Supplementary MaterialsS1 Fig: Parallelism and selectivity results from the feasibility study of the SMN-ECL assay qualification. SMN protein was found in platelets. We display that SMN protein levels in C/C-allele mice, which model a slight form of SMA, were high in neonatal stage, decreased in the 1st 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 possess implications Linezolid supplier for the measurement of SMN protein induction in whole blood in response to SMN-upregulating therapy. Intro Spinal Muscular Atrophy (SMA) is definitely a genetically inherited neuromuscular disease, happening in approximately 1 in 11,000 live births, and is the leading genetic cause of death in babies and toddlers (examined in [1]). Individuals exhibit severe proximal muscle mass weakness and atrophy due low levels of survival motor neuron protein (SMN), which mainly leads to degeneration of alpha-motor neurons from the anterior horn. SMA presents as a continuous spectrum of symptoms that are clinically classified into four types of SMA dependent upon motor milestones accomplished during development [2]. The most severe form of the disease, Werdnig-Hoffmann disease or Type I SMA, presents early in existence; most babies are never able to sit individually and have a life expectancy of less than two years [3]. The intermediate form of the disease, Type II SMA (also known as Dubowitz disease), typically presents after 6 months of age. Type II individuals accomplish the ability to stay seated individually but are never able to walk individually. Survival rates for Type II were reported as 98.5% at 5 years and 68.5% at 25 years [4]. Type III SMA, also known as Kugelberg-Welander syndrome, is definitely Linezolid supplier a milder form of the disease with symptoms typically showing after children are already standing up and walking. Life expectancy does not differ from the general human population [4,5]. Type IV SMA has the mildest symptoms and presents in the second or third decade of existence [6,7]. SMA is the result of a deletion or mutation in the survival engine neuron 1 (gene manifestation, aiming to improve splicing and increase the production of functional, full length SMN protein [12C14]. SMN is definitely a ubiquitously indicated, intracellular protein known to be critically involved in snRNP assembly and the formation of additional cellular RNPs comprising coding and non-coding RNAs (examined in [15]). In addition, skeletal muscle mass dietary fiber development and restoration processes are sensitive to low levels of SMN protein [16C18]. Several organizations are exploring a variety of methods, including modulation of splicing, to upregulate manifestation of SMN protein; these attempts include a quantity of programs for novel antisense oligonucleotides, gene delivery vectors, and small molecules that are at various stages of preclinical and Linezolid supplier clinical development. As therapeutics aimed at increasing Linezolid supplier SMN protein levels progress through clinical development, there is an increasing need for a pharmacodynamic marker to access target engagement and select the optimal dose for the therapeutics. To support successful clinical development of a treatment for SMA, it is important to be able to readily and accurately measure SMN protein in an accessible tissue, such as whole blood. Existing methods to measure SMN protein levels include an Elecsys platform-based assay developed by Roche Diagnostics to measure SMN in whole blood [19], an electrochemiluminescence immunoassay (ECLIA) to measure SMN in buccal cells [20], an SMN-ELISA developed to measure SMN in peripheral blood mononuclear cells [21], and western blotting and a homogeneous time-resolved fluorescence (HTRF) assay [12] to measure SMN protein in tissues homogenates. Here we report the development of a sensitive electrochemiluminescence (ECL)-based immunoassay for measuring SMN protein in as little as 5L of Rabbit Polyclonal to PKA-R2beta (phospho-Ser113) whole blood. After validating the assay according to FDA guidelines, the assay was used to better understand SMN protein expression, stability and variability over time in whole blood of healthy individuals and SMA patients in non-interventional clinical studies at University of Utah and Jasper Clinic. These findings, in addition to the development of the SMN-ECL immunoassay capable of sensitively measuring SMN protein in whole blood, Linezolid supplier have a direct relevance for clinical development of SMA therapeutics. Materials and Methods SMN-ECL immunoassay protocol The SMN-ECL immunoassay was developed in a sandwich immunoassay format. The calibrator for the assay was recombinant human SMN produced in (Enzo ADI-NBP-201)..