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

Genetic 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.A 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.The 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.This 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

Evaluation of Peripheral Blood Mononuclear Cell Processing and Analysis for Survival Motor Neuron Protein

<div><h3>Objectives</h3><p>Survival 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.</p> <h3>Methods</h3><p>Several 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.</p> <h3>Results</h3><p>Delaying 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.</p> <h3>Conclusions</h3><p>The 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.</p> </div

Study SMAF-001: SMN in SMA patients and carriers.

<p>SMN protein (normalized by total protein) was evaluated in Type 2 and 3 SMA patients and Carriers for differences by motor function and stability of signal with lysate storage. <b>A</b>: SMN protein classified by Type and Carrier status completely overlapped between groups. When adjusted by age there was a significant difference in SMN between patients and adult carriers (p<0.001) but not between SMA Types (p = 0.75). <b>B</b>: SMN levels differentiated by current motor function appeared to distinguish between sitters and ambulatory patients (P<0.05), with the exception of a 1.4 year old recently diagnosed child who could sit with assistance. When controlling for age this trend was not statistically significant (p = 0.97). <b>C</b>: SMN levels were plotted against subject age and there was a trend towards lower SMN levels in older individuals. The correlations for age-related decline were different between SMA and Carriers, with R²-values at 0.65 (p<0.005) and 0.30 (p<0.05) respectively. Arrows depict subjects taking valproic acid, a drug with putative SMN-upregulating effects. <b>D</b>: Samples from each subject were processed and frozen as lysates for storage for 48 h, 1 month, 3 months, and 6 months. Comparability of signals between the 48 h timepoint and subsequent timepoints was generally high, with R²-values of 0.79–0.94 for each timepoints. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050763#pone-0050763-g008" target="_blank">Figure 8A–B</a> error bars indicate the minimum and maximum values while the horizontal bar indicates the median value. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050763#pone-0050763-g008" target="_blank">Figure 8D</a> the trendlines depict R²-values.</p

Study 4: Subcellular PBMC populations and SMN signal.

<p>SMN levels were analyzed by CD+ cell subtype at the 7d timepoint from Study 4. <b>A</b>: Analysis of SMN by PBMC cell subpopulation revealed that the CD14+ population had statistically significant reductions in SMN levels. <b>B</b>: Fractionation of PBMCs with normalization by cell count showed no differences in SMN signal in group analysis. <b>C</b>: Evaluation of total soluble protein levels by CD+ population revealed that CD14+ cells had double the protein concentrations of CD4+, CD8+, CD19+, and CD56+ cells. This differential is sufficient to drive variability in situations that cause CD14+ populations to fluctuate. <b>D</b>: SMN levels (normalized by protein) show consistently lower levels in CD14+ fractions compared to all other fractions, with differences up to 7x within individual PBMC subpopulations. <b>E</b>: SMN in individuals as measured by cell counts were also variable ranging up to 3.5x between individuals’ subcellular populations, but was overall less variable than protein normalized SMN measures. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050763#pone-0050763-g005" target="_blank">Figures 5A–C</a> the bodies of the boxplots indicate the first and third quartiles, while the horizontal bar indicates the median. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050763#pone-0050763-g005" target="_blank">Figures 5D–E</a> error bars depict standard deviation.</p

Study SMAF-001 demographics.

<p>SMA Type and Carrier status was self-reported by subjects or their consenting parents. Subjects identified as Type 2/3 were included in the Type 3 group.</p

Study 2: Impact of post-isolation delay and cell freezing.

<p>SMN signals were evaluated in PBMCs from 4 individuals that were analyzed with 45 minute, 2 h and 24 h delays after cell isolation. A subsample from each timepoint was frozen to assess post-freezing viability and SMN signals. <b>A:</b> Cell viability was generally lower in PBMCs that had been frozen, ranging from 88–95% viability compared to 93–98% in unfrozen cells. Statistical comparisons were made to the 0 h timepoint. <b>B</b>: The comparative recovery of viable PBMCs after freezing relative to fresh samples was only ∼40–60% at all timepoints, suggesting a major loss of cells in the freezing process. <b>C</b>: Delaying the processing of isolated PBMCs to lysates had no impact on protein concentrations through delays of 2 h, however there was again a trend for increased protein concentrations in samples left for 24 h. <b>D</b>: SMN levels (normalized to protein concentrations) in unfrozen PBMCs were generally similar across all timepoints, despite wide variability in signals. <b>E</b>: Analysis of SMN by cell counts revealed that SMN signals in unfrozen cells tended to increase with post-isolation delays. Frozen cell SMN signals generally seemed to decrease over time compared to both frozen cells processed with minimal delays or compared to unfrozen cells. Signals from frozen cells with 24 h post-isolation delays were lower than unfrozen cells. Error bars represent minimum and maximum values. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050763#pone-0050763-g002" target="_blank">Figure 2</a> the bodies of the boxplots indicate the first and third quartiles, while the horizontal bar indicates the median.</p

Study 1: Impact of short-term PBMC processing delays.

<p>PBMCs were collected via CPT tubes from 3 individuals and processed with delays of 0, 45 minutes, 2 h and 24 h prior PBMC isolation by centrifugation. <b>A:</b> Cell viability was similar at all timepoints for all subjects, ranging from 94–99%. <b>B:</b> Cell counts were consistent through 45 minutes, but were significantly reduced by 30–40% with delays of 2 h and 24 h compared to the 0 h timepoint. <b>C:</b> Total soluble protein was consistent with up to 2 h processing delays, however at 24 h there was a trend towards increased protein concentrations by up to 40%. <b>D</b>: SMN levels with 24 h processing delays were accordingly reduced when normalized by total protein. <b>E</b>: SMN levels by cell counts were similar with all delays examined, albeit with trends for higher variability than the SMN signal generated by protein normalization. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050763#pone-0050763-g001" target="_blank">Figure 1</a> error bars indicate the minimum and maximum values while the horizontal bar indicates the median value.</p