(626) Optimization of electroporation for the effective delivery of genes to the airways of mice and pigs

Background: Electroporation is the application of mild electric fields to cells (and tissue) using short pulse durations that transiently permeabilizes the plasma membrane to allow delivery of otherwise impermeant drugs and nucleic acids. We have previously shown that this approach is effective for in vivo delivery. Pulses can be applied across the chest of anesthetized animals (including mice, rats, ferrets, rabbits, and pigs) after intratracheal delivery of plasmid DNA, resulting in safe and effective gene transfer. High level gene transfer and expression has been seen throughout all cell types in the lung, including airway and alveolar epithelial cells, endothelial cells, smooth muscle cells, and fibroblasts. In the airways of mice, we have shown previously that plasmids expressing GFP or a GFP-CFTR fusion protein express in a high percentage of ciliated and non-ciliated airway epithelial cells throughout the lung. If this method is to be used ultimately to treat CF, we must evaluate its efficacy for functional airway gene delivery of CFTR and its use in a large animal model. We are extending our studies to evaluate functional expression of CFTR in CF mouse models using electroporation and to determine the distribution of gene expression in the lungs of adult, 50-75kg pigs.

Methods: CFTR, green fluorescent protein (GFP), and a GFP-CFTR fusion gene were cloned into plasmids behind the cytomegalovirus (CMV) immediate early promoter or the human ubiquitin (UbC) promoter and delivered to wild type, G542X, or W1282X mouse lungs by electroporation. 100 μg of plasmid in 50 μL of buffered saline was delivered to each lightly anesthetized mice by aspiration, and eight 200-V/cm square-wave electric pulses of 10 msec duration were applied across the chest. Two to 30 days later, lungs were either perfused and inflation fixed for histological analysis or trachea were removed for functional CFTR studies using Ussing chamber experiments. For pigs, animals were anesthetized, ventilated, and 50 mls of DNA (2 mg/ml in buffered saline) was delivered through a bronchoscope to desired lobe, followed by 10 seconds of positive pressure ventilation. Conformable defibrillator pads were placed on either side of the chest and a field of 150V/cm was applied across the chest in 8 pulses of 150 usec duration each. Animals were recovered and gene expression was evaluated 2 days later in inflation fixed lungs using immunofluorescence. All studies were done in accordance with the Guide for Care and Use of Laboratory animals and with IACUC approval.

Results: Gene delivery of GFP-expressing plasmids to the lungs of pigs resulted in abundant gene expression throughout both large and small airways of the animals. As in mice, expression was detected in airway epithelial cells, as well as in alveolar cells in the parenchyma. The approach was safe and led to no trauma or injury to the animals. Evaluation of CFTR activity in wild type and nonsense mice is ongoing.

Conclusions: Our results so far indicate that electroporation is an effective method of gene delivery to the airway epithelium in both small (mouse) and large (pig) animal models.

Acknowledgements: This work was supported by research grants from the Cystic Fibrosis Foundation (DAD and JDL).

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