Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality. Most SMA cases are associated with the low levels of SMN owing to deletion of Survival Motor Neuron 1 (SMN1). SMN2, a nearly identical copy of SMN1, fails to compensate for the loss of SMN1 due to predominant skipping of exon 7. Hence, correction of aberrant splicing of SMN2 exon 7 holds the potential for cure of SMA. Here we report an 8-mer antisense oligonucleotide (ASO) to have a profound stimulatory response on correction of aberrant splicing of SMN2 exon 7 by binding to a unique GC-rich sequence located within intron 7 of SMN2. We confirm that the splicing-switching ability of this short ASO comes with a high degree of specificity and reduced off-target effect compared to larger ASOs targeting the same sequence. We further demonstrate that a single low nanomolar dose of this 8-mer ASO substantially increases the levels of SMN and a host of factors including Gemin 2, Gemin 8, ZPR1, hnRNP Q and Tra2-β1 known to be down-regulated in SMA. Our findings underscore the advantages and unmatched potential of very short ASOs in splicing modulation in vivo.
survival motor neuron (SMN); SMN1; SMN2; alternative splicing; intron 7; exon 7; ISS-N1; GC-rich sequence; antisense oligonucleotide (ASO); 8-mer ASO; SMA
Humans have two nearly identical copies of the Survival Motor Neuron (SMN) gene, SMN1 and SMN2. In spinal muscular atrophy (SMA), SMN2 is not able to compensate for the loss of SMN1 due to exclusion of exon 7. Here we describe a novel inhibitory element located immediately downstream of the 5′ splice site in intron 7. We call this element intronic splicing silencer N1 (ISS-N1). Deletion of ISS-N1 promoted exon 7 inclusion in mRNAs derived from the SMN2 minigene. Underlining the dominant role of ISS-N1 in exon 7 skipping, abrogation of a number of positive cis elements was tolerated when ISS-N1 was deleted. Confirming the silencer function of ISS-N1, an antisense oligonucleotide against ISS-N1 restored exon 7 inclusion in mRNAs derived from the SMN2 minigene or from endogenous SMN2. Consistently, this oligonucleotide increased the levels of SMN protein in SMA patient-derived cells that carry only the SMN2 gene. Our findings underscore for the first time the profound impact of an evolutionarily nonconserved intronic element on SMN2 exon 7 splicing. Considering that oligonucleotides annealing to intronic sequences do not interfere with exon-junction complex formation or mRNA transport and translation, ISS-N1 provides a very specific and efficient therapeutic target for antisense oligonucleotide-mediated correction of SMN2 splicing in SMA.
Spinal Muscular Atrophy (SMA) is an autosomal recessive disease that leads to specific loss of motor neurons. It is caused by deletions or mutations of the survival of motor neuron 1 gene (SMN1). The remaining copy of the gene, SMN2, generates only low levels of the SMN protein due to a mutation in SMN2 exon 7 that leads to exon skipping.
To correct SMN2 splicing, we use Adenovirus type 5–derived vectors to express SMN2-antisense U7 snRNA oligonucleotides targeting the SMN intron 7/exon 8 junction. Infection of SMA type I–derived patient fibroblasts with these vectors resulted in increased levels of exon 7 inclusion, upregulating the expression of SMN to similar levels as in non–SMA control cells.
These results show that Adenovirus type 5–derived vectors delivering U7 antisense oligonucleotides can efficiently restore full-length SMN protein and suggest that the viral vector-mediated oligonucleotide application may be a suitable therapeutic approach to counteract SMA.
Exon skipping induced by gene mutations is a common mechanism responsible for many genetic diseases. A practical approach to correct the aberrant splicing of defective genes is to use antisense oligonucleotides (ASOs). The recognition of splice sites and the regulation of splicing involve multiple positive or negative cis-acting elements and trans-acting factors. Base-pairing of ASOs to a negative element in a targeted pre-mRNA blocks the binding of splicing repressors to this cis-element and/or disrupts an unfavorable secondary structure; as a result, the ASO restores exon inclusion. For example, we have recently shown that appropriate 2’-O-(2-methoxyethyl) (MOE) phosphorothioate-modified ASOs can efficiently correct survival motor neuron 2 (SMN2) exon 7 splicing in a cell-free splicing assay, in cultured human cells—including patient fibroblasts—and in both peripheral tissues and the CNS of SMA mouse models. These ASOs are promising drug leads for SMA therapy.
