Large Animal Models: Bridging the Gap
Authors: Stephen L. Hart
Journal: Molecular Therapy (2003)

Ten years ago, it was anticipated widely that cystic fibro- sis (CF) would be one of the first diseases to be treated by gene therapy. The CF gene, the cystic fibrosis transmem- brane conductance regulator (CFTR), had been cloned [1Rommens, J. M., Ianuzzi, M. C., Kerem, B.-S., et al. (1989). Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 245: 1059-1065.Google Scholar, 2Kerem, B.-S., Rommens, J. M., Buchanan, et al. (1989). Identification of the cystic fibrosis gene: genetic analysis. Science 245: 1073-1080.Google Scholar, 3Riordan, J. R., Rommens, J. M., Kerem, B.-S., et al. (1989). Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245:1066-1073.Google Scholar], and its function as a cAMP-regulated chloride channel had been established [4Welsh, M. J., and Smith, A. E. (1993). Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis. Cell 73: 1251-1254.Google Scholar]. Disease-causing mutations in the CFTR gene sequence were identified [3Riordan, J. R., Rommens, J. M., Kerem, B.-S., et al. (1989). Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245:1066-1073.Google Scholar] and CFTR knockout mice had become available [5Snouwaert, J. N., Brigman, K. K., Latour, A. M., et al. (1992). An animal model for cystic fibrosis made by gene targeting. Science 257: 1083-1088.Google Scholar], allowing gene augmentation strategies to be evaluated [6Whitsett, J. A., Dey, C. R., Stripp, B. R., et al. (1992). Human cystic fibrosis transmembrane conductance regulator directed to respiratory epithelial cells of trans- genic mice. Nat. Genet. 2: 13-20.Google Scholar, 7Alton, E. W., Middleton, P. G., Caplen, N. J., et al. (1993). Noninvasive liposome-mediated gene delivery can correct the ion transport defect in cystic fibrosis mutant mice. Nat. Genet. 5: 135-142.Google Scholar, 8Hyde, S. C., Gill, D. R., Higgins, C. F., et al. (1993). Correction of the ion transport defect in cystic fibrosis transgenic mice by gene therapy. Nature 362: 250-255.Google Scholar]. Moreover, many features were thought to predispose CF to treatment by gene thera- py. Low efficiency of lung transfection was hoped to be clin- ically beneficial, due to a chloride-transport bystander effect in the epithelium. Furthermore, CFTR is expressed from a housekeeping promoter and non-CF expression levels are low [9Yoshimura, K., Nakamura, H., Trapnell, B. C., et al. (1991). The cystic fibrosis gene has a “housekeeping”-type promoter and is expressed at low levels in cells of epithelial origin. J. Biol. Chem. 266: 9140-9144.Google Scholar]. In addition, the lung epithelium is readily accessible, minimizing the need for cell targeting, and it is also a con- tained system, such that very little administered vector reaches the circulation. At the last count, twenty-seven clinical trials were under- way worldwide using adenovirus, adenovirus-associated virus, or formulations of cationic liposomes. However, results published to date have generally been disappointing relative to the high expectations [10Griesenbach, U., Ferrari, S., Geddes, D.M., et al. (2002). Gene therapy progress and prospects: cystic fibrosis. Gene Ther. 9: 1344-1350.Google Scholar]. The universal con- founding feature has been low levels of CFTR expression that were not sustained for more than a few days. There is a range of obstacles to gene transfer in the lung that have been iden- tified. The viscous mucus barrier in the CF lung is problem- atic for all vector systems. Access of vectors to the target cells is limited by the localization of their receptors to basolateral membranes. Adenoviral vectors retargeted to apical recep- tors are still inefficient, due to the physical barriers of the glycocalyx. Most cationic liposome vectors exhibit poor effi- ciency of plasmid transfer across the nuclear envelope, restricting their transfection of nondividing cells, the status of most airway epithelial cells. New synthetic vector formu- lations are emerging with enhanced efficiency in airway epithelium in rodent gene transfer studies [11Gautam, A., Densmore, C. L., Golunski, E., et al. (2001). Transgene expression in mouse airway epithelium by aerosol gene therapy with PEI-DNA complexes. Mol. Ther. 3: 551-556.Google Scholar, 12Jenkins, R. G., Herrick, S. E., Kinnon, C., et al. (2000). An Integrin-Targeted Nonviral Vector for Pulmonary Gene Therapy. Gene Ther. 7: 393-400.Google Scholar]. Problems remain, however, with key issues such as the immunological and inflammatory response to both synthetic and viral vec- tors. In addition, there remains some doubt over the appro- priate target cell, with evidence that gene delivery to sub- mucosal glands may be necessary—presenting further prob- lems of accessibility. Finally, the required efficiency of gene transfer may be much higher than what was thought origi- nally [13Johnson, L. G., Boyles, S. E., Wilson, et al. (1995). Normalization of raised sodium absorption and raised calcium-mediated chloride secretion by adenovirus-mediated expression of cystic fibrosis transmembrane conductance regulator in primary human cystic fibrosis airway epithelial cells. J. Clin. Invest. 