Next Generation Exon 51 Skipping Antisense Oligonucleotides for Duchenne Muscular Dystrophy.

In the last two decades, antisense oligonucleotides (AONs) that induce corrective exon skipping have matured as promising therapies aimed at tackling the dystrophin deficiency that underlies the severe and progressive muscle fiber degeneration in Duchenne muscular dystrophy (DMD) patients. Pioneering first generation exon 51 skipping AONs like drisapersen and eteplirsen have more recently been followed up by AONs for exons 53 and 45, with, to date, a total of four exon skipping AON drugs having reached (conditional) regulatory US Food and Drug Administration (FDA) approval for DMD. Nonetheless, considering the limited efficacy of these drugs, there is room for improvement. The aim of this study was to develop more efficient [2'-O-methyl-modified phosphorothioate (2'OMePS) RNA] AONs for DMD exon 51 skipping by implementing precision chemistry as well as identifying a more potent target binding site. More than a hundred AONs were screened in muscle cell cultures, followed by a selective comparison in the hDMD and hDMDdel52/mdx mouse models. Incorporation of 5-methylcytosine and position-specific locked nucleic acids in AONs targeting the drisapersen/eteplirsen binding site resulted in 15-fold higher exon 51 skipping levels compared to drisapersen in hDMDdel52/mdx mice. However, with similarly modified AONs targeting an alternative site in exon 51, 65-fold higher skipping levels were obtained, restoring dystrophin up to 30% of healthy control. Targeting both sites in exon 51 with a single AON further increased exon skipping (100-fold over drisapersen) and dystrophin (up to 40%) levels. These dystrophin levels allowed for normalization of creatine kinase (CK) and lactate dehydrogenase (LDH) levels, and improved motor function in hDMDdel52/mdx mice. As no major safety observation was obtained, the improved therapeutic index of these next generation AONs is encouraging for further (pre)clinical development.

An in vivo accelerated developmental myelination model for testing promyelinating therapeutics.

BACKGROUND: Therapeutic agents stimulating the process of myelination could be beneficial for the treatment of demyelinating diseases, such as multiple sclerosis. The efficient translation of compounds promoting myelination in vitro to efficacy in vivo is inherently time-consuming and expensive. Thyroid hormones accelerate the differentiation and maturation of oligodendrocytes, thereby promoting myelination. Systemic administration of the thyroid hormone thyroxine (T4) accelerates brain maturation, including myelination, during early postnatal development. The objective of this study was to validate an animal model for rapid testing of promyelinating therapeutic candidates for their effects on early postnatal development by using T4 as a reference compound. METHODS: Daily subcutaneous injections of T4 were given to Sprague Dawley rat pups from postnatal day (PND) 2 to PND10. Changes in white matter were determined at PND10 using diffusion tensor magnetic resonance imaging (DTI). Temporal changes in myelination from PND3 to PND11 were also assessed by quantifying myelin basic protein (MBP) expression levels in the brain using the resonance Raman spectroscopy/enzyme-linked immunosorbent assay (RRS-ELISA) and quantitative immunohistochemistry. RESULTS: DTI of white matter tracts showed significantly higher fractional anisotropy in the internal capsule of T4-treated rat pups. The distribution of total FA values in the forebrain was significantly shifted towards higher values in the T4-treated group, suggesting increased myelination. In vivo imaging data were supported by in vitro observations, as T4 administration significantly potentiated the developmental increase in MBP levels in brain lysates starting from PND8. MBP levels in the brain of animals that received treatment for 9 days correlated with the FA metric determined in the same pups in vivo a day earlier. Furthermore, accelerated developmental myelination following T4 administration was confirmed by immunohistochemical staining for MBP in coronal brain sections of treated rat pups. CONCLUSIONS: T4-treated rat pups had increased MBP expression levels and higher MRI fractional anisotropy values, both indications of accelerated myelination. This simple developmental myelination model affords a rapid test of promyelinating activity in vivo within several days, which could facilitate in vivo prescreening of candidate therapeutic compounds for developmental hypomyelinating diseases. Further research will be necessary to assess the utility of this platform for screening promyelination compounds in more complex demyelination disease models, such us multiple sclerosis.

