Spinal Muscular Atrophy Gene Research Articles

The spinal muscular atrophy gene product regulates actin dynamics.

Spinal Muscular Atrophy (SMA) is a neuromuscular disease caused by low levels of the Survival of Motoneuron (SMN) protein. SMN interacts with and regulates the actin-binding protein profilin2a, thereby influencing actin dynamics. Dysfunctional actin dynamics caused by SMN loss disrupts neurite outgrowth, axonal pathfinding, and formation of functional synapses in neurons. Whether the SMN protein directly interacts with and regulates filamentous (F-) and monomeric globular (G-) actin is still elusive. In a quantitative single cell approach, we show that SMN loss leads to dysregulated F-/G-actin fractions. Furthermore, quantitative assessment of cell morphology suggests an F-actin organizational defect. Interestingly, this is mediated by an interaction of SMN with G- and F-actin. In co-immunoprecipitation, in-vitro pulldown and co-localization assays, we elucidated that this interaction is independent of the SMN-profilin2a interaction. Therefore, we suggest two populations being relevant for functional actin dynamics in healthy neurons: SMN-profilin2a-actin and SMN-actin. Additionally, those two populations may influence each other and therefore regulate binding of SMN to actin. In SMA, we showed a dysregulated co-localization pattern of SMN-actin which could only partially rescued by SMN restoration. However, dysregulation of F-/G-actin fractions was reduced by SMN restoration. Taken together, our results suggest a novel molecular function of SMN in binding to actin independent from SMN-profilin2a interaction.

U1 snRNA interactions with deep intronic sequences regulate splicing of multiple exons of spinal muscular atrophy genes.

The U1 small nuclear RNA (snRNA) forms ribonucleoprotein particles (RNPs) such as U1 snRNP and U1-TAF15 snRNP. U1 snRNP is one of the most studied RNPs due to its critical role in pre-mRNA splicing in defining the 5' splice site (5'ss) of every exon through direct interactions with sequences at exon/intron junctions. Recent reports support the role of U1 snRNP in all steps of transcription, namely initiation, elongation, and termination. Functions of U1-TAF15 snRNP are less understood, though it associates with the transcription machinery and may modulate pre-mRNA splicing by interacting with the 5'ss and/or 5'ss-like sequences within the pre-mRNA. An anti-U1 antisense oligonucleotide (ASO) that sequesters the 5' end of U1 snRNA inhibits the functions of U1 snRNP, including transcription and splicing. However, it is not known if the inhibition of U1 snRNP influences post-transcriptional regulation of pre-mRNA splicing through deep intronic sequences. We examined the effect of an anti-U1 ASO that sequesters the 5' end of U1 snRNA on transcription and splicing of all internal exons of the spinal muscular atrophy (SMA) genes, SMN1 and SMN2. Our study was enabled by the employment of a multi-exon-skipping detection assay (MESDA) that discriminates against prematurely terminated transcripts. We employed an SMN2 super minigene to determine if anti-U1 ASO differently affects splicing in the context of truncated introns. We observed substantial skipping of multiple internal exons of SMN1 and SMN2 triggered by anti-U1 treatment. Suggesting a role for U1 snRNP in interacting with deep intronic sequences, early exons of the SMN2 super minigene with truncated introns were resistant to anti-U1 induced skipping. Consistently, overexpression of engineered U1 snRNAs targeting the 5'ss of early SMN1 and SMN2 exons did not prevent exon skipping caused by anti-U1 treatment. Our results uncover a unique role of the U1 snRNA-associated RNPs in splicing regulation executed through deep intronic sequences. Findings are significant for developing novel therapies for SMA based on deep intronic targets.

