A standardised protocol for blood and cerebrospinal fluid collection and processing for biomarker research in ataxia.

The European Spinocerebellar Ataxia Type 3/Machado-Joseph Disease Initiative (ESMI) is a consortium established with the ambition to set up the largest European longitudinal trial-ready cohort of Spinocerebellar Ataxia Type 3/Machado-Joseph Disease (SCA3/MJD), the most common autosomal dominantly inherited ataxia worldwide. A major focus of ESMI has been the identification of SCA3/MJD biomarkers to enable future interventional studies. As biosample collection and processing variables significantly impact the outcomes of biomarkers studies, biosampling procedures standardisation was done previously to study visit initiation. Here, we describe the ESMI consensus biosampling protocol, developed within the scope of ESMI, that ultimately might be translated to other neurodegenerative disorders, particularly ataxias, being the first step to protocol harmonisation in the field.

PolyQ-expanded ataxin-3 protein levels in peripheral blood mononuclear cells correlate with clinical parameters in SCA3: a pilot study.

In view of upcoming clinical trials, quantitative molecular markers accessible in peripheral blood are of critical importance as prognostic or pharmacodynamic markers in genetic neurodegenerative diseases such as Spinocerebellar Ataxia Type 3 (SCA3), in particular for signaling target engagement. In this pilot study, we focused on the quantification of ataxin-3, the protein altered in SCA3, in human peripheral blood mononuclear cells (PBMCs) acquired from preataxic and ataxic SCA3 mutation carriers as well as healthy controls, as a molecular marker directly related to SCA3 pathophysiology. We established two different highly sensitive TR-FRET-based immunoassays to measure the protein levels of either total full-length, non-expanded and expanded, ataxin-3 or specifically polyQ-expanded ataxin-3. In PBMCs, a clear discrimination between SCA3 mutation carrier and controls were seen measuring polyQ-expanded ataxin-3 protein level. Additionally, polyQ-expanded ataxin-3 protein levels correlated with disease progression and clinical severity as assessed by the Scale for the Assessment and Rating of Ataxia. Total full-length ataxin-3 protein levels were directly influenced by the expression levels of the polyQ-expanded ataxin-3 protein, but were not correlated with clinical parameters. Assessment of ataxin-3 levels in fibroblasts or induced pluripotent stem cells allowed to distinguish mutation carriers from controls, thus providing proof-of-principle validation of our PBMC findings across cell lines. Total full-length or polyQ-expanded ataxin-3 protein was not detectable by TR-FRET assays in other biofluids like plasma or cerebrospinal fluid, indicating the need for ultra-sensitive assays for these biofluids. Standardization studies revealed that tube systems, blood sampling, and PBMC preparation may influence ataxin-3 protein levels indicating a high demand for standardized protocols in biomarker studies. In conclusion, the polyQ-expanded ataxin-3 protein is a promising candidate as a molecular target engagement marker in SCA3 in future clinical trials, determinable even in-easily accessible-peripheral blood biomaterials. These results, however, require validation in a larger cohort and further standardization of modifying conditions.

Clinical-grade regulatory T cells: Comparative analysis of large-scale expansion conditions.

Recent clinical trials have indicated the high potential of regulatory T cells (Tregs) in the prevention of acute and chronic graft-versus-host disease (GvHD) after hematopoietic stem cell transplantation, but immune interventions require large numbers of Tregs. With respect to their limited natural occurrence, development and optimization of protocols for large-scale expansion of clinical-grade Tregs are essential if considered for therapeutic use. We compared different clinical-grade large-scale expansion protocols for repetitive transfer of large numbers of Tregs in clinical trials for the prevention of acute and/or chronic GvHD. Donor Tregs were isolated using magnetic-activated cell sorting (MACS) technology with good manufacturing practice-compliant devices. CD8 and CD19 depletion followed by CD25 enrichment resulted in the isolation of CD4+CD25+CD127- Tregs with a mean purity of 77%. Cell populations were expanded ex vivo using X-Vivo 15 (±rapamycin), TexMACS (±rapamycin), and CellGro DC (±rapamycin) in the presence of interleukin-2. The highest rates of expansion of clinical-grade Tregs were observed for X-Vivo 15 and CellGro DC without rapamycin in compared with all other expansion media tested. The suppressive capacity of the expanded Treg population was maintained under all conditions investigated. Our data suggest that expansion with CellGro provides data comparable to those obtained with TexMACS or X-Vivo 15 with rapamycin, although all three conditions did not provide the same propagation rate as X-Vivo 15 alone. With respect to functionality, phenotype, and stability, CellGro DC medium represents a reasonable alternative for good manufacturing practice-compatible large-scale ex vivo expansion.