Varathalingam, A., Pringle, I. A., Cheng, S. H., Gill, D. R. & Hyde, S. C.
Molecular Therapy, 9, S177Download Back
Non-viral mediated gene transfer is being investigated as an approach for the treatment of lung diseases such as Cystic Fibrosis (CF). However, an important limiting factor is the inflammatory responses generated by such vectors in patients and animal models. Gene delivery of plasmid DNA complexed with cationic lipid to the murine lung results in increased levels of pro-inflammatory cytokines, which includes tumour necrosis factor alpha (TNF-alpha) (Scheule et al, 1997, Human Gene Therapy, 8: 689-707). Clinical studies have also indicated an inflammatory response following dosing of plasmid DNA/lipid complexes to the lungs of patients with CF (Alton et al, 1999, Lancet, 353 947). It is believed that the inflammation occurs, at least in part, in response to the unmethylated bacterial CpG sequences delivered to the cytoplasm of the target cells which results in the activation of Nuclear Factor kappa beta (NF-kB). NF-kB is a key regulator of inflammatory and immune responses and is essential for the transcription of multiple pro-inflammatory molecules including TNF-alpha, interleukin 12 (IL-12) and interferons (IFN) and has become a target for anti-inflammatory treatment. Previous studies have employed an NF-kB decoy strategy using synthetic oligodeoxynucleotides (ODNs) to block the binding of NF-kB to the promoter region of its targeted genes following systemic delivery of non-viral vectors to the mouse lung. The addition of the oligodeoxynucleotides inhibited TNF-alpha in a dose-dependent manner (Tan et al, 2002, Molecular Therapy, 804-812). This study investigates the effect of NF-kB decoy oligodeoxynucleotides on the inflammatory response following topical delivery of Genzyme lipid GL67 to the mouse lung. Female BALB/c mice were dosed intranasally with 40µg of luciferase expressing plasmid pCIKLux and 40µg of synthetic single stranded phosphothioate ODN, containing an NF-kB consensus binding sequence (S+) complexed with cationic lipid GL67 in total volume of 100µl. A mutated form of the ODN (S11), containing a scrambled sequence of S+ with no NF-kB consensus site was used as a control. Mouse lungs were harvested 24hrs after dosing and luciferase and pro-inflammatory cytokine levels were measured. With topical delivery of the S+/pCIKLux/GL67 complex, there was an 80% and 70% reductions in TNF-alpha (ANOVA with Fisher's PLSD, p=0.0001) and IFN-gamma (p=0.00370) respectively compared to dosing with pCIKLux/GL67 and NaÔve. The ODNs did not have a significant effect on transgene expression when dosed with either S+/pCIKLux/GL67 (Mann Witney U test, p=0.75), or S11/pCIKLux/GL67 (p=0.2). This type of approach may allow reduced inflammation in the lung following delivery of GL67 complexes. Reduction in inflammation may also lead to improved duration of transgene expression by avoiding cytokine induced promoter attenuation. Further experiments are in progress to detect cytokine levels induced by intransanal delivery of PEI/pDNA complexes co-delivered with NF-kB ODNs.