Targeted high-efficiency, homogeneous myocardial gene transfer

Abstract

Myocardial gene therapy continues to show promise as a tool for investigation and treatment of cardiac disease. Progress toward clinical approval has been slowed by limited in vivo delivery methods. We investigated the problem in a porcine model, with an objective of developing a method for high efficiency, homogeneous myocardial gene transfer that could be used in large mammals, and ultimately in humans. Eighty-one piglets underwent coronary catheterization for delivery of viral vectors into the left anterior descending artery and/or the great cardiac vein. The animals were followed for 5 or 28 days, and then transgene efficiency was quantified from histological samples. The baseline protocol included treatment with VEGF, nitroglycerin, and adenosine followed by adenovirus infusion into the LAD. Gene transfer efficiency varied with choice of viral vector, with use of VEGF, adenosine, or nitroglycerin, and with calcium concentration. The best results were obtained by manipulation of physical parameters. Simultaneous infusion of adenovirus through both left anterior descending artery and great cardiac vein resulted in gene transfer to 78+/-6% of myocytes in a larger target area. This method was well tolerated by the animals. We demonstrate targeted, homogeneous, high efficiency gene transfer using a method that should be transferable for eventual human usage.

Figure 1

(A) Fluoroscopic image of catheter position during the gene transfer procedure into the LAD. The arrowhead indicates the balloon catheter position. (B) X-gal stained gross and microscopic sections of Adβgal exposed hearts after gene transfer using the baseline protocol (VEGF 0.5 μg/ml, TNG 250 μg/ml, adenosine 5 mg/ml and 1.0 mM calcium). (C) X-gal stained gross and microscopic sections after exposure to AdNull control using the baseline protocol. Microscopic sections are counterstained with hematoxylin-eosin and magnified 400X.

Figure 2

(A) Effect of cGMP-dependent vascular permeability agents on gene transfer efficacy. (B) Effect of calcium and adenosine. In both graphs, the transgene efficiency was determined as the percentage of X-gal positive cells within the grossly blue area. (n = 6 for baseline, n = 3 for all other groups, * signifies p ≤ 0.01).

Figure 3

Effects of coronary flow characteristics. (A) Effect of coronary flow rate and virus contact time. (B) Effect of utilizing great cardiac vein in virus delivery. (C) Comparison of delivery method to the gross volume of myocardium expressing the transgene. (n = 6 for baseline and LAD/GCV groups, n = 3 for all other groups, * signifies p ≤ 0.01).

Figure 4

(A) Fluoroscopic image of catheter position during the gene transfer procedure with simultaneous infusion through both LAD and GCV. Arrowheads indicate the balloon catheter positions. (B) X-gal stained gross and microscopic sections of Adβgal exposed hearts after gene transfer using the LAD and GCV simultaneous perfusion protocol (Magnification 400X). Slides are stained with X-gal and counterstained with hematoxylin-eosin.

Figure 5

Microscopic section of Ad-null infused heart shows minimal perivascular inflammatory infiltrate without damage to adjacent myocytes.