Exon skipping; antisense oligonucleotide; MOE; splicing; SMN2; SMA; ESS; ISS; cis-acting element; in vitro splicing assay; minigene; exon 7 inclusion; RT-PCR; ICV; ICV infusion; mouse tissue; spinal cord; CNS
Spinal muscular atrophy (SMA) is a recessive neuromuscular disorder caused by the homozygous loss of the SMN1 gene. The human SMN2 gene has a C-to-T transition at position +6 of exon 7 and thus produces exon 7-skipping mRNAs. However, we observed an unexpectedly high level of exon 7-containing SMN2 transcripts as well as SMN protein in testis of smn−/− SMN2 transgenic mice. Using affinity chromatography, we identified several SMN RNA-associating proteins in mouse testis and human HeLa cells, including hnRNP Q. The major hnRNP Q isoform, Q1, directly bound SMN exon 7 in the vicinity of nucleotide +6. Overexpression of hnRNP Q1 promoted the inclusion of exon 7 in SMN2, probably by activating the use of its upstream 3′ splice site. However, the minor isoforms Q2/Q3 could antagonize the activity of hnRNP Q1 and induced exon 7 exclusion. Intriguingly, enhanced exon 7 inclusion was also observed upon concomitant depletion of three hnRNP Q isoforms. Thus, differential expression of hnRNP Q isoforms may result in intricate control of SMN precursor mRNA splicing. Here, we demonstrate that hnRNP Q is a splicing modulator of SMN, further underscoring the potential of hnRNP Q as a therapeutic target for SMA.
Humans have two nearly identical copies of the survival motor neuron (SMN ) gene, SMN1 and SMN2. Homozygous loss of SMN1 causes spinal muscular atrophy (SMA). SMN2 is unable to prevent the disease due to skipping of exon 7. Using a systematic approach of in vivo selection, we have previously demonstrated that a weak 5′ splice site (ss) serves as the major cause of skipping of SMN2 exon 7. Here we show the inhibitory impact of RNA structure on the weak 5′ ss of exon 7. We call this structure terminal stem–loop 2 (TSL2). Confirming the inhibitory nature of TSL2, point mutations that destabilize TSL2 promote exon 7 inclusion in SMN2, whereas strengthening of TSL2 promotes exon 7 skipping even in SMN1. We also demonstrate that TSL2 negatively affects the recruitment of U1snRNP at the 5′ ss of exon 7. Using enzymatic structure probing, we confirm that the sequence at the junction of exon 7/intron 7 folds into TSL2 and show that mutations in TSL2 cause predicted structural changes in this region. Our findings reveal for the first time the critical role of RNA structure in regulation of alternative splicing of human SMN.
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 (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.
Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations in the SMN1 gene that result in a deficiency of SMN protein. One approach to treat SMA is to use antisense oligonucleotides (ASOs) to redirect the splicing of a paralogous gene, SMN2, to boost production of functional SMN. Injection of a 2′-O-2-methoxyethyl–modified ASO (ASO-10-27) into the cerebral lateral ventricles of mice with a severe form of SMA resulted in splice-mediated increases in SMN protein and in the number of motor neurons in the spinal cord, which led to improvements in muscle physiology, motor function and survival. Intrathecal infusion of ASO-10-27 into cynomolgus monkeys delivered putative therapeutic levels of the oligonucleotide to all regions of the spinal cord. These data demonstrate that central nervous system–directed ASO therapy is efficacious and that intrathecal infusion may represent a practical route for delivering this therapeutic in the clinic.
