Publications

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SMN Protein Can Be Reliably Measured in Whole Blood with an Electrochemiluminescence (ECL) Immunoassay: Implications for Clinica
Spinal muscular atrophy (SMA) is caused by defects in the survival motor neuron 1 (SMN1) gene that encodes survival motor neuron (SMN) protein. The majority of therapeutic approaches currently in clinical development for SMA aim to increase SMN protein expression and there is a need for sensitive methods able to quantify increases in SMN protein levels in accessible tissues. We have developed a sensitive electrochemiluminescence (ECL)- based immunoassay for measuring SMN protein in whole blood with a minimum volume requirement of 5μL. The SMN-ECL immunoassay enables accurate measurement of SMN in whole blood and other tissues. Using the assay, we measured SMN protein in whole blood from SMA patients and healthy controls and found that SMN protein levels were associated with SMN2 copy number and were greater in SMA patients with 4 copies, relative to those with 2 and 3 copies. SMN protein levels did not vary significantly in healthy individuals over a four-week period and were not affected by circadian rhythms. Almost half of the SMN protein was found in platelets.We show that SMN protein levels in C/C-allele mice, which model a mild form of SMA, were high in neonatal stage, decreased in the first few weeks after birth, and then remained stable throughout the adult stage. Importantly, SMN protein levels in the CNS correlated with SMN levels measured in whole blood of the C/C-allele mice. These findings have implications for the measurement of SMN protein induction in whole blood in response to SMN-upregulating therapy.
2.95 MB SMN Protein Can Be Reliably Measured in Whole Blood with an Electrochemiluminescence (ECL) Immunoassay: Implications for Clinica
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Baseline Results of the NeuroNEXT Spinal Muscular Atrophy Infant Biomarker Study
Objective: This study prospectively assessed putative promising biomarkers for use in assessing infants with spinal muscular atrophy (SMA). Methods: This prospective, multi-center natural history study targeted the enrollment of SMA infants and healthy control infants less than 6 months of age. Recruitment occurred at 14 centers within the NINDS National Network for Excellence in Neuroscience Clinical Trials (NeuroNEXT) Network. Infant motor function scales and putative electrophysiological, protein and molecular biomarkers were assessed at baseline and subsequent visits.
481.5 KB Baseline Results of the NeuroNEXT Spinal Muscular Atrophy Infant Biomarker Study
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Biomarker for Spinal Muscular Atrophy: Expression of SMN in Peripheral Blood of SMA Patients and Healthy Controls
Spinal muscular atrophy is caused by a functional deletion of SMN1 on Chromosome 5, which leads to a progressive loss of motor function in affected patients. SMA patients have at least one copy of a similar gene, SMN2, which produces functional SMN protein, although in reduced quantities. The severity of SMA is variable, partially due to differences in SMN2 copy numbers. Here, we report the results of a biomarker study characterizing SMA patients of varying disease severity. SMN copy number, mRNA and Protein levels in whole blood of patients were measured and compared against a cohort of healthy controls. The results show differential regulation of expression of SMN2 in peripheral blood between patients and healthy subjects.
551.23 KB Biomarker for Spinal Muscular Atrophy: Expression of SMN in Peripheral Blood of SMA Patients and Healthy Controls
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Assays for the Identification and Prioritization of Drug Candidates for Spinal Muscular Atrophy
Spinal muscular atrophy (SMA) is an autosomal recessive genetic disorder resulting in degeneration of a-motor neurons of the anterior horn and proximal muscle weakness. It is the leading cause of genetic mortality in children younger than 2 years. It affects *1 in 11,000 live births. In 95% of cases, SMA is caused by homozygous deletion of the SMN1 gene. In addition, all patients possess at least one copy of an almost identical gene called SMN2. A single point mutation in exon 7 of the SMN2 gene results in the production of low levels of full-length survival of motor neuron (SMN) protein at amounts insufficient to compensate for the loss of the SMN1 gene. Although no drug treatments are available for SMA, a number of drug discovery and development programs are ongoing, with several currently in clinical trials. This review describes the assays used to identify candidate drugs for SMA that modulate SMN2 gene expression by various means. Specifically, it discusses the use of highthroughput screening to identify candidate molecules from primary screens, as well as the technical aspects of a number of widely used secondary assays to assess SMN messenger ribonucleic acid (mRNA) and protein expression, localization, and function. Finally, it describes the process of iterative drug optimization utilized during preclinical SMA drug development to identify clinical candidates for testing in human clinical trials.
