[28 Model System for Developing Gene Therapy Approaches for Myocardial Ischemia Reperfusion Injury

By Jusan Yang, Teresa C. Ritchie,* and John F. Engelhardt Introduction

Ischemic heart diseases arise from a number of clinical conditions, including transient coronary artery occlusion, thrombotic stroke, cardiac surgery, and heart transplantation. Cardiac muscle damage from these episodes is attributable to a period of ischemia followed by reperfusion with oxygenated blood.1 Reactive oxygen species (ROS) formed during reperfusion may cause direct tissue damage and/or may act as second messengers in redox-sensitive signal transduction pathways. Direct tissue damage caused by ROS is thought to be an important component of immediate necrosis in the ischemic zone, whereas ROS-activated signal transduction pathways are thought to initiate a cascade of events leading to apoptosis of cardiac myocytes.2-4 Although endogenous free radical-scavenging enzymes are capable of clearing damaging ROS generated during normal cellular metabolism, abnormal ischemia-reperfusion episodes can lead to an overwhelming production of ROS that exceeds cellular metabolic resources to clear these toxic compounds. This is reflected by a variety of pathologic features such as myocardial stunning, reperfusion arrhythmias, impaired reflow, and calcium overload.1'5

Potentially, therapeutic interventions to block ROS-sensitive signal transduction pathways can prevent a major component of ischemia-reperfusion (IR) damage. Current treatments for myocardial IR injury include the administration of exogenous antioxidant reagents, such as nonenzymatic (e.g., vitamins A, E, and C) and enzymatic ROS scavengers [e.g., superoxide dismutase (SOD) and cata-lase]. Although protective effects have been observed with these pharmaceutical reagents, the results have been highly variable. For example, both positive and negative effects were observed with enzymatic ROS scavengers, most likely due to limited uptake of exogenous enzymes at the relevant cellular targets or

* Deceased.

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dose-dependent toxicity.5"11 An alternative method for altering the cellular redox environment and protecting the myocardium from IR damage is the direct delivery of transgenes expressing redox-scavenging enzymes.12 The development of gene therapy techniques to deliver such genes to heart muscle will help refine therapeutic approaches by allowing for direct modulation of pathophysiologically relevant ROS pathways at appropriate subcellular sites.

Unique features of the heart render it an organ presenting both opportunity and challenge for the application of gene therapy strategies. Historically, gene transfer to cardiomyocytes has been largely unsuccessful, because systemic administration of viral vectors into the circulation primarily results in their delivery to the liver and lung.13 This chapter discusses technical aspects of our efforts to determine the most effective mode of delivery of viral vectors to cardiomyocytes and the application of these techniques in gene therapy for ischemia-reperfusion injury in the heart. Methods are also presented for validating the rat IR model, and for assessing the efficacy of transgene expression in reducing the size of an IR-induced infarct.

Choice of Gene Transfer Vector and Mode of Delivery

In the development of strategies for gene delivery to cardiac muscle, several issues must be considered. First is the choice of an appropriate viral vector system for this organ. Potential barriers for applying gene therapy techniques include the efficiency of gene transfer, the duration of transgene expression, and organ inflammation that may follow infection with viral gene therapy vectors. Another issue is the mode of delivery necessary to achieve widespread transgene expression in heart muscle, but that is preferably restricted to this organ. In preliminary stages of the development of this model system, two recombinant viral vectors expressing green fluorescent protein (GFP) reporter genes, adenovirus and adeno-associated virus (AAV), were evaluated. Both vectors have been widely used in gene transfer applications and possess different advantages for specific applications. Adenovirus exhibits a wide tropism for many cells and tissues, and achieves high-level, although short-term, transgene expression. A disadvantage with firstgeneration El-deleted adenoviral vectors includes cellular inflammatory responses

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13 M. Y. Alexander, K. A. Webster, P. H. McDonald, and H. M. Prentice, Clin. Exp. Pharmacol. Physiol. 26,661 (1999).

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