Spinal muscular atrophy (SMA) is an autosomal-recessive disorder characterized by α-motor neuron loss in the spinal cord anterior horn. SMA results from deletion or mutation of the Survival Motor Neuron 1 gene (SMN1) and retention of SMN2. A single nucleotide difference between SMN1 and SMN2 results in exclusion of exon 7 from the majority of SMN2 transcripts, leading to decreased SMN protein levels and development of SMA. A series of splice enhancers and silencers regulate incorporation of SMN2 exon 7; these splice motifs can be blocked with antisense oligomers (ASOs) to alter SMN2 transcript splicing. We have evaluated a morpholino (MO) oligomer against ISS-N1 [HSMN2Ex7D(−10,−29)], and delivered this MO to postnatal day 0 (P0) SMA pups (Smn−/−, SMN2+/+, SMN▵7+/+) by intracerebroventricular (ICV) injection. Survival was increased markedly from 15 days to >100 days. Delayed CNS MO injection has moderate efficacy, and delayed peripheral injection has mild survival advantage, suggesting that early CNS ASO administration is essential for SMA therapy consideration. ICV treatment increased full-length SMN2 transcript as well as SMN protein in neural tissue, but only minimally in peripheral tissue. Interval analysis shows a decrease in alternative splice modification over time. We suggest that CNS increases of SMN will have a major impact on SMA, and an early increase of the SMN level results in correction of motor phenotypes. Finally, the early introduction by intrathecal delivery of MO oligomers is a potential treatment for SMA patients.
There is at present no cure or effective therapy for spinal muscular atrophy (SMA), a neurodegenerative disease that is the leading genetic cause of infant mortality. SMA usually results from loss of the SMN1 (survival of motor neuron 1) gene, which leads to selective motor neuron degeneration. SMN2 is nearly identical to SMN1 but has a nucleotide replacement that causes exon 7 skipping, resulting in a truncated, unstable version of the SMA protein. SMN2 is present in all SMA patients, and correcting SMN2 splicing is a promising approach for SMA therapy. We identified a tetracycline-like compound, PTK-SMA1, which stimulates exon 7 splicing and increases SMN protein levels in vitro and in vivo in mice. Unlike previously identified molecules that stimulate SMN production via SMN2 promoter activation or undefined mechanisms, PTK-SMA1 is a unique therapeutic candidate in that it acts by directly stimulating splicing of exon 7. Synthetic small-molecule compounds such as PTK-SMA1 offer an alternative to antisense oligonucleotide therapies that are being developed as therapeutics for a number of disease-associated splicing defects.
Humans have two nearly identical copies of the Survival Motor Neuron (SMN) gene: SMN1 and SMN2. The two SMN genes code for identical proteins; however, SMN2 predominantly generates a shorter transcript due to skipping of exon 7, the last coding exon. Skipping of SMN2 exon 7 leads to production of a truncated SMN protein that is highly unstable. The inability of SMN2 to compensate for the loss of SMN1 results in spinal muscular atrophy (SMA), the second most prevalent genetic cause of infant mortality. Since SMN2 is almost universally present in SMA patients, correction of SMN2 exon 7 splicing holds the promise for cure. Consistently, SMN2 exon 7 splicing has emerged as one of the best studied splicing systems in humans. The vast amount of recent literature provides a clue that SMN2 exon 7 splicing is regulated by an intron definition mechanism, which does not require cross-exon communication as prerequisite for exon inclusion. Our conclusion is based on the prominent role of intronic cis-elements, some of them have emerged as the frontrunners among potential therapeutic targets of SMA. Further, the widely expressed T-cell-restricted intracellular antigen-1 (TIA1), a member of the glutamine rich domain containing RNA-binding proteins, has recently been found to regulate SMN exon 7 splicing by binding to intron 7 sequences away from the 5′ splice site (ss). These findings make a strong argument for an “intron definition model,” according to which regulatory sequences within a downstream intron are capable of enforcing exon inclusion even in the absence of a defined upstream 3′ ss of an alternatively spliced exon.
