Delivery of Sleeping Beauty Transposons to Dog Liver for Gene Therapy

DESCRIPTION (provided by applicant): The overall goal of Discovery Genomics, Inc. is to develop the Sleeping Beauty transposon system for human gene therapy. To achieve this to treat hemophilia, we must be able to direct uptake and long-term expression of therapeutic genes in targeted tissues such as the liver. Only one method of plasmid delivery has been effective for high-level gene expression in the liver in mice - the rapid, high-pressure delivery known as hydrodynamic injection. In the mouse, hydrodyna mic delivery of DNA requires injection of a large volume (10% vol/wt) of a DNA solution through the tail vein in less than 10 seconds. For larger animals, this procedure is conjectured to be unacceptable on a whole animal basis. In this highly focused proj ect we propose to develop catheter-based, local hydrodynamic delivery to the liver of dogs that will require minimal surgery and amounts of therapeutic DNA. These features are especially important for the delivery of Factor IX and Factor VIII-expressing tr ansposons for treatment of hemophilia. Recognizing that delivery to only one cell type in the liver (and other organs) is not possible, we will to couple physical and biological controls over gene expression to our transgenic constructs that will reduce th eir expression in hematopoietic cells to reduce undesirable immunological responses. This project will dovetail with our ongoing SBIR project to deliver Factor VIII and IX genes in dogs via open-chest surgery for treatment of hemophilia. The Specific Aims of the project are to 1) develop appropriate transposons with reporter genes and recovery cassettes that can be used to evaluate the efficacy of hydrodynamic delivery in dogs, 2) determine an optimal, catheter-based delivery procedure that can be used to i nject DNA with minimal surgical intervention into canine livers, and 3) demonstrate that long-term gene expression will result from transposition of Sleeping Beauty transposons into canine hepatocytes using the procedure(s) developed in Aim 2. Publi c Health Relevance: Delivery of Sleeping Beauty Transposons to Dog Liver for Gene Therapy. There currently is no efficient and effective method for delivery of Sleeping Beauty transposons to liver in humans for gene therapy. The goal of this project is to develop a transposon delivery system that will allow hydrodynamic delivery of SB transposons in dogs as a model large animal for humans.