Transcriptome- and proteome-wide effects of a circular RNA encompassing four early exons of the spinal muscular atrophy genes

Spinal muscular atrophy (SMA) genes, SMN1 and SMN2 (hereinafter referred to as SMN1/2), produce multiple circular RNAs (circRNAs), including C2A–2B–3–4 that encompasses early exons 2A, 2B, 3 and 4. C2A-2B-3-4 is a universally and abundantly expressed circRNA of SMN1/2. Here we report the transcriptome- and proteome-wide effects of overexpression of C2A–2B–3–4 in inducible HEK293 cells. Our RNA-Seq analysis revealed altered expression of ~ 15% genes (4172 genes) by C2A–2B–3–4. About half of the affected genes by C2A–2B–3–4 remained unaffected by L2A–2B–3–4, a linear transcript encompassing exons 2A, 2B, 3 and 4 of SMN1/2. These findings underscore the unique role of the structural context of C2A–2B–3–4 in gene regulation. A surprisingly high number of upregulated genes by C2A–2B–3–4 were located on chromosomes 4 and 7, whereas many of the downregulated genes were located on chromosomes 10 and X. Supporting a cross-regulation of SMN1/2 transcripts, C2A–2B–3–4 and L2A–2B–3–4 upregulated and downregulated SMN1/2 mRNAs, respectively. Proteome analysis revealed 61 upregulated and 57 downregulated proteins by C2A–2B–3–4 with very limited overlap with those affected by L2A–2B–3–4. Independent validations confirmed the effect of C2A–2B–3–4 on expression of genes associated with chromatin remodeling, transcription, spliceosome function, ribosome biogenesis, lipid metabolism, cytoskeletal formation, cell proliferation and neuromuscular junction formation. Our findings reveal a broad role of C2A–2B–3–4, and expands our understanding of functions of SMN1/2 genes.

Molecular-genetic study of <i>SMN1</i> and <i>SMN2</i> genes associated with spinal muscular atrophy in individuals with infertility prior to in vitro fertilization

Introduction. The rate of infertile couples comprises 17–24 % and tends to increase, with more of them turning to assisted reproductive technologies (ART). In case a required result is achieved, future parents-to-be prepare for birth of a healthy child in the family, but each couple may face a birth of offspring with severe genetic disorders including spinal muscular atrophy (SMA).Aim: to access a carriage rate for SMN1 gene exon 7 deletion associated with SMA and SMN2 gene copy number in married couples planning childbirth using in vitro fertilization (IVF).Materials and Methods. There were enrolled 170 couples (340 subjects) suffering from infertility and referred for IVF in the Sverdlovsk region (SR) of Russia. The search for deletions/duplications in the SMN1 and SMN2 genes was carried out by quantitatively analyzing number of gene copies using the SALSA MLPA Probemix P460 commercial kit (MRC-Holland, the Netherlands). The Hardy–Weinberg ratio was used to calculate estimated rate of homozygous carriers with SMN1 deletions in the next generation.Results. Among 340 patients, a deletion of exon 7 in the SMN1 gene (one copy out of two) was found in 9 individuals (3 males and 6 females) unrelated to marital relations. Thus, only 9 out of 340 examined subjects carry such gene mutation associated with SMA, with total rate of carriage comprised 2.65 % (1/38). Given the number of IVF procedures performed in SR, it may be assumed that the probability for birth of a sick child in such couple comprises at least 1:6410. A number of patients had SMN1 gene duplications – 9 (5.29 %) males and 4 (2.35 %) females baring 3 copies of the SMN1 gene. In addition, the majority of study participants (54 %) turned out to have 3 copies of the SMN2 gene.Conclusion. The rate of SMA carriage in married couples planning a pregnancy with aid of ART corresponds to the general population reaching 1:38 level. It is believed necessary that all couples entering the IVF program should be examined for carriage of SMN1 gene mutations to assess SMA risk in offspring.

Synergistic Effect of an Antisense Oligonucleotide and Small Molecule on Splicing Correction of the Spinal Muscular Atrophy Gene.

