BIBLIOS

Document type
Academic documents

Document subtype
PhD thesis

Title
NOVEL PERSONALIZED THERAPIES FOR CYSTIC FIBROSIS IN A PAEDIATRIC POPULATION

Participants in the publication
Juliana LF Roda (Author)
Margarida D Amaral (Co-adviser)
Dep. Química e Bioquímica
Guiomar G Oliveira (Co-adviser)

Summary
Cystic Fibrosis (CF) is the most common life-limiting autosomal recessive disease among Caucasians. This genetic disease is caused by variants in the CF transmembrane conductance regulator (CFTR) gene which encodes a chloride (Cl-) channel in the apical plasma membrane of epithelial cells. Variants in the CFTR gene cause abnormally viscous secretions responsible for a wide variability of clinical manifestation in different organs. New approved drugs that target specific gene CFTR variants have entered clinical practice but do not apply to all of the 2,000 CFTR variants reported, excluding individuals with rare variants. These CFTR modulators are divided in two main groups: potentiators (Ivacaftor) and correctors (Lumacaftor, Tezacaftor and Elexacaftor). The main objective of this doctoral work is to characterize clinically and genetically a small paediatric cohort of individuals with CF for a potential personalized therapeutic intervention with CFTR modulators using intestinal organoids. A cohort of 23 paediatric people with CF (pwCF) undergoing follow-up at Hospital Pediátrico – Centro Hospitalar e Universitário de Coimbra had their clinical and genetic features analysed in order to determine which ones are candidates to CFTR modulators. All individuals have the p.Phe508del variant in at least one allele. Fifteen pwCF were p.Phe508del-homozygous and are eligible for dual therapy (Luma/Teza+Iva) and for triple therapy (Teza+Iva+Elexa). pwCF with c.579+1G>T (n=2), p.Gln685ThrfsX4 (n=1) variants and a novel variant c.3321dup (n=1) have minimal function variant and a classic phenotype which also makes them eligible to triple therapy. Patients with a residual function variant: p.Arg334Trp (n= 3) and p.Pro5Leu (n=1) have a less severe phenotype, however in Portugal, they have at present no available therapy. Genetic and molecular characterization of pwCF poses an important step not just for CF diagnosis and prognosis which is tightly correlated with the clinical phenotype, but also for the eligibility of CFTR modulator drugs. In this cohort of 23 paediatric pwCF the association of faecal calprotectin (a biomarker of intestinal inflammation) with the clinical characteristics was analysed. Seventeen (17/23) pwCF had elevated faecal calprotectin, and the median value was 88μg/g (IQR=178μg/g). Higher faecal calprotectin levels were observed in the pancreatic insufficient (PI) group (101vs30 μg/g, P=0.027). No significant association between elevated faecal calprotectin and nutritional status or gastrointestinal symptoms was found. In 11 of these individuals, rectal biopsies were obtained and unspecific focal rectal inflammation was found in four individuals (4/11). This finding was associated with elevated faecal calprotectin (p=0.015). Sensitivity was 100% and specificity was 86%. In pwCF analysed here, elevated faecal calprotectin was frequent, particularly if PI, and it was present in individuals with histologic evidence of rectal inflammation. Faecal calprotectin may be an indicator of asymptomatic rectal inflammation in pwCF. Rectal biopsies from the same 11 pwCF were used in order to determine CFTR residual function and to predict specific responses to approved CFTR modulator drugs. CFTRmediated Cl- secretion was assessed by Ussing chamber intestinal voltage measurements (IVM). In parallel, intestinal organoids were prepared, cultured and analysed using the Forskolin-Induced Swelling (FIS) assay by confocal microscopy, before and after incubation with modulators, namely: Iva, Teza/Iva or Elexa/Teza/Iva. The eight pwCF with rare variants and p.Phe508del in the other allele, were included: three with p.Arg334Trp and one with p.Pro5Leu (who have an atypical phenotype and in fact, exhibit CFTR residual function in both IVM and FIS assays); two with c.579+1G>T, one with p.Gln685ThrfsX4 and one with the variant c.3321dup (who have a classical CF phenotype and no CFTR function in IVM and FIS assays). Three pwCF who were homozygous for p.Phe508del with variable clinical phenotype also participated, two had no residual CFTR function (and one with no conclusive analysis). CFTR was rescued by Teza/Iva in organoids with p.Arg334Trp and p.Pro5Leu variants. However, this combination failed to rescue CFTR function in c.579+1G>T, p.Gln685ThrfsX4, c.3321dup and p.Phe508del-homozygous organoids. Triple therapy Elexa/Teza/Iva significantly rescued CFTR in organoids from five pwCF: two with c.579+1G>T, one with p.Arg334Trp and two p.Phe508del -homozygous. Our results illustrate the value of using intestinal organoids to predict individual responses to approved CFTR modulator drugs, indicating that individuals with CF bearing p.Arg334Trp or p.Pro5Leu variants may benefit from treatment with double therapy of CFTR modulators. A more significant clinical benefit is expected for the triple therapy in pwCF carrying the p.Phe508del variant in at least one allele.


Institution
UNIVERSIDADE DE COIMBRA

Publication Identifiers

Address
Coimbra, Portugal


Keywords
Cystic Fibrosis Paediatric patients

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