1.15 MB Assays for the Identification and Prioritization of Drug Candidates for Spinal Muscular Atrophy
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A Comparison of Three Electrophysiological Methods for the Assessment of Disease Status in a Mild Spinal Muscular Atrophy Mouse
Objectives: There is a need for better, noninvasive quantitative biomarkers for assessing the rate of progression and possible response to therapy in spinal muscular atrophy (SMA). In this study, we compared three electrophysiological measures: compound muscle action potential (CMAP) amplitude, motor unit number estimate (MUNE), and electrical impedance myography (EIM) 50 kHz phase values in a mild mouse model of spinal muscular atrophy, the Smn1c/c mouse. Methods: Smn1c/c mice (N = 11) and wild type (WT) animals (2/2, N = 13) were measured on average triweekly until approximately 1 year of age. Measurements included CMAP, EIM, and MUNE of the gastrocnemius muscle as well as weight and front paw grip strength. At the time of sacrifice at one year, additional analyses were performed on the animals including serum survival motor neuron (SMN) protein levels and muscle fiber size. Results: Both EIM 50 kHz phase and CMAP showed strong differences between WT and SMA animals (repeated measures 2- way ANOVA, P,0.0001 for both) whereas MUNE did not. Both body weight and EIM showed differences in the trajectory over time (p,0.001 and p = 0.005, respectively). At the time of sacrifice at one year, EIM values correlated to motor neuron counts in the spinal cord and SMN levels across both groups of animals (r = 0.41, p = 0.047 and r = 0.57, p = 0.003, respectively), while CMAP did not. Motor neuron number in Smn1c/c mice was not significantly reduced compared to WT animals. Conclusions: EIM appears sensitive to muscle status in this mild animal model of SMA. The lack of a reduction in MUNE or motor neuron number but reduced EIM and CMAP values support that much of the pathology in these animals is distal to the cell body, likely at the neuromuscular junction or the muscle itself.
384.72 KB A Comparison of Three Electrophysiological Methods for the Assessment of Disease Status in a Mild Spinal Muscular Atrophy Mouse
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VEGF, VEGF-C, and VEGF-D Increases in Aqueous Humor Samples in a LPS Induced Model of Uveitis in New Zealand White Rabbits
VEGF, VEGF-C, and VEGF-D levels increased significantly in LPS treated eyes of NZW and are indicative of an inflammatory response. Ophthalmic exams, cell counts, and protein increases in LPS treated eyes support increased inflammation in this rabbit model of uveitis. The significantly increased levels of VEGF-C and VEGF-D in AH are new findings.
116.2 KB VEGF, VEGF-C, and VEGF-D Increases in Aqueous Humor Samples in a LPS Induced Model of Uveitis in New Zealand White Rabbits
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Optimization of peripheral blood mononuclear cell processing for SMN protein signal analysis
SMN protein levels in peripheral blood mononuclear cells (PBMCs) have potential to become a key pharmacodynamic (PD) marker of drug efficacy in SMA clinical trials that utilize SMN-targeted approaches. Detailed analysis of variability and factors affecting SMN protein levels in PBMCs are required to allow interpretation of SMN signals in trials. Factors like sample processing delays, subject age, meals, and respiratory infections were evaluated for their ability to impact SMN protein levels in PBMCs. A best practice protocol for SMN ELISA analysis of PBMCs was developed based on these studies as well as prior experience.
138.97 KB Optimization of peripheral blood mononuclear cell processing for SMN protein signal analysis
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Development of SMA mouse model plasma protein biomarker assays
Several putative SMA plasma protein biomarkers have been reported, based on association with motor function. These proteins have been identified as potential markers of disease state and progression, and may serve as pharmacodynamic (PD) markers that respond to SMN changes in target tissues. With data from SMA clinical trials pending, SMA mouse models provide a useful screening tool for PD markers.
298.83 KB Development of SMA mouse model plasma protein biomarker assays
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Ocular Pharmacokinetics of a Novel Loteprednol
Sponsor publication, PK completed by PharmOptima
604.31 KB Ocular Pharmacokinetics of a Novel Loteprednol