We are developing a gene based therapeutic for boys with Duchenne muscular dystrophy (DMD). DMD only affects boys since it is on the X-chromosome which determines the sex of the baby. It causes muscle weakness due to a defective gene that results in the loss of an important muscle protein called Dystrophin. Presently there is no effective cure for DMD and only limited treatment options. Prednisone, a steroid with potentially serious side effects, provides partial benefit, but it does have significant side-effects after longterm use. To develop a cure, we are attempting to manipulate and replace the defective gene into two animal models of DMD with a smaller but functional dystrophin gene (micro-dystrophin). The microdystrophin gene will be transferred into muscle by adeno-associated virus (AAV). This virus does not cause any known human disease, making it appropriate for safe use in a clinical trial. The premise for this gene therapy proposal is that clinically meaningful results can be achieved by vascular delivery in patients with Duchenne muscular dystrophy (DMD). Furthermore, we think it is likely that FDA will move in a stepwise fashion toward vascular delivery whereby regional vascular delivery will precede systemic vascular delivery for clinical trials. This proposal revolves around this central point and gene delivery to the legs through the femoral artery is the starting point. The first goal for this project (Aim 1) is to define the best adeno-associated virus (AAV) serotype to cross the vascular barrier when delivered through the femoral artery of the mdx mouse. We will first define the best AAV serotype that crosses the vascular barrier carrying micro-dystrophin driven by a truncated muscle creatine kinase (MCK) promoter delivered through the femoral artery of the mdx mouse. The dog environment for gene transfer more closely matches clinical challenges because of size and immunogenicity related to outbreeding. Success in the dog will establish dosing and delivery parameters more closely simulating clinical trials and define guidelines for immunosuppression. Next, we will define the immunosuppression regimen required in the dog model using wild-type dogs to develop assays, and then the GRMD model to define the immunosuppression reagent ideal for expression of micro-dystrophin (Aim 2). Finally, the optimal AAV serotype carrying micro-dystrophin in the mouse will be perfused into the femoral artery of the golden retriever muscular dystrophy (GRMD) dog (Aim 3).
