ISS-N1 makes the first FDA-approved drug for spinal muscular atrophy

Spinal muscular atrophy (SMA) is one of the leading genetic diseases of children and infants. SMA is caused by deletions or mutations of Survival Motor Neuron 1 (SMN1) gene. SMN2, a nearly identical copy of SMN1, cannot compensate for the loss of SMN1 due to predominant skipping of exon 7. While various regulatory elements that modulate SMN2 exon 7 splicing have been proposed, intronic splicing silencer N1 (ISS-N1) has emerged as the most promising target thus far for antisense oligonucleotide-mediated splicing correction in SMA. Upon procuring exclusive license from the University of Massachussets Medical School in 2010, Ionis Pharmaceuticals (formerly ISIS Pharamaceuticals) began clinical development of Spinraza™ (synonyms: Nusinersen, IONIS-SMNRX, ISIS-SMNRX), an antisense drug based on ISS-N1 target. Spinraza™ showed very promising results at all steps of the clinical development and was approved by US Food and Drug Administration (FDA) on December 23, 2016. Spinraza™ is the first FDA-approved treatment for SMA and the first antisense drug to restore expression of a fully functional protein via splicing correction. The success of Spinraza™ underscores the potential of intronic sequences as promising therapeutic targets and sets the stage for further improvement of antisense drugs based on advanced oligonucleotide chemistries and delivery protocols

Effective Brief-Writing for California Appellate Courts

This Article suggests an approach to appellate brief writing that will insure compliance with the rules of court and a clear expression of the advocate\u27s position in the issues. The author describes both the specific techniques of effective brief writing and the technical format used in writing briefs for California appellate courts. Providing practical tools and techniques for the practitioner, the author argues that adherence to these techniques will significantly improve the quality of appellate practice in California

Novel Functions of the Survival Motor Neuron Protein

The Survival Motor Neuron (SMN) protein is a multi-functional protein that participates in a wide variety of critical pathways. Low levels of SMN cause spinal muscular atrophy (SMA), the most common genetic cause of infant mortality. While the role of SMN in the assembly of small nuclear ribonucleoproteins (snRNPs) has been well characterized, many of its other diverse functions have not been thoroughly explored. Here, we examine the critical role of SMN in the growth and development of male mammalian sex organs. We show that low levels of SMN in a mild mouse model of SMA cause impaired testis development, degenerated seminiferous tubules, reduced sperm count, and low fertility. Underscoring an increased requirement for SMN expression, wild type testis showed extremely high levels of SMN protein compared to other tissues. The testis phenotype is linked to increased apoptosis in seminiferous tubules and extreme perturbations in the testis transcriptome. We examine the RNA binding function of SMN by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) to identify RNA sequence and structural motif(s) of SMN. Our results reveal a combination of sequence motifs and structural contexts that drive the specificity of RNA-SMN interactions. Our results of truncation and substitution experiments suggest a requirement for multiple contacts between SMN and RNA to maintain the high affinity. We demonstrate that both affinity and specificity of RNA-SMN interaction are influenced by salt concentrations. To identify in vivo RNA targets of SMN, we performed crosslinking and immunoprecipitation coupled with high-throughput sequencing (HITS-CLIP). HITS-CLIP identified a variety of RNA targets of SMN with an enrichment of mRNAs participating in a number of pathways, including ribosome function and actin cytoskeleton regulation. In order to determine whether expression levels of target RNAs are regulated by SMN, we performed knockdown of SMN levels followed by RNA-Seq. SPON2, LAMB2, and EEF1A2 in particular were all predicted by HITS-CLIP to be bound by SMN and were downregulated upon SMN knockdown, indicating a direct regulatory role for SMN on expression of these genes