Global Rhes knockout in the Q175 Huntington's disease mouse model.

Huntington's disease (HD) results from an expansion mutation in the polyglutamine tract in huntingtin. Although huntingtin is ubiquitously expressed in the body, the striatum suffers the most severe pathology. Rhes is a Ras-related small GTP-binding protein highly expressed in the striatum that has been reported to modulate mTOR and sumoylation of mutant huntingtin to alter HD mouse model pathogenesis. Reports have varied on whether Rhes reduction is desirable for HD. Here we characterize multiple behavioral and molecular endpoints in the Q175 HD mouse model with genetic Rhes knockout (KO). Genetic RhesKO in the Q175 female mouse resulted in both subtle attenuation of Q175 phenotypic features, and detrimental effects on other kinematic features. The Q175 females exhibited measurable pathogenic deficits, as measured by MRI, MRS and DARPP32, however, RhesKO had no effect on these readouts. Additionally, RhesKO in Q175 mixed gender mice deficits did not affect mTOR signaling, autophagy or mutant huntingtin levels. We conclude that global RhesKO does not substantially ameliorate or exacerbate HD mouse phenotypes in Q175 mice.

Detailed genetic and functional analysis of the hDMDdel52/mdx mouse model.

Duchenne muscular dystrophy (DMD) is a severe, progressive neuromuscular disorder caused by reading frame disrupting mutations in the DMD gene leading to absence of functional dystrophin. Antisense oligonucleotide (AON)-mediated exon skipping is a therapeutic approach aimed at restoring the reading frame at the pre-mRNA level, allowing the production of internally truncated partly functional dystrophin proteins. AONs work in a sequence specific manner, which warrants generating humanized mouse models for preclinical tests. To address this, we previously generated the hDMDdel52/mdx mouse model using transcription activator like effector nuclease (TALEN) technology. This model contains mutated murine and human DMD genes, and therefore lacks mouse and human dystrophin resulting in a dystrophic phenotype. It allows preclinical evaluation of AONs inducing the skipping of human DMD exons 51 and 53 and resulting in restoration of dystrophin synthesis. Here, we have further characterized this model genetically and functionally. We discovered that the hDMD and hDMDdel52 transgene is present twice per locus, in a tail-to-tail-orientation. Long-read sequencing revealed a partial deletion of exon 52 (first 25 bp), and a 2.3 kb inversion in intron 51 in both copies. These new findings on the genomic make-up of the hDMD and hDMDdel52 transgene do not affect exon 51 and/or 53 skipping, but do underline the need for extensive genetic analysis of mice generated with genome editing techniques to elucidate additional genetic changes that might have occurred. The hDMDdel52/mdx mice were also evaluated functionally using kinematic gait analysis. This revealed a clear and highly significant difference in overall gait between hDMDdel52/mdx mice and C57BL6/J controls. The motor deficit detected in the model confirms its suitability for preclinical testing of exon skipping AONs for human DMD at both the functional and molecular level.

Rapid and robust patterns of spontaneous locomotor deficits in mouse models of Huntington's disease.

Huntington's disease (HD) is an inherited neurodegenerative disorder characterized by severe disruption of cognitive and motor functions, including changes in posture and gait. A number of HD mouse models have been engineered that display behavioral and neuropathological features of the disease, but gait alterations in these models are poorly characterized. Sensitive high-throughput tests of fine motor function and gait in mice might be informative in evaluating disease-modifying interventions. Here, we describe a hypothesis-free workflow that determines progressively changing locomotor patterns across 79 parameters in the R6/2 and Q175 mouse models of HD. R6/2 mice (120 CAG repeats) showed motor disturbances as early as at 4 weeks of age. Similar disturbances were observed in homozygous and heterozygous Q175 KI mice at 3 and 6 months of age, respectively. Interestingly, only the R6/2 mice developed forelimb ataxia. The principal components of the behavioral phenotypes produced two phenotypic scores of progressive postural instability based on kinematic parameters and trajectory waveform data, which were shared by both HD models. This approach adds to the available HD mouse model research toolbox and has a potential to facilitate the development of therapeutics for HD and other debilitating movement disorders with high unmet medical need.

