Avoiding the Nuclear Barrier: The Development of mRNA as a Gene Transfer Agent.

Painter H, Davies LA, Sumner-Jones SG, Hyde SC, Gill DR

Pediatric Pulmonology, 40 S28 281

The North American Cystic Fibrosis Conference, Baltimore, 2005

Gene therapy is being considered as a possible treatment for cystic fibrosis (CF) lung disease. In the terminally differentiated cells of the airway epithelium, the nuclear membrane is a potential obstacle for many non-viral gene transfer agents (GTAs). One candidate GTA that avoids this barrier is messenger RNA (mRNA), which does not need to enter the nucleus before being translated into protein in the cytoplasm. This study investigated the use of mRNA as a GTA in the mouse nasal epithelium, a tissue that contains respiratory epithelial cell types similar to target cells lining the human airways. Naked plasmid DNA (pDNA) expressing luciferase (pCIKLux) was used as a comparator. Delivery of 150µg mRNA expressing the luciferase reporter gene (lux-mRNA) via nasal perfusion (n = 7) resulted in luciferase activity of 534 ± 377 RLU/mg protein (mean ± SEM), two orders of magnitude above that of naÔve animals (1.12 ± 0.54 RLU/mg protein) (p = 0.0054). Encouragingly, as little as 12.5µg mRNA resulted in detectable luciferase expression (p = 0.0297). Furthermore, at all doses tested, there was no statistical difference in luciferase activity between lux-mRNA or pDNA. A time course study revealed that lux-mRNA directed luciferase expression as early as 1 hour (p = 0.0013) and for at least 48 hours (p = 0.0001) after dosing, but was at background levels by 7 days (p = 0.8007). In contrast, expression from naked plasmid DNA was not seen until 6 hours after delivery (p < 0.0001), and a low level of luciferase activity was still detected at 7 days (p = 0.0377). Since the target cells in the airway epithelium are either slowly dividing or terminally differentiated, a successful GTA must be amenable to repeated administration without loss of efficacy. To test this, mice (n = 8) were dosed with 25µg lux-mRNA a total of four times (at 7 day intervals) and nasal tissue harvested 12 hours after the final dose. Luciferase activity was not significantly different to that of animals receiving only one dose of lux-mRNA (p = 0.99), suggesting that mRNA can be re-administered to the nasal epithelium in mice without loss of efficacy. To identify the transfected cells in the nasal epithelium, green fluorescent protein-expressing mRNA (GFP-mRNA) or pDNA (pEGFPN1) was delivered to the mouse nose (n = 3). A proportion of GFP-positive cells were found in the respiratory epithelium (mRNA = 36%; pDNA = 25%), confirming that therapeutically relevant cells were being transfected. Many more GFP-positive cells were observed in the mRNA dosed animals (149 ± 60 cells/60 sections) compared to animals dosed with plasmid DNA (9 ± 5 cells/60 sections). Interestingly, the mRNA-transfected cells produced a fainter signal, suggesting that more cells express GFP after mRNA transfection, but at a lower level per cell compared to pDNA. These results demonstrate that mRNA can efficiently and repeatedly transfect the cells of the nasal epithelium resulting in expression levels comparable to that observed with naked pDNA in this model. We are currently evaluating a CFTR-mRNA construct as a suitable GTA for CF treatment.