Beating primary ciliary dyskinesia

• Main Author: Salvado, Beatriz Alexandra Adrião
• Publication Date: 2023
• Format: Master thesis
• Language: eng
• Source: Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
• Download full: http://hdl.handle.net/10362/161485
• topic: Motile Cilia; Primary ciliary dyskinesia (PCD); CCDC40; LNPs formulated with cmRNA; Zebrafish; Ciências Médicas

Abstract Primary ciliary dyskinesia (PCD) is a genetic disorder caused by malfunction or absence of motile cilia, which play a key role in the clearance of mucus, bacteria and debris from the respiratory tract. Consequently, individuals with PCD often experience recurrent respiratory infections and bronchiectasis. PCD arises from mutations in a wide array of genes responsible for motile cilia structure and function. In this work, we tried to identify zebrafish mutants from four PCD genes, namely DNAH 5, DNAH 11, CCDC39 and CCNO, previously created by using a CRISPR-Cas9 approach to mimic human PCD mutations in the homologous site of the zebrafish genome. Since we were not able to find any homozygous mutants from these selected genes during the time-course of my Master project, we focused on F1 ccdc40+/- zebrafish mutants already identified to generate homozygous mutant larvae to administer a novel mRNA-based therapy enclosed on lipidic nanoparticles produced by Ethris GmbH. To come as close as possible to a viable treatment for human PCD patients, we tested a topical delivery in zebrafish larvae. Hence, zebrafish larvae were placed in zebrafish medium enriched with formulated LNPS with CCDC40 cmRNA at 30 hpf. The incubation period was tested (1 day, 2 days or 4 days of incubation) and two different mRNA concentrations of 10 and 20 ng/μL were investigated. We concluded that an increase in cilia beating frequency (CBF) was accomplished by incubating ccdc40-/- larvae for 4 days with LNPS formulated with CCDC40 cmRNA at 20 ng/μL. These conditions enabled a partial recovery of ciliary movement in small localized areas of the olfactory pit of the zebrafish. Our conclusions are that tests should continue to increase the concentration under the duration of 4 days of incubation with renewal of therapy at 2 days.