In Vivo Gene Delivery
Our laboratory has also developed methods for extracellular delivery of non-viral vectors. It has been demonstrated by multiple labs that injection of naked DNA into muscle results in high level expression of gene product. Over the past 15 years, electroporation has been coupled with the direct injection approach in muscle and liver of living animals and levels of expression have been shown to increase up to a thousand-fold. Using electroporation, we have also demonstrated gene transfer and expression in the muscle, cornea, heart, kidney, vasculature, and lungs of mice and rats. We have designed unique electrode systems for the vasculature that allows us to use limiting amounts of DNA in the transfer process, yet yields nanogram amounts of gene product per mm of treated vessel. All cell layers of the vasculature express gene product, and we have been able to restrict gene expression to specific cell types within the blood vessel by using DNA nuclear targeting sequences.
Vascular gene transfer using electroporation. The top panel shows that there are no histological differences between untreated vessels (left) and electroporated vessels (right). The lower two sections show vessels that received GFP-expressing plasmids in longitudinal or cross section. Note that all cell layers receive DNA and express gene product.
Most of our recent attention has focused on the lung. We have developed methods to transfer DNA via the airways using either surface electrodes that are placed on the chest wall or using a bronchoscope as an internal point source. As in the vasculature, multiple cell layers and all cell types express gene product. We have used several different cell-specific DNA nuclear targeting sequences to deliver genes to all cell types in the lung, but restrict nuclear import of the DNA and subsequent gene expression to airway smooth muscle cells, endothelial cells, or alveolar type II epithelial cells.
Electroporation is an effective method for pulmonary gene delivery. Rats were anesthetized and GFP-expressing plasmids were delivered to the lung via the airways and electroporated using electrodes on either side of the chest. Three days later, gene expression was visualized. No trauma, inflammation, or lung injury are associated with the technique.
The technique is safe, rapid, causes no trauma or injury, and most importantly, causes no inflammation. We have used this approach to deliver ion channel genes to the lung to both prevent and treat models of acute lung injury and well as to deliver genes that regulate smooth muscle contraction in several mouse models of asthma. Apart from showing success in treating these diseases in small animal models, we have begun studies applying pulmonary electroporation to a large animal model and have confirmed our studies in smaller animals that this is a highly safe and effective approach for high level gene delivery.« back to all projects