Loss Of Survival Motor Neuron Research Articles

Protective effects of butyrate-based compounds on a mouse model for spinal muscular atrophy

Proximal spinal muscular atrophy (SMA) is a childhood-onset degenerative disease resulting from the selective loss of motor neurons in the spinal cord. SMA is caused by the loss of SMN1 (survival motor neuron 1) but retention of SMN2. The number of copies of SMN2 modifies disease severity in SMA patients as well as in mouse models, making SMN2 a target for therapeutics development. Sodium butyrate (BA) and its analog (4PBA) have been shown to increase SMN2 expression in SMA cultured cells. In this study, we examined the effects of BA, 4PBA as well as two BA prodrugs—glyceryl tributyrate (BA3G) and VX563—on the phenotype of SMNΔ7 SMA mice. Treatment with 4PBA, BA3G and VX563 but not BA beginning at PND04 significantly improved the lifespan and delayed disease end stage, with administration of VX563 also improving the growth rate of these mice. 4PBA and VX563 improved the motor phenotype of SMNΔ7 SMA mice and prevented spinal motor neuron loss. Interestingly, neither 4PBA nor VX563 had an effect on SMN expression in the spinal cords of treated SMNΔ7 SMA mice; however, they inhibited histone deacetylase (HDAC) activity and restored the normal phosphorylation states of Akt and glycogen synthase kinase 3β, both of which are altered by SMN deficiency in vivo. These observations show that BA-based compounds with favorable pharmacokinetics ameliorate SMA pathology possibly by modulating HDAC and Akt signaling.

Low levels of Survival Motor Neuron protein are sufficient for normal muscle function in the SMNΔ7 mouse model of SMA.

Spinal Muscular Atrophy (SMA) is an autosomal recessive disorder characterized by loss of lower motor neurons. SMA is caused by deletion or mutation of the Survival Motor Neuron 1 (SMN1) gene and retention of the SMN2 gene. The loss of SMN1 results in reduced levels of the SMN protein. SMN levels appear to be particularly important in motor neurons; however SMN levels above that produced by two copies of SMN2 have been suggested to be important in muscle. Studying the spatial requirement of SMN is important in both understanding how SMN deficiency causes SMA and in the development of effective therapies. Using Myf5-Cre, a muscle-specific Cre driver, and the Cre-loxP recombination system, we deleted mouse Smn in the muscle of mice with SMN2 and SMNΔ7 transgenes in the background, thus providing low level of SMN in the muscle. As a reciprocal experiment, we restored normal levels of SMN in the muscle with low SMN levels in all other tissues. We observed that decreasing SMN in the muscle has no phenotypic effect. This was corroborated by muscle physiology studies with twitch force, tetanic and eccentric contraction all being normal. In addition, electrocardiogram and muscle fiber size distribution were also normal. Replacement of Smn in muscle did not rescue SMA mice. Thus the muscle does not appear to require high levels of SMN above what is produced by two copies of SMN2 (and SMNΔ7).

Systems biology investigation of cAMP modulation to increase SMN levels for the treatment of spinal muscular atrophy.

Spinal muscular atrophy (SMA), a leading genetic cause of infant death worldwide, is an autosomal recessive disorder caused by the loss of SMN1 (survival motor neuron 1), which encodes the protein SMN. The loss of SMN1 causes a deficiency in SMN protein levels leading to motor neuron cell death in the anterior horn of the spinal cord. SMN2, however, can also produce some functional SMN to partially compensate for loss of SMN1 in SMA suggesting increasing transcription of SMN2 as a potential therapy to treat patients with SMA. A cAMP response element was identified on the SMN2 promoter, implicating cAMP activation as a step in the transcription of SMN2. Therefore, we investigated the effects of modulating the cAMP signaling cascade on SMN production in vitro and in silico. SMA patient fibroblasts were treated with the cAMP signaling modulators rolipram, salbutamol, dbcAMP, epinephrine and forskolin. All of the modulators tested were able to increase gem formation, a marker for SMN protein in the nucleus, in a dose-dependent manner. We then derived two possible mathematical models simulating the regulation of SMN2 expression by cAMP signaling. Both models fit well with our experimental data. In silico treatment of SMA fibroblasts simultaneously with two different cAMP modulators resulted in an additive increase in gem formation. This study shows how a systems biology approach can be used to develop potential therapeutic targets for treating SMA.

Morpholino antisense oligonucleotides targeting intronic repressor Element1 improve phenotype in SMA mouse models.

