fully functional protein. SMN1 and SMN2 differ by 10 nucleotides, however, none of these non-polymorphic differences alter the SMN coding sequence. Rather, due to a subtle disruption of a critical pre-mRNA processing regulatory element, SMN2 transcripts are alternatively spliced. Therefore, by increasing the inclusion of SMN2 exon 7 at the pre- mRNA splicing step, this would result in the production of increased levels of full-length SMN transcript, and in turn, fully functional SMN protein. In this study, the goal was to develop a trans-splicing system that results in an increase in full-length SMN expression from the SMN2 gene. Recently a therapeutic approach has been developed referred to as trans-splicing. Conceptually, this strategy relies upon pre- mRNA splicing occurring between two different molecules: 1) the endogenous mutant RNA and 2) the therapeutic RNA that provides the correct RNA sequence via a trans-splicing event. We have identified trans-splicing RNAs that efficiently increase SMN full- length expression and result in elevated levels of SMN protein. The trans-splicing RNAs were initially examined and expressed from a plasmid-backbone and shown to re-direct splicing from an 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 re-directed to produce increased levels of full-length SMN and total SMN protein levels were similarly elevated following the delivery of trans-splicing RNAs. These experiments shed light on potential therapeutic avenues for SMA, but provide a mechanist tool that could be used to address a number of genetic disorders that are due to an alternative splicing event.