Genetic and cellular therapy for cardiovascular disease
Donald D. Glower, M.D and Carmelo Milano, M.D.
Our laboratory has been interested in developing novel therapies for cardiovascular disease for the past 6 years. In collaboration with Drs. Robert Lefkowitz, and Walter Koch, the beta-adrenergic signaling pathway has been a target of intense investigation and gene therapy. Initial work in the mid 1990s elucidated changes in intracellular signaling associated with cardiomyopathy. Beta-adrenergic receptor density is decreased in the myocardium of cardiomyopathic animals, and existing beta-receptors are desensitized by the phosphorylation of the intracellular portion of the beta-receptor by increased levels of beta-adrenergic receptor kinase. Initial work in our laboratory involved transgenic mice constructed to overexpress the beta-receptor, which were crossbred with cardiomyopathic mice. The resulting strains showed improved cardiac contractility and survival. Since then, various targets of the intracellular signaling system have been targeted for genetic therapy. In particular, an peptide inhibitor of the beta-adrenergic receptor kinase (BARKct), developed by Walter Koch, Ph.D., has been extensively utilized to rescue animals from heart failure. We showed in a rabbit model of ischemic heart failure that delivery of the BARKct transgene via an adenoviral vector could improve LV contractility by improving intracellular adenylyl cyclase activity. Our method of transgene delivery has evolved over the past 5 years. Initially, an aortic cross clamp followed by intraventricular injection of adenovirus yielded adequate global transgene expression. Over the two years, we have had success at selective left circumflex or right coronary artery catheterization and direct intracoronary adenoviral delivery. Current projects include the use of BARKct gene therapy to prevent RV dysfunction following PA banding, and the use of newer vectors that produce longer-term transgene expression. In the lab of Dr. Carmelo Milano, we are investigating the ability to perform genetic therapy at the time of cardiopulmonary bypass. The advantages of this approach include selective myocardial expression of a transgene, and controlled conditions, which optimize gene transfer. Currently we are utilizing an adenoviral vector administered to the arrested heart via an anterograde cardioplegia catheter to attain transgene expression. Current projects include over-expression of the beta-adrenergic receptor in normal hearts at the time of CPB, time course experiments to ascertain the duration of transgene expression utilizing this method of gene therapy, and physiologic assessment to determine the hemodynamic effects of beta-adrenergic receptor overexpression in porcine myocardium following CPB. Collaboration with Doris Taylor, Ph.D. has been ongoing for several years. We have been interested in the use of autologous skeletal myoblasts for myocardial regeneration following myocardial infarction. Biopsies taken from the soleus muscle provide a source of myoblasts (myocyte precursor cells), which are then grown in culture until adequate cells are obtained. These cells are then injected into the area of infarction, and eventually form a discrete island cells within the scar. Work in rabbits showed that indices of ventricular function (both systolic and diastolic) improve following autologous cell transplantation. Our choice of autologous cells (as opposed to embryonic stem cells, adult or fetal cardiocytes) is multifold. Immunosuppression is not required for autologous transplantation, in contrast to the other methods. Myoblasts, similar to skeletal muscle, are fairly resistant to ischemic injury, and are therefore stand some chance of survival when injected into a scar. Finally, myoblasts are not fully differentiated, leaving open the possibility of differentiation into cardiac-like myocytes. Interestingly, it appears that the transplanted cells show some features of cardiac myocytes, such as the presence of intercalating disks. Current projects include electrical mapping of the transplanted cells, use of genetically engineered myoblasts to improve graft survival, and detailed characterization of the cell phenotype. We are also investigating cell transplantation in larger animal models.
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