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by the loss of Survival Motor Neuron-1 (SMN1). In all SMA patients, a nearly identical copy gene called SMN2 is present, which produces low levels of functional protein owing to an alternative splicing event. To prevent exon-skipping, we have targeted an intronic repressor, Element1 (E1), located upstream of SMN2 exon 7 using Morpholino-based antisense oligonucleotides (E1(MO)-ASOs). A single intracerebroventricular injection in the relatively severe mouse model of SMA (SMNΔ7 mouse model) elicited a robust induction of SMN protein, and mean life span was extended from an average survival of 13 to 54 days following a single dose, consistent with large weight gains and a correction of the neuronal pathology. Additionally, E1(MO)-ASO treatment in an intermediate SMA mouse (SMN(RT) mouse model) significantly extended life span by ∼700% and weight gain was comparable with the unaffected animals. While a number of experimental therapeutics have targeted the ISS-N1 element of SMN2 pre-mRNA, the development of E1 ASOs provides a new molecular target for SMA therapeutics that dramatically extends survival in two important pre-clinical models of disease.

P.6.5 Improved antisense oligonucleotide design to suppress aberrant SMN2 gene transcript processing

Spinal muscular atrophy (SMA) is caused by loss of the Survival Motor Neuron 1 (SMN1) gene, resulting in reduced SMN protein. Humans possess the additional SMN2 gene (or genes) that does produce low level of full length SMN, but cannot adequately compensate for loss of SMN1 due to aberrant splicing. The majority of SMN2 gene transcripts lack exon 7 and the resultant SMNΔ7 mRNA is translated into an unstable and non-functional protein. Splice intervention therapies to promote exon 7 retention and increase amounts of full-length SMN2 transcript offer great potential as a treatment for SMA patients. Several splice silencing motifs in SMN2 have been identified as potential targets for antisense oligonucleotide mediated splice modification. A strong splice silencer is located downstream of exon 7 in SMN2 intron 7. Antisense oligonucleotides targeting this motif promoted SMN2 exon 7 retention in the mature SMN2 transcripts, with increased SMN expression detected in SMA fibroblasts. We report here systematic optimisation of phosphorodiamidate morpholino oligonucleotides (PMO) that promote exon 7 retention to levels that rescued the phenotype in a severe mouse model of SMA after intracerebroventricular delivery. Furthermore, the PMO gives the longest survival reported to date after a single dosing by ICV.

Spinal muscular atrophy: a motor neuron disorder or a multi‐organ disease?

Spinal muscular atrophy (SMA) is an autosomal recessive disorder that is the leading genetic cause of infantile death. SMA is characterized by loss of motor neurons in the ventral horn of the spinal cord, leading to weakness and muscle atrophy. SMA occurs as a result of homozygous deletion or mutations in Survival Motor Neuron-1 (SMN1). Loss of SMN1 leads to a dramatic reduction in SMN protein, which is essential for motor neuron survival. SMA disease severity ranges from extremely severe to a relatively mild adult onset form of proximal muscle atrophy. Severe SMA patients typically die mostly within months or a few years as a consequence of respiratory insufficiency and bulbar paralysis. SMA is widely known as a motor neuron disease; however, there are numerous clinical reports indicating the involvement of additional peripheral organs contributing to the complete picture of the disease in severe cases. In this review, we have compiled clinical and experimental reports that demonstrate the association between the loss of SMN and peripheral organ deficiency and malfunction. Whether defective peripheral organs are a consequence of neuronal damage/muscle atrophy or a direct result of SMN loss will be discussed.

Improved Antisense Oligonucleotide Design to Suppress Aberrant SMN2 Gene Transcript Processing: Towards a Treatment for Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is caused by loss of the Survival Motor Neuron 1 (SMN1) gene, resulting in reduced SMN protein. Humans possess the additional SMN2 gene (or genes) that does produce low level of full length SMN, but cannot adequately compensate for loss of SMN1 due to aberrant splicing. The majority of SMN2 gene transcripts lack exon 7 and the resultant SMNΔ7 mRNA is translated into an unstable and non-functional protein. Splice intervention therapies to promote exon 7 retention and increase amounts of full-length SMN2 transcript offer great potential as a treatment for SMA patients. Several splice silencing motifs in SMN2 have been identified as potential targets for antisense oligonucleotide mediated splice modification. A strong splice silencer is located downstream of exon 7 in SMN2 intron 7. Antisense oligonucleotides targeting this motif promoted SMN2 exon 7 retention in the mature SMN2 transcripts, with increased SMN expression detected in SMA fibroblasts. We report here systematic optimisation of phosphorodiamidate morpholino oligonucleotides (PMO) that promote exon 7 retention to levels that rescued the phenotype in a severe mouse model of SMA after intracerebroventricular delivery. Furthermore, the PMO gives the longest survival reported to date after a single dosing by ICV.

