Non-invasive multi-omics biomarkers in immune cells subtypes for personalized medicine in childhood asthma and molecular mechanisms of non-response to inhaled corticosteroids

Asthma is the most common chronic disease among children and affects more than 10 % of the childhood population. Children respond reasonably well to standard therapy with inhaled corticosteroids (ICS), however, there is large inter-individual variability, and a substantial proportion of children has only partly controlled disease. More than 20% of these patients will suffer from severe symptoms despite regular use of ICS or develop adverse drug reactions (ADRs). The Global Initiative for Asthma (GINA) is trying to resolve the problems of inefficient ICS and of ICS therapy associated ADRs by seeking for biomarkers guided personalized treatment of patients enrolled in GINA step2. Together with our partners in International Pharmacogenomics in Childhood Asthma consortium (PiCA), we have recently performed so far worldwide largest Genome Wide Association Study (GWAs) in more than 3000 asthma patients and identified several candidate single nucleotide polymorphisms (SNPs) and genes associated with ICS response, including SNPs near genes IL1RL1, CACNA2D3, WNT5A and ORMDL3, as best potential genetic biomarkers. The aim of the proposed study is to elucidate the molecular mechanisms of non-response to ICS (inhaled corticosteroids) in asthma patients, and to identify biomarkers for predicting response to ICS treatment. To this purpose, we will integrate genomic (GWAs), expression (RNA-seq, qRT-PCR), proteomic, epigenetic (DNA methylation) and regulatory (non-coding RNA profiling, alternative intron excision) data with bioinformatics tools (Gene Ontology -GO). Within the PiCa consortium, our Slovenian cohort of mild and moderate asthma patients (GINA step 2 treatment) is the only one that has availability of RNA for expression analysis. Moreover, RNA has been isolated from naïve patients before starting ICS therapy and then again after 4-6 weeks after treatment, representing a worldwide unique biobank containing DNA / RNA / protein samples isolated from the same patients before and during therapy. For those patients, clinical data were collected 4-6 weeks after introduction of ICS treatment. The follow up study revealed 30% of ICS nonresponders in our cohort. The originality of the proposed study is assured as this would be the first comprehensive transcriptomic analysis of different tissues using NGS (RNAseq) prior to treatment with ICS, combined with other »omics« data. The highly innovative part of the project is high resolution immune cell profiling and transcriptome/proteome/epigenome differential analysis (responders vs non-responders) in immune cell subtypes using 1. Single cell RNA sequencing in whole blood cells; 2. Deconvolution analysis of RNAseq data from whole blood cells; 3. Cell sorting and RNAseq in best candidate cell subtypes. Best candidate primary immune cells subtypes (initially CD8+ T cells and ILC2 cells) will bi isolated from responders and non-responders, grown in functional cell models, stimulated with patient’s specific clinically verified allergens and treated with corticosteroids. Development of functional cell models that mimic in vivo treatment of patients will enable validation of best candidate biomarkers (genes/proteins) discovered by multi-omics study in patients cohorts (in vivo data) using the techniques for manipulation of the target gene expression such as CRISPR to get more deep understanding of signaling pathways and molecular mechanisms involved in ICS treatment response. Opposite from the most previous studies that have been largely based on tissues obtained by autopsy, including muscle, pulmonary epithelium, and fibroblasts focus, our functional studies of molecular mechanisms and biological pathways of corticosteroid (non)responsiveness will focus on cells derived from tissues that could be such as nasal swab and blood, to discover biomarkers that could be realistically translated into clinical practice for personalized medicine in childhood asthma.