Diverse role of survival motor neuron protein

The multifunctional Survival Motor Neuron (SMN) protein is required for the survival of all organisms of the animal kingdom. SMN impacts various aspects of RNA metabolism through the formation and/or interaction with ribonucleoprotein (RNP) complexes. SMN regulates biogenesis of small nuclear RNPs, small nucleolar RNPs, small Cajal body-associated RNPs, signal recognition particles and telomerase. SMN also plays an important role in DNA repair, transcription, pre-mRNA splicing, histone mRNA processing, translation, selenoprotein synthesis, macromolecular trafficking, stress granule formation, cell signaling and cytoskeleton maintenance. The tissue-specific requirement of SMN is dictated by the variety and the abundance of its interacting partners. Reduced expression of SMN causes spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. SMA displays a broad spectrum ranging from embryonic lethality to an adult onset. Aberrant expression and/or localization of SMN has also been associated with male infertility, inclusion body myositis, amyotrophic lateral sclerosis and osteoarthritis. This review provides a summary of various SMN functions with implications to a better understanding of SMA and other pathological conditions

High-affinity RNA targets of the Survival Motor Neuron protein reveal diverse preferences for sequence and structural motifs

The Survival Motor Neuron (SMN) protein is essential for survival of all animal cells. SMN harbors a nucleic acid-binding domain and plays an important role in RNA metabolism. However, the RNA-binding property of SMN is poorly understood. Here we employ iterative in vitro selection and chemical structure probing to identify sequence and structural motif(s) critical for RNA–SMN interactions. Our results reveal that motifs that drive RNA–SMN interactions are diverse and suggest that tight RNA–SMN interaction requires presence of multiple contact sites on the RNA molecule. We performed UV crosslinking and immunoprecipitation coupled with high-throughput sequencing (HITS-CLIP) to identify cellular RNA targets of SMN in neuronal SH-SY5Y cells. Results of HITS-CLIP identified a wide variety of targets, including mRNAs coding for ribosome biogenesis and cytoskeleton dynamics. We show critical determinants of ANXA2 mRNA for a direct SMN interaction in vitro. Our data confirms the ability of SMN to discriminate among close RNA sequences, and represent the first validation of a direct interaction of SMN with a cellular RNA target. Our findings suggest direct RNA– SMN interaction as a novel mechanism to initiate the cascade of events leading to the execution of SMN-specific functions

Knowledge sources and SME internationalization

Masteroppgave(MSc) in Master of Science in Business, Strategy - Handelshøyskolen BI, 2017, Masteroppgave(MSc) in Master of Science in Business, International business - Handelshøyskolen BI, 2017Our thesis focuses on the knowledge that small and medium firms need to internationalize. Our study reviews relevant literature on internationalization and combines several theoretical lenses to take an integrated approach to analyzing how knowledge sources are relevant to SME internationalization. This includes ideas from the knowledge-based view, network theory, theory of international new ventures, upper echelon theory, and organizational learning theory. Thus, our approach is a broader, and high level discussion of how knowledge sources relate to internationalization. Through a mixed-method approach using exploratory surveys and interviews, our analysis helped us create a greater understanding of the knowledge sources involved in knowledge-intensive, high-tech, SME internationalization and resulted in the identification of several key knowledge sources that are thought to be associated with a higher international performance. Customers, international partnerships, prior management and employee international knowledge and experience, and government resources, such as export agencies/consultants, are identified as the most important knowledge sources to consider. It is proposed that optimal combinations of knowledge resources, built using these sources, can enhance firm capabilities, leading SMEs to greater levels of internationalization

Human Survival Motor Neuron genes generate a vast repertoire of circular RNAs

Circular RNAs (circRNAs) perform diverse functions, including the regulation of transcription, translation, peptide synthesis, macromolecular sequestration and trafficking. Inverted Alu repeats capable of forming RNA:RNA duplexes that bring splice sites together for backsplicing are known to facilitate circRNA generation. However, higher limits of circRNAs produced by a single Alu-rich gene are currently not predictable due to limitations of amplification and analyses. Here, using a tailored approach, we report a surprising diversity of exon-containing circRNAs generated by the Alu-rich Survival Motor Neuron (SMN) genes that code for SMN, an essential multifunctional protein in humans. We show that expression of the vast repertoire ofSMN circRNAs is universal. Several of the identified circRNAs harbor novel exons derived from both intronic and intergenic sequences. A comparison with mouse Smn circRNAs underscored a clear impact of primate-specific Alu elements on shaping the overall repertoire of human SMN circRNAs. We show the role of DHX9, an RNA helicase, in splicing regulation of several SMN exons that are preferentially incorporated into circRNAs. Our results suggest self- and cross-regulation of biogenesis of various SMN circRNAs. These findings bring a novel perspective towards a better understanding of SMN gene function

