Towards Gene Therapy for Cystic Fibrosis: Bio-Distribution of GL67A/pGM169 DNA and mRNA Following Aerosol Delivery to the Mouse Lung.

Pringle IA, Davies LA, Lawton AE, Painter H, Evans N, Green A-M, Stoneham S, McCormick D, Coles RL, Nunez-Alonso GA, Scheule RK, Cheng SH, Gill DR, Hyde SC

Molecular Therapy, 16 S341


The American Society of Gene Therapy Annual Conference, Boston, 2008

We are undertaking clinical trials for the development of a gene therapy for Cystic Fibrosis (CF) lung disease.

Clinical studies are planned for the aerosol delivery Genzyme lipid (GL67A)/plasmid DNA (pDNA) complexes with a view to achieving clinical benefit in the lungs of patients with CF. In order to minimise CpG-related inflammatory responses in the lung we generated a CpG-free plasmid (pGM169) expressing human CFTR. While aerosol delivery should largely restrict GL67A/pGM169 DNA deposition and gene expression to the lung, regulatory agencies require that vector bio-distribution in non-target organs is quantified.

This is particularly important given the inherent risk concerned with long-term integration of the vector in germline tissues. Therefore in support of our clinical trial application we assessed the duration of pGM169 expression in the mouse lung, evidence of pGM169 expression in other organs and the level of pDNA deposition in mouse internal organs. To facilitate these studies we developed two highly sensitive Taqman assays to detect pDNA (Taqman PCR) and mRNA (Taqman RT-PCR) from pGM169. Both of the assays have sensitivities in the near single copy number range and were unaffected by normal levels of total DNA and total RNA concentrations.

The GL67A/pGM169 formulation (60mls; 150mg pGM169) was aerosolised to adult mice (BALB/c, 12 male, 12 female) with a Pari LC+ nebuliser. At day 1, 14, 28 and 56 post-delivery the internal organs of 3 male and 3 female mice were harvested and total DNA and total RNA extracted for analysis by quantitative TaqMan PCR and RT-PCR.

Consistently high levels of pGM169 mRNA were detected in the lungs for the duration of the study (10-20% endogenous murine CFTR) and there was no statistical difference between the levels of gene expression at any time point (Kruskal-Wallis P>0.05). No pGM169 mRNA was detected in any other organ (spleen, liver, gonads) at any time-point, indicating that gene expression was restricted to the lung. Plasmid DNA was readily detected in the lung at d1 (mean 1600ng per lung), but only a mean of 15ng was detected at d28 and d56. In other organs (spleen, liver and gonads) only very low levels of pDNA were detected at d1-d14, around 6-8 logs lower than the lung.

These levels were so low that we cannot rule out the possibility that they were simply derived from lung contamination during harvesting. By d28 most samples were negative and by d56 there was no pDNA detectable in any organ other than the lung, therefore reducing the risk of permanent germline gene transfer.

These data confirm that despite persistent high levels of lung gene expression for at least 8 weeks following aerosol delivery, no pGM169 mRNA or DNA was detected in organs other than the lung. These data enhance the safety profile of aerosol delivery of our GL67A/pGM169 clinical trial formulation.