SMN1; SMN2; SMA; TIA1; Splicing; ISS-N1; hnRNPA1
Proximal spinal muscular atrophy (SMA) is a neuromuscular disease caused by low levels of the survival motor neuron (SMN) protein. The reduced SMN levels are due to loss of the survival motor neuron-1 (SMN1) gene. Humans carry a nearly identical SMN2 gene that generates a truncated protein, due to a C to T nucleotide alteration in exon 7 that leads to inefficient RNA splicing of exon 7. This exclusion of SMN exon 7 is central to the onset of the SMA disease, however, this offers a unique therapeutic intervention in which corrective splicing of the SMN2 gene would restore SMN function. Exon 7 splicing is regulated by a number of exonic and intronic splicing regulatory sequences and trans-factors that bind them. A better understanding of the way SMN pre-mRNA is spliced has lead to the development of targeted therapies aimed at correcting SMN2 splicing. As therapeutics targeted toward correction of SMN2 splicing continue to be developed available SMA mouse models can be utilized in validating their potential in disease treatment.
Spinal Muscular Atrophy (SMA); Survival Motor Neuron (SMN); pre-mRNA Splicing; Intronic Splicing Enhancer (ISE); Exonic Splicing Enhancer (ESE); RNA Therapy
A significant proportion of disease-causing mutations affect precursor-mRNA splicing, inducing skipping of the exon from the mature transcript. Using F9 exon 5, CFTR exon 12 and SMN2 exon 7 models, we characterized natural mutations associated to exon skipping in Haemophilia B, cystic fibrosis and spinal muscular atrophy (SMA), respectively, and the therapeutic splicing rescue by using U1 small nuclear RNA (snRNA). In minigene expression systems, loading of U1 snRNA by complementarity to the normal or mutated donor splice sites (5′ss) corrected the exon skipping caused by mutations at the polypyrimidine tract of the acceptor splice site, at the consensus 5′ss or at exonic regulatory elements. To improve specificity and reduce potential off-target effects, we developed U1 snRNA variants targeting non-conserved intronic sequences downstream of the 5′ss. For each gene system, we identified an exon-specific U1 snRNA (ExSpeU1) able to rescue splicing impaired by the different types of mutations. Through splicing-competent cDNA constructs, we demonstrated that the ExSpeU1-mediated splicing correction of several F9 mutations results in complete restoration of secreted functional factor IX levels. Furthermore, two ExSpeU1s for SMA improved SMN exon 7 splicing in the chromosomal context of normal cells. We propose ExSpeU1s as a novel therapeutic strategy to correct, in several human disorders, different types of splicing mutations associated with defective exon definition.
Proximal spinal muscular atrophy (SMA) is a neuromuscular disease caused by low levels of the survival motor neuron (SMN) protein. In humans there are two nearly identical SMN genes, SMN1 and SMN2. The SMN2 gene generates a truncated protein, due to a C to T nucleotide alteration in exon 7, which leads to inefficient RNA splicing of exon 7. This exclusion of SMN exon 7 is central to the onset of the SMA disease. Exon 7 splicing is regulated by a number of exonic and intronic splicing regulatory sequences and the trans-factors that bind them. Here we identify conserved intronic sequences in the SMN genes. Five regions were examined due to conservation and their proximity to exons 6 through 8. Using mutagenesis two conserved elements located in intron 7 of the SMN genes that affect exon 7 splicing have been identified. Additional analysis of one of these regions showed decreased inclusion of exon 7 in SMN transcripts when deletions or mutations were introduced. Furthermore, multimerization of this conserved region was capable of restoring correct SMN splicing. Together these results describe a novel intronic splicing enhancer sequence located in the final intron of the SMN genes. This discovery provides insight into the splicing of the SMN genes using conserved intonic sequence as a tool to uncover regions of importance in pre-messenger RNA splicing. A better understanding of the way SMN pre-mRNA is spliced can lead to the development of new therapies.
Spinal Muscular Atrophy (SMA); Survival Motor Neuron (SMN); pre-mRNA Splicing; Intronic Splicing Enhancer
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, ∼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.