Spinal muscular atrophy (SMA) is treated by increasing the level of Survival Motor Neuron (SMN) protein through correction of SMN2 exon 7 skipping or exogenous expression of SMN through gene therapy. Currently available therapies have multiple shortcomings, including poor body-wide distribution, invasive delivery, and potential negative consequences due to high doses needed for clinical efficacy. Here we test the effects of a combination treatment of a splice-correcting antisense oligonucleotide (ASO) Anti-N1 with the small compounds risdiplam and branaplam. We show that a low-dose treatment of Anti-N1 with either compound produces a synergistic effect on the inclusion of SMN2 exon 7 in SMA patient fibroblasts. Using RNA-Seq, we characterize the transcriptomes of cells treated with each compound as well as in combination. Although high doses of each individual treatment trigger widespread perturbations of the transcriptome, combination treatment of Anti-N1 with risdiplam and branaplam results in minimal disruption of gene expression. For individual genes targeted by the 3 compounds, we observe little to no additive effects of combination treatment. Overall, we conclude that the combination treatment of a splice-correcting ASO with small compounds represents a promising strategy for achieving a high level of SMN expression while minimizing the risk of off-target effects.

Gene therapy for selected neuromuscular and trinucleotide repeat disorders - An insight to subsume South Asia for multicenter clinical trials.

In this article, the authors discuss how they utilized the genetic mutation data in Sri Lankan Duchenne muscular dystrophy (DMD), Spinal muscular atrophy (SMA), Spinocerebellar ataxia (SCA) and Huntington's disease (HD) patients and compare the available literature from South Asian countries to identifying potential candidates for available gene therapy for DMD, SMA, SCA and HD patients. Rare disease patients (n=623) with the characteristic clinical findings suspected of HD, SCA, SMA and Muscular Dystrophy were genetically confirmed using Multiplex Ligation Dependent Probe Amplification (MLPA), and single plex PCR. A survey was conducted in the "Wiley database on Gene Therapy Trials Worldwide" to identify DMD, SMA, SCA, and HD gene therapy clinical trials performed worldwide up to April 2021. In order to identify candidates for gene therapy in other neighboring countries we compared our findings with available literature from India and Pakistan which has utilized the same molecular diagnostic protocol to our study. From the overall cohort of 623 rare disease patients with the characteristic clinical findings suspected of HD, SCA, SMA and Muscular Dystrophy, n=343 (55%) [Muscular Dystrophy- 65%; (DMD-139, Becker Muscular Dystrophy -BMD-11), SCA type 1-3-53% (SCA1-61,SCA2- 23, SCA3- 39), HD- 52% (45) and SMA- 34% (22)] patients were positive for molecular diagnostics by MLPA and single plex PCR. A total of 147 patients in Sri Lanka amenable to available gene therapy; [DMD-83, SMA-15 and HD-49] were identified. A comparison of Sri Lankan finding with available literature from India and Pakistan identified a total of 1257 patients [DMD-1076, SMA- 57, and HD-124] from these three South Asian Countries as amenable for existing gene therapy trials. DMD, SMA, and HD gene therapy clinical trials (113 studies) performed worldwide up to April 2021 were concentrated mostly (99%) in High Income Countries (HIC) and Upper Middle-Income Countries (UMIC). However, studies on the potential use of anti-sense oligonucleotides (ASO) for treatment of SCAs have yet to reach clinical trials. Most genetic therapies for neurodegenerative and neuromuscular disorders have been evaluated for efficacy primarily in Western populations. No multicenter gene therapy clinical trial sites for DMD, SMA and HD in the South Asian region, leading to lack of knowledge on the safety and efficacy of such personalized therapies in other populations, including South Asians. By fostering collaboration between researchers, clinicians, patient advocacy groups, government and industry in gene therapy initiatives for the inherited-diseases community in the developing world would link the Global North and Global South and breathe life into the motto "Together we can make a difference".

