Difficulties of gene therapy

Most patients with cystic fibrosis die from respiratory failure due to progressive lung destruction caused by chronic bacterial infection and inflammation. The genetic basis of cystic fibrosis was resolved in 1989 when the CFTR (cystic fibrosis transmembrane conductance regulator) gene was cloned and disease-associated mutations were identified. CFTR encodes an epithelial chloride channel that helps regulate salt and water transport. Within a year of this landmark discovery, scientists showed that by providing a normal copy of the CFTR cDNA sequence to damaged cells they could fix the genetic defect. Early studies suggested that the correction of as few as 6–10% of epithelia might be therapeutic. The directness and simplicity of such an approach had great appeal, and over the next 12 years many basic and applied experiments were completed. However, the goal of attaining clinically relevant and persistent correction of the cystic fibrosis defect in humans has not been achieved. The fundamental principle of gene therapy for cystic fibrosis is to deliver a normal copy of the CFTR DNA to airway epithelia using a delivery vehicle (vector). Other technologies, such as the targeted repair of DNA or RNA, are also being investigated. Most gene therapy approaches in cystic fibrosis involve the delivery of the vector to the lungs by instillation or as an aerosol. Thus the delivery vehicle is applied to the apical surface of the cells. Delivery via the vascular system might also be possible. Whatever the mechanism used, scientists recognise the respiratory epithelium as one of the most challenging targets for the practical application of gene therapy (figure). Many different cell types make up this complex epithelium, and the key types that must be targeted remain unknown. Since the pathology of cystic fibrosis is primarily confined to the airways, the cells of the conducting airways rather than the alveoli are the main therapeutic targets. However, the airway epithelium is in constant contact with the environment and has evolved a vast array of mechanisms and responses to keep microbes and other environmental challenges, including the vectors of gene transfer, from entering the host. Although many vector systems work in the laboratory, translation of these results into successful clinical trials has proven much more challenging. Difficulties include delivery of the vector to the cell, lack of persistent gene expression in targeted cells, and immune responses to viral gene products, transgenes, or cells targeted by the vectors. Furthermore, mice with deletions of the CFTR gene or with common human CFTR mutations do not develop lung disease like people. There is not, therefore, a good animal model in which to test new therapies. Early attempts to deliver the gene used DNA, alone or complexed with lipids or other molecular conjugates. Viral vectors, especially those derived from adenovirus or adenoassociated virus (AAV), have also been studied extensively. Basic studies in cystic fibrosis gene therapy are now largely centred at the intersections between virology, cell biology, and immunology. Incremental progress has come with the completion of detailed studies of the interactions between the host cell and various vector systems. Scientists now know that the receptors for many viral vectors are polarised to the basolateral membrane of airway epithelia. Appropriate models of polarised epithelia that mimic these barriers must therefore be used for in-vitro and in-vivo studies. Progress has been made in reduction of immune responses by further deletions of viral genes in the vectors and through pharmacological modification of host responses. To attain persistent CFTR gene expression, without the need for repeated administration, studies using integrating AAV, lentiviral, and retroviral systems have begun. As the barriers to gene transfer become well known, new strategies to overcome them are emerging. For example, the barrier of polarity of viral receptors away from the apical cell surface can be overcome by use of vector formulations that include calcium chelators or other agents that transiently disrupt epithelial tight junctions, thereby allowing access to the basolateral membrane (figure). New approaches to redirect vectors to receptors on the apical surface are also being implemented. For example, AAV serotypes 5 and 6 bind and enter airway epithelia from the apical surface, and new ligands can be engineered into the fibre or capsid proteins of adenovirus to target apical receptors. In primate and non-primate lentiviral vectors, the protein ligands in the viral envelope can be changed by a process termed “pseudotyping”. Proteins from other enveloped viruses are substituted using this approach to redirect the binding and fusion of these integrating vectors to the apical surface. Finally, we must identify what progenitor cell types should be targeted to attain long-term expression. Now that many of the fundamental barriers to successful gene transfer are understood, the eventual application of the technique as a therapy for cystic fibrosis seems promising. Airway epithelium and vectors for gene transfer