TSUNAMI: an antisense method to phenocopy splicing-associated diseases in animals

Antisense oligonucleotides (ASOs) are versatile molecules that can be designed to specifically alter splicing patterns of target pre-mRNAs. Here we exploit this feature to phenocopy a genetic disease. Spinal muscular atrophy (SMA) is a motor neuron disease caused by loss-of-function mutations in the SMN1 gene. The related SMN2 gene expresses suboptimal levels of functional SMN protein due to alternative splicing that skips exon 7; correcting this defect-e.g., with ASOs-is a promising therapeutic approach. We describe the use of ASOs that exacerbate SMN2 missplicing and phenocopy SMA in a dose-dependent manner when administered to transgenic Smn(-/-) mice. Intracerebroventricular ASO injection in neonatal mice recapitulates SMA-like progressive motor dysfunction, growth impairment, and shortened life span, with alpha-motor neuron loss and abnormal neuromuscular junctions. These SMA-like phenotypes are prevented by a therapeutic ASO that restores correct SMN2 splicing. We uncovered starvation-induced splicing changes, particularly in SMN2, which likely accelerate disease progression. These results constitute proof of principle that ASOs designed to cause sustained splicing defects can be used to induce pathogenesis and rapidly and accurately model splicing-associated diseases in animals. This approach allows the dissection of pathogenesis mechanisms, including spatial and temporal features of disease onset and progression, as well as testing of candidate therapeutics

New Samarium and Neodymium based admixed ferromagnets with near zero net magnetization and tunable exchange bias field

Rare earth based intermetallics, SmScGe and NdScGe, are shown to exhibit near zero net magnetization with substitutions of 6 to 9 atomic percent of Nd and 25 atomic percent of Gd, respectively. The notion of magnetic compensation in them is also elucidated by the crossover of zero magnetization axis at low magnetic fields (less than 103 Oe) and field-induced reversal in the orientation of the magnetic moments of the dissimilar rare earth ions at higher magnetic fields. These magnetically ordered materials with no net magnetization and appreciable conduction electron polarization display an attribute of an exchange bias field, which can be tuned. The attractively high magnetic ordering temperatures of about 270 K, underscore the importance of these materials for potential applications in spintronics.Comment: 6 page text + 5 figure

Genetic structuring across alternative life history tactics and small spatial scales in brown trout (Salmo trutta)

Facultative migration occurs when, in response to prevailing conditions, individuals in a population may (or may not) undertake a migration. The brown trout (Salmo trutta) is a species that exhibits facultative migration, where some individuals within populations may move to mainstem rivers (fluvial–adfluvial migration), lakes (lacustrine–adfluvial migration), estuaries (partial anadromy) or sea (anadromy) to feed, while others remain resident. This study attempts to separate two alternative hypotheses for the population structuring that underpins the expression of facultative migration in this species: (a) that anadromous and nonanadromous fish comprise two gene pools; (b) that individual genetic variation or individual variation in gene–environment interactions is responsible for the expression of different life‐history tactics within the same gene pool. The study design involved sampling and analyses of anadromous and nonanadromous brown trout from three independent tributary rivers known to produce (sea‐run) trout within the same catchment. Results indicate that, in all cases, population genetic divergence was linked to geographical location and not to life‐history tactics. Two genetically distinct coexisting population pairs were identified in two separate tributaries. Despite similar environmental conditions in both tributaries, the frequency of each life‐history tactic (anadromy vs. nonanadromous) within these population pairs differed significantly. The results of this study support the hypothesis that facultative migration in brown trout is likely to be driven by a quantitative threshold trait, where the threshold value varies both among populations and among individuals within populations

A functional SNP in the regulatory region of the decay-accelerating factor gene associates with extraocular muscle pareses in myasthenia gravis

Complement activation in myasthenia gravis (MG) may damage muscle endplate and complement regulatory proteins such as decay-accelerating factor (DAF) or CD55 may be protective. We hypothesize that the increased prevalence of severe extraocular muscle (EOM) dysfunction among African MG subjects reported earlier may result from altered DAF expression. To test this hypothesis, we screened the DAF gene sequences relevant to the classical complement pathway and found an association between myasthenics with EOM paresis and the DAF regulatory region c.-198C>G SNP (odds ratio=8.6; P=0.0003). This single nucleotide polymorphism (SNP) results in a twofold activation of a DAF 5′-flanking region luciferase reporter transfected into three different cell lines. Direct matching of the surrounding SNP sequence within the DAF regulatory region with the known transcription factor-binding sites suggests a loss of an Sp1-binding site. This was supported by the observation that the c.-198C>G SNP did not show the normal lipopolysaccharide-induced DAF transcriptional upregulation in lymphoblasts from four patients. Our findings suggest that at critical periods during autoimmune MG, this SNP may result in inadequate DAF upregulation with consequent complement-mediated EOM damage. Susceptible individuals may benefit from anti-complement therapy in addition to immunosuppression

