SMN Protein Can Be Reliably Measured in Whole Blood with an Electrochemiluminescence (ECL) Immunoassay: Implications for Clinical Trials

Spinal muscular atrophy (SMA) is caused by defects in the survival motor neuron 1 (SMN1) gene that encodes survival motor neuron (SMN) protein. The majority of therapeutic approaches currently in clinical development for SMA aim to increase SMN protein expression and there is a need for sensitive methods able to quantify increases in SMN protein levels in accessible tissues. We have developed a sensitive electrochemiluminescence (ECL)-based immunoassay for measuring SMN protein in whole blood with a minimum volume requirement of 5μL. The SMN-ECL immunoassay enables accurate measurement of SMN in whole blood and other tissues. Using the assay, we measured SMN protein in whole blood from SMA patients and healthy controls and found that SMN protein levels were associated with SMN2 copy number and were greater in SMA patients with 4 copies, relative to those with 2 and 3 copies. SMN protein levels did not vary significantly in healthy individuals over a four-week period and were not affected by circadian rhythms. Almost half of the SMN protein was found in platelets. We show that SMN protein levels in C/C-allele mice, which model a mild form of SMA, were high in neonatal stage, decreased in the first few weeks after birth, and then remained stable throughout the adult stage. Importantly, SMN protein levels in the CNS correlated with SMN levels measured in whole blood of the C/C-allele mice. These findings have implications for the measurement of SMN protein induction in whole blood in response to SMN-upregulating therapy

Astrocytes refine cortical connectivity at dendritic spines

During cortical synaptic development, thalamic axons must establish synaptic connections despite the presence of the more abundant intracortical projections. How thalamocortical synapses are formed and maintained in this competitive environment is unknown. Here, we show that astrocyte-secreted protein hevin is required for normal thalamocortical synaptic connectivity in the mouse cortex. Absence of hevin results in a profound, long-lasting reduction in thalamocortical synapses accompanied by a transient increase in intracortical excitatory connections. Three-dimensional reconstructions of cortical neurons from serial section electron microscopy (ssEM) revealed that, during early postnatal development, dendritic spines often receive multiple excitatory inputs. Immuno-EM and confocal analyses revealed that majority of the spines with multiple excitatory contacts (SMECs) receive simultaneous thalamic and cortical inputs. Proportion of SMECs diminishes as the brain develops, but SMECs remain abundant in Hevin-null mice. These findings reveal that, through secretion of hevin, astrocytes control an important developmental synaptic refinement process at dendritic spines. DOI: http://dx.doi.org/10.7554/eLife.04047.00

SMN protein is required throughout life to prevent spinal muscular atrophy disease progression.

Spinal muscular atrophy (SMA) is caused by the loss of the survival motor neuron 1 (SMN1) gene function. The related SMN2 gene partially compensates but produces insufficient levels of SMN protein due to alternative splicing of exon 7. Evrysdi™ (risdiplam), recently approved for the treatment of SMA, and related compounds promote exon 7 inclusion to generate full-length SMN2 mRNA and increase SMN protein levels. SMNΔ7 type I SMA mice survive without treatment for ~17 days. SMN2 mRNA splicing modulators increase survival of SMN∆7 mice with treatment initiated at postnatal day 3 (PND3). To define SMN requirements for adult mice, SMNΔ7 mice were dosed with an SMN2 mRNA splicing modifier from PND3 to PND40, then dosing was stopped. Mice not treated after PND40 showed progressive weight loss, necrosis, and muscle atrophy after ~20 days. Male mice presented a more severe phenotype than female mice. Mice dosed continuously did not show disease symptoms. The estimated half-life of SMN protein is 2 days indicating that the SMA phenotype reappeared after SMN protein levels returned to baseline. Although SMN protein levels decreased with age in mice and SMN protein levels were higher in brain than in muscle, our studies suggest that SMN protein is required throughout the life of the mouse and is especially essential in adult peripheral tissues including muscle. These studies indicate that drugs such as risdiplam will be optimally therapeutic when given as early as possible after diagnosis and potentially will be required for the life of an SMA patient

Discovery of novel small molecule inhibitors of VEGF expression in tumor cells using a cell-based high throughput screening platform.

Current anti-VEGF (Vascular Endothelial Growth Factor A) therapies to treat various cancers indiscriminately block VEGF function in the patient resulting in the global loss of VEGF signaling which has been linked to dose-limiting toxicities as well as treatment failures due to acquired resistance. Accumulating evidence suggests that this resistance is at least partially due to increased production of compensatory tumor angiogenic factors/cytokines. VEGF protein production is differentially controlled depending on whether cells are in the normal homeostatic state or in a stressed state, such as hypoxia, by post-transcriptional regulation imparted by elements in the 5\u27 and 3\u27 untranslated regions (UTR) of the VEGF mRNA. Using the Gene Expression Modulation by Small molecules (GEMS™) phenotypic assay system, we performed a high throughput screen to identify low molecular weight compounds that target the VEGF mRNA UTR-mediated regulation of stress-induced VEGF production in tumor cells. We identified a number of compounds that potently and selectively reduce endogenous VEGF production under hypoxia in HeLa cells. Medicinal chemistry efforts improved the potency and pharmaceutical properties of one series of compounds resulting in the discovery of PTC-510 which inhibits hypoxia-induced VEGF expression in HeLa cells at low nanomolar concentration. In mouse xenograft studies, oral administration of PTC-510 results in marked reduction of intratumor VEGF production and single agent control of tumor growth without any evident toxicity. Here, we show that selective suppression of stress-induced VEGF production within tumor cells effectively controls tumor growth. Therefore, this approach may minimize the liabilities of current global anti-VEGF therapies

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 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

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

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 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 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