Molecular Therapy, 15 S1 S361
The American Society of Gene Therapy Annual Conference, Seattle, 2007
The lung is an important target for a variety of gene therapy applications and aerosol delivery of gene transfer agents (GTAs) is a practical route for vector administration in the clinic.
The cationic polymer polyethyleneimine (PEI) is effective following aerosolisation to the lung. It has been proposed that PEI possesses a buffering (proton sponge) capacity, which may protect plasmid DNA (pDNA) from lysosomal degradation. In addition, PEI has many attributes that make it suitable for aerosol gene therapy; it is chemically uncomplicated, inexpensive, easy to produce and relatively straightforward to complex with pDNA. However, little is known about the stability of pDNA/PEI aerosol formulations after complexation, and we have investigated this under a variety of storage conditions.
Plasmid pCIKLux expressing luciferase was complexed with branched 25kDa PEI (N:P of 10:1) at an initial concentration of 0.2mg/ml in sterile water.Aliquots of pCIKLux/PEI complexes were stored at -20°C, 4°C, 20°C and 37°C over a period of 6 months, and the biological efficacy of stored samples was quantified at intervals, following transient transfection of HEK 293T cells in culture and subsequent analysis of luciferase activity. Gene expression at each time point was compared to expression mediated by freshly prepared (control) pCIKLux/PEI complexes. Storage at -20°C was associated with precipitation of the complexes upon thawing that resulted in almost complete loss of biological efficacy. No precipitation or aggregation of complexes was observed with samples stored at between 4°C and 37°C, although a temperature and time-dependent loss of biological activity was observed. All samples retained biological efficacy similar to control samples during the first 7 days of storage. However, by 36 days, expression mediated by samples stored at 37°C and 20°C had fallen to 19% and 56% of control levels respectively. In contrast, samples stored at 4°C retained complete biological efficacy even after storage for 180 days.
To demonstrate the suitability of stored pDNA/PEI complexes for aerosol administration we have recently initiated aerosol delivery studies utilising a mouse lung model. Formulations containing pCIKLux/PEI at 0.2mg/ml in sterile water were prepared and stored at 4°C prior to aerosol delivery to the lungs of female BALB/c mice (8-16wks) via whole body aerosol exposure (10ml aerosol). Aerosol delivery of complexes stored at 4°C for 7 days resulted in modest levels of lung luciferase expression (7.95 ± 1.12 RLU/mg) that were equivalent to expression seen in mice exposed to aerosols immediately following complexation of pDNA and PEI (7.93 ± 1.30 RLU/mg) (p=0.99 ANOVA). Studies looking at longer storage periods are ongoing.
These initial results demonstrate that under appropriate storage conditions, stable pDNA/PEI formulations can be prepared and stored for extended periods prior to utilisation in gene therapy studies. This is an important step towards developing PEI as a robust GTA for use in the clinic.