Strain-Specific Differences in Pulmonary Gene Transfer Efficiency: Relevance for Toxicology Studies in Mice.

Legakis G, Causton B, Zhu J, Nikki Newman, Farley R, Meng C, Lloyd CM, Alton EW, Griesenbach U

Molecular Therapy, Vol 19, S1, Abstract 272

The American Society of Gene and Cell Therapy Annual Conference, Seattle, 2011

As part of our ongoing clinical trial programme aimed at assessing, if non-viral gene transfer improves lung disease severity in cystic fibrosis (CF) patients, the UK CF Gene Therapy Consortium is currently conducting toxicology studies to support a multi-dose non-viral gene therapy trial in CF subjects. As part of this work, preliminary experiments determined significantly lower (approximately one log) lung transfection efficiency in outbred ICR(CD-1) mice (commonly used for toxicology studies conducted by contract research organisations) compared to inbred Balb/C mice (commonly used for pre-clinical gene transfer experiments) following nebulisation with pCIKLuc (regulated by a CMV promoter/enhancer). Here, we reproduced and confirmed these initial findings. We next extended these shssowing that strain-specific differences were maintained when using other non-viral vectors such as PEI, plasmids regulated by eukaryotic promoters or when transfecting the mouse nasal epithelium, which is a good model for human airways. To understand these strain specific-differences we first compared inflammatory responses after bolus administration of GL67A/pCIKlux complexes to the mouse lung by nasal sniffing. As previously reported nasal sniffing of GL67A/pDNA caused lung inflammation, but on histological assessment 24 hr after transfection there were no differences between the two strains. The pro-inflammatory chemokines KC and MIP1a were significantly (p<0.05) increased in broncho-alveolar lavage fluid of both strains 6 and 24 hrs after gene transfer. Interestingly, levels of both cytokines were significantly higher in Balb/C compared to CD-1 mice at both time-points (KC (pg/ml) shown as example, Balb/C 6 hr: 896±438, CD1 6 hr: 438±67, Balb/C 24 hr: 644±97, CD1 24 hr: 255±33, n=16/group). It is currently unclear if and how differences in cytokine expression relate to differences in transfection efficiency and additional studies have to be performed. We next compared gene expression in CD1/Balb/C F1 hybrids to the parental strains. Interestingly, the F1 hybrids had intermediate levels of gene expression and were significantly different from both parental strains. In conclusion, the mouse strain affects transfection efficiency. In the short-term, these underline the importance of careful selection of suitable mouse strains before embarking on toxicology studies and has led us to perform our toxicology study in Balb/C mice rather than CD1 mice. In the longer-term, understanding the reasons for these differences may inform key factors involved in airway gene transfer and may be informative for improving gene transfer in man.