Restoration of SMN to Emx-1 expressing cortical neurons is not sufficient to provide benefit to a severe mouse model of Spinal Muscular Atrophy

Spinal Muscular Atrophy (SMA), an autosomal recessive neuromuscular disorder, is a leading genetic cause of infant mortality. SMA is caused by the homozygous loss of Survival Motor Neuron-1 (SMN1). However, low, but essential, levels of SMN protein are produced by a nearly identical copy gene called SMN2. Detailed analysis of neuromuscular junctions in SMA mice has revealed a selective vulnerability in a subset of muscle targets, suggesting that while SMN is reduced uniformly, the functional deficits manifest sporadically. Additionally, in severe SMA models, it is becoming increasing apparent that SMA is not restricted solely to motor neurons. Rather, additional tissues including the heart, vasculature, and the pancreas contribute to the complete SMA-associated pathology. Recently, transgenic models have been utilized to examine the tissue-specific requirements of SMN, including selective depletion and restoration of SMN in motor neurons. To determine whether the cortical neuronal populations expressing the Emx-1 promoter are involved in SMA pathology, we generated a novel SMA mouse model in which SMN expression was specifically induced in Emx-1 expressing cortical neurons utilizing an Emx-1-Cre transgene. While SMN expression was robust in the central nervous system as expected, SMA mice did not live longer. Weight and time-to-right motor function were not significantly improved.

Decreasing Disease Severity in Symptomatic,Smn−/−;SMN2+/+, Spinal Muscular Atrophy Mice Following scAAV9-SMN Delivery

Spinal muscular atrophy (SMA), an autosomal recessive neuromuscular disorder, is the leading genetic cause of infant mortality. SMA is caused by the homozygous loss of Survival Motor Neuron-1 (SMN1). In humans, a nearly identical copy gene is present, SMN2. SMN2 is retained in all SMA patients and encodes the same protein as SMN1. However, SMN1 and SMN2 differ by a silent C-to-T transition at the 5' end of exon 7, causing alternative splicing of SMN2 transcripts and low levels of full-length SMN. SMA is monogenic and therefore well suited for gene-replacement strategies. Recently, self-complementary adeno-associated virus (scAAV) vectors have been used to deliver the SMN cDNA to an animal model of disease, the SMNΔ7 mouse. In this study, we examine a severe model of SMA, Smn(-/-);SMN2(+/+), to determine whether gene replacement is viable in a model in which disease development begins in utero. Using two delivery paradigms, intracerebroventricular injections and intravenous injections, we delivered scAAV9-SMN and demonstrated a two to four fold increase in survival, in addition to improving many of the phenotypic parameters of the model. This represents the longest extension in survival for this severe model for any therapeutic intervention and suggests that postsymptomatic treatment of SMA may lead to significant improvement of disease severity.

Direct central nervous system delivery provides enhanced protection following vector mediated gene replacement in a severe model of Spinal Muscular Atrophy

Spinal Muscular Atrophy (SMA), an autosomal recessive neuromuscular disorder, is the leading genetic cause of infant mortality. SMA is caused by the homozygous loss of Survival Motor Neuron-1 (SMN1). SMA, however, is not due to complete absence of SMN, rather a low level of functional full-length SMN is produced by a nearly identical copy gene called SMN2. Despite SMN’s ubiquitous expression, motor neurons are preferentially affected by low SMN levels. Recently gene replacement strategies have shown tremendous promise in animal models of SMA. In this study, we used self-complementary Adeno Associated Virus (scAAV) expressing full-length SMN cDNA to compare two different routes of viral delivery in a severe SMA mouse model. This was accomplished by injecting scAAV9-SMN vector intravenously (IV) or intracerebroventricularly (ICV) into SMA mice. Both routes of delivery resulted in a significant increase in lifespan and weight compared to untreated mice with a subpopulation of mice surviving more than 200days. However, the ICV injected mice gained significantly more weight than their IV treated counterparts. Likewise, survival analysis showed that ICV treated mice displayed fewer early deaths than IV treated animals. Collectively, this report demonstrates that route of delivery is a crucial component of gene therapy treatment for SMA.

