Towards gene therapy for primary ciliary dyskinesia

Abstract

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.