A multimodal approach to identify clinically relevant biomarkers to comprehensively monitor disease progression in a mouse model of pediatric neurodegenerative disease.

While research has accelerated the development of new treatments for pediatric neurodegenerative disorders, the ability to demonstrate the long-term efficacy of these therapies has been hindered by the lack of convincing, noninvasive methods for tracking disease progression both in animal models and in human clinical trials. Here, we unveil a new translational platform for tracking disease progression in an animal model of a pediatric neurodegenerative disorder, CLN6-Batten disease. Instead of looking at a handful of parameters or a single "needle in a haystack", we embrace the idea that disease progression, in mice and patients alike, is a diverse phenomenon best characterized by a combination of relevant biomarkers. Thus, we employed a multi-modal quantitative approach where 144 parameters were longitudinally monitored to allow for individual variability. We use a range of noninvasive neuroimaging modalities and kinematic gait analysis, all methods that parallel those commonly used in the clinic, followed by a powerful statistical platform to identify key progressive anatomical and metabolic changes that correlate strongly with the progression of pathological and behavioral deficits. This innovative, highly sensitive platform can be used as a powerful tool for preclinical studies on neurodegenerative diseases, and provides proof-of-principle for use as a potentially translatable tool for clinicians in the future.

Using a State-of-the-Art Toolbox to Evaluate Molecular and Functional Readouts of Antisense Oligonucleotide-Induced Exon Skipping in mdx Mice.

Duchenne muscular dystrophy (DMD) is a severe childhood muscle disease primarily caused by the lack of functional dystrophin at the muscle fiber membranes. Multiple therapeutic approaches are currently in (pre)clinical development, aimed at restoring expression of (truncated) dystrophin. Key questions in this phase relate to route of drug administration, dose regimen, and levels of dystrophin required to improve muscle function. A series of studies applying antisense oligonucleotides (AONs) in the mdx mouse model for DMD has been reported over the last two decades, claiming a variable range of exon skipping and increased dystrophin levels correlated to some functional improvement. The aim of this study was to compare the efficacy of subcutaneous (SC) versus intravenous (IV) dosing routes of an mdx-specific AON at both the molecular and functional level, using state-of-the-art quantitative technologies, including digital droplet polymerase chain reaction, capillary Western immunoassay, magnetic resonance imaging, and automated kinematic analysis. The majority of all readouts we quantified, both molecular and functional, showed that IV dosing of the AON had a more pronounced beneficial effect than SC dosing in mdx mice. Last, but not least, the more quantitative molecular and functional data obtained in this study suggest that low levels of dystrophin protein of at least 2.5% of wild type may already have a beneficial effect on muscle leakiness and may improve motor performance of mdx mice.

Evaluation of the Effect of Bariatric Surgery-Induced Weight Loss on Knee Gait and Cartilage Degeneration.

The objective of the study was to investigate the effects of bariatric surgery-induced weight loss on knee gait and cartilage degeneration in osteoarthritis (OA) by combining magnetic resonance imaging (MRI), gait analysis, finite element (FE) modeling, and cartilage degeneration algorithm. Gait analyses were performed for obese subjects before and one-year after the bariatric surgery. FE models were created before and after weight loss for those subjects who did not have severe tibio-femoral knee cartilage loss. Knee cartilage degenerations were predicted using an adaptive cartilage degeneration algorithm which is based on cumulative overloading of cartilage, leading to iteratively altered cartilage properties during OA. The average weight loss was 25.7±11.0 kg corresponding to a 9.2±3.9 kg/m2 decrease in body mass index (BMI). External knee rotation moment increased, and minimum knee flexion angle decreased significantly (p < 0.05) after weight loss. Moreover, weight loss decreased maximum cartilage degeneration by 5±23% and 13±11% on the medial and lateral tibial cartilage surfaces, respectively. Average degenerated volumes in the medial and lateral tibial cartilage decreased by 3±31% and 7±32%, respectively, after weight loss. However, increased degeneration levels could also be observed due to altered knee kinetics. The present results suggest that moderate weight loss changes knee kinetics and kinematics and can slow-down cartilage degeneration for certain patients. Simulation results also suggest that prediction of cartilage degeneration is subject-specific and highly depend on the altered gait loading, not just the patient's weight.