Here, we report a long-distance interaction (LDI) as a critical regulator of alternative splicing of Survival Motor Neuron 2 (SMN2) exon 7, skipping of which is linked to spinal muscular atrophy (SMA), a leading genetic disease of children and infants. We show that this LDI is linked to a unique intra-intronic structure that we term internal stem through LDI-1 (ISTL1). We used site-specific mutations and Selective 2′-Hydroxyl Acylation analyzed by Primer Extension to confirm the formation and functional significance of ISTL1. We demonstrate that the inhibitory effect of ISTL1 is independent of hnRNP A1/A2B1 and PTB1 previously implicated in SMN2 exon 7 splicing. We show that an antisense oligonucleotide-mediated sequestration of the 3′ strand of ISTL1 fully corrects SMN2 exon 7 splicing and restores high levels of SMN and Gemin2, a SMN-interacting protein, in SMA patient cells. Our results also reveal that the 3′ strand of ISTL1 and upstream sequences constitute an inhibitory region that we term intronic splicing silencer N2 (ISS-N2). This is the first report to demonstrate a critical role of a structure-associated LDI in splicing regulation of an essential gene linked to a genetic disease. Our findings expand the repertoire of potential targets for an antisense oligonucleotide-mediated therapy of SMA.
Controlling the patterns of splicing of specific genes is an important goal in the development of new therapies. We have shown that the splicing of a refractory exon, SMN2 exon 7, could be increased in fibroblasts derived from patients with spinal muscular atrophy by using bifunctional targeted oligonucleotide enhancers of splicing (TOES) oligonucleotides that anneal to the exon and contain a ‘tail’ of enhancer sequences that recruit activating proteins. We show here that there are striking agreements between the effects of oligonucleotides on splicing in vitro and on both splicing and SMN2 protein expression in patient-derived fibroblasts, indicating that the effects on splicing are the major determinant of success. Increased exon inclusion depends on the number, sequence and chemistry of the motifs that bind the activator protein SRSF1, but it is not improved by increasing the strength of annealing to the target site. The optimal oligonucleotide increases protein levels in transfected fibroblasts by a mean value of 2.6-fold (maximum 4.6-fold), and after two rounds of transfection the effect lasted for a month. Oligonucleotides targeted to the upstream exon (exon 6 in SMN) are also effective. We conclude that TOES oligonucleotides are highly effective reagents for restoring the splicing of refractory exons and can act across long introns.
Loss-of-function mutations in SMN1 cause spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. The related SMN2 gene expresses suboptimal levels of functional SMN protein, due to a splicing defect. Many SMA patients reach adulthood, and there is also adult-onset (type IV) SMA. There is currently no animal model for adult-onset SMA, and the tissue-specific pathogenesis of post-developmental SMN deficiency remains elusive. Here, we use an antisense oligonucleotide (ASO) to exacerbate SMN2 mis-splicing. Intracerebroventricular ASO injection in adult SMN2-transgenic mice phenocopies key aspects of adult-onset SMA, including delayed-onset motor dysfunction and relevant histopathological features. SMN2 mis-splicing increases during late-stage disease, likely accelerating disease progression. Systemic ASO injection in adult mice causes peripheral SMN2 mis-splicing and affects prognosis, eliciting marked liver and heart pathologies, with decreased IGF1 levels. ASO dose–response and time-course studies suggest that only moderate SMN levels are required in the adult central nervous system, and treatment with a splicing-correcting ASO shows a broad therapeutic time window. We describe distinctive pathological features of adult-onset and early-onset SMA.