Transgenic inactivation of murine myostatin does not decrease the severity of disease in a model of Spinal Muscular Atrophy

Spinal Muscular Atrophy (SMA) is a devastating neurodegenerative disease and is a leading genetic cause of infantile death. SMA is caused by the homozygous loss of Survival Motor Neuron-1 (SMN1). The presence of a nearly identical copy gene called SMN2 has led to the development of several strategies that are designed to elevate SMN levels, and it is clear that SMN2 is an important modifier gene. However, the possibility exists that SMN-independent strategies to lessen the severity of the SMA phenotype could provide insight into disease development as well as aid in the identification of potential therapeutic targets. Muscle enhancement has been considered an interesting target for a variety of neurodegenerative diseases, including SMA. Previously we have shown in SMA mice that delivery of recombinant follistatin resulted in an extension in survival and a general lessening of disease severity. Follistatin is known to functionally block myostatin (MSTN), a potent inhibitor of muscle development. However, follistatin is a multifaceted protein involved in a variety of cellular pathways. To determine whether MSTN inhibition was the primary pathway associated with the previously reported follistatin results, we generated an animal model of SMA in which Mstn was genetically inactivated. In this report we characterize the novel SMA/Mstn model and demonstrate that Mstn inactivation does not significantly enhance muscle development in neonatal animals, nor does it result in an amelioration of the SMA phenotype.

Bifunctional RNAs Targeting the Intronic Splicing Silencer N1 Increase SMN Levels and Reduce Disease Severity in an Animal Model of Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss of survival motor neuron-1 (SMN1). A nearly identical copy gene, SMN2, is present in all SMA patients. Although the SMN2 coding sequence has the potential to produce full-length SMN, nearly 90% of SMN2-derived transcripts are alternatively spliced and encode a truncated protein. SMN2, however, is an excellent therapeutic target. Previously, we developed antisense-based oligonucleotides (bifunctional RNAs) that specifically recruit SR/SR-like splicing factors and target a negative regulator of SMN2 exon-7 inclusion within intron-6. As a means to optimize the antisense sequence of the bifunctional RNAs, we chose to target a potent intronic repressor downstream of SMN2 exon 7, called intronic splicing silencer N1 (ISS-N1). We developed RNAs that specifically target ISS-N1 and concurrently recruit the modular SR proteins SF2/ASF or hTra2β1. RNAs were directly injected in the brains of SMA mice. Bifunctional RNA injections were able to elicit robust induction of SMN protein in the brain and spinal column of neonatal SMA mice. Importantly, hTra2β1-ISS-N1 and SF2/ASF-ISS-N1 bifunctional RNAs significantly extended lifespan and increased weight in the SMNΔ7 mice. This technology has direct implications for SMA therapy and provides similar therapeutic strategies for other diseases caused by aberrant splicing.

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

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.

Combination of SMNTrans-Splicing and a Neurotrophic Factor Increases the Life Span and Body Mass in a Severe Model of Spinal Muscular Atrophy

Spinal muscular atrophy (SMA), a neurodegenerative disease, is the second most common genetic disorder and the leading genetic cause of infantile death. SMA arises from the loss of Survival Motor Neuron-1 (SMN1), leading to degeneration of lower motor neurons and, consequently, the atrophy of voluntary muscles. A duplicated copy gene called SMN2 exists in humans. SMN2 is unable to fully compensate for the loss of SMN1 because it produces very low levels of functional SMN protein due to an alternative splicing event. A C/T transition in SMN2 exon 7 results in a transcript lacking exon 7 and, therefore, creates a truncated SMN protein that cannot fully compensate for the loss of SMN1. However, SMN2 is an ideal target for therapeutic strategies that redirect this critical splicing event. Previously, we developed the first trans-splicing strategy to increase the full-length mRNA and functional SMN protein from the SMN2 gene. To improve the trans-splicing efficacy, we then developed a single-vector system that expressed a trans-splicing RNA (tsRNA) and an antisense blocking the downstream splice site. This single vector greatly enhanced trans-splicing of SMN2 transcripts in vitro and in vivo. In this report, we have added a neurotrophic factor [insulin-like growth factor (IGF)-1] to this single vector to determine whether neuroprotection and SMN induction provide greater protection in an SMA animal model. Intracerebroventricular injection of the trans-splicing/IGF vector significantly increased SMN protein in brain and spinal cord of SMAΔ7 mice and lessened the severity of disease in a more severe mouse model as evidenced by an extension of life span and increased body mass.

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.