Acquisition and reversal of visual discrimination learning in APPSwDI/Nos2-/- (CVN) mice.

Studies of cognitive behavior in rodent models of Alzheimer's disease (AD) are the mainstay of academic and industrial efforts to find effective treatments for this disorder. However, in the majority of such studies, the nature of rodent behavioral tests is considerably different from the setting associated with cognitive assessments of individuals with AD. The recently developed touchscreen technique provides a more translational way of rodent cognitive testing because the stimulus (images in different locations on the screen) and reaction (touch) are similar to those employed in human test routines, such as the Cambridge Neuropsychological Test Automated Battery. Here, we used Visual Discrimination and Reversal of Visual Discrimination touchscreen tasks to assess cognitive performance of APPSwDI/Nos2-/- (CVN) mice, which express mutated human APP and have a homozygous deletion of the Nos2 gene. We revealed that CVN mice made more first-time errors and received more correction trials than WT mice across both discrimination and reversal phases, although mutation effect size was larger during the latter phase. These results indicate sensitivity of touchscreen-based measurements to AD-relevant mutations in CVN mice and warrant future touchscreen experiments aimed at evaluating other cognitive and motivational phenotypes in this AD mouse model.

Effects of mop handle height on shoulder muscle activity and perceived exertion during floor mopping using a figure eight method.

The aim of this study was to investigate effects of mop handle height on electromyographic (EMG) activities of the shoulder muscles and perceived exertion for the shoulder area during floor mopping using a figure eight method. An experimental study with 13 cleaners was conducted using surface EMG and category ratio (CR-10) scale. EMG activity was recorded unilaterally from the upper trapezius, infraspinatus, anterior and middle deltoid muscles. Each subject performed four trials of mopping and each trial consisted of using a different mop handle height (mop adjustment at the level of shoulder, chin, nose and eye) in randomized order. EMG data were normalized to a percentage of maximal voluntary contraction (%MVC). The muscle activities were assessed by estimating the 10th, 50th and 90th percentiles of the amplitude probability distribution function (APDF) of the EMG signals and analysed by linear mixed model analysis. Results showed that shoulder muscle activity was significantly lower when the mop handle height was adjusted to shoulder level or chin level as compared to eye level. These findings were supported by subjective ratings of exertion. It seems that mop handle height adjustment between shoulder and chin level may be recommended as a basis for figure eight mopping.

Nonlinear state-space modeling of human motion using 2-D marker observations.

A novel method for the estimation of human kinematics, based on state-space modeling, is proposed. The state consists of the positions, orientations, velocities, and accelerations of an articulated model. Estimation is performed using the unscented Kalman filter (UKF) algorithm with a fixed-interval smoother. Impulsive acceleration at floor contact of the foot is estimated by implementing a contact constraint in the UKF evolution model. The constraint inserts an acceleration impulse into the model state. The estimation method was applied to marker-based motion analysis in a motion laboratory. Validation measurements were performed with a rigid test device and with human gait. A triaxial accelerometer was used to evaluate acceleration estimates. Comparison between the proposed method and the extended Kalman smoother showed a clear difference in the quality of estimates during impulsive accelerations. The proposed approach enables estimation of human kinematics during both continuous and transient accelerations. The approach provides a novel way of estimating acceleration at foot initial contact, and thus enables more accurate evaluation of loading from the beginning of the floor contact.

