Translation reinitiation after uORFs does not fully protect mRNAs from nonsense-mediated decay.

It is estimated that nearly 50% of mammalian transcripts contain at least one upstream open reading frame (uORF), which are typically one to two orders of magnitude smaller than the downstream main ORF. Most uORFs are thought to be inhibitory as they sequester the scanning ribosome, but in some cases allow for translation reinitiation. However, termination in the 5' UTR at the end of uORFs resembles premature termination that is normally sensed by the nonsense-mediated mRNA decay (NMD) pathway. Translation reinitiation has been proposed as a method for mRNAs to prevent NMD. Here, we test how uORF length influences translation reinitiation and mRNA stability in HeLa cells. Using custom 5' UTRs and uORF sequences, we show that reinitiation can occur on heterologous mRNA sequences, favors small uORFs, and is supported when initiation occurs with more initiation factors. After determining reporter mRNA half-lives in HeLa cells and mining available mRNA half-life data sets for cumulative predicted uORF length, we conclude that translation reinitiation after uORFs is not a robust method for mRNAs to prevent NMD. Together, these data suggest that the decision of whether NMD ensues after translating uORFs occurs before reinitiation in mammalian cells.

Conditional deletion of SMN in cell culture identifies functional SMN alleles.

Spinal muscular atrophy (SMA) is caused by mutation or deletion of survival motor neuron 1 (SMN1) and retention of SMN2 leading to SMN protein deficiency. We developed an immortalized mouse embryonic fibroblast (iMEF) line in which full-length wild-type Smn (flwt-Smn) can be conditionally deleted using Cre recombinase. iMEFs lacking flwt-Smn are not viable. We tested the SMA patient SMN1 missense mutation alleles A2G, D44V, A111G, E134K and T274I in these cells to determine which human SMN (huSMN) mutant alleles can function in the absence of flwt-Smn. All missense mutant alleles failed to rescue survival in the conditionally deleted iMEFs. Thus, the function lost by these mutations is essential to cell survival. However, co-expression of two different huSMN missense mutants can rescue iMEF survival and small nuclear ribonucleoprotein (snRNP) assembly, demonstrating intragenic complementation of SMN alleles. In addition, we show that a Smn protein lacking exon 2B can rescue iMEF survival and snRNP assembly in the absence of flwt-Smn, indicating exon 2B is not required for the essential function of Smn. For the first time, using this novel cell line, we can assay the function of SMN alleles in the complete absence of flwt-Smn.

Intragenic complementation of amino and carboxy terminal SMN missense mutations can rescue Smn null mice.

Spinal muscular atrophy is caused by reduced levels of SMN resulting from the loss of SMN1 and reliance on SMN2 for the production of SMN. Loss of SMN entirely is embryonic lethal in mammals. There are several SMN missense mutations found in humans. These alleles do not show partial function in the absence of wild-type SMN and cannot rescue a null Smn allele in mice. However, these human SMN missense allele transgenes can rescue a null Smn allele when SMN2 is present. We find that the N- and C-terminal regions constitute two independent domains of SMN that can be separated genetically and undergo intragenic complementation. These SMN protein heteromers restore snRNP assembly of Sm proteins onto snRNA and completely rescue both survival of Smn null mice and motor neuron electrophysiology demonstrating that the essential functional unit of SMN is the oligomer.

Early Inflammation in Muscular Dystrophy Differs between Limb and Respiratory Muscles and Increases with Dystrophic Severity.

Duchenne muscular dystrophy (DMD) is a genetic, degenerative, striated muscle disease exacerbated by chronic inflammation. Mdx mice in the genotypic DMD model poorly represent immune-mediated pathology observed in patients. Improved understanding of innate immunity in dystrophic muscles is required to develop specific anti-inflammatory treatments. Here, inflammation in mdx mice and the more fibrotic utrn+/-;mdx Het model was comprehensively investigated. Unbiased analysis showed that mdx and Het mice contain increased levels of numerous chemokines and cytokines, with further increased in Het mice. Chemokine and chemokine receptor gene expression levels were dramatically increased in 4-week-old dystrophic quadriceps muscles, and to a lesser extent in diaphragm during the early injury phase, and had a small but consistent increase at 8 and 20 weeks. An optimized direct immune cell isolation method prevented loss of up to 90% of macrophages with density-dependent centrifugation previously used for mdx flow cytometry. Het quadriceps contain higher proportions of neutrophils and infiltrating monocytes than mdx, and higher percentages of F4/80Hi, but lower percentages of F4/80Lo cells and patrolling monocytes compared with Het diaphragms. These differences may restrict regenerative potential of dystrophic diaphragms, increasing pathologic severity. Fibrotic and inflammatory gene expression levels are higher in myeloid cells isolated from Het compared with mdx quadriceps, supporting Het mice may represent an improved model for testing therapeutic manipulation of inflammation in DMD.