A novel human-specific splice isoform alters the critical C-terminus of Survival Motor Neuron protein

Spinal muscular atrophy (SMA), a leading genetic disease of children and infants, is caused by mutations or deletions of Survival Motor Neuron 1 (SMN1) gene. SMN2, a nearly identical copy of SMN1, fails to compensate for the loss of SMN1 due to skipping of exon 7. SMN2predominantly produces SMNΔ7, an unstable protein. Here we report exon 6B, a novel exon, generated by exonization of an intronic Alu-like sequence of SMN. We validate the expression of exon 6B-containing transcripts SMN6B and SMN6BΔ7 in human tissues and cell lines. We confirm generation of SMN6B transcripts from both SMN1 and SMN2. We detect expression of SMN6B protein using antibodies raised against a unique polypeptide encoded by exon 6B. We analyze RNA-Seq data to show that hnRNP C is a potential regulator of SMN6B expression and demonstrate that SMN6B is a substrate of nonsense-mediated decay. We show interaction of SMN6B with Gemin2, a critical SMN-interacting protein. We demonstrate that SMN6B is more stable than SMNΔ7 and localizes to both the nucleus and the cytoplasm. Our finding expands the diversity of transcripts generated from human SMN genes and reveals a novel protein isoform predicted to be stably expressed during conditions of stress

Oxidative Stress Triggers Body-Wide Skipping of Multiple Exons of the Spinal Muscular Atrophy Gene

Humans carry two nearly identical copies of Survival Motor Neuron gene: SMN1 and SMN2. Loss of SMN1 leads to spinal muscular atrophy (SMA), the most frequent genetic cause of infant mortality. While SMN2 cannot compensate for the loss of SMN1 due to predominant skipping of exon 7, correction of SMN2 exon 7 splicing holds the promise of a cure for SMA. Previously, we used cell-based models coupled with a multi-exon-skipping detection assay (MESDA) to demonstrate the vulnerability of SMN2 exons to aberrant splicing under the conditions of oxidative stress (OS). Here we employ a transgenic mouse model and MESDA to examine the OS-induced splicing regulation of SMN2 exons. We induced OS using paraquat that is known to trigger production of reactive oxygen species and cause mitochondrial dysfunction. We show an overwhelming co-skipping of SMN2 exon 5 and exon 7 under OS in all tissues except testis. We also show that OS increases skipping of SMN2 exon 3 in all tissues except testis. We uncover several new SMN2 splice isoforms expressed at elevated levels under the conditions of OS. We analyze cis-elements and transacting factors to demonstrate the diversity of mechanisms for splicing misregulation under OS. Our results of proteome analysis reveal downregulation of hnRNP H as one of the potential consequences of OS in brain. Our findings suggest SMN2 as a sensor of OS with implications to SMA and other diseases impacted by low levels of SMN protein

Severe impairment of male reproductive organ development in a low SMN expressing mouse model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is caused by low levels of survival motor neuron (SMN), a multifunctional protein essential for higher eukaryotes. While SMN is one of the most scrutinized proteins associated with neurodegeneration, its gender-specific role in vertebrates remains unknown. We utilized a mild SMA model (C/C model) to examine the impact of low SMN on growth and development of mammalian sex organs. We show impaired testis development, degenerated seminiferous tubules, reduced sperm count and low fertility in C/C males, but no overt sex organ phenotype in C/C females. Underscoring an increased requirement for SMN expression, wild type testis showed extremely high levels of SMN protein compared to other tissues. Our results revealed severe perturbations in pathways critical to C/C male reproductive organ development and function, including steroid biosynthesis, apoptosis, and spermatogenesis. Consistent with enhanced apoptosis in seminiferous tubules of C/C testes, we recorded a drastic increase in cells with DNA fragmentation. SMN was expressed at high levels in adult C/C testis due to an adult-specific splicing switch, but could not compensate for low levels during early testicular development. Our findings uncover novel hallmarks of SMA disease progression and link SMN to general male infertility