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. 2016 Mar 8;11(3):e0150640.
doi: 10.1371/journal.pone.0150640. eCollection 2016.

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

Phillip Zaworski  1 , Katharine M von Herrmann  2 , Shannon Taylor  1 , Sara S Sunshine  2 , Kathleen McCarthy  2 , Nicole Risher  3 , Tara Newcomb  4 , Marla Weetall  3 , Thomas W Prior  5 , Kathryn J Swoboda  4 , Karen S Chen  2 , Sergey Paushkin  2
Affiliations

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

Phillip Zaworski et al. PLoS One. .

Abstract

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.

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Conflict of interest statement

Competing Interests: PZ and ST are employees of PharmOptima, Inc. NR and MW are employees of PTC Therapeutics. KM is a current employee of F. Hoffmann-La Roche, but was an employee of the Spinal Muscular Atrophy Foundation at the time of this research. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. SMN protein stability in whole blood: short term, long term, and freeze / thaw events.
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%).
Fig 2
Fig 2. SMN protein levels in capillary and venous blood obtained over time from healthy volunteers did not vary significantly.
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).
Fig 3
Fig 3. SMN protein levels in SMA patient and control whole blood samples.
(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 SMN2. In patients over 2 months of age, SMN levels were significantly greater in SMA patient samples with 4 SMN2 copies relative to those with 2 and 3 SMN2 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).
Fig 4
Fig 4. SMN protein levels in tissues of C/C-allele and WT mice measured by SMN-ECL and SMN-ELISA.
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.
Fig 5
Fig 5. SMN protein levels in WT and C/C-allele SMA mice of different ages.
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.
Fig 6
Fig 6. Correlation of SMN protein levels between tissues.
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.
See this image and copyright information in PMC

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