95: 1377-1382.Google Scholar]. The fact that successful gene augmentation studies in CF mice have not been predictive of the outcome of clinical tri- als is a perplexing finding. While studies of murine models have led to tremendous advances in our understanding of CF, they have been poor models of respiratory gene transfer in man, because they do not develop the severe inflamma- tory response and thickened mucus associated with CF lung disease [14G rubb, B. R., and Boucher, R. C. (1999). Pathophysiology of gene-targeted mouse models for cystic fibrosis. Physiol. Rev. 79: S193-214.Google Scholar]. Anatomical and physiological features underpin these species differences. Mice lack bronchial submucosal glands, leading to the production of fewer secretions in their lungs compared to humans [15Pack, R. J., Al-Ugaily, L. H., and Morris, G. (1981). The cells of the tracheobronchial epithelium of the mouse: a quantitative light and electron microscope study. J. Anat. 132: 71-84.Google Scholar]. In addition, Cl- secretion in the murine lung is performed predominantly by an alterna- tive Cl- channel, not the CFTR channel, and CFTR mRNA levels are lower throughout the murine pulmonary epitheli- um than in the lungs of man [16Rochelle, L. G., Li, D. C., Ye, H., et al. (2000). Distribution of ion transport mRNAs throughout murine nose and lung. Am. J. Physiol. Lung Cell. Mol. Physiol. 279: L14- 24.Google Scholar]. Therefore, model systems that are more representative of the barriers to gene transfer in the human airway are needed to develop protocols and vectors for gene therapy of CF. The report in this issue by Emerson et al., from the UK CF Gene Therapy Consortium (www.cfgenetherapy.org.uk), investigates gene transfer in the sheep lung as an alternative large animal model for respiratory gene transfer. The size, anatomy, and physiology of sheep and human lungs are similar, and human and sheep CFTR share similarities of sequence and developmental expression. Naked DNA, or a formulation of DNA and the cationic liposome GL-67/DOPE, was deposited in specific lung segments by bronchoscopy. Levels of the reporter gene, chloramphenicol transacetylase (CAT), were assessed quantitatively at the levels of both pro- tein and mRNA, and the inflammatory response was assessed at increasing DNA dosage levels. The authors com- pared their findings in the sheep with previous murine stud- ies using similar vectors and with a similar study in the pig lung performed with a different, nonviral vector system [17Cunningham, S., Meng, Q. H., Klein, N., et al. (2002). Evaluation of a porcine model for pulmonary gene transfer using a novel synthetic vector. J. Gene Med. 4: 438-446.Google Scholar]. One of the more striking differences between the studies in sheep and mice was that transfections with GL67 along with 1mM or 2 mM DNA, produced 500 or 4,400-fold less CAT protein, respectively, than was achieved in mice. Further, CAT protein levels in mouse lung after GL67 transfection were ten-fold higher than naked DNA transfection, while in sheep the difference was only three fold. By contrast, naked DNA performed equally as well as GL67 in clinical studies [18Zabner, J., Cheng, S. H., Meeker, D., et al. (1997). Comparison of DNA-lipid com- plexes and DNA alone for gene transfer to cystic fibrosis airway epithelia in vivo. J. Clin Invest. 100: 1529-1537.Google Scholar]. While further data is required for a comparison of sheep and pig lung transfections, both studies highlight how sheep and/or pig models may help bridge the gap between results in murine models of CF and results in the clinic. The use of large animal models potentially heralds a new direction and new opportunities in preclinical studies of CF gene therapy, particularly in the key area of vector development. At the moment, however, these are not models for CF lung disease. In the future, cloning technology may facilitate the development of CF models in sheep or pigs models. Their similarities to man in lung anatomy and their CFTR expression patterns in submucosal glands provide hope that they may develop lung disease similar to that seen in CF patients.