Lower impulsive loadings following intensive weight loss after bariatric surgery in level and stair walking: a preliminary study.

BACKGROUND: There are currently very few of studies which have evaluated the role of bariatric surgery in joint loadings and changes in gait. We wanted to examine how impulsive loading would change level and stair walking in severely or morbidly obese subjects after they had undergone bariatric surgery and weight loss. METHODS: Thirteen female and three male adults aged between 30 and 63 years, cleared for Roux-en-Y gastric bypass, were recruited into this study. All subjects were severely or morbidly obese i.e., body mass index was >35 kg/m(2). The measurement methods consisted of triaxial skin mounted accelerometers and ground reaction force (GRF); conducted at two different predetermined gait speeds. RESULTS: The average weight loss was 27.4 (SD8.7) kg after 8.8 (SD3.9) months of follow-up period. Most of the absolute GRF parameters decreased in proportion to weight loss. However, medio-lateral GRF parameters decreased more than expected. The general trend in the knee accelerations demonstrated lower impulsive loadings in both axial and horizontal directions after weight loss. We did not observe any significant changes in stair walking. CONCLUSIONS: Weight loss after bariatric surgery not only induces a simple mass-related adaptation in gait but also achieves mechanical plasticity in gait strategy.

Kinematic and kinetic changes in obese gait in bariatric surgery-induced weight loss.

This study examines the effects of a radical bariatric surgery-induced weight loss on the gait of obese subjects. We performed a three-dimensional motion analysis of lower limbs, and collected force platform data in the gait laboratory to calculate knee and hip joint moments. Subjects (n=13) performed walking trials in the laboratory before and 8.8 months (SD 4.2) after the surgical procedure at two gait speeds (1.2m/s and 1.5m/s). The average weight loss was 26.7kg (SD 9.2kg), corresponding to 21.5% (SD 6.8%) of the initial weight. We observed a decrease in step width at both gait speeds, but no changes in relative double support or swing time or stride length. A significant decrease was noted in the absolute values of peak knee abductor, peak knee flexor and peak hip extensor moments. However, the moment values normalized by the body weight and height remained unchanged in most cases. Thus, we conclude that weight loss reduces hip and knee joint moments in proportion to the amount of weight lost.

Method for testing motion analysis laboratory measurement systems.

This paper proposes a method for comparing data from accelerometers, optical based 3D motion capture systems, and force platforms (FPs) in the context of spatial and temporal differences. Testing method is based on the motion laboratory accreditation test (MLAT), which can be used to test FP and camera based motion capture components of a motion analysis laboratory. This study extends MLAT to include accelerometer data. Accelerometers were attached to a device similar to the MLAT rod. The elevation of the rod from the plane of the floor is computed and compared with the force platform vector orientation and the rod orientation obtained by optical motion capture system. Orientation of the test device is achieved by forming nonlinear equation group, which describes the components of the measured accelerations. Solution for this equation group is estimated by using the Gauss-Newton method. This expanded MLAT procedure can be used in the laboratory setting were either FP, camera based motion capture, or any other motion capture system is used along with accelerometer measurements.

Postural control and thigh muscle activity in men with knee osteoarthritis.

The aim of this study was to examine the standing balance and the function of vastus medialis (VM) and biceps femoris (BF) muscles with surface electromyography (EMG). Fifty-four subjects with uni- or bilateral knee osteoarthritis (OA) (aged 50-69 years) and 53 age-matched randomly selected clinically and radiologically healthy men participated in this study. Postural control was assessed on a force platform with a bipedal stance with eyes open (EO) and closed (EC) and a monopedal stance with EO. The balance parameters, mean sway velocity, velocity along AP and ML axes, elliptical area, standard deviation of center of pressure, average radial displacement, mean frequency and frequency domain balance parameters and different power spectral density frequency bands were determined. Root mean square (RMS) for EMG amplitude, mean EMG frequency (f(EMG,mean)) and median EMG frequency (f(EMG,med)) of motor unit activity were calculated from the normalized EMG data. During bipedal stance with EC and EO, there were no significant differences in balance parameters between groups, but during bipedal stance with EO, the RMS in VM was about 56% higher (p<0.05) in subjects with knee OA than in the control subjects and the values of f(EMG,mean) and f(EMG,med) were about 48% higher (p<0.05) in control subjects than subjects with knee OA. It is concluded that subjects with knee OA do not have any standing balance deficit, but they do exhibit increased muscle activity in VM muscle compared to control subjects.

