The investigators want to study the JAK-inhibitors and their impact on the immune system and evaluate the potential of a gene-therapeutic strategy
The investigators want to study ex vivo the effect of JAK-inhibitors on the transcriptional profile and immune cell landscape in patients with inborn errors of the JAK-STAT pathway and the ex vivo evaluation of the feasibility of a gene therapeutic approach for STAT1 GOF. Following aspects will be compared: * To study pSTAT, transcriptional profile and cytokine production on bulk and sorted peripheral blood cell populations following stimulation in the presence or absence of different jakinibs * To evaluate to what extent jakinibs can normalize the transcriptional in different cell types (or not and identify blind spots of this treatment strategy) * To evaluate ex vivo the impact of a gene therapeutic approach for STAT1 GOF.
Study Type
INTERVENTIONAL
Allocation
NON_RANDOMIZED
Purpose
TREATMENT
Masking
NONE
Enrollment
20
Blood/serum samples will be collected during routine clinical visits at the time of planned peripheral venous blood sampling. Samples will be processed and either used immediately (flow cytometry-based cell sorting, gDNA extraction, in vitro functional assays, primary cell culture or single-cell applications) or stored for later analysis.
University Hospitals Leuven,
Leuven, Vlaams-Brabant, Belgium
RECRUITINGChange in STAT phosphorylation levels in peripheral blood mononuclear cells (PBMCs) after cytokine stimulation with and without JAK inhibitor exposure
Quantification via Median fluorescence intensity (MFI) of phosphorylated STAT1, STAT3, and STAT5 using multiparameter flow cytometry in bulk PBMCs and sorted T cells, B cells, NK cells, and monocyte subsets after standardized cytokine stimulation (e.g., IFNα, IFNγ, IL-6, IL-2) with or without JAK inhibitor exposure. Outcomes reported as fold-change relative to baseline.
Time frame: From time of inclusion to 24 months
Change in transcriptional profiles of immune cell subsets during JAK inhibitor treatment
Differential gene expression assessed by single-cell RNA sequencing of PBMCs. Outcome is reported as the number of differentially expressed genes (adjusted p\<0.05) at different sampling timepoints (n=3)
Time frame: From time of inclusion to 24 months
Impact of ex vivo gene therapeutic correction in STAT1 gain-of-function patient-derived PBMCs
On-target editing efficiency measured as percentage of corrected alleles by targeted sequencing.
Time frame: 24 months from inclusion
Mutation-specific differences in response to JAK inhibitor treatment
Comparison of cytokine-induced phosphorylation (MFI of pSTAT1, pSTAT3, pSTAT5) and transcriptional responses (differential expression and pathway enrichment) between patients harboring distinct STAT1 gain-of-function variants. Outcomes reported as half-inhibitory concentration IC50 and area under the curve AUC in comparison to baseline
Time frame: From time of inclusion to 24 months
Impact of our gene therapeutic approach on cell viability
Cell viability will be measured with MTT-assay
Time frame: From time of inclusion to 24 months
This platform is for informational purposes only and does not constitute medical advice. Always consult a qualified healthcare professional.
Off target events in our ex vivo gene therapeutic approach
Off-target events detected by GUIDE-seq and confirmed by amplicon sequencing.
Time frame: 24 months
Transcriptional correction of our ex vivo gene therapeutic approach
Transcriptional correction measured by single-cell RNA sequencing (fold-change of interferon pathway gene expression).
Time frame: 24 months
Functional differentiation capacity of gene therapy-corrected cells
Functional differentiation capacity of corrected cells assessed by flow cytometry (percentages of T, B, NK, and monocyte subsets).
Time frame: 24 months