Dual SMN inducing therapies can rescue survival and motor unit function in symptomatic ∆7SMA mice.

Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by survival motor neuron (SMN) protein deficiency which results in motor neuron loss and muscle atrophy. SMA is caused by a mutation or deletion of the survival motor neuron 1 (SMN1) gene and retention of the nearly identical SMN2 gene. SMN2 contains a C to T change in exon 7 that results in exon 7 exclusion from 90% of transcripts. SMN protein lacking exon 7 is unstable and rapidly degraded. The remaining full-length transcripts from SMN2 are insufficient for normal motor neuron function leading to the development of SMA. Three different therapeutic approaches that increase full-length SMN (FL-SMN) protein production are approved for treatment of SMA patients. Studies in both animal models and humans have demonstrated increasing SMN levels prior to onset of symptoms provides the greatest therapeutic benefit. Treatment of SMA, after some motor neuron loss has occurred, is also effective but to a lesser degree. The SMN∆7 mouse model is a well characterized model of severe or type 1 SMA, dying at 14 days of age. Here we treated three groups of ∆7SMA mice starting before, roughly during, and after symptom onset to determine if combining two mechanistically distinct SMN inducing therapies could improve the therapeutic outcome both before and after motor neuron loss. We found, compared with individual therapies, that morpholino antisense oligonucleotide (ASO) directed against ISS-N1 combined with the small molecule compound RG7800 significantly increased FL-SMN transcript and protein production resulting in improved survival and weight of ∆7SMA mice. Moreover, when give late symptomatically, motor unit function was completely rescued with no loss in function at 100 days of age in the dual treatment group. We have therefore shown that this dual therapeutic approach successfully increases SMN protein and rescues motor function in symptomatic ∆7SMA mice.

What Genetics Has Told Us and How It Can Inform Future Experiments for Spinal Muscular Atrophy, a Perspective.

Proximal spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disorder characterized by motor neuron loss and subsequent atrophy of skeletal muscle. SMA is caused by deficiency of the essential survival motor neuron (SMN) protein, canonically responsible for the assembly of the spliceosomal small nuclear ribonucleoproteins (snRNPs). Therapeutics aimed at increasing SMN protein levels are efficacious in treating SMA. However, it remains unknown how deficiency of SMN results in motor neuron loss, resulting in many reported cellular functions of SMN and pathways affected in SMA. Herein is a perspective detailing what genetics and biochemistry have told us about SMA and SMN, from identifying the SMA determinant region of the genome, to the development of therapeutics. Furthermore, we will discuss how genetics and biochemistry have been used to understand SMN function and how we can determine which of these are critical to SMA moving forward.

Mild SMN missense alleles are only functional in the presence of SMN2 in mammals.

Spinal muscular atrophy (SMA) is caused by reduced levels of full-length SMN (FL-SMN). In SMA patients with one or two copies of the Survival Motor Neuron 2 (SMN2) gene there are a number of SMN missense mutations that result in milder-than-predicted SMA phenotypes. These mild SMN missense mutation alleles are often assumed to have partial function. However, it is important to consider the contribution of FL-SMN as these missense alleles never occur in the absence of SMN2. We propose that these patients contain a partially functional oligomeric SMN complex consisting of FL-SMN from SMN2 and mutant SMN protein produced from the missense allele. Here we show that mild SMN missense mutations SMND44V, SMNT74I or SMNQ282A alone do not rescue mice lacking wild-type FL-SMN. Thus, missense mutations are not functional in the absence of FL-SMN. In contrast, when the same mild SMN missense mutations are expressed in a mouse containing two SMN2 copies, functional SMN complexes are formed with the small amount of wild-type FL-SMN produced by SMN2 and the SMA phenotype is completely rescued. This contrasts with SMN missense alleles when studied in C. elegans, Drosophila and zebrafish. Here we demonstrate that the heteromeric SMN complex formed with FL-SMN is functional and sufficient to rescue small nuclear ribonucleoprotein assembly, motor neuron function and rescue the SMA mice. We conclude that mild SMN missense alleles are not partially functional but rather they are completely non-functional in the absence of wild-type SMN in mammals.

Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy.

