Utility of Survival Motor Neuron ELISA for Spinal Muscular Atrophy Clinical and Preclinical Analyses

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.

Systemic, postsymptomatic antisense oligonucleotide rescues motor unit maturation delay in a new mouse model for type II/III spinal muscular atrophy

“…ADI-900-209; Enzo Life Sciences) according to the manufacturer's protocol. This ELISA was developed by the SMA community as an improved alternative to Western blots (63). This kit detects both human and mouse SMN protein.…”

Assays for the Identification and Prioritization of Drug Candidates for Spinal Muscular Atrophy

“…Also, ELISAs utilizing antibody pairs with different preferences for human or mouse epitopes (e.g., 11708) can yield results that bias the measurement toward detection of one species over another. 41,154 Overall, the careful selection of antibodies and buffer conditions would enable the creation of ELISAs suitable for initial to downstream screens for SMN in cells, depending on the therapeutic concept for a given drug program.…”

“…109,122,125,133 SMN is expressed ubiquitously, but its levels vary greatly across tissues, with the lowest reported levels being in the muscle, nerve, and spinal cord and with the skin and blood at up to 50-fold higher levels. 154 The broad range of SMN protein levels by tissue is further complicated by data on differences in cellular subpopulations such as motor neurons, which have been reported to be inherently deficient in the full-length protein due to inefficient splicing of SMN exon 7. 154,167 Another factor that can impact SMN protein levels in screening from cell lines or testing clinical specimens is age.…”

“…154 The broad range of SMN protein levels by tissue is further complicated by data on differences in cellular subpopulations such as motor neurons, which have been reported to be inherently deficient in the full-length protein due to inefficient splicing of SMN exon 7. 154,167 Another factor that can impact SMN protein levels in screening from cell lines or testing clinical specimens is age. Lines of evidence using quantitative assays suggest that SMN expression across species and tissue type usually decline with age.…”

“…Lines of evidence using quantitative assays suggest that SMN expression across species and tissue type usually decline with age. 154,161,168 Whether this decline is potentiated by disease stage and severity in patients remains unclear, without larger quantitative studies. In summary, factors influencing the measurement of SMN protein in samples and specimens include tissue and cell type, buffer conditions, and age of the specimen or cell line donor should also be weighed when designing any new screening assays.…”