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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 be used to perform adenoviral or AAV-mediated gene therapy injections. The time of sample collection has to be adjusted to the transgene expression peak, which depends on the viral vector used. For example, when performing adenoviral gene transfers, the time of the sample collection is set to 6 days after the gene transfer.

1. Medication

1. Administer a daily dose of 200 mg of amiodarone and 2.5 mg of bisoprolol to prevent fatal ventricular arrhythmias. The medication begins 1 week before the ischemia operation and is continued daily until the follow-up.
2. In addition, administer peroral doses of clopidogrel (300 mg) and acetylsalicylic acid (300 mg) to the animals 1 day before the ischemia operation to prevent acute in-stent thrombosis after the stent placement.
3. Administer 100 mg of lidocaine and 2.5 mL of (246 mg/mL) MgSO₄ intravenously to the animals at the beginning of the ischemia operation to prevent ventricular arrhythmias.
4. Administer intramuscular injection of cefuroxime (500 mg) at the beginning of each operation for infection prophylaxis.
5. Administer 30 mg of enoxaparin intravenously at the beginning of the ischemia operations and subcutaneously after the operation procedure for thrombosis prevention.
6. For anesthesia and analgesia, administer 1.5 mL of atropin, 6 mL of azaperone (40 mg/mL), propofol 20 mg/mL, at a rate of 15 mg/kg/h, and fentanyl 50 µg/mL at a rate of 10 µg/kg/h. Drug dosages were the same for each pig. Refer to local animal use guidelines for dose administration.
7. Anesthetize the animals during all operations. All the operations should be performed in a sterile environment using a sterile technique.

2. Transthoracic echocardiography

1. Perform transthoracic echocardiography before ischemia operation, gene transfer, and euthanasia to evaluate any detectable pericardial fluid and determine the myocardial strain.
2. Place the transducer in the third or fourth intercostal space under the armpit of the pig to access parasternal short-axis views at the mitral valve level, papillary muscle, and apical levels (Video 1). The marker of the transducer should point to the sternum of the pig. To save a clip, press Acquire.

3. Endovascular operations under fluoroscopic guidance

1. Perform left ventricle cine imaging after coronary angiograms before ischemia operation, gene transfer and tissue collection.
2. Preparation of the operation
   1. Prepare for the operations by sedating the pigs with an intramuscular injection of 1.5 mL of atropine and 6 mL of azaperone.
   2. After the sedation, induce general propofol and fentanyl anesthesia for the angiographic procedures to the pigs with doses of 15 mg/kg/h and 10 µg/kg/h, respectively.
      NOTE: The pigs are anesthetized for the entire procedure.
   3. Support the ventilation by intubation and ventilator and monitor the vital physiological parameters, such as ECG and respiratory parameters.
3. Introducer sheath placement
   1. Place an introducer sheath into the right femoral artery for all the operations as standard practice in cardiology. Use ultrasound to track the femoral artery and pierce it with an entry needle (18 G).
      NOTE: Use an 8F introducer sheath for intramyocardial gene transfers and a 6F sheath for all other operations. Introduce the guidewire of the sheath through the needle to thread the artery and hold the guidewire still while removing the needle.
   2. Insert the introducer sheath along the guidewire, and when placed, remove the guidewire and administer 1.25 mg of sublingual dinitrate to the pig to induce coronary vasodilation.
4. Coronary angiography
   1. Perform coronary angiography directly before ischemia operation, gene transfer, and tissue collection. The machinery needed for the angiograms is shown in Figure 2.
   2. Use a 6F catheter under fluoroscopic guidance with an iodine contrast agent to image the right coronary artery and the left ascending coronary artery (Video 2).
5. Left ventricle cine imaging under rest and dobutamine stress
   1. Administer a 21 mL bolus of iodine contrast agent into the left ventricle via a 5F pigtail catheter using an auto-injector. First, set the bolus duration at 3 s and the total volume for 21 mL. Then, press Single and Yes.
   2. Calculate the ejection fraction by the measurement software of the angiographic workstation. To perform the calculation, select Ventricular Analysis of the image in question. Scroll the image to select a time frame, one in diastole and one in systole. Select a tool to draw ventricular outlines of each time frame.
      NOTE: The software now calculates the ejection fraction and stroke volume by the Simpson's method. The ejection fraction measurement is performed during rest and under dobutamine-induced stress.
6. Stress imaging
   1. Dose dobutamine intravenously in escalating doses from 10 µg/kg/min to 20 µg/kg/min for the dobutamine-induced stress imaging until the target heart rate of 160 bpm is reached. Then, perform the cine imaging.
7. Ischemia operation
   1. Place a bottleneck stent into the left coronary artery (LAD) 14 days before the gene transfer to induce chronic myocardial ischemia. After the bottleneck stent placement, check whether the bottleneck stent is placed correctly, restricting the coronary blood flow.
      NOTE: The bottleneck stent is placed on a dilation catheter and consists of a bare-metal stent covered by a polytetrafluoroethylene tube formed in a bottleneck shape to reduce coronary blood flow⁹.
8. Defining the stent size
   1. Choose the size of the stent, either 3.0/3.5/4.0 x 8 mm, according to the size of the left ascending coronary artery in the angiogram by using the automatic measurement software in the angiographic workstation (Video 3)¹².
9. Stent placement
   1. Place a coil to the left coronary artery and glide the bottleneck stent to the LAD, placing it distally to the first diagonal.
   2. Inflate the stent to nominal pressure in the artery using an in-deflator with a stent-to-lumen ratio of 1.3, anchoring the bottleneck in place. After an additional 15 s, deflate the stent and retract the equipment from the artery.
      NOTE: Confirm the correct placement of the bottleneck stent by angiogram.

