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Time Post Infection (Weeks)

Fig. 1. Recombinant AAV gene transfer after direct myocardial injection. Photomicrographs (left) show GFP expression in control uninfected and rAV.EGFP-infected myocardium at 1 to 32 weeks postinfection (as marked). Right: Morphometry was used to quantify the extent of gene transfer. Results depict the mean (┬▒SEM) for four animals.

to residual viral proteins expressed in the target cells. AAV, on the other hand, is a nonpathogenic parvovirus that elicits no immune reactions and mediates long-term, stable transgene expression, particularly in skeletal muscle.14 However, the transduction efficiency with AAV is variable among tissue types and can be limited by the anatomical organization of the tissue target and/or the abundance of viral receptors. Furthermore, unlike adenoviral vectors, which provide immediate transient high levels of expression, AAV vectors have a substantial lag time for maximal transgene expression. Both adenoviral and AAV transduction of heart muscle were evaluated via direct cardiac muscle injection, subpericardial infusion, and coronary artery infusion.

GFP reporter gene expression in heart muscle was achieved with both recombinant AAV (rAV.EGFP) and first-generation recombinant adenoviral (rAd.GFP, E1/E3 deleted) vectors. With AAV, transgene expression in rat heart increased over time. GFP expression can be detected 1 week postinfection with AAV, peaks by 3-5 weeks postinfection, and reaches 30% of the cardiomyocytes in the area of the intramyocardial injection (Fig. 1). Hearts infected with the rAV.EGFP vector exhibited no evidence of any inflammatory response in regions associated with transgene expression at any of the time points evaluated (data not shown). A major disadvantage with AAV-mediated gene delivery for IR injury in the heart was the limited local expression at the site of injection. Furthermore, efforts to increase distribution of the virus by either subpericardial or coronary artery infusion were ineffective (data not shown). In contrast to AAV, recombinant adenovirus mediated a greater level of transgene expression, which was evenly distributed after coronary

14 J. Yang, W. Zhou, Y. Zhang, T. Zidon, T. Ritchie, and J. F. Engelhardt, J. Virol. 73,9468 (1999).

Control

3 Days

1 Week

2 Weeks

4 Weeks

6 Weeks

Fig. 2. Recombinant adenoviral infection of the heart by coronary artery infusion. Photomicrographs show GFP fluorescent images in control uninfected and rAd.GFP-infected myocardium 3 days to 6 weeks after infection by coronary artery infusion (as marked).

artery infusion (Fig. 2). When comparing the three modes of delivery (direct injection, subpericardial injection, and coronary artery infusion) the highest efficiency of delivery was achieved with coronary artery infusion. With coronary artery infusion of recombinant adenovirus (rAd), GFP reporter expression was predominantly confined to the left ventricular wall, which is the area of ischemia produced by transient coronary artery occlusion in our rat IR model. Thus, transgene expression with rAd includes the majority of the risk area. rAd-mediated transgene expression was achieved as early as 3 days, and approximately 50-80% of the left ventricle was transduced by 1 week after infection. Maximal expression was seen at 1 week with a significant decline by 2-4 weeks. Complete loss of transgene expression was noted by 6 weeks. In contrast to rAAV, large T cell-mediated inflammatory responses were noted with this El-deleted adenovirus vector by 2 weeks postinfection. These cellular inflammatory responses are likely responsible for the decline in transgene expression that occurs 2 weeks after infection.

On the basis of these preliminary experiments, the model system described in this chapter utilizes coronary artery infusion of adenovirus 3 days before the initiation of experimental ischemia-reperfusion to avoid complications with cellular immune reactions. Ultimately, the development of gene therapy approaches for the heart will require the use of "gutted" rAd, in which all the viral genes except the long terminal repeats (LTRs) and if signal packaging sequence are deleted. This approach will overcome most of the disadvantages associated with the cellular immune responses to this vector.

Rat Model of Transient Ischemia-Reperfusion in Heart

The rat model system for ischemia-reperfusion in the heart involves the surgical implantation of "occluder" and "releaser" sutures around the left main coronary artery, using a modified procedure by Himori and Matsuura.15 The sutures exit the body wall and are left accessible after the surgery (Figs. 3 and 4). Infection with viral vectors can be performed during the surgical procedure before placement of the sutures. After 3 days of postoperative recovery, IR is performed by manipulation of the sutures in anesthetized or conscious animals. In this model, 1 hr of ischemia is followed by 5 hr of reperfusion, using alterations in the electrocardiogram (EKG) to confirm ischemia and reperfusion, and the serum level of creatine kinase (CK) to assess IR damage. As shown in Fig. 5, left coronary artery occlusion produces an ST-T wave depression or elevation of more than 0.1 mV 1 min after the initiation of ischemia. This change is maintained during the entire ischemic period and is used to confirm the success of the occlusion technique. The normalization of the S-T segment is observed 1-5 min after release of the occlusion and confirms the success of reperfusion. The serum level of CK (Fig. 6) increases approximately 6- to 7-fold after ischemia-reperfusion injury. Serum CK levels before IR were similar to those of control animals that had not undergone surgery 3 days previously, suggesting that this experimental model does not invoke detectable levels of damage that might otherwise complicate experiments.

On the basis of these results, the protocol described for the IR model system utilizes 1 hr of ischemia and 5 hr of reperfusion. The short, 3-day interval between viral infection and analysis also assures that no cellular inflammatory responses to adenovirus have occurred during the experimental window.

Experimental Methods

Surgical Methods

All animal surgery for the development of this model is performed under a protocol approved by the University of Iowa Animal Care and Use Committee, and conforms to the National Institutes of Health (NIH, Bethesda, MD) guidelines. All surgical procedures described should be performed by aseptic technique with sterilized instruments, materials, and buffers.

Materials

Ketamine (Fort Dodge Animal Health, Fort Dodge, IA) Xylazine (Phoenix Pharmaceutical, St. Joseph, MO) Phosphate-buffered saline (PBS), 1 x Normal saline Small animal surgical board

15 N. Himori and a. Matsuura, Am. J. Physiol. 256, H1719 (1989).

Fig. 4. Placement of the occluder and releaser sutures. The placement of the occluder suture is shown in a dissected rat heart (A) and in a schematic illustration of the heart (B). The left atrium is used as a landmark, because the left main coronary artery runs along its medial side. The occluder suture is paced just at the lower edge of the atrium about 2-3 mm lateral on either side of its medial margin [large arrow in (B), marked by asterisks (**)], and at a depth of about 2 mm. Placement of the occluder suture around the left main coronary artery (LCA) is marked by a dashed line in (C). The drawing in (C) illustrates the knots tied in the occluder and releaser sutures during surgery. The occluder suture runs just under the LCA (marked by an arrow), as indicated by a dashed line. LA, Left atrium; LV, left ventricle; PA, pulmonary artery; RA, right atrium; RV, right ventricle; O, occluder suture; R, releaser suture.

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