Novel molecular approaches to cystic fibrosis gene therapy

Abstract

Gene therapy holds promise for the treatment of a range of inherited diseases, such as cystic fibrosis. However, efficient delivery and expression of the therapeutic transgene at levels sufficient to result in phenotypic correction of cystic fibrosis pulmonary disease has proved elusive. There are many reasons for this lack of progress, both macroscopically in terms of airway defence mechanisms and at the molecular level with regard to effective cDNA delivery. This review of approaches to cystic fibrosis gene therapy covers these areas in detail and highlights recent progress in the field. For gene therapy to be effective in patients with cystic fibrosis, the cDNA encoding the cystic fibrosis transmembrane conductance regulator protein must be delivered effectively to the nucleus of the epithelial cells lining the bronchial tree within the lungs. Expression of the transgene must be maintained at adequate levels for the lifetime of the patient, either by repeat dosage of the vector or by targeting airway stem cells. Clinical trials of gene therapy for cystic fibrosis have demonstrated proof of principle, but gene expression has been limited to 30 days at best. Results suggest that viral vectors such as adenovirus and adeno-associated virus are unsuited to repeat dosing, as the immune response reduces the effectiveness of each subsequent dose. Nonviral approaches, such as cationic liposomes, appear more suited to repeat dosing, but have been less effective. Current work regarding non-viral gene delivery is now focused on understanding the mechanisms involved in cell entry, endosomal escape and nuclear import of the transgene. There is now increasing evidence to suggest that additional ligands that facilitate endosomal escape or contain a nuclear localization signal may enhance liposome-mediated gene delivery. Much progress in this area has been informed by advances in our understanding of the mechanisms by which viruses deliver their genomes to the nuclei of host cells.

Figure 1. Schematic diagram demonstrating methodology used to construct first generation E1 deletion adenovirus vectors

E1-expressing cells, such as HEK-293 cells, are co-transfected with genomic adenovirus DNA and a shuttle plasmid containing the expression cassette with flanking sequences derived from sequences immediately upstream and downstream of the E1 region. The genomic adenovirus DNA is modified to confer a selection advantage for recombinant virus. Recombination between the plasmid and genomic DNA results in the generation of an adenovirus vector with an expression cassette replacing the E1 region. ITRs and the packaging signal are represented in purple. Reprinted from Advances in Virus Research, vol. 55, M. M. Hitt and F. L. Graham, “Adenovirus vectors for human gene therapy”, pp. 479–505, © 2000, with permission from Elsevier.

Figure 2. Chemical structures of lipids commonly used in the formation of cationic liposomes, highlighting hydrophilic head-groups, linker regions and hydrophobic lipid anchors

Head-groups vary in degree of positive charge; the co-lipid DOPE has a neutral head-group. Lipid anchors consist of either two hydroxyalkyl chains or a modified cholesterol component (DC-Chol and GL-67™).

Figure 3. Cell-entry mechanisms for cationic liposome–plasmid DNA complexes

The two major pathways are by endocytosis (A) and direct fusion with the cell membrane (B). The endocytic pathway results in the formation of endosomes, which can lead to eventual destruction of plasmid DNA before release into the cytoplasm. Reprinted from Bioscience Reports, vol. 22(2), © 2002, “Cationic liposome-mediated gene delivery in vivo” by N. Smyth Templeton, Fig. 3, with kind permission of both Springer Sciences and Business Media, and the author, Nancy Smyth Templeton.

Figure 4. Schematic representation of nuclear import process involving importin α and importin β

Reprinted from Advanced Drug Delivery Reviews, vol. 34, C. W. Pouton, “Nuclear import of polypeptides, polynucleotides and supramolecular complexes”, pp. 51–64, © 1998, with permission from Elsevier.

Figure 5. Adenovirus 2 entry into host cells during infection

Following binding to CAR and α_v-integrin, clathrin-mediated endocytosis occurs. The virus particle is taken up into the acidic endosome, but escapes to the cytosol. Transport to the nuclear pore complex along the microtubules is dynein/dynactin-dependent. The virus then docks to CAN/Nup214 on the nuclear pore complex, disassembles and recruits histone H1. The adenovirus genome and core proteins are then imported with the H1 import factors importin β and importin 7. Reproduced from the Journal of Gene Medicine, “Adenovirus endocytosis”, O. Meier and U. F. Greber, pp. 451–462, 2003, with permission. © John Wiley and Sons Limited.