Publication
Using zebrafish to restore motile cilia function in development and disease
| Summary: | Primary ciliary dyskinesia (PCD) is a rare genetic disorder characterized by motile cilia dysfunction in multiple structures of the human body. PCD arises from mostly recessive mutations in genes associated with cilia structure, causing the dysfunction of motile cilia and consequently leading to impaired mucociliary clearance in the airway and recurrent respiratory infections. Identifying targeted therapies is crucial, because this disease causes irreversible damage to the respiratory tract. This study used zebrafish as an in vivo PCD model since this vertebrate contains epithelial multiciliated cells in the olfactory pit (OP) which mimic the structure and movement of those on the human airway. Due to a shared gene pool with humans, this animal model enables the study of PCD-associated mutations through genetic knockout, using CRISPR-Cas9 technology, or through genetic knockdown, by microinjection of antisense morpholino (MO) oligonucleotides into one-cell stage embryos. The first goal of this project was to analyze the temporal gene expression patterns of ccdc39 and ccdc40, two zebrafish OP genes that code for the CCDC39/CCDC40 protein complex required for cilia structure and motility. Based on these results, the second goal was to test mRNA-based therapy delivery in the MO zebrafish model. Gene expression analysis showed that there was an increase of ccdc39 expression at 33 hours post-fertilization (hpf), while ccdc40 increased at 28 hpf. Human CCDC39 mRNA therapy (0.2 μg/μl) was delivered by bath at 28 hpf, in ccdc39 knockdown larvae, preceding the endogenous expression increase, and removed after 50 hours. The MO-microinjected larvae had a nearly complete absence of healthy ciliary movement in the OP which was significantly improved by restoring up to 46% of OP area exhibiting healthy movement after topical therapy delivery. This study confirmed that zebrafish is a relevant exploratory PCD model and tested initial therapeutic approaches for translation into future PCD patients. |
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| Main Authors: | Neves,Mariana Pereira |
| Subject: | Primary Ciliary Dyskinesia motile cilia zebrafish gene expression mRNA therapy |
| Year: | 2025 |
| Country: | Portugal |
| Document type: | master thesis |
| Access type: | embargoed access |
| Associated institution: | Universidade de Lisboa |
| Language: | English |
| Origin: | Repositório da Universidade de Lisboa |
| Summary: | Primary ciliary dyskinesia (PCD) is a rare genetic disorder characterized by motile cilia dysfunction in multiple structures of the human body. PCD arises from mostly recessive mutations in genes associated with cilia structure, causing the dysfunction of motile cilia and consequently leading to impaired mucociliary clearance in the airway and recurrent respiratory infections. Identifying targeted therapies is crucial, because this disease causes irreversible damage to the respiratory tract. This study used zebrafish as an in vivo PCD model since this vertebrate contains epithelial multiciliated cells in the olfactory pit (OP) which mimic the structure and movement of those on the human airway. Due to a shared gene pool with humans, this animal model enables the study of PCD-associated mutations through genetic knockout, using CRISPR-Cas9 technology, or through genetic knockdown, by microinjection of antisense morpholino (MO) oligonucleotides into one-cell stage embryos. The first goal of this project was to analyze the temporal gene expression patterns of ccdc39 and ccdc40, two zebrafish OP genes that code for the CCDC39/CCDC40 protein complex required for cilia structure and motility. Based on these results, the second goal was to test mRNA-based therapy delivery in the MO zebrafish model. Gene expression analysis showed that there was an increase of ccdc39 expression at 33 hours post-fertilization (hpf), while ccdc40 increased at 28 hpf. Human CCDC39 mRNA therapy (0.2 μg/μl) was delivered by bath at 28 hpf, in ccdc39 knockdown larvae, preceding the endogenous expression increase, and removed after 50 hours. The MO-microinjected larvae had a nearly complete absence of healthy ciliary movement in the OP which was significantly improved by restoring up to 46% of OP area exhibiting healthy movement after topical therapy delivery. This study confirmed that zebrafish is a relevant exploratory PCD model and tested initial therapeutic approaches for translation into future PCD patients. |
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