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In JoVE (1)
Other Publications (6)
- Molecular Therapy : the Journal of the American Society of Gene Therapy
- Molecular Therapy : the Journal of the American Society of Gene Therapy
- Biochemical and Biophysical Research Communications
- PloS One
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Wiley Interdisciplinary Reviews. RNA
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Articles by Tristan H. Coady in JoVE
JoVE Clinical and Translational Medicine
Доставка терапевтических агентов Через Intracerebroventricular (ICV) и внутривенного (IV) инъекций у мышей
1Department of Molecular Microbiology and Immunology, Bond Life Sciences Center, University of Missouri, 2Department of Biological Sciences, Columbia University, 3Department of Veterinary Pathobiology, Bond Life Sciences Center, University of Missouri
В этой статье показано два совершенно разных методов инъекций: 1) в головном мозге (intracerebroventricular) и 2) системные (внутривенное) введение терапевтических агентов в центральной нервной системы у новорожденных мышей.
Other articles by Tristan H. Coady on PubMed
Stimulating Full-length SMN2 Expression by Delivering Bifunctional RNAs Via a Viral Vector
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.
Restoration of SMN Function: Delivery of a Trans-splicing RNA Re-directs SMN2 Pre-mRNA Splicing
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.
The Wallerian Degeneration Slow (Wld(s)) Gene Does Not Attenuate Disease in a Mouse Model of Spinal Muscular Atrophy
Spinal muscular atrophy (SMA) is a severe neuromuscular disease characterized by loss of spinal alpha-motor neurons, resulting in the paralysis of skeletal muscle. SMA is caused by deficiency of survival motor neuron (SMN) protein levels. Recent evidence has highlighted an axon-specific role for SMN protein, raising the possibility that axon degeneration may be an early event in SMA pathogenesis. The Wallerian degeneration slow (Wld(s)) gene is a spontaneous dominant mutation in mice that delays axon degeneration by approximately 2-3 weeks. We set out to examine the effect of Wld(s) on the phenotype of a mouse model of SMA. We found that Wld(s) does not alter the SMA phenotype, indicating that Wallerian degeneration does not directly contribute to the pathogenesis of SMA development.
Development of a Single Vector System That Enhances Trans-splicing of SMN2 Transcripts
RNA modalities are developing as a powerful means to re-direct pathogenic pre-mRNA splicing events. Improving the efficiency of these molecules in vivo is critical as they move towards clinical applications. Spinal muscular atrophy (SMA) is caused by loss of SMN1. A nearly identical copy gene called SMN2 produces low levels of functional protein due to alternative splicing. We previously reported a trans-splicing RNA (tsRNA) that re-directed SMN2 splicing. Now we show that reducing the competition between endogenous splices sites enhanced the efficiency of trans-splicing. A single vector system was developed that expressed the SMN tsRNA and a splice-site blocking antisense (ASO-tsRNA). The ASO-tsRNA vector significantly elevated SMN levels in primary SMA patient fibroblasts, within the central nervous system of SMA mice and increased SMN-dependent in vitro snRNP assembly. These results demonstrate that the ASO-tsRNA strategy provides insight into the trans-splicing mechanism and a means of significantly enhancing trans-splicing activity in vivo.
Trans-splicing-mediated Improvement in a Severe Mouse Model of Spinal Muscular Atrophy
Spinal muscular atrophy is a leading genetic cause of infantile death and occurs in approximately 1/6000 live births. SMA is caused by the loss of Survival Motor Neuron-1 (SMN1), however, all patients retain at least one copy of a nearly identical gene called SMN2. While SMN2 and SMN1 are comprised of identical coding sequences, the majority of SMN2 transcripts are alternatively spliced, encoding a truncated protein that is unstable and nonfunctional. Considerable effort has focused upon modulating the SMN2 alternative splicing event since this would produce more wild-type protein. Recently we reported the development of an optimized trans-splicing system that involved the coexpression of a SMN2 trans-splicing RNA and an antisense RNA that blocks a downstream splice site in SMN2 pre-mRNA. Here, we demonstrate that in vivo delivery of the optimized trans-splicing vector increases an important SMN-dependent activity, snRNP assembly, in disease-relevant tissue in the SMA mouse model. A single injection of the vector into the intracerebral-ventricular space in SMA neonates also lessens the severity of the SMA phenotype in a severe SMA mouse model, extending survival approximately 70%. Collectively, these results provide the first in vivo demonstration that SMN2 trans-splicing leads to a lessening of the severity of the SMA phenotype and provide evidence for the power of this strategy for reprogramming genetic diseases at the pre-mRNA level.
SMN in Spinal Muscular Atrophy and SnRNP Biogenesis
Ribonucleoprotein (RNP) complexes function in nearly every facet of cellular activity. The spliceosome is an essential RNP that accurately identifies introns and catalytically removes the intervening sequences, providing exquisite control of spatial, temporal, and developmental gene expressions. U-snRNPs are the building blocks for the spliceosome. A significant amount of insight into the molecular assembly of these essential particles has recently come from a seemingly unexpected area of research: neurodegeneration. Survival motor neuron (SMN) performs an essential role in the maturation of snRNPs, while the homozygous loss of SMN1 results in the development of spinal muscular atrophy (SMA), a devastating neurodegenerative disease. In this review, the function of SMN is examined within the context of snRNP biogenesis and evidence is examined which suggests that the SMN functional defects in snRNP biogenesis may account for the motor neuron pathology observed in SMA.
