Towards gene therapy for primary ciliary dyskinesia

摘要

Primary ciliary dyskinesia (PCD) describes a family of raregenetic disorders affecting ciliary motility in several organsystems. The respiratory defects that can lead to lung failure,however, are most concerning. New treatments forPCD are needed that prevent progressive lung damage andwe aim to develop gene therapy to achieve this. DNAH5 isthe gene most frequently mutated in PCD. It encodes alarge 500kDa structural protein with ATPase activity thatpowers ciliary movement. PCD rarity, the shortage of genotypedpatient samples and lack of suitable animal modelsmeans that better model systems are needed in which tostudy gene therapies. A further challenge is that gene transferof DNAH5 will require an efficient non-viral deliveryvector capable of packaging this gene, which is too largefor commonly used viral vectors. To model PCD we haveused RNA interference to silence DNAH5 in normalhuman bronchial epithelial cells grown in air-liquid interfacecultures. Cells were transduced with a lentivirusexpressing an shRNA for DNAH5. Silencing of DNAH5expression was demonstrated and preliminary evidencethat the cilia were immotile.We have cloned DNAH5 frommRNA of human ciliated cells into a mammalian expressionvector and sequenced it. The DNAH5 clone expressedboth mRNA and protein in transfected cells. Transfectionswere performed with a nanocomplex formulation comprisingliposomes and targeting peptides that we havedeveloped for DNAH5 gene transfer to the respiratoryepithelium.We are now able to investigate PCD gene therapyusing these models, DNAH5 constructs and vectordelivery system.