Delivery of bifunctional RNAs that target an intronic repressor and increase SMN levels in an animal model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a motor neuron disease caused by the loss of survival motor neuron-1 (SMN1). A nearly identical copy gene, SMN2, is present in all SMA patients, which produces low levels of functional protein. Although the SMN2 coding sequence has the potential to produce normal, full-length SMN, approximately 90% of SMN2-derived transcripts are alternatively spliced and encode a truncated protein lacking the final coding exon (exon 7). SMN2, however, is an excellent therapeutic target. Previously, we developed bifunctional RNAs that bound SMN exon 7 and modulated SMN2 splicing. To optimize the efficiency of the bifunctional RNAs, a different antisense target was required. To this end, we genetically verified the identity of a putative intronic repressor and developed bifunctional RNAs that target this sequence. Consequently, there is a 2-fold mechanism of SMN induction: inhibition of the intronic repressor and recruitment of SR proteins via the SR recruitment sequence of the bifunctional RNA. The bifunctional RNAs effectively increased SMN in human primary SMA fibroblasts. Lead candidates were synthesized as 2'-O-methyl RNAs and were directly injected in the central nervous system of SMA mice. Single-RNA injections were able to illicit a robust induction of SMN protein in the brain and throughout the spinal column of neonatal SMA mice. In a severe model of SMA, mean life span was extended following the delivery of bifunctional RNAs. This technology has direct implications for the development of an SMA therapy, but also lends itself to a multitude of diseases caused by aberrant pre-mRNA splicing.

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

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.

A Negatively-Acting Bifunctional RNA Increases Survival Motor Neuron in vitro and in vivo

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.

Nonsense-mediated messenger RNA decay of survival motor neuron 1 causes spinal muscular atrophy

Autosomal recessive proximal spinal muscular atrophy (SMA) is a neurodegenerative disorder resulting from functional loss of survival motor neuron 1 (SMN1). Homozygous absence of SMN1 due to deletion or gene conversion accounts for about 96% of SMA cases. In the remaining 4%, subtle SMN1 mutations are commonly identified. Here, we describe two novel intragenic SMN1 mutations in three type I SMA individuals: a point mutation in exon 3 (c.469C > T) and a substitution in intron 4 (c.628-140A > G). In-vivo splicing assays demonstrated that the intronic substitution creates a novel splice donor site, culminating in aberrant splicing and insertion of 65 bp from intron 4 between exons 4 and 5 in SMN1 transcripts (c.627_628ins65). Both mutations render SMN1 transcripts susceptible to nonsense-mediated mRNA decay (NMD), resulting in mRNA degradation, insufficient SMN protein levels and development of an SMA phenotype. Treatment of patient cell lines with the translation inhibitors puromycin and emetine markedly increased the levels of mutant SMN1 transcripts. A similar effect was observed after siRNA-mediated knockdown of UPF1, a factor essential for NMD. This study provides first evidence that NMD of SMN1 transcripts is responsible for the molecular basis of disease in a subset of SMA patients.

An SMA Project Report: Neural Cell-Based Assays Derived from Human Embryonic Stem Cells

Human embryonic stem (ES) cells are promising resources for developing new treatments for neurodegenerative diseases. Spinal muscular atrophy (SMA) is one of the leading causes of childhood paralysis and infant mortality. SMA is caused by inactivation of the survival motor neuron-1 (SMN1) gene. The nearly identical SMN2 gene contains a silent polymorphism that disrupts splicing and as a result cannot compensate for loss of SMN1. The SMA Project was established by the National Institute of Neurological Disorders and Stroke (NINDS) as a pilot effort to establish a fully transparent coalition between academics, industry, and government to create a centralized network of shared resources and information to identify and test new SMA therapeutics. As one of the funded projects, the work described here tested the feasibility of generating a SMA cell-based assay using neural lineages derived from human ES cells approved for National Institutes of Health (NIH)-funded research. Minigene cassettes were constructed, employing firefly luciferase or green fluorescent protein (GFP) as reporters for splicing efficiency of SMN1 and/or SMN2 under the control of the SMN1, SMN2, or cytomegalovirus (CMV) promoters. Transient transfection of proliferating neuroprogenitors in a 96-well format with plasmid DNA or adenoviral vectors showed differential levels that correlated with the splicing minigene and the promoter used; luciferase activities with SMN1 splicing minigenes were higher than SMN2, and the CMV promoter generated higher levels of activity than the SMN1 and SMN2 promoters. Our results indicate that human ES cell-derived neuroprogenitors provide a promising new primary cell source for assays of new therapeutics for neurodegenerative diseases.