Structural and functional characterization of capsid binding by anti-AAV9 monoclonal antibodies from infants after SMA gene therapy

Success in the treatment of infants with spinal muscular atrophy (SMA) underscores the potential of vectors based on adeno-associated virus (AAV). However, a major obstacle to the full realization of this potential is pre-existing natural and therapy-induced anti-capsid humoral immunity. Structure-guided capsid engineering is one possible approach to surmounting this challenge but necessitates an understanding of capsid-antibody interactions at high molecular resolution. Currently, only mouse-derived monoclonal antibodies (mAbs) are available to structurally map these interactions, which presupposes that mouse and human-derived antibodies are functionally equivalent. In this study, we have characterized the polyclonal antibody responses of infants following AAV9-mediated gene therapy for SMA and recovered 35 anti-capsid mAbs from the abundance of switched-memory B (smB) cells present in these infants. For 21 of these mAbs, seven from each of three infants, we have undertaken functional and structural analysis measuring neutralization, affinities, and binding patterns by cryoelectron microscopy (cryo-EM). Four distinct patterns were observed akin to those reported for mouse-derived mAbs, but with early evidence of differing binding pattern preference and underlying molecular interactions. This is the first human and largest series of anti-capsid mAbs to have been comprehensively characterized and will prove to be powerful tools for basic discovery and applied purposes.

Gene Therapy in ALS and SMA: Advances, Challenges and Perspectives.

Gene therapy is defined as the administration of genetic material to modify, manipulate gene expression or alter the properties of living cells for therapeutic purposes. Recent advances and improvements in this field have led to many breakthroughs in the treatment of various diseases. As a result, there has been an increasing interest in the use of these therapies to treat motor neuron diseases (MNDs), for which many potential molecular targets have been discovered. MNDs are neurodegenerative disorders that, in their most severe forms, can lead to respiratory failure and death, for instance, spinal muscular atrophy (SMA) or amyotrophic lateral sclerosis (ALS). Despite the fact that SMA has been known for many years, it is still one of the most common genetic diseases causing infant mortality. The introduction of drugs based on ASOs-nusinersen; small molecules-risdiplam; and replacement therapy (GRT)-Zolgensma has shown a significant improvement in both event-free survival and the quality of life of patients after using these therapies in the available trial results. Although there is still no drug that would effectively alleviate the course of the disease in ALS, the experience gained from SMA gene therapy gives hope for a positive outcome of the efforts to produce an effective and safe drug. The aim of this review is to present current progress and prospects for the use of gene therapy in the treatment of both SMA and ALS.

The phospho-landscape of the survival of motoneuron protein (SMN) protein: relevance for spinal muscular atrophy (SMA).

Spinal muscular atrophy (SMA) is caused by low levels of the survival of motoneuron (SMN) Protein leading to preferential degeneration of lower motoneurons in the ventral horn of the spinal cord and brain stem. However, the SMN protein is ubiquitously expressed and there is growing evidence of a multisystem phenotype in SMA. Since a loss of SMN function is critical, it is important to decipher the regulatory mechanisms of SMN function starting on the level of the SMN protein itself. Posttranslational modifications (PTMs) of proteins regulate multiple functions and processes, including activity, cellular trafficking, and stability. Several PTM sites have been identified within the SMN sequence. Here, we map the identified SMN PTMs highlighting phosphorylation as a key regulator affecting localization, stability and functions of SMN. Furthermore, we propose SMN phosphorylation as a crucial factor for intracellular interaction and cellular distribution of SMN. We outline the relevance of phosphorylation of the spinal muscular atrophy (SMA) gene product SMN with regard to basic housekeeping functions of SMN impaired in this neurodegenerative disease. Finally, we compare SMA patient mutations with putative and verified phosphorylation sites. Thus, we emphasize the importance of phosphorylation as a cellular modulator in a clinical perspective as a potential additional target for combinatorial SMA treatment strategies.

Internal Introns Promote Backsplicing to Generate Circular RNAs from Spinal Muscular Atrophy Gene.