BACKGROUND: Spinal muscular atrophy type 1 (SMA1) is a progressive, monogenic motor neuron disease with an onset during infancy that results in failure to achieve motor milestones and in death or the need for mechanical ventilation by 2 years of age. We studied functional replacement of the mutated gene encoding survival motor neuron 1 (SMN1) in this disease. METHODS: Fifteen patients with SMA1 received a single dose of intravenous adeno-associated virus serotype 9 carrying SMN complementary DNA encoding the missing SMN protein. Three of the patients received a low dose (6.7×1013 vg per kilogram of body weight), and 12 received a high dose (2.0×1014 vg per kilogram). The primary outcome was safety. The secondary outcome was the time until death or the need for permanent ventilatory assistance. In exploratory analyses, we compared scores on the CHOP INTEND (Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders) scale of motor function (ranging from 0 to 64, with higher scores indicating better function) in the two cohorts and motor milestones in the high-dose cohort with scores in studies of the natural history of the disease (historical cohorts). RESULTS: As of the data cutoff on August 7, 2017, all 15 patients were alive and event-free at 20 months of age, as compared with a rate of survival of 8% in a historical cohort. In the high-dose cohort, a rapid increase from baseline in the score on the CHOP INTEND scale followed gene delivery, with an increase of 9.8 points at 1 month and 15.4 points at 3 months, as compared with a decline in this score in a historical cohort. Of the 12 patients who had received the high dose, 11 sat unassisted, 9 rolled over, 11 fed orally and could speak, and 2 walked independently. Elevated serum aminotransferase levels occurred in 4 patients and were attenuated by prednisolone. CONCLUSIONS: In patients with SMA1, a single intravenous infusion of adeno-associated viral vector containing DNA coding for SMN resulted in longer survival, superior achievement of motor milestones, and better motor function than in historical cohorts. Further studies are necessary to confirm the safety and efficacy of this gene therapy. (Funded by AveXis and others; ClinicalTrials.gov number, NCT02122952 .).

Complete sequencing of the SMN2 gene in SMA patients detects SMN gene deletion junctions and variants in SMN2 that modify the SMA phenotype.

Spinal muscular atrophy (SMA) is a progressive motor neuron disease caused by loss or mutation of the survival motor neuron 1 (SMN1) gene and retention of SMN2. We performed targeted capture and sequencing of the SMN2, CFTR, and PLS3 genes in 217 SMA patients. We identified a 6.3 kilobase deletion that occurred in both SMN1 and SMN2 (SMN1/2) and removed exons 7 and 8. The deletion junction was flanked by a 21 bp repeat that occurred 15 times in the SMN1/2 gene. We screened for its presence in 466 individuals with the known SMN1 and SMN2 copy numbers. In individuals with 1 SMN1 and 0 SMN2 copies, the deletion occurred in 63% of cases. We modeled the deletion junction frequency and determined that the deletion occurred in both SMN1 and SMN2. We have identified the first deletion junction where the deletion removes exons 7 and 8 of SMN1/2. As it occurred in SMN1, it is a pathogenic mutation. We called variants in the PLS3 and SMN2 genes, and tested for association with mild or severe exception patients. The variants A-44G, A-549G, and C-1897T in intron 6 of SMN2 were significantly associated with mild exception patients, but no PLS3 variants correlated with severity. The variants occurred in 14 out of 58 of our mild exception patients, indicating that mild exception patients with an intact SMN2 gene and without modifying variants occur. This sample set can be used in the association analysis of candidate genes outside of SMN2 that modify the SMA phenotype.

Natural history of infantile-onset spinal muscular atrophy.

OBJECTIVE: Infantile-onset spinal muscular atrophy (SMA) is the most common genetic cause of infant mortality, typically resulting in death preceding age 2. Clinical trials in this population require an understanding of disease progression and identification of meaningful biomarkers to hasten therapeutic development and predict outcomes. METHODS: A longitudinal, multicenter, prospective natural history study enrolled 26 SMA infants and 27 control infants aged <6 months. Recruitment occurred at 14 centers over 21 months within the NINDS-sponsored NeuroNEXT (National Network for Excellence in Neuroscience Clinical Trials) Network. Infant motor function scales (Test of Infant Motor Performance Screening Items [TIMPSI], The Children's Hospital of Philadelphia Infant Test for Neuromuscular Disorders, and Alberta Infant Motor Score) and putative physiological and molecular biomarkers were assessed preceding age 6 months and at 6, 9, 12, 18, and 24 months with progression, correlations between motor function and biomarkers, and hazard ratios analyzed. RESULTS: Motor function scores (MFS) and compound muscle action potential (CMAP) decreased rapidly in SMA infants, whereas MFS in all healthy infants rapidly increased. Correlations were identified between TIMPSI and CMAP in SMA infants. TIMPSI at first study visit was associated with risk of combined endpoint of death or permanent invasive ventilation in SMA infants. Post-hoc analysis of survival to combined endpoint in SMA infants with 2 copies of SMN2 indicated a median age of 8 months at death (95% confidence interval, 6, 17). INTERPRETATION: These data of SMA and control outcome measures delineates meaningful change in clinical trials in infantile-onset SMA. The power and utility of NeuroNEXT to provide "real-world," prospective natural history data sets to accelerate public and private drug development programs for rare disease is demonstrated. Ann Neurol 2017;82:883-891.

