Abstract
ObjectivesGenetic defects leading to the reduction of the survival motor neuron protein (SMN) are a causal factor for Spinal Muscular Atrophy (SMA). While there are a number of therapies under evaluation as potential treatments for SMA, there is a critical lack of a biomarker method for assessing efficacy of therapeutic interventions, particularly those targeting upregulation of SMN protein levels. Towards this end we have engaged in developing an immunoassay capable of accurately measuring SMN protein levels in blood, specifically in peripheral blood mononuclear cells (PBMCs), as a tool for validating SMN protein as a biomarker in SMA.MethodsA sandwich enzyme-linked immunosorbent assay (ELISA) was developed and validated for measuring SMN protein in human PBMCs and other cell lysates. Protocols for detection and extraction of SMN from transgenic SMA mouse tissues were also developed.ResultsThe assay sensitivity for human SMN is 50 pg/mL. Initial analysis reveals that PBMCs yield enough SMN to analyze from blood volumes of less than 1 mL, and SMA Type I patients' PBMCs show ∼90% reduction of SMN protein compared to normal adults. The ELISA can reliably quantify SMN protein in human and mouse PBMCs and muscle, as well as brain, and spinal cord from a mouse model of severe SMA.ConclusionsThis SMN ELISA assay enables the reliable, quantitative and rapid measurement of SMN in healthy human and SMA patient PBMCs, muscle and fibroblasts. SMN was also detected in several tissues in a mouse model of SMA, as well as in wildtype mouse tissues. This SMN ELISA has general translational applicability to both preclinical and clinical research efforts.
Highlights
Spinal Muscular Atrophy (SMA) is a progressive neuromuscular disease typified by severe proximal weakness and degeneration of alpha motor neurons in the anterior horn of the spinal cord [1,2]
Humans carry a second nearly identical copy of the survival motor neuron protein (SMN) gene called SMN2 [5]. Both the Survival of Motor Neuron 1 (SMN1) and SMN2 genes express SMN protein, the amount of functional full-length protein produced by SMN2 is much less than that produced by SMN1 [5,6,7]
SMN2 cannot completely compensate for the loss of the SMN1 gene, patients with milder forms of SMA generally have higher SMN2 copy numbers, and this phenomenon has been recapitulated in several transgenic mouse models of SMA [9,10]
Summary
Spinal Muscular Atrophy (SMA) is a progressive neuromuscular disease typified by severe proximal weakness and degeneration of alpha motor neurons in the anterior horn of the spinal cord [1,2]. SMN2 cannot completely compensate for the loss of the SMN1 gene, patients with milder forms of SMA generally have higher SMN2 copy numbers, and this phenomenon has been recapitulated in several transgenic mouse models of SMA [9,10]. This inverse relationship between SMA severity and SMN2 expression provides a strong biological rationale for developing SMA therapeutics that upregulate SMN expression
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