Molecular Therapy, 9 S1 S187
The American Society of Gene Therapy Annual Conference, Minneapolis, 2004
One of the significant barriers to non-viral gene transfer in vivo is penetration of the nuclear membrane. mRNA, which is translated in the cytoplasm and therefore does not need to negotiate nuclear transport, is being evaluated for gene transfer in vivo.
A plasmid expressing firefly luciferase (pSP64-lux-Poly(A)) was constructed and linearised to serve as a template for in vitro transcription (yield was 2.0mg RNA per 1ml reaction). HEK293T cells were transfected with 2µg mRNA (encoding luciferase) complexed with 4µg cationic lipid (DOTAP) and luciferase expression was observed 9 hours after transfection, confirming the integrity of the luciferase mRNA.
For in vivo delivery, luciferase-expressing mRNA was complexed with Genzyme lipid GL67 (80µg RNA/60nmol GL67) and dosed to the lungs of female BALB/c mice (6-8 weeks) via intranasal instillation. Lungs were harvested at 3, 6, 12, and 24 hours after delivery (n = 5-6 per group). At 3 hours, mean luciferase expression was five-fold greater than that of naive animals (mRNA: 0.236 ± 0.089 RLU/mg protein, naÔve: 0.047 ± 0.019 RLU/mg protein) (ANOVA with Fisher's PLSD, p = 0.0001). mRNA dependent reporter gene expression decreased with time and was lower than expression from luciferase expressing plasmid DNA. In vivo delivery was also demonstrated following perfusion to the mouse nose.
Naked mRNA (150µg), mRNA/DOTAP (100µg) or mRNA/DOTAP-Chol (25µg) was delivered to female BALB/c mice (n=7). The reagents were delivered in a total volume of 100µl and perfused over 15 minutes. Nasal tissue was harvested and measured for luciferase activity after 4 hours. Naked mRNA generated a mean luciferase activity approximately 500 fold over values of mRNA/DOTAP, mRNA/DOTAP-Chol and naive groups (ANOVA, p = 0.0001). The lack of expression seen with the mRNA/lipid complexes in the nasal epithelium could be due to the route of entry for these complexes via endocytosis and/or the release of the mRNA into the cytoplasm may take longer than 4 hours.
This study demonstrates that mRNA can be used to deliver and express transgenes in the mouse lung and nose. Gene replacement therapy is being considered as a potential treatment for Cystic Fibrosis, where absence of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein from the respiratory epithelium results in fatal lung disease. As only a small amount of CFTR expression may be needed per cell to achieve normal levels (Trapnell et al, 1991, PNAS 88:6565-6569), the use of mRNA might offer advantages over other gene transfer agents, depending on the number of cells transfected in vivo. Time course and dose response studies of mRNA expression in the mouse nose are underway.
Further studies using GFP mRNA will investigate the number and type of cells transfected.