Nucleic Acid-Based Therapeutic Approach for Spinal and Bulbar Muscular Atrophy and Related Neurological Disorders

The recent advances in nucleic acid therapeutics demonstrate the potential to treat hereditary neurological disorders by targeting their causative genes. Spinal and bulbar muscular atrophy (SBMA) is an X-linked and adult-onset neurodegenerative disorder caused by the expansion of trinucleotide cytosine-adenine-guanine repeats, which encodes a polyglutamine tract in the androgen receptor gene. SBMA belongs to the family of polyglutamine diseases, in which the use of nucleic acids for silencing a disease-causing gene, such as antisense oligonucleotides and small interfering RNAs, has been intensively studied in animal models and clinical trials. A unique feature of SBMA is that both motor neuron and skeletal muscle pathology contribute to disease manifestations, including progressive muscle weakness and atrophy. As both motor neurons and skeletal muscles can be therapeutic targets in SBMA, nucleic acid-based approaches for other motor neuron diseases and myopathies may further lead to the development of a treatment for SBMA. Here, we review studies of nucleic acid-based therapeutic approaches in SBMA and related neurological disorders and discuss current limitations and perspectives to apply these approaches to patients with SBMA

Systemic Versus CNS Delivery Of MOE Antisense Oligonucleotide to Correct Defective Splicing in a Severe Mouse Model of Spinal Muscular Atrophy

Spinal Muscular Atrophy (SMA) is a genetic disease characterized by progressive degeneration of motor neurons in the spinal cord, leading to muscle weakness and atrophy. SMA is caused by deletion or mutations in the Survival-of-motor neuron(SMN1) gene. The paralogous SMN2 gene, present in one or more copies in all SMA patients, attenuates SMA severity, but expresses low levels of full-length SMN protein, due to alternative splicing that results in inefficient inclusion of exon 7. Increasing SMN2 exon 7 inclusion to express more full-length, functional SMN protein in motor neurons is a promising approach to treat SMA. We previously reported a 2′-O-(2-methoxyethyl) (MOE) phosphorothioate 18mer antisense oligonucleotide (ASO) that targets a splicing-repressor binding site in intron 7. By preventing binding of the repressor (hnRNP A1), the ASO promotes efficient SMN2exon 7 inclusion in liver and kidneys of transgenic mice after systemic administration. It is generally believed that SMN restoration in spinal-cord motor neurons is necessary and sufficient to cure SMA. However, cardiac defects were recently reported in both severe SMA patients and mouse models. These defects might reflect autonomic dysfunction; alternatively, they could be caused by factors outside the CNS. In the latter scenario, peripheral SMN restoration might be necessary for therapy.We sought to compare the therapeutic effects of systemic restoration versus CNS restoration of the SMN protein in neonates of a severe mouse model (Smn-/-; hSmn2+/0) that survives 10 days. To increase SMN levels in the CNS, we directly injected the ASO into a cerebral lateral ventricle at postnatal day 1 (P1); to increase SMN levels in peripheral tissues, we subcutaneously injected the ASO into P0-P3 neonates. Surprisingly, neonatal systemic administration robustly rescued SMA mice, and was much more effective than intracerebroventricular administration alone; a single neonatal subcutaneous injection extended the median lifespan by 25-fold. The majority of the rescued mice had no motor defects, and showed increased motor-neuron numbers and normal neuromuscular-junction morphology. Remarkably, some of the SMA mice treated systemically at P0-P3 have so far survived for 1 year and are still vigorous. Our data not only demonstrate an effective drug candidate, but also reveal the importance of SMN restoration outside the CNS for treatment of severe SMA. The mechanisms underlying the striking effectiveness of SMN restoration in peripheral tissues will be discussed

Complement membrane attack is required for endplate damage and clinical disease in passive experimental myasthenia gravis in Lewis rats

Myasthenia gravis (MG) is a debilitating and potentially fatal neuromuscular disease characterized by the generation of autoantibodies reactive with nicotinic acetylcholine receptors (AChR) that cause loss of AChR from the neuromuscular endplate with resultant failure of neuromuscular transmission. A role for complement (C) in the pathology of human MG has been suggested based upon identification of C activation products in plasma and deposited at the endplate in MG. In the rat model, experimental autoimmune MG (EAMG), C depletion or inhibition restricts clinical disease, further implicating C in pathology. The mechanisms by which C activation drives pathology in MG and EAMG are unclear. Here we provide further evidence implicating C and specifically the membrane attack complex (MAC) in the Lewis rat passive EAMG model of MG. Rats deficient in C6, an essential component of the MAC, were resistant to disease induction and endplate destruction was reduced markedly compared to C6-sufficient controls. After reconstitution with C6, disease severity and endplate destruction in the C6-deficient rats was equivalent to that in controls. The data confirm the essential role of the MAC in the destruction of the endplate in EAMG and raise the prospect of specific MAC inhibition as an alternative therapy in MG patients resistant to conventional treatments