Development of a Single Vector System that Enhances Trans-Splicing of SMN2 Transcripts

Coady, Tristan H.; Shababi, Monir; Passini, Marco A.; Lorson, Christian L.

doi:10.1371/journal.pone.0003468

Cited by 74 publications

(67 citation statements)

References 28 publications

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“…Plasmids and Cloning pM13, pMU3, Int3 constructs, and Int4 11 antisense are described previously (Coady et al 2008(Coady et al , 2007. The Int4 11 was cloned into BsrGI-MluI sites of Int3 construct to make Int3-4 11 .…”

Section: Methodsmentioning

confidence: 99%

“…SMNΔ7 mice (Smn −/− , SMN2 +/+ , SMNΔ7 +/+ ) were genotyped at the day of birth (P1) as described previously and injected at P2 (Coady et al 2008;Shababi et al 2010). A single ICV injection was performed as previously described (Baughan et al 2009;Coady et al 2008;Coady and Lorson 2010;Passini and Wolfe 2001;Shababi et al 2010). The injection stock solution contained 10 μg of Int3 plasmid DNA, 1 μl of D-(+)-glucose 20% (w/v) (Sigma, St. Louis, MO), 2 μl of 2.5-kDa linear polyethylenimine (PEI) homopolymer (150 mM; Polysciences Inc., Warrington, PA), and 1 μl of trypan blue (4%) saline (Sigma).…”

Section: Animal Injectionsmentioning

confidence: 99%

“…Our initial trans-splicing RNA (tsRNA) targeted intron 6 and replaced SMN2 exon 7 with the functional exon 7 and thus, increased the fulllength SMN expression from the SMN2 gene (Coady et al 2007). To move towards an in vivo application, we improved the efficacy of the trans-splicing vector by expressing an antisense cassette that binds to the splice acceptor site of the downstream exon and thereby reducing the competition between the trans-splicing targeted region of the RNA and the proximal splice site (Coady et al 2008;Coady and Lorson 2010).…”

Section: Introductionmentioning

confidence: 99%

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Optimization of SMN Trans-Splicing Through the Analysis of SMN Introns

Shababi

Lorson

2011

J Mol Neurosci

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Spinal muscular atrophy (SMA), a neurodegenerative disease, is the leading genetic cause of infantile death and is caused by the loss of survival motor neuron 1 (SMN1). Humans carry a duplicated copy gene, SMN2, which produces very low levels of functional protein due to an alternative splicing event. This splicing difference is the reason that SMN2 cannot prevent SMA development when SMN1 is deleted. SMN2 generates a transcript lacking exon 7 and consequently gives rise to an unstable truncated SMN protein that cannot protect from SMA. To increase full-length SMN protein, we utilize a strategy referred to as trans-splicing. This strategy relies upon pre-mRNA splicing occurring between two separate molecules: (1) the endogenous target RNA and (2) the therapeutic RNA that provides the correct RNA sequence via a trans-splicing event. The initial trans-splicing RNA targeted intron 6 and replaced exon 7 with the SMN1 exon 7 in SMN2 pre-mRNA. To determine the most efficient intron for SMN trans-splicing event, a panel of trans-splicing RNA molecules was constructed. Each trans-splicing RNA molecule targets a specific intron within the SMN2 pre-mRNA and based on the target intron, replaces the downstream exons including exon 7. These constructs were examined by RT-PCR, immunofluorescence, and Western blotting. We have identified intron 3 as the most efficient intron to support trans-splicing in cellular assays. The intron 3 trans-splicing construct targets intron 3 and replaces exons 4-7 and was distinguished based on its ability to produce the highest level of the trans-spliced RNA and full-length SMN protein in SMA patient fibroblasts. The efficiency of the intron 3 construct was further improved by addition of an antisense that blocks the 3' splice site at the intron 4/exon 5 junction. Most importantly, intracerebroventricular injection of the Int3 construct into SMNΔ7 mice elevated the SMN protein levels in the central nervous system. This research demonstrates an alternative platform to correct genetic defects, including SMN expression and examines the molecular basis for trans-splicing.

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Section: Methodsmentioning

confidence: 99%

Section: Animal Injectionsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Optimization of SMN Trans-Splicing Through the Analysis of SMN Introns

Shababi

Lorson

2011

J Mol Neurosci

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“…A second approach is to modulate the endogenous SMN2 gene with small molecules that activate the SMN2 promoter (9)(10)(11)(12)(13)(14)(15)(16) or correct the SMN2 pre-mRNA splicing pattern (10,(17)(18)(19)(20)(21)(22). The alteration of SMN2 splicing also can be realized with antisense oligonucleotides and transsplicing RNAs (23)(24)(25)(26)(27). However, while modulating SMN2 in vitro increased SMN levels and reconstituted nuclear gems in SMA cell lines, efficacy studies with small molecule drugs have not translated to measurable improvements in the clinic (28).…”

Section: Introductionmentioning

confidence: 99%

CNS-targeted gene therapy improves survival and motor function in a mouse model of spinal muscular atrophy

Passini¹,

Bu²,

Roskelley³

et al. 2010

J. Clin. Invest.

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Spinal muscular atrophy (SMA) is a neuromuscular disease caused by a deficiency of survival motor neuron (SMN) due to mutations in the SMN1 gene. In this study, an adeno-associated virus (AAV) vector expressing human SMN (AAV8-hSMN) was injected at birth into the CNS of mice modeling SMA. Western blot analysis showed that these injections resulted in widespread expression of SMN throughout the spinal cord, and this translated into robust improvement in skeletal muscle physiology, including increased myofiber size and improved neuromuscular junction architecture. Treated mice also displayed substantial improvements on behavioral tests of muscle strength, coordination, and locomotion, indicating that the neuromuscular junction was functional. Treatment with AAV8-hSMN increased the median life span of mice with SMA-like disease to 50 days compared with 15 days for untreated controls. Moreover, injecting mice with SMA-like disease with a human SMN-expressing self-complementary AAV vector -a vector that leads to earlier onset of gene expression compared with standard AAV vectors -led to improved efficacy of gene therapy, including a substantial extension in median survival to 157 days. These data indicate that CNS-directed, AAV-mediated SMN augmentation is highly efficacious in addressing both neuronal and muscular pathologies in a severe mouse model of SMA.

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“…Such settings were reported for epidermolysis bullosa (COL7A1, KRT14, PLEC) (Murauer et al, 2010;Wally et al, 2010;Wally et al, 2008), Duchenne muscular dystrophy (Lorain et al, 2010), cystic fibrosis (Liu et al, 2002;Song et al, 2009), frontotemporal dementia with parkinsonism (Rodriguez-Martin et al, 2009), severe combined immunodeficiency (Zayed et al, 2007), spinal muscular atrophy (Coady et al, 2007), sickle cell anemia and ß-thalassemia (Kierlin-Duncan and Sullenger, 2007). First in vivo assays showed the functionality of SMaRT in a mouse model of spinal muscular atrophy (Coady et al, 2008;Coady and Lorson, 2010). However, SMaRT has also been shown to be functional for a number of other approaches like in vivo imaging (Walls et al, 2008), antibody and therapeutic protein production (Wang et al, 2009;Iwasaki et al, 2009) and suicide therapy in squamous cell carcinoma .…”

Section: Spliceosome Mediated Rna Trans-splicingmentioning

confidence: 79%