Gait and muscle activation changes in men with knee osteoarthritis.

The aim was to examine the biomechanics of level- and stair-walking in men with knee osteoarthritis (OA) at different pre-determined gait speeds and to compare the results with those obtained from healthy control subjects. Special emphasis was placed on the estimation of joint loading. Fifty-four men with knee OA (50-69 years) and 53 healthy age- and sex-matched controls were enrolled in the study. The participants walked barefoot in the laboratory (1.2 m/s+/-5%), corridor (1.2; 1.5 and 1.7 m/s+/-5%), and climbing and coming down stairs (0.5 and 0.8 m/s+/-5%) separately. Joint loading was assessed with skin mounted accelerometers (SMAs) attached just above and below the more affected knee joint. The 3-D ground reaction forces (GRFs) and muscle activation with surface-electromyography (EMG) from vastus medialis (VM) and biceps femoris (BF) were also measured simultaneously. There were no differences in SMA variables between groups during level-walking, but maximal loading rate (LR(max)) was higher bilaterally in the controls (P<.05). Patients loaded their lower extremity more forcefully especially during stair descent at faster speed. The distinctions in muscle activation both at level- and stair ambulation in VM and BF muscles revealed that the patients used different strategies to execute the same walking tasks. It is concluded that the differences in measured SMA and GRF parameters between the knee OA patients and the controls were only minor at constant gait speeds. It is speculated that the faster speeds in the stair descent subjected the compensatory mechanisms to the maximum highlighting the differences between groups.

Reproducibility of loading measurements with skin-mounted accelerometers during walking.

OBJECTIVE: To examine reproducibility of load measurements with skin-mounted accelerometers (SMAs) during walking. DESIGN: Reliability study. SETTING: A motion analysis laboratory. PARTICIPANTS: Ten healthy young men. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Two triaxial accelerometers were fixed to the subjects' skin above and below the knee joint. The subjects walked barefoot at their preferred speed and at a constant speed (1.3m/s, +/-5%) in a gait laboratory and along a corridor. The same protocol was repeated over 2 days. Initial peak acceleration (IPA), peak-to-peak (PP) acceleration, and maximal and average acceleration transient rates (ATRs) were calculated. The coefficient of variation (CV) and Pearson linear correlation coefficient were calculated to measure reproducibility of SMA load measurements. RESULTS: IPA and PP acceleration had good interday repeatability (CV <15%). The repeatability of average ATR and maximal ATR parameters was generally not acceptable. The loading variables obtained from ground reaction forces and SMA measurements during gait revealed high linear correlations, indicating that with SMA measurements it is possible to predict certain ground reaction force loading parameters. CONCLUSIONS: SMAs are practical for use in clinical environments to collect acceleration data that may be used to estimate joint loads.

High-resolution QRS fiducial point corrections in sparsely sampled ECG recordings.

A model based high-resolution QRS fiducial point correction algorithm, which is suitable for sparsely sampled electrocardiogram (ECG) recordings, is presented. The presented method can be divided into three steps. First, the initial QRS fiducial points are estimated by using ordinary interpolation methods. Then, the data points of each QRS are extracted and centered in time and the shape of the QRS complex is estimated by nonlinearly fitting a double exponential function to the extracted data points. Finally, the estimated model and its derivative are linearly fitted to the data points of each QRS complex separately and new fiducial point estimates are obtained. The proposed method is tested with simulations and real ECG data. As a result, it is observed that the proposed method is also suitable for asymmetric QRS complexes unlike, e.g., the commonly used cubic spline interpolation method.