Large Animal Model for Evaluating the Efficacy of the Gene Therapy in Ischemic Heart

Summary

Myocardial gene therapy for ischemic heart disease holds great promise for future therapeutics. Here, we introduce a large animal model for evaluating the efficacy of gene therapy in the ischemic heart.

Abstract

Coronary artery disease is one of the significant causes of mortality and morbidity worldwide. Despite the progression of current therapeutics, a considerable proportion of coronary artery disease patients remain symptomatic. Gene therapy-mediated therapeutic angiogenesis offers a novel therapeutic method for improving myocardial perfusion and relieving symptoms. Gene therapy with different angiogenic factors has been studied in few clinical trials. Due to the novelty of the method, the progress of myocardial gene therapy is a continuous path from bench to bedside. Therefore, large animal models are needed for evaluating the safety and efficacy. The more the large animal model identifies the original disease and the endpoints used in clinics, the more predictable outcomes are from clinical trials. Here, we introduce a large animal model for evaluating the efficacy of the gene therapy in the ischemic porcine heart. We use clinically relevant imaging methods such as ultrasound imaging and ¹⁵H₂O-PET. For targeting the gene transfers into the desired area, electroanatomical mapping is used. The aim of this method is: (1) to mimic chronic coronary artery disease, (2) to induce therapeutic angiogenesis at hypoxic areas of the heart, and (3) to evaluate the safety and efficacy of the gene therapy by using relevant endpoints.

Introduction

Coronary artery disease is accountable for the vast proportion of mortality and disease burden worldwide¹. Current treatment strategies are percutaneous interventions, pharmacological treatment, and bypass surgery². However, despite the progression of these current therapeutics, many patients suffer from so-called refractory angina, underlining the unmet need for novel treatment approaches³. Gene therapy-mediated therapeutic angiogenesis could target this patient group.

Myocardial gene therapy is most often delivered by using different viral vectors, most commonly replication-deficient adenovirus⁴. As therapeutic genes, various angiogenic growth factors are used. The most substantially studied angiogenic growth factors are the vascular endothelial growth factors (VEGFs) that mediate their angiogenic signaling through vascular endothelial growth factor receptors (VEGFRs) and their co-receptors⁵. Several clinical trials have proved the benefit and safety of cardiac gene therapy and made this novel treatment method a realistic option for treating ischemic heart diseases^6,7. However, this concept still needs enhancement of the therapeutic genes and viral vectors put to the test in large animal models before entering the clinics. The pig has been frequently used as a laboratory animal since its heart is very similar to the human heart. The size of the cardiovascular system of a pig allows the usage of similar catheter inventions as used in humans. All imaging modalities available for humans can be used in pigs⁸.

There are several large animal models for chronic ischemia. The most commonly used is the ameroid constrictor model^9,10,11. The downside of this method is the invasiveness since thoracotomy is needed to access the coronary vasculature. Previously in our group, a mini-invasive bottleneck stent model for chronic myocardial ischemia has been developed¹². This method is also used in this manuscript to induce myocardial ischemia.

The usability of ultrasound imaging has evolved substantially despite the age of the imaging modality. For example, myocardial strain is still mainly in research usage due to its novelty. Myocardial strain reflects changes in the contractile function of the heart better than the traditional M-mode ejection fraction measurement¹³. Thus, here in the large animal model, myocardial strain measurement is utilized. To evaluate the function of the heart, cardiac output is also measured by cine imaging of the left ventricle during angiography. Cardiac output is measured both at rest and under dobutamine-induced stress to evaluate myocardial function under stress.

In addition to the measurements of the heart function, information on myocardial perfusion is essential in gene therapy studies aiming at therapeutic angiogenesis. In this animal model, animals are imaged with ¹⁵O-labeled radiowater positron emission tomography (¹⁵H₂O-PET) as this is the golden standard for measuring myocardial perfusion. ¹⁵H₂O-PET has been previously validated for measuring perfusion of ischemic porcine heart¹⁴.

Thus, the methods and modalities mentioned above constitute an excellent perspective for evaluating the efficacy of gene therapy in the ischemic heart.

Protocol

The experiments presented here are performed using about 10-week-old female domestic pigs and are approved by the Animal Experiment Board in Finland. Animals weigh 30-40 kg at the beginning of the protocol, allowing the same procedural equipment and imaging modalities as possible for humans. Chronic ischemia is induced 14 days before the gene transfer, and the follow-up time after the gene transfer depends on the viral vector used. The study protocol is shown in Figure 1. This protocol can b…

Representative Results

The success of the ischemia operation can be confirmed with this protocol by coronary angiogram and by determining the hypokinetic area by transthoracic ultrasound (Figure 1) before proceeding to the gene delivery. The state of the coronary occlusion can be evaluated by coronary angiogram, and the electroanatomical mapping ensures the ischemic and hibernating areas. The efficacy of the gene therapy can be analyzed by measuring the circumferential strain, ejection …

Discussion

The timepoints of this protocol may be modified according to the viral vector used. Also, the immunohistological analyses may be selected according to the therapeutic gene. It is also possible to add more timepoints and endpoints to the protocol if needed.

This protocol comprises stages, which are essential to succeed and impossible to correct afterward. First, if one fails to induce appropriate ischemia, the animal must be excluded from further procedures and analyses. Standardization of the …

Materials

1% PFA	VWR	VWRC28794.295	Prepared from paraformaldehyde powder
15 % sucrose	VWR	VWRC27480.294	Prepared from solid sucrose
4% PFA	VWR	VWRC28794.295	Prepared from paraformaldehyde powder
5 F pigtail catheter	Cordis	534-550S
6 F catheter AR2	Cordis	670-112-00
6 F introducer sheath	Cordis	504-606X
8 F introducer sheath	Cordis	504-608X
Acetylsalicylic acid			Varying producer
Amiodarone			Varying producer
Angiographic station	GE Healthcare
Angiolaboratory set	Mölnlycke		designed for the needs of our angiolaboratory, contains sterile drapes, cups and swabs
Bisoprolol			Varying producer
Cefuroxime			Varying producer
Clopidogrel			Varying producer
Coroflex Blue stent	B.Braun Medical	5029012	Catalog number depends on stent size
Crile forceps
Cyclotron	GE Healthcare
Dobutamine			Varying producer
Electroanatomical mapping system	Biologics Delivery Systems, Johnson & Johnson company
Enoxaparin			Varying producer
Fentanyl			Varying producer
Intramyocardial injection catheter	Johnson & Johnson
Iodine contrast agent	Iomeron
Kitchen knife			Varying producer
Lidocaine			Varying producer
Liquid nitrogen			Varying producer
MgSO4			Varying producer
Needle 18 G	Cordis	12-004943
Perfusion pump
PET-CT scanner	Siemens Healthcare
Polytetrafluoroethylene tube
Propofol			Varying producer
Scalpel no 11	VWR	SWAN0503
Sublingual dinitrate	Takeda
Ultrasound machine	Philips