Antisense oligonucleotides and spinal muscular atrophy: skipping along.

Antisense oligonucleotides (ASOs) can be used to alter the splicing of a gene and either restore production of a required protein or eliminate a toxic product. In this issue of Genes & Development, Hua and colleagues (pp. 1634-1644) show that ASOs directed against an intron splice silencer (ISS) in the survival motor neuron 2 (SMN2) gene alter the amount of full-length SMN transcript in the nervous system, restoring SMN to levels that could correct spinal muscular atrophy (SMA).

SMN expression is required in motor neurons to rescue electrophysiological deficits in the SMNΔ7 mouse model of SMA.

Proximal spinal muscular atrophy (SMA) is the most frequent cause of hereditary infant mortality. SMA is an autosomal recessive neuromuscular disorder that results from the loss of the Survival Motor Neuron 1 (SMN1) gene and retention of the SMN2 gene. The SMN2 gene produces an insufficient amount of full-length SMN protein that results in loss of motor neurons in the spinal cord and subsequent muscle paralysis. Previously we have shown that overexpression of human SMN in neurons in the SMA mouse ameliorates the SMA phenotype while overexpression of human SMN in skeletal muscle had no effect. Using Cre recombinase, here we show that either deletion or replacement of Smn in motor neurons (ChAT-Cre) significantly alters the functional output of the motor unit as measured with compound muscle action potential and motor unit number estimation. However ChAT-Cre alone did not alter the survival of SMA mice by replacement and did not appreciably affect survival when used to deplete SMN. However replacement of Smn in both neurons and glia in addition to the motor neuron (Nestin-Cre and ChAT-Cre) resulted in the greatest improvement in survival of the mouse and in some instances complete rescue was achieved. These findings demonstrate that high expression of SMN in the motor neuron is both necessary and sufficient for proper function of the motor unit. Furthermore, in the mouse high expression of SMN in neurons and glia, in addition to motor neurons, has a major impact on survival.

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).

SMN deficiency disrupts gastrointestinal and enteric nervous system function in mice.

The 2007 Consensus Statement for Standard of Care in Spinal Muscular Atrophy (SMA) notes that patients suffer from gastroesophageal reflux, constipation and delayed gastric emptying. We used two mouse models of SMA to determine whether functional GI complications are a direct consequence of or are secondary to survival motor neuron (Smn) deficiency. Our results show that despite normal activity levels and food and water intake, Smn deficiency caused constipation, delayed gastric emptying, slow intestinal transit and reduced colonic motility without gross anatomical or histopathological abnormalities. These changes indicate alterations to the intrinsic neural control of gut functions mediated by the enteric nervous system (ENS). Indeed, Smn deficiency led to disrupted ENS signaling to the smooth muscle of the colon but did not cause enteric neuron loss. High-frequency electrical field stimulation (EFS) of distal colon segments produced up to a 10-fold greater contractile response in Smn deficient tissues. EFS responses were not corrected by the addition of a neuronal nitric oxide synthase inhibitor indicating that the increased contractility was due to hyperexcitability and not disinhibition of the circuitry. The GI symptoms observed in mice are similar to those reported in SMA patients. Together these data suggest that ENS cells are susceptible to Smn deficiency and may underlie the patient GI symptoms.

A large animal model of spinal muscular atrophy and correction of phenotype.