adult-onset SMA; pathology; SMN2; spinal muscular atrophy; splicing
Humans have two near identical copies of Survival Motor Neuron gene: SMN1 and SMN2. Loss of SMN1 coupled with the predominant skipping of SMN2 exon 7 causes spinal muscular atrophy (SMA), a neurodegenerative disease. SMA patient cells devoid of SMN1 provide a powerful system to examine splicing pattern of various SMN2 exons. Until now, similar system to examine splicing of SMN1 exons was unavailable. We have recently screened several patient cell lines derived from various diseases, including SMA, Alzheimer’s disease, Parkinson’s disease and Batten disease. Here we report a Batten disease cell line that lacks functional SMN2, as an ideal system to examine pre-mRNA splicing of SMN1. We employ a multiple-exon-skipping detection assay (MESDA) to capture simultaneously skipping of multiple exons. Our results show surprising diversity of splice isoforms and reveal novel splicing events that include skipping of exon 4 and co-skipping of three adjacent exons of SMN. Contrary to the general belief, MESDA captured oxidative-stress induced skipping of SMN1 exon 5 in several cell types, including non-neuronal cells. We further demonstrate that the predominant SMN2 exon 7 skipping induced by oxidative stress is modulated by a combinatorial control that includes promoter sequence, endogenous context, and the weak splice sites. We also show that an 8-mer antisense oligonucleotide blocking a recently described GC-rich sequence prevents SMN2 exon 7 skipping under the conditions of oxidative stress. Our findings bring new insight into splicing regulation of an essential housekeeping gene linked to neurodegeneration and infant mortality.
Spinal muscular atrophy (SMA) is caused by low survival motor neuron (SMN) levels and patients represent a clinical spectrum due primarily to varying copies of the survival motor neuron-2 (SMN2) gene. Patient and animals studies show that disease severity is abrogated as SMN levels increase. Since therapies currently being pursued target the induction of SMN, it will be important to understand the dosage, timing and cellular requirements of SMN for disease etiology and potential therapeutic intervention. This requires new mouse models that can induce SMN temporally and/or spatially. Here we describe the generation of two hypomorphic Smn alleles, SmnC-T-Neo and Smn2B-Neo. These alleles mimic SMN2 exon 7 splicing, titre Smn levels and are inducible. They were specifically designed so that up to three independent lines of mice could be generated, herein we describe two. In a homozygous state each allele results in embryonic lethality. Analysis of these mutants indicates that greater than 5% of Smn protein is required for normal development. The severe hypomorphic nature of these alleles is caused by inclusion of a loxP-flanked neomycin gene selection cassette in Smn intron 7, which can be removed with Cre recombinase. In vitro and in vivo experiments demonstrate these as inducible Smn alleles. When combined with an inducible Cre mouse, embryonic lethality caused by low Smn levels can be rescued early in gestation but not late. This provides direct genetic evidence that a therapeutic window for SMN inductive therapies may exist. Importantly, these lines fill a void for inducible Smn alleles. They also provide a base from which to generate a large repertoire of SMA models of varying disease severities when combined with other Smn alleles or SMN2-containing mice.
Spinal muscular atrophy (SMA) is a progressive neurodegenerative disease associated with low levels of the essential survival motor neuron (SMN) protein. Reduced levels of SMN is due to the loss of the SMN1 gene and inefficient splicing of the SMN2 gene caused by a C>T mutation in exon 7. Global analysis of the severe SMNΔ7 SMA mouse model revealed altered splicing and increased levels of the hypoxia-inducible transcript, Hif3alpha, at late stages of disease progression. Severe SMA patients also develop respiratory deficiency during disease progression. We sought to evaluate whether hypoxia was capable of altering SMN2 exon 7 splicing and whether increased oxygenation could modulate disease in a severe SMA mouse model. Hypoxia treatment in cell culture increased SMN2 exon 7 skipping and reduced SMN protein levels. Concordantly, the treatment of SMNΔ7 mice with hyperoxia treatment increased the inclusion of SMN2 exon 7 in skeletal muscles and resulted in improved motor function. Transfection splicing assays of SMN minigenes under hypoxia revealed that hypoxia-induced skipping is dependent on poor exon definition due to the SMN2 C>T mutation and suboptimal 5′ splice site. Hypoxia treatment in cell culture led to increased hnRNP A1 and Sam68 levels. Mutation of hnRNP A1-binding sites prevented hypoxia-induced skipping of SMN exon 7 and was found to bind both hnRNP A1 and Sam68. These results implicate hypoxic stress as a modulator of SMN2 exon 7 splicing in disease progression and a coordinated regulation by hnRNP A1 and Sam68 as modifiers of hypoxia-induced skipping of SMN exon 7.
