Use of antisense oligonucleotides to enhance exon 7 incorporation in the pre-mRNA splicing of SMN2

Abstract only availableSpinal muscular atrophy (SMA) is a neurodegenerative disorder that is relatively common in humans and is caused by loss of the survival motor neuron 1 (SMN1) gene. SMA is the leading cause of hereditary infant mortality by causing anterior horn cell degeneration in the spinal cord resulting in trunk and limb paralysis. The survival motor neuron 2 (SMN2) gene is located proximally to the SMN1 gene on chromosome 5q and the two genes are almost identical. Interestingly, only mutations in SMN1 cause SMA, whereas mutations in SMN2 have no clinical consequence. A differential pre-mRNA splicing event results in SMN2s failure to compensate fully for SMN1 mutations. Exon 7 is excised during SMN2 RNA splicing and this causes expression of a non-functional gene product. SMN2 produces the SMN protein at low levels (~10% compared to SMN1) but not enough to compensate for the loss of SMN1. Previous studies have shown that the use of antisense oligonucleotides can significantly decrease recognition of the 3' splice site of exon 8, resulting in an increase in increased levels exon 7 inclusion in SMN2-derived transcripts. Here, we have developed in vivo expression vectors that generate antisense oligonucleotides spanning two regions of the SMN2 transcript: a repressor exon 7 splicing “repressor” region and the exon 8 splice acceptor site. We are adding a pol III terminating sequence downstream of the antisense sequences to ensure that the clones are only making short oligos. If successful, these vectors could be used to increase SMN protein expression in an SMA context and may open a new area of SMA therapeutics, as well as providing a fundamental basis for treating other genetic disorders where splicing events are the main cause of disease.Molecular Biology Progra

Genetic Differentiation of Lymphoblastic Sarcoma Histocompatibility Sites in DBA/1J and C57BL/6 Mice

Author Institution: Department of Biological Sciences, Kent State UniversityA chemically induced, 20-methylcholanthrene, lymphoblastic sarcoma, in DBA/1J mice was used to determine the histocompatibility of the tumor between the DBA/1J and C57BL/GJ strains. Although 8 different histocompatibility sites exist between the 2 strains, this work demonstrated that these 8 sites were transmitted and expressed as a 5 unit discrete factor phenotypic variation. Fi x Fi hybrid animals were tested through the Fg generation and showed that only 5 linkage groups are involved in the transmission of the various histocompatibility sites. Survivors of the implanted F9 generation were used as progenitors for the Fi0 generation. Tumor take frequencies with the Fi0 generation demonstrated that the inheritance of tumor susceptibility behaved as a discrete function of a 5 unit inheritance factor

SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss of motor neurons in patients with null mutations in the SMN1 gene. The almost identical SMN2 gene is unable to compensate for this deficiency because of the skipping of exon 7 during pre-messenger RNA (mRNA) processing. Although several splicing factors can modulate SMN2 splicing in vitro, the physiological regulators of this disease-causing event are unknown. We found that knockout of the splicing factor SAM68 partially rescued body weight and viability of SMAΔ7 mice. Ablation of SAM68 function promoted SMN2 splicing and expression in SMAΔ7 mice, correlating with amelioration of SMA-related defects in motor neurons and skeletal muscles. Mechanistically, SAM68 binds to SMN2 pre-mRNA, favoring recruitment of the splicing repressor hnRNP A1 and interfering with that of U2AF65 at the 3' splice site of exon 7. These findings identify SAM68 as the first physiological regulator of SMN2 splicing in an SMA mouse model

Building Dynamic Capabilities through Digital Innovation Units? - An analysis of their contribution and the spill-over effects to the main organization

To meet the challenge of digital transformation and remain competitive in an increasingly volatile business environment, many incumbent companies have set up digital innovation units (DIUs). Despite a steadily growing body of knowledge, research explaining the value contribution of such units is still at an early stage. Drawing on empirical data from a multiple case study, we adopt a dynamic capabilities perspective to better understand how DIUs also contribute to building dynamic capabilities as part of their role in the digital transformation of the main organization. To this end, we examined the DIUs of six manufacturing companies and were able to identify several DIU activities and skills that feed into the development and expansion of dynamic capabilities. Moreover, with respect to these activities and skills, we also uncovered positive spillover effects from the DIU to the main organization

Enhancement of SMN protein levels in a mouse model of spinal muscular atrophy using novel drug-like compounds

Spinal muscular atrophy (SMA) is a neurodegenerative disease that causes progressive muscle weakness, which primarily targets proximal muscles. About 95% of SMA cases are caused by the loss of both copies of the SMN1 gene. SMN2 is a nearly identical copy of SMN1, which expresses much less functional SMN protein. SMN2 is unable to fully compensate for the loss of SMN1 in motor neurons but does provide an excellent target for therapeutic intervention. Increased expression of functional full-length SMN protein from the endogenous SMN2 gene should lessen disease severity. We have developed and implemented a new high-throughput screening assay to identify small molecules that increase the expression of full-length SMN from a SMN2 reporter gene. Here, we characterize two novel compounds that increased SMN protein levels in both reporter cells and SMA fibroblasts and show that one increases lifespan, motor function, and SMN protein levels in a severe mouse model of SMA

