Towards gene therapy for primary ciliary dyskinesia

Primary ciliary dyskinesia is a genetic disorder where patients develop lung disease as they are unable to clear airway infections effectively. There is currently no treatment for the underlying genetic defect. This thesis describes advances towards the development of a gene therapy targeting the airway disease in PCD patients with DNAH5 mutations, the most common cause of PCD. Little work has been done in the field so many challenges remain. DNAH5 is a large gene and the cDNA has not been cloned. In addition PCD models are currently inadequate. Finally, non-viral vectors for airway gene delivery produce sub-optimal levels of transgene expression and more efficient viral vectors cannot package the large DNAH5 cDNA. To address these issues functional DNAH5 cDNA was cloned from healthy airway cells and the cDNA validated by sequencing and expression studies. Lentiviral shRNA transduction of healthy cells was used to knock down DNAH5 in airway cells semi-immortalised with BMI-1, a proto-oncogene that allows extended growth capacity of cells whilst retaining their mucociliary differentiation potential. The novel cell lines lacked cilia motility as is seen in patient cells. Minicircle DNA, in the context of airway gene delivery, was found to enhance gene expression in vitro and in vivo. A non-viral vector was optimised, characterised and used to deliver DNAH5 coding minicircle DNA to PCD models but poor transfection efficiency of DNAH5 prevented functional correction. Transfection of smaller genes was efficient so the vector in its current form could be useful for gene therapy treatment of the majority of PCD causing genes. Studies are needed to determine and overcome the bottlenecks in the efficient transfection of large transgenes to help advance PCD gene therapy

A Nanosensor Toolbox for Rapid, Label-Free Measurement of Airway Surface Liquid and Epithelial Cell Function

Ciliated lung epithelial cells and the airway surface liquid (ASL) comprise one of the body's most important protective systems. This system is finely tuned, and perturbations to ASL rheology, ASL depth, ASL pH, the transepithelial potential, and the cilia beat frequency are all associated with disease pathology. Further, these apparently distinct properties interact with each other in a complex manner. For example, changes in ASL rheology can result from altered mucin secretion, changes in ASL pH, or changes in ASL depth. Thus, one of the great challenges in trying to understand airway pathology is that the properties of the ASL/epithelial cell system need to be assessed near-simultaneously and without perturbing the sample. Here, we show that nanosensor probes mounted on a scanning ion conductance microscope make this possible for the first time, without any need for labeling. We also demonstrate that ASL from senescence-retarded human bronchial epithelial cells retains its native properties. Our results demonstrate that by using a nanosensor approach, it is possible to pursue faster, more accurate, more coherent, and more informative studies of ASL and airway epithelia in health and disease

Delivery of ENaC siRNA to epithelial cells mediated by a targeted nanocomplex: a therapeutic strategy for cystic fibrosis

The inhibition of ENaC may have therapeutic potential in CF airways by reducing sodium hyperabsorption, restoring lung epithelial surface fluid levels, airway hydration and mucociliary function. The challenge has been to deliver siRNA to the lung with sufficient efficacy for a sustained therapeutic effect. We have developed a self-assembling nanocomplex formulation for siRNA delivery to the airways that consists of a liposome (DOTMA/DOPE; L), an epithelial targeting peptide (P) and siRNA (R). LPR formulations were assessed for their ability to silence expression of the transcript of the gene encoding the α-subunit of the sodium channel ENaC in cell lines and primary epithelial cells, in submerged cultures or grown in air-liquid interface conditions. LPRs, containing 50 nM or 100 nM siRNA, showed high levels of silencing, particularly in primary airway epithelial cells. When nebulised these nanocomplexes still retained their biophysical properties and transfection efficiencies. The silencing ability was determined at protein level by confocal microscopy and western blotting. In vivo data demonstrated that these nanoparticles had the ability to silence expression of the α-ENaC subunit gene. In conclusion, these findings show that LPRs can modulate the activity of ENaC and this approach might be promising as co-adjuvant therapy for cystic fibrosis

Effective silencing of ENaC by siRNA delivered with epithelial-targeted nanocomplexes in human cystic fibrosis cells and in mouse lung

Introduction Loss of the cystic fibrosis transmembrane conductance regulator in cystic fibrosis (CF) leads to hyperabsorption of sodium and fluid from the airway due to upregulation of the epithelial sodium channel (ENaC). Thickened mucus and depleted airway surface liquid (ASL) then lead to impaired mucociliary clearance. ENaC regulation is thus a promising target for CF therapy. Our aim was to develop siRNA nanocomplexes that mediate effective silencing of airway epithelial ENaC in vitro and in vivo with functional correction of epithelial ion and fluid transport. Methods We investigated translocation of nanocomplexes through mucus and their transfection efficiency in primary CF epithelial cells grown at air–liquid interface (ALI).Short interfering RNA (SiRNA)-mediated silencing was examined by quantitative RT-PCR and western analysis of ENaC. Transepithelial potential (Vt), short circuit current (Isc), ASL depth and ciliary beat frequency (CBF) were measured for functional analysis. Inflammation was analysed by histological analysis of normal mouse lung tissue sections. Results Nanocomplexes translocated more rapidly than siRNA alone through mucus. Transfections of primary CF epithelial cells with nanocomplexes targeting αENaC siRNA, reduced αENaC and βENaC mRNA by 30%. Transfections reduced Vt, the amiloride-sensitive Isc and mucus protein concentration while increasing ASL depth and CBF to normal levels. A single dose of siRNA in mouse lung silenced ENaC by approximately 30%, which persisted for at least 7 days. Three doses of siRNA increased silencing to approximately 50%. Conclusion Nanoparticle-mediated delivery of ENaCsiRNA to ALI cultures corrected aspects of the mucociliary defect in human CF cells and offers effective delivery and silencing in vivo

Receptor-targeted liposome-peptide-siRNA nanoparticles represent a novel and efficient therapeutic approach to prevent conjunctival fibrosis.

There is increasing evidence that the Myocardin-related transcription factor/Serum response factor (MRTF/SRF) pathway plays a key role in fibroblast activation and that knocking down MRTF can lead to reduced scarring and fibrosis. Here, we have developed a receptor-targeted liposome-peptide-siRNA nanoparticle as a non-viral delivery system for MRTF-B siRNA in conjunctival fibrosis. Using 50 nM siRNA, the MRTF-B gene was efficiently silenced by 76% and 72% with LYR and LER nanoparticles, respectively. The silencing efficiency was low when non-targeting peptides or siRNA alone or liposome-siRNA alone were used. LYR and LER nanoparticles also showed higher silencing efficiency than PEGylated LYR-P and LER-P nanoparticles. The nanoparticles were not cytotoxic using different liposomes, targeting peptides, and 50 nM siRNA. Three-dimensional fibroblast-populated collagen matrices were also used as a functional assay to measure contraction in vitro, and showed that MRTF-B LYR nanoparticles completely blocked matrix contraction after a single transfection treatment. In conclusion, this is the first study to develop and show that receptor-targeted liposome-peptide-siRNA nanoparticles represent an efficient and safe non-viral siRNA delivery system that could be used to prevent fibrosis after glaucoma filtration surgery and other contractile scarring conditions in the eye

Combined exome and whole-genome sequencing identifies mutations in ARMC4 as a cause of primary ciliary dyskinesia with defects in the outer dynein arm.

Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous ciliopathy disorder affecting cilia and sperm motility. A range of ultrastructural defects of the axoneme underlie the disease, which is characterised by chronic respiratory symptoms an