Human survival motor neuron 1 (SMN1) codes for SMN, an essential housekeeping protein involved in most aspects of RNA metabolism. Deletions or mutations of SMN1 lead to spinal muscular atrophy (SMA), a devastating neurodegenerative disease linked to a high rate of infant mortality. SMN2, a near identical copy of SMN1 present in humans, cannot compensate for the loss of SMN1 due to predominant skipping of SMN2 exon 7. Restoration of SMN by splicing modulation of SMN2 exon 7 or gene replacement are currently approved therapies of SMA. Human SMN genes produce a vast repertoire of circular RNAs (circRNAs). However, the mechanism of SMN circRNA generation has not yet been examined in detail. For example, it remains unknown if forward splicing impacts backsplicing that generates circRNAs containing multiple exons. Here, we employed SMN as a model system to examine the impact of intronic sequences on the generation of circRNAs. We performed our experiments in HeLa cells transiently transfected with minigenes expressing three abundantly represented circRNAs containing two or more SMN exons. We observed an enhanced rate of circRNA generation when introns joining exons to be incorporated into circRNAs were present as compared to the intronless context. These results underscore the stimulatory effect of forward splicing in the generation of circRNAs containing multiple exons. These findings are consistent with the reported low abundance of SMN circRNAs comprised of single exons. We confirmed our findings using inducible HEK 293 cells stably expressing the SMN circRNAs. Our results support the role of the exon junction complex in the generation of the exon-only-containing circRNAs. We showed that SMN circRNAs were preferentially localized in the cytoplasm. These findings provide new insights regarding our understanding of circRNA generation and open avenues to uncover novel functions of the SMN genes.

Central and peripheral delivered AAV9-SMN are both efficient but target different pathomechanisms in a mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by loss of the SMN1 gene and low SMN protein levels. Although lower motor neurons are a primary target, there is evidence that peripheral organ defects contribute to SMA. Current SMA gene therapy and clinical trials use a single intravenous bolus of the blood-brain-barrier penetrant scAAV9-cba-SMN by either systemic or central nervous system (CNS) delivery, resulting in impressive amelioration of the clinical phenotype but not a complete cure. The impact of scAAV9-cba-SMN treatment regimens on the CNS as well as on specific peripheral organs is yet to be described in a comparative manner. Therefore, we injected SMA mice with scAAV9-cba-SMN either intravenously (IV) for peripheral SMN restoration or intracerebroventricularly (ICV) for CNS-focused SMN restoration. In our system, ICV injections increased SMN in peripheral organs and the CNS while IV administration increased SMN in peripheral tissues only, largely omitting the CNS. Both treatments rescued several peripheral phenotypes while only ICV injections were neuroprotective. Surprisingly, both delivery routes resulted in a robust rescue effect on survival, weight, and motor function, which in IV-treated mice relied on peripheral SMN restoration but not on targeting the motor neurons. This demonstrates the independent contribution of peripheral organs to SMA pathology and suggests that treatments should not be restricted to motor neurons.

2020 Update to Spinal Muscular Atrophy Management in Saudi Arabia.

Novel therapeutic strategies have shown some promise in treating spinal muscular atrophy (SMA). However, the outcomes and acceptance of these new strategies are yet to be explored. We aimed to investigate physicians' opinions and perceptions toward management strategies of SMA across Saudi Arabia. This is a cross-sectional survey using a self-administered, structured questionnaire sent to physicians who care for SMA patients during the Saudi Pediatric Neurology Society annual conference. A total of 72 clinicians of different neurological subspecialties were included. 48.6% prescribed nusinersen to their patients, with 39% of them having patients started on nusinersen. Though, 8.3% prescribed onasemnogene abeparvovec for 1–3 patients, while none of their patients started on the treatment. 64.3% stated that the only treatment available for SMA in their settings is supportive care. Around 69.4% described having a moderate to high knowledge on SMA gene therapy, and 79.2% would recommend it. 48.6% confirmed they would prescribe gene therapy at the age of 6 months, and 78.3% would prescribe it for type-I SMA. Pediatric neurologists are receptive to novel and innovative therapies for SMA in Saudi Arabia. However, the high treatment acquisition cost, strict regulations, logistical issues, and budget constraints delay their adoption and implementation.