Spinal muscular atrophy (SMA) is an inherited motor neuron disease caused by homozygous loss of the Survival Motor Neuron 1 (SMN1) gene. In the absence of SMN1, inefficient inclusion of exon 7 in transcripts from the nearly identical SMN2 gene results in ubiquitous SMN decrease but selective motor neuron degeneration. Here we investigated whether cell type-specific differences in the efficiency of exon 7 splicing contribute to the vulnerability of SMA motor neurons. We show that normal motor neurons express markedly lower levels of full-length SMN mRNA from SMN2 than do other cells in the spinal cord. This is due to inefficient exon 7 splicing that is intrinsic to motor neurons under normal conditions. We also find that SMN depletion in mammalian cells decreases exon 7 inclusion through a negative feedback loop affecting the splicing of its own mRNA. This mechanism is active in vivo and further decreases the efficiency of exon 7 inclusion specifically in motor neurons of severe-SMA mice. Consistent with expression of lower levels of full-length SMN, we find that SMN-dependent downstream molecular defects are exacerbated in SMA motor neurons. These findings suggest a mechanism to explain the selective vulnerability of motor neurons to loss of SMN1.
Alternative splicing of the pyruvate kinase M gene involves a choice between mutually exclusive exons 9 and 10. Use of exon 10 to generate the M2 isoform is crucial for aerobic glycolysis (the Warburg effect) and tumour growth. We previously demonstrated that splicing enhancer elements that activate exon 10 are mainly found in exon 10 itself, and deleting or mutating these elements increases the inclusion of exon 9 in cancer cells. To systematically search for new enhancer elements in exon 10 and develop an effective pharmacological method to force a switch from PK-M2 to PK-M1, we carried out an antisense oligonucleotide (ASO) screen. We found potent ASOs that target a novel enhancer in exon 10 and strongly switch the splicing of endogenous PK-M transcripts to include exon 9. We further show that the ASO-mediated switch in alternative splicing leads to apoptosis in glioblastoma cell lines, and this is caused by the downregulation of PK-M2, and not by the upregulation of PK-M1. These data highlight the potential of ASO-mediated inhibition of PK-M2 splicing as therapy for cancer.
alternative splicing; antisense oligonucleotides; cancer
Proximal spinal muscular atrophy (SMA) is a neuromuscular disorder for which there is no available therapy. SMA is caused by loss or mutation of the survival motor neuron 1 gene, SMN1, with retention of a nearly identical copy gene, SMN2. In contrast to SMN1, most SMN2 transcripts lack exon 7. This alternatively spliced transcript, Δ7-SMN, encodes a truncated protein that is rapidly degraded. Inhibiting this degradation may be of therapeutic value for the treatment of SMA. Recently aminoglycosides, which decrease translational fidelity to promote readthrough of termination codons, were shown to increase SMN levels in patient cell lines. Amid uncertainty concerning the role of SMN's C-terminus, the potential of translational readthrough as a therapeutic mechanism for SMA is unclear. Here, we used stable cell lines to demonstrate the SMN C-terminus modulates protein stability in a sequence-independent manner that is reproducible by translational readthrough. Geneticin (G418) was then identified as a potent inducer of the Δ7-SMN target sequence in vitro through a novel quantitative assay amenable to high throughput screens. Subsequent treatment of patient cell lines demonstrated that G418 increases SMN levels and is a potential lead compound. Furthermore, treatment of SMA mice with G418 increased both SMN protein and mouse motor function. Chronic administration, however, was associated with toxicity that may have prevented the detection of a survival benefit. Collectively, these results substantiate a sequence independent role of SMN's C-terminus in protein stability and provide the first in vivo evidence supporting translational readthrough as a therapeutic strategy for the treatment of SMA.