SMN protein stability in whole blood: short term, long term, and freeze / thaw events.

<p>Whole blood of healthy subjects was used in the study. (A) SMN protein was measured in previously frozen, undiluted whole blood samples incubated at 4°C or at room temperature. (B) SMN protein was measured in undiluted whole blood samples of two subjects stored at -80°C or at -20°C. (C) SMN protein levels were measured in samples of two subjects that went through freeze-thaw cycles. *FDA acceptance criteria (below 85%).</p

Correlation of SMN protein levels between tissues.

<p>SMN protein levels were correlated in WT and C/C-allele SMA mice between (A) whole blood and brain and (B) whole blood and spinal cord.</p

SMN protein levels in tissues of C/C-allele and WT mice measured by SMN-ECL and SMN-ELISA.

<p>Protein levels were measured in the spinal cord of C/C-allele and WT mice using (A) SMN-ECL and (B) SMN-ELISA. Both assays showed a statistically significant difference in SMN levels between WT and C/C-allele mice (p < 0.0001). (C) SMN protein levels in the whole blood of C/C-allele, WT and heterozygous mice measured by SMN-ECL.</p

SMN protein levels in SMA patient and control whole blood samples.

<p>(A) SMN levels with respect to age in all subjects. (B) SMN protein levels were measured in SMA patients with 2, 3 and 4 copies of <i>SMN2</i>. In patients over 2 months of age, SMN levels were significantly greater in SMA patient samples with 4 <i>SMN2</i> copies relative to those with 2 and 3 <i>SMN2</i> copies (p = 0.0001). (C) SMN was also measured in three control samples and SMN levels were found to be significantly greater in the control samples relative to levels in SMA patients over 2 months of age (p < 0.0001).</p

SMN protein levels in WT and C/C-allele SMA mice of different ages.

<p>SMN protein levels were measured by SMN-ECL at various days post-birth in (A) brain, (B) whole blood, (C) spinal cord, and (D) gastrocnemius muscle.</p

SMN only detected in cerebral spinal fluid samples containing hemoglobin.

<p>The CSF samples obtained from healthy volunteers were concentrated prior to analysis, and the sensitivity of the SMN-ECL immunoassay was 0.3 pg/mL. Hemoglobin was measured using a hemoglobin immunoassay (Bethyl Laboratories E88-135). Approximately 3 pg of SMN correspond to 10,000 ng of hemoglobin in 1 mL of whole blood from a healthy adult. LLQ: lower limit of quantification.</p

SMN protein levels in capillary and venous blood obtained over time from healthy volunteers did not vary significantly.

<p>Venous (A, B) and capillary (C) whole blood samples were obtained at 0, 4, 6, 24, 48, 72 hours and 1, 2, 3, 4 weeks from five healthy individuals. Fig 2B is an expanded version of Fig 2A. (D) SMN protein levels in capillary blood correlated significantly with SMN levels in venous blood (r² = 0.76, p < 0.0001).</p

SMN protein was found in various cell types in whole blood.

<p>Platelets, red blood cells, PBMCs and reticulocytes were separated by a fractionation procedure.</p

Normalization of Patient-Identified Plasma Biomarkers in SMNΔ7 Mice following Postnatal SMN Restoration

<div><p>Introduction and Objective</p><p>Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disorder. SMA is caused by homozygous loss of the <i>SMN1</i> gene and retention of the <i>SMN2</i> gene resulting in reduced levels of full length SMN protein that are insufficient for motor neuron function. Various treatments that restore levels of SMN are currently in clinical trials and biomarkers are needed to determine the response to treatment. Here, we sought to investigate in SMA mice a set of plasma analytes, previously identified in patients with SMA to correlate with motor function. The goal was to determine whether levels of plasma markers were altered in the SMNΔ7 mouse model of SMA and whether postnatal SMN restoration resulted in normalization of the biomarkers.</p><p>Methods</p><p>SMNΔ7 and control mice were treated with antisense oligonucleotides (ASO) targeting ISS-N1 to increase SMN protein from <i>SMN2</i> or scramble ASO (sham treatment) via intracerebroventricular injection on postnatal day 1 (P1). Brain, spinal cord, quadriceps muscle, and liver were analyzed for SMN protein levels at P12 and P90. Ten plasma biomarkers (a subset of biomarkers in the SMA-MAP panel available for analysis in mice) were analyzed in plasma obtained at P12, P30, and P90.</p><p>Results</p><p>Of the eight plasma biomarkers assessed, 5 were significantly changed in sham treated SMNΔ7 mice compared to control mice and were normalized in SMNΔ7 mice treated with ASO.</p><p>Conclusion</p><p>This study defines a subset of the SMA-MAP plasma biomarker panel that is abnormal in the most commonly used mouse model of SMA. Furthermore, some of these markers are responsive to postnatal SMN restoration. These findings support continued clinical development of these potential prognostic and pharmacodynamic biomarkers.</p></div

Analytes that are not responsive to SMN.

<p>ASO-Het mice were considered controls for statistical comparison using One-way ANOVA and Dunnett's multiple comparisons test (GraphPad Prism, La Jolla CA). COMP was significantly reduced in ASO-SMA (n = 12) and SMA (n = 13) compared with ASO-Het (n = 10). There was no significant difference between Het (n = 5) and ASO-Het mice for COMP. AXL and CHI3LI were unchanged in all groups compared with ASO-Het mice. ** <0.01, *** <0.001.</p