Polymerase chain reaction detection methods of Survival Motor Neuron genes: a review

Two SMN (survival motor neuron) genes are presented in the human genome: SMN1, which present the telomeric gene whose homozygous deletion or mutation like gene conversion, causes spinal muscular atrophy (SMA), and SMN2, the centromeric version whose copy number modulates the phenotype of SMA These genes are commonly detected by Polymerase Chain reaction-based methods, and these are MLPA (Multiplex ligation-dependent probe amplification), qPCR (quantitative Polymerase chain reaction) and PCR-RFLP (Polymerase chain reaction-Restriction fragment length polymorphism). This paper reviews the current standing of the most common PCR methods used in the detection of spinal muscular atrophy genes. MLPA, qPCR, and PCR-RFLP currently represent the most common methods of choice for the detection of mutations, especially for deletion and duplication mutations.

New Treatments in Spinal Muscular Atrophy: Positive Results and New Challenges

Spinal muscular atrophy (SMA) is one of the most common autosomal recessive diseases with progressive weakness of skeletal and respiratory muscles, leading to significant disability. The disorder is caused by mutations in the survival motor neuron 1 (SMN1) gene and a consequent decrease in the SMN protein leading to lower motor neuron degeneration. Recently, Food and Drug Administration (FDA) and European Medical Agency (EMA) approved the antisense oligonucleotide nusinersen, the first SMA disease-modifying treatment and gene replacement therapy by onasemnogene abeparvovec. Encouraging results from phase II and III clinical trials have raised hope that other therapeutic options will enter soon in clinical practice. However, the availability of effective approaches has raised up ethical, medical and financial issues that are routinely faced by the SMA community. This review covers the available data and the new challenges of SMA therapeutic strategies.

Result of carrier screening for spinal muscular atrophy among 3049 reproductive-age individuals from Yunnan region

To perform carrier screening for spinal muscular atrophy (SMA) among 3049 reproductive-age individuals from Yunnan region and determine the copy number of survival motor neuron (SMN) gene and carrier frequencies. Multiplex ligation-dependent probe amplification (MLPA) was used to determine the copy number of exon 7 of SMN1 and SMN2 genes and identify those with a single copy of SMN1 gene. Prenatal diagnosis was performed for couples whom were both found to be SMA carriers. In total 62 SMA carriers were identified among the 3049 subjects, which yielded a carrier frequency of 1 in 49 (2.03%). No statistical difference was found in the carrier frequency between males and females (1.91% vs. 2.30%, P>0.05). Respectively, 1.3% (41/3049) and 0.69% (21/3049) of the carriers were caused by heterozygous deletion and conversion of the SMN1 gene. The average copy number for SMN1 alleles was 1.99. Two couples were found to be both as SMA carriers, for whom the birth of an affected fetus was avoided by prenatal diagnosis. No difference was found in the carrier frequency of SMA-related mutations between the two genders in Yunnan region, which was in keeping to an autosomal recessive inheritance pattern. Determination of the carrier frequency for SMA and SMN gene variants may provide a basis for genetic counseling and prenatal diagnosis for the disease.

Sam68 binds Alu-rich introns in SMN and promotes pre-mRNA circularization.

The Spinal Muscular Atrophy (SMA) gene SMN was recently duplicated (SMN1 and SMN2) in higher primates. Furthermore, invasion of the locus by repetitive elements almost doubled its size with respect to mouse Smn, in spite of an almost identical protein-coding sequence. Herein, we found that SMN ranks among the human genes with highest density of Alus, which are evolutionary conserved in primates and often occur in inverted orientation. Inverted repeat Alus (IRAlus) negatively regulate splicing of long introns within SMN, while promoting widespread alternative circular RNA (circRNA) biogenesis. Bioinformatics analyses revealed the presence of ultra-conserved Sam68 binding sites in SMN IRAlus. Cross-link-immunoprecipitation (CLIP), mutagenesis and silencing experiments showed that Sam68 binds in proximity of intronic Alus in the SMN pre-mRNA, thus favouring circRNA biogenesis in vitro and in vivo. These findings highlight a novel layer of regulation in SMN expression, uncover the crucial impact exerted by IRAlus and reveal a role for Sam68 in SMN circRNA biogenesis.

