786. Development of Zero-CpG Plasmids with Reduced Inflammatory Responses Following Delivery of Lipid/pDNA Complexes to the Mouse Lung

Abstract

Nonviral gene therapy is being developed for the treatment of Cystic Fibrosis (CF) lung disease. To date, such approaches have been limited by the poor duration of expression observed and a have also resulted in inflammatory responses following delivery of lipid/ plasmid DNA (pDNA) complexes to the lungs of mice and human subjects. This inflammatory response appears to be due in part to the detection by toll-like receptor 9 of CpG motifs present in pDNA. The majority of first generation pDNA vectors used in pre-clinical and clinical studies contained a large number of immune-stimulatory CpG motifs and one approach to reducing inflammation is to reduce or completely remove CpGs from the pDNA. Therefore we have constructed two novel series of pDNA vectors. Second generation pDNA vectors were constructed from elements with a low CpG content (such as the Genzyme ΔCpG plasmid backbone), which reduced the overall number of CpGs by approximately 50%. Third generation plasmids were constructed from elements that contained no CpG motifs (ΔCpG R6K origin, ΔCpG ZeoR, Murine CMV enhancer, human EF1a promoter) to produce zero-CpG plasmids. Versions of these plasmids that expressed luciferase reporter gene were complexed with Genzyme lipid 67 and delivered to mouse lung by nasal instillation (BALB/c, n=10, 80μg pDNA/100μl). At 24-hours post-dosing, bronchoalveolar lavage fluid (BALF) was collected and the level of IL-12, IFN-γ, TNF-α and total cells was determined. Untreated controls mice had very low levels of all four markers (0%). Mice that received CpG-rich first generation plasmid complexes had elevated levels of all four inflammatory markers (100%). Delivery of second generation (CpG-reduced) plasmids to the mouse lung did not significantly reduce levels IL-12 (105%), TNF-α (60%), IFN-γ (86%) or total cells (80%) (Mann-Whitney U P>0.05). However, following delivery of third generation (zero- CpG) plasmid complexes, levels of IL-12 (10%), TNF-α (0%), IFN-γ (4%) and total cells (14%) were indistinguishable from untreated control mice (P<0.01). Furthermore, at 24-hours post- dosing, luciferase activity in the lung was 10-fold higher from third generation plasmids compared with all other treated groups (P<0.001). In addition, expression from the zero-CpG plasmids persisted for at least 60 days post-dosing while the CpG containing plasmids resulted in only transient expression in the mouse lung (<7 days). These studies suggest that partial reduction of CpG content is no sufficient to reduce the inlfammation observed in mouse lung airway gene transfer models. However the lack of an inflammatory response following delivery of zero-CpG plasmids suggests that these vectors represent a significant advance in terms of safety and efficacy of nonviral delivery to the lung. Further studies outlining the development and testing of clinically relevant zero-CpG will also be presented.