OBJECTIVES: Spinal muscular atrophy (SMA) is caused by reduced levels of survival motor neuron (SMN) protein, which results in motoneuron loss. Therapeutic strategies to increase SMN levels including drug compounds, antisense oligonucleotides, and scAAV9 gene therapy have proved effective in mice. We wished to determine whether reduction of SMN in postnatal motoneurons resulted in SMA in a large animal model, whether SMA could be corrected after development of muscle weakness, and the response of clinically relevant biomarkers. METHODS: Using intrathecal delivery of scAAV9 expressing an shRNA targeting pig SMN1, SMN was knocked down in motoneurons postnatally to SMA levels. This resulted in an SMA phenotype representing the first large animal model of SMA. Restoration of SMN was performed at different time points with scAAV9 expressing human SMN (scAAV9-SMN), and electrophysiology measurements and pathology were performed. RESULTS: Knockdown of SMN in postnatal motoneurons results in overt proximal weakness, fibrillations on electromyography indicating active denervation, and reduced compound muscle action potential (CMAP) and motor unit number estimation (MUNE), as in human SMA. Neuropathology showed loss of motoneurons and motor axons. Presymptomatic delivery of scAAV9-SMN prevented SMA symptoms, indicating that all changes are SMN dependent. Delivery of scAAV9-SMN after symptom onset had a marked impact on phenotype, electrophysiological measures, and pathology. INTERPRETATION: High SMN levels are critical in postnatal motoneurons, and reduction of SMN results in an SMA phenotype that is SMN dependent. Importantly, clinically relevant biomarkers including CMAP and MUNE are responsive to SMN restoration, and abrogation of phenotype can be achieved even after symptom onset.

Improving single injection CSF delivery of AAV9-mediated gene therapy for SMA: a dose-response study in mice and nonhuman primates.

Spinal muscular atrophy (SMA) is the most frequent lethal genetic neurodegenerative disorder in infants. The disease is caused by low abundance of the survival of motor neuron (SMN) protein leading to motor neuron degeneration and progressive paralysis. We previously demonstrated that a single intravenous injection (IV) of self-complementary adeno-associated virus-9 carrying the human SMN cDNA (scAAV9-SMN) resulted in widespread transgene expression in spinal cord motor neurons in SMA mice as well as nonhuman primates and complete rescue of the disease phenotype in mice. Here, we evaluated the dosing and efficacy of scAAV9-SMN delivered directly to the cerebral spinal fluid (CSF) via single injection. We found widespread transgene expression throughout the spinal cord in mice and nonhuman primates when using a 10 times lower dose compared to the IV application. Interestingly, in nonhuman primates, lower doses than in mice can be used for similar motor neuron targeting efficiency. Moreover, the transduction efficacy is further improved when subjects are kept in the Trendelenburg position to facilitate spreading of the vector. We present a detailed analysis of transduction levels throughout the brain, brainstem, and spinal cord of nonhuman primates, providing new guidance for translation toward therapy for a wide range of neurodegenerative disorders.

A single administration of morpholino antisense oligomer rescues spinal muscular atrophy in mouse.

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+/+, SMNΔ7+/+) 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.

Spinal muscular atrophy: development and implementation of potential treatments.

In neurodegenerative disorders, effective treatments are urgently needed, along with methods to determine whether treatment worked. In this review, we discuss the rapid progress in the understanding of recessive proximal spinal muscular atrophy and how this is leading to exciting potential treatments of the disease. Spinal muscular atrophy is caused by loss of the survival motor neuron 1 (SMN1) gene and reduced levels of SMN protein. The critical downstream targets of SMN deficiency that result in motor neuron loss are not known. However, increasing SMN levels has a marked impact in mouse models, and these therapeutics are rapidly moving toward clinical trials. Promising preclinical therapies, the varying degree of impact on the mouse models, and potential measures of treatment effect are reviewed. One key issue discussed is the variable outcome of increasing SMN at different stages of disease progression.

Spinal muscular atrophy: why do low levels of survival motor neuron protein make motor neurons sick?

Many neurogenetic disorders are caused by the mutation of ubiquitously expressed genes. One such disorder, spinal muscular atrophy, is caused by loss or mutation of the survival motor neuron1 gene (SMN1), leading to reduced SMN protein levels and a selective dysfunction of motor neurons. SMN, together with partner proteins, functions in the assembly of small nuclear ribonucleoproteins (snRNPs), which are important for pre-mRNA splicing. It has also been suggested that SMN might function in the assembly of other ribonucleoprotein complexes. Two hypotheses have been proposed to explain the molecular dysfunction that gives rise to spinal muscular atrophy (SMA) and its specificity to a particular group of neurons. The first hypothesis states that the loss of SMN's well-known function in snRNP assembly causes an alteration in the splicing of a specific gene (or genes). The second hypothesis proposes that SMN is crucial for the transport of mRNA in neurons and that disruption of this function results in SMA.