P.367Comparative analysis of single stranded rAAV vectors expressing human SMN1 in correcting spinal muscular atrophy in mice

Spinal muscular atrophy (SMA) is the most frequent genetic cause of infant death. It is an autosomal recessive neuromuscular disorder due to mutations in the SMN1 gene, leading to a selective loss of spinal cord -motor neurons and proximal muscle weakness. Remarkable progress has been made in the last years on the development of therapeutic approaches for SMA, including gene therapy. In particular, intravenous administration of self-complementary (sc) recombinant AAV9-SMN1 vectors have shown efficacy in several animal models and a clinical trial in type 1 SMA patients. In the present study, we evaluated the therapeutic efficacy of single stranded (ss) rAAV9 vectors expressing the human SMN1 coding sequence under various regulatory sequences in the Smn2B/- mouse model. Vectors were administrated by a single intracerebroventricular (ICV) injection at 8E12 vg/kg in newborn mice and compared for phenotype rescue. The ssAAV9-SMN1 vectors were able to increase the survival rate of Smn2B/- mice up to 1 year with differences in the median lifespan, compared to 26 days in non-treated mutant mice. Notably, this treatment increased the number of spinal motor neurons and improved the morphology of neuromuscular junctions close to normal. These results demonstrate the importance of regulatory sequences in vector design and the feasibility of SMA gene therapy by administration of ssAAV-SMN1 vectors.

August is a banner month for drug approvals

While others vacationed last month, scientists and regulators at the US Food and Drug Administration were busy. The agency approved 9 new drugs in August—more than a third of the drugs it has approved so far this year. And hearteningly for chemists who work in drug discovery, all 9 are small molecules. Through Aug. 29, the FDA approved 26 new drugs, compared with 34 in the same period in 2018 and 31 in the first eight months of 2017. The agency approved a record 59 drugs in 2018.S mall molecules are dominating this year’s new drugs. Of the 26 approved, 20—over 75%—are small molecules. Four are antibodies, one is a peptide, and one is a Botox-like botulinum toxin. Not included in the tally is the Novartis spinal muscular atrophy gene therapy Zolgensma, which the FDA doesn’t consider a drug. Sixty-four percent of the drugs approved in 2018 were small molecules.

AAV9 Vector: a Novel modality in gene therapy for spinal muscular atrophy.

Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, is characterized by the deterioration of alpha motor neurons in the brainstem and spinal cord. Currently, there is no cure for SMA, which calls for an urgent need to explore affordable and effective therapies and to maximize patients' independence and quality of life. Adeno-associated virus (AAV) vector, one of the most promising and well-investigated vehicles for delivering transgenes, is a compelling candidate for gene therapy. Some of the hallmarks of AAVs are their nonpathogenicity, inability to incur an immune response, potential to achieve robust transgene expression, and varied tropism for several tissues of the body. Recently, these features were harnessed in a clinical trial conducted by AveXis in SMA patients, where AAV9 was employed as a vehicle for one-time administration of the SMN gene, the causative gene in SMA. The trial demonstrated remarkable improvements in motor milestones and rates of survival in the patients. This review focuses on the advent of SMA gene therapy and summarizes different preclinical studies that were conducted leading up to the AAV9-SMA trial in SMA patients.

Evaluation of Cell-Penetrating Peptide Delivery of Antisense Oligonucleotides for Therapeutic Efficacy in Spinal Muscular Atrophy.

Antisense oligonucleotides (ASOs) are a widely used form of gene therapy, which is translatable to multiple disorders. A major obstacle for ASO efficacy is its bioavailability for in vivo and in vitro studies. To overcome this challenge we use cell-penetrating peptides (CPPs) for systemic delivery of ASOs. One of the most advanced clinical uses of ASOs is for the treatment of spinal muscular atrophy (SMA). In this chapter, we describe the techniques used for in vitro screening and analysing in vivo biodistribution of CPP-conjugated ASOs targeting the survival motor neuron 2, SMN2, the dose-dependent modifying gene for SMA.