Abstract
ObjectivesSurvival Motor Neuron (SMN) protein levels may become key pharmacodynamic (PD) markers in spinal muscular atrophy (SMA) clinical trials. SMN protein in peripheral blood mononuclear cells (PBMCs) can be quantified for trials using an enzyme-linked immunosorbent assay (ELISA). We developed protocols to collect, process, store and analyze these samples in a standardized manner for SMA clinical studies, and to understand the impact of age and intraindividual variability over time on PBMC SMN signal.MethodsSeveral variables affecting SMN protein signal were evaluated using an ELISA. Samples were from healthy adults, adult with respiratory infections, SMA patients, and adult SMA carriers.ResultsDelaying PBMCs processing by 45 min, 2 hr or 24 hr after collection or isolation allows sensitive detection of SMN levels and high cell viability (>90%). SMN levels from PBMCs isolated by EDTA tubes/Lymphoprep gradient are stable with processing delays and have greater signal compared to CPT-collected samples. SMN signal in healthy individuals varies up to 8x when collected at intervals up to 1 month. SMN signals from individuals with respiratory infections show 3–5x changes, driven largely by the CD14 fraction. SMN signal in PBMC frozen lysates are relatively stable for up to 6 months. Cross-sectional analysis of PBMCs from SMA patients and carriers suggest SMN protein levels decline with age.ConclusionsThe sources of SMN signal variability in PBMCs need to be considered in the design and of SMA clinical trials, and interpreted in light of recent medical history. Improved normalization to DNA or PBMC subcellular fractions may mitigate signal variability and should be explored in SMA patients.
Highlights
Spinal Muscular Atrophy (SMA) is a progressive and largely pediatric neuromuscular disease that is the primary genetic cause of death in infants and toddlers
Studies 1 and 2: Impact of Processing Delays on Survival Motor Neuron (SMN) Signal and peripheral blood mononuclear cells (PBMCs) Yields In Study 1 whole blood was collected from three healthy individuals in CPT tubes and processed to PBMCs immediately or with delays prior to PBMC isolation of 45 min, 2 h, or 24 h at room temperature
While viable cell numbers did not decrease with processing delays, the number of PBMCs isolated with 2 h and 24 h processing delays declined by,30% compared to immediately processed samples (Figure 1A–B)
Summary
Spinal Muscular Atrophy (SMA) is a progressive and largely pediatric neuromuscular disease that is the primary genetic cause of death in infants and toddlers. Patients with milder forms of SMA are able to sit (Type 2), walk (Type 3), and have normal or near normal lifespans – the majority of patients are adolescents or older [3,4,5,7]. SMA is genetically unique, as the disease is caused by loss of the Survival of Motor Neuron 1 (SMN1) gene but a near perfect phenocopy gene (SMN2), can modify disease phenotype and partially compensate for the loss of functional SMN protein. Patients with milder forms of disease generally have more copies of the SMN2 gene [8]
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