Salt Concentration and Liposomal DNA Therapeutic Formulations.

Nicholls P, Ballantyne JA, Davies LA, Hyde SC, Gill DR

Molecular Therapy, 17 S1 S227

The American Society of Gene Therapy Annual Conference, San Diego, 2009

Plasmid DNA (pDNA) complexed with the cationic lipid GL67A (a combination of GL67 (Genzyme), DOPE and DMPEPEG5000) has shown promise for lung gene therapy.

Clinical trials are underway in individuals with cystic fibrosis, where pDNA/GL67A will be delivered to the lung by aerosol. The development of complex non-viral formulations depends on the optimization of many factors to achieve the required delivery characteristics such as particle size, transfection efficiency and target specificity. One component of a non-viral formulation that is often ignored is the plasmid DNA, where the preparation and handling is commonly regarded as simple, robust and standardised. However minor impurities of the pDNA, which are frequently overlooked, can have dramatic effects on the final formulation.

Differences in pDNA performance can result from different manufacturing processes, despite pDNA being prepared to identical specifications; thus the method of pDNA production has become an important factor in ensuring consistency. Whilst striving for increased purity of pDNA we have observed a previously unreported phenomenon where pDNA/liposome mixing properties are adversely affected. Plasmid DNA (5.28mg/ml) supplied by Aldevron in Water For Injection (WFI) was mixed with GL67A and complexes allowed to form using standard mixing protocols.

Unexpectedly, complex precipitation and aggregation were observed immediately on mixing, subsequently preventing efficient aerosolization of the material for lung delivery. A series of plasmid DNA and GL67A mixing studies revealed that when plasmid DNA was prepared with a low level of sodium chloride remaining in the final buffer it formulated much more effectively than when pDNA was prepared in WFI. The increased conductivity correlated with decreased precipitation both upon mixing and after incubation for 24 hours. Thus we confirmed that counter ion levels can have drastic effects on the formulation of plasmid DNA therapeutics. We also showed that preparation of pDNA under conditions of low ionic strength can lead to changes in the structure of the DNA, despite the preservation of supercoiled forms.

Therefore we caution that as the technology for DNA production processes continues to improve, particularly in the proprietary polishing stage of production, the effects on final DNA purity should not be ignored, as negative as well as positive effects on formulation performance may result.