How intelligence interviewees mentally identify relevant information

This research explored how intelligence interviewees mentally identify the relevant information at their disposal. We theorized that interviewees estimate the interviewer's objectives based on how they frame any attempt to solicit information. Then interviewees organize the information they possess into item designations that pragmatically correspond to the perceived interviewer-objective. The more an interviewer specifies what they want to know, the more the interviewee will mentally designate information items corresponding with that objective. To examine the theory, we conducted two identical experiments wherein participants assumed the role of an informant with one of two dispositions. They were to be cooperative or resistant when undergoing an interview. The interviewer posed specific or ambiguous questions. In Study 1 (N = 210), interviewees identified applicable information items based on their interviewer's questions. And interviewees answered their interviewer's questions in Study 2 (N = 199). We aimed to demonstrate that question type influences mental designations and disposition affects disclosures. Disposition had a stronger influence on interviewees' disclosure than when reasoning about what the interviewer wants to know. But contrary to our expectations, mental designation preferences indicated that interviewees generally assume interviewers want to know complete details, irrespective of question specificity. We suggest avenues for future research

Building Adaptive Capacity for Volatile Business Environments: A Longitudinal Study of the Establishment of a Digital Innovation Unit

To meet the challenge of digital transformation and remain competitive in an increasingly volatile business environment many incumbent companies have set up digital innovation units (DIU). Despite a steady increase in the body of knowledge, current research paints a rather static picture of DIUs due to a lack of studies that have collected data over an extended period of time to examine DIU-related processes. Based on empirical data from an in-depth, longitudinal case study with an incumbent machine manufacturer we examine the process of building a DIU and how the main organization can develop and expand its adaptive capacity (AC) in the process. We identify three mechanisms that positively impact the expansion of AC as well as five disturbing factors. Furthermore, we give first implications and point at the research avenue of the relationship between AC and trust

Morphological characteristics of motor neurons do not determine their relative susceptibility to degeneration in a mouse model of severe spinal muscular atrophy

Spinal muscular atrophy (SMA) is a leading genetic cause of infant mortality, resulting primarily from the degeneration and loss of lower motor neurons. Studies using mouse models of SMA have revealed widespread heterogeneity in the susceptibility of individual motor neurons to neurodegeneration, but the underlying reasons remain unclear. Data from related motor neuron diseases, such as amyotrophic lateral sclerosis (ALS), suggest that morphological properties of motor neurons may regulate susceptibility: in ALS larger motor units innervating fast-twitch muscles degenerate first. We therefore set out to determine whether intrinsic morphological characteristics of motor neurons influenced their relative vulnerability to SMA. Motor neuron vulnerability was mapped across 10 muscle groups in SMA mice. Neither the position of the muscle in the body, nor the fibre type of the muscle innervated, influenced susceptibility. Morphological properties of vulnerable and disease-resistant motor neurons were then determined from single motor units reconstructed in Thy.1-YFP-H mice. None of the parameters we investigated in healthy young adult mice - including motor unit size, motor unit arbor length, branching patterns, motor endplate size, developmental pruning and numbers of terminal Schwann cells at neuromuscular junctions - correlated with vulnerability. We conclude that morphological characteristics of motor neurons are not a major determinant of disease-susceptibility in SMA, in stark contrast to related forms of motor neuron disease such as ALS. This suggests that subtle molecular differences between motor neurons, or extrinsic factors arising from other cell types, are more likely to determine relative susceptibility in SMA

The discursive function of additives in interaction

The insertion of additives such as too has been argued to be obligatory (in affirmative sentences) if the immediate context contains a suitable antecedent such that the presupposition triggered by additives is satisfied. However, the obligatoriness of additives has been found to be gradient and their insertion to depend on contextual factors. While most research has focused on comprehension, the present study examines the production of additives and the extent to which they are obligatory by manipulating the factors Similarity and Turn Distance. We furthermore explored whether not using additives even in obligatory environments could be an instance of diverging (i.e. socially distancing) from the antecedent speaker. For this purpose we investigated whether speakers would omit additives when interacting with an impolite antecedent speaker. Overall, the results of our two experiments suggest that (i) in line with previous results on similarity, speakers tend to utter additives more frequently when their utterance’s content more closely matches the content of a previously formulated utterance; and (ii) speakers use additives more frequently when the matching utterance directly precedes their utterance. Furthermore, the results of experiment II suggest that (iii) speakers deliberately drop the use of additives when doing so would allow them to signal divergence from an impolite speaker. Our findings lend support to models in which speakers use additives as a discourse managing tool to organise the discourse and maintain discourse coherence