4. PET imaging

NOTE: One day before the gene transfer, perform rest and stress ¹⁵O-labeled radiowater PET/CT scans (requires hospital environment and radiological technicians).

1. Reference imaging
   1. Perform computed tomography (CT) scans before rest and stress imaging. Use the CT information for attenuation correction.
2. ¹⁵O-labeled radiowater imaging
   1. Perform rest and stress imaging using an 800 MBq ¹⁵H₂O bolus.
3. Stress imaging
   1. Perform stress imaging with a further 800 MBq ¹⁵O-water bolus after a suitable radioactive decay of 12 min.
      NOTE: Hyperemia is induced by adenosine (200 µg/kg/min intravenous), as described previously¹².

5. Gene transfer

1. Electroanatomical mapping
   1. Proceed to electroanatomical mapping after a coronary angiogram and functional measurements (echocardiography, LV cine imaging).
   2. Introduce a mapping catheter to the left ventricle via femoral sheath in fluoroscopic guidance.
      NOTE: Register about 100-150 points around the left ventricle with the mapping catheter to create the electroanatomical map.
2. Finishing the electroanatomical map
   1. Delete the outlier points to ensure a more reliable electroanatomical map of the left ventricle.
   2. Do this by selecting Clip Planes of the map and delete the points that differ from the points forming the ventricular shape. Next, select Trajectories for the map view and delete the points that have traveled horizontally during point registering.
      NOTE: Make sure the remaining points cover the left ventricle and register more points if needed.
3. Gene transfer injections
   1. Introduce an intramyocardial injection catheter to the left ventricle via the femoral sheath under fluoroscopic guidance. Set the injection needle length to 3 mm.
4. Criteria for intramyocardial injections
   1. Guide the gene transfers by electroanatomical mapping system and target the injections into viable but hypokinetic areas of the left ventricle.
      NOTE: For viability, use a unipolar voltage over 5 mV as a criterion. For hypokinesia, select a local linear shortening (LLS) as low as available, at least below 12% but preferably below 6%¹³.
5. Intramyocardial injections
   1. During 30 s, inject the vector material into the point of selection (step 5.4) and keep the injection needle inside the myocardium for an additional 5 s before retracting to prevent backflow to the left ventricle.

6. Euthanasia and sample collection

NOTE: After the coronary angiogram and ejection fraction measurements described in steps 3.4.1 and 3.5.2, respectively, administer 50 mL of saturated potassium chloride intravenously to the anesthetized pig.

1. Perfusion fixation of the heart
   1. Harvest the heart from the thoracic cavity. Rinse with water. Place an 18 G needle above the aortic valve and attach the needle to a perfusion pump. Perfuse the heart with 750 mL of 1% paraformaldehyde (PFA).
2. Sample collection
   1. Slice the heart into 1 cm thick slices using a sharp kitchen knife. Collect the samples from the gene transfer area into 4% PFA and liquid nitrogen.
      NOTE: To harvest negative controls, collect a control sample from the posterior wall of the left ventricle.
3. Safety tissue collection
   1. Harvest samples from remote tissues, such as the lung, liver, kidney, spleen, and ovaries. Take samples into 4% PFA and liquid nitrogen.

7. Sample storing

1. Store the samples for staining in 4% PFA for 48 h at 4 °C.
   NOTE: Replace the PFA daily with a fresh liquid.
   1. After 48 h, replace the PFA with 15% sucrose in deionized water. Store for at least 24 h before embedding the samples into paraffin blocks. Snap frozen samples are stored at -70 °C.

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 fraction, and myocardial perfusion by ¹⁵H₂O-PET (Figure 3). The tissue samples can be collected directly from the gene transfer area by comparing the heart to the electroanatomical map. The transgene expression and therapeutic angiogenesis (Figure 4) can be evaluated through immunohistological analysis by analyzing the number of positive cells after beta-galactosidase staining and by analyzing the myocardial capillary area after CD31 staining. In addition, the safety of the gene therapy may be assessed by diagnostic imaging (pericardium effusion assessment by echocardiography), immunohistology, and distribution analysis.

Figure 1: Study protocol. Ischemia is induced 14 days before the gene transfer. ¹⁵H₂O-PET imaging is performed 1 day before gene transfer and before euthanasia and sample collection. The time of the sample collection depends on the viral vector and therapeutic gene used. When using adenoviral vectors, the second ¹⁵H₂O-PET is on the 5^th day, and the time of sample collection is on the 6^th day, respectively. Please click here to view a larger version of this figure.

Figure 2: Angiolaboratory setup. The machines needed for coronary interventions: ultrasound machine, ventilator, and angiographic station, from left to right. Please click here to view a larger version of this figure.

Figure 3: Representative image of circumferential strain, and ¹⁵H₂O-PET and an electroanatomical map from the ischemic heart. ¹⁵H₂O-PET: red color represents the area of maximal perfusion, and blue indicates the area of hypoperfusion. Electroanatomical map: Brown dots in the electroanatomical map represent the injection sites. The red color indicates hypokinetic areas of the left ventricle, whereas purple indicates the area of normal contractability. Please click here to view a larger version of this figure.

Figure 4: Representative image of β-galactosidase and PECAM-1 stainings. β-galactosidase is expressed in AdLacZ transduced hearts and can be used to show transgene expression. PECAM-1 staining is used to detect myocardial capillaries and to analyze the capillary area. The lower row represents the area distant from the gene transfer. Scale bar in β-galactosidase stainings: 200 µm. Scale bar in PECAM-1 stainings: 100 µm. Please click here to view a larger version of this figure.

Video 1: Transthoracic echocardiography short-axis view. Please click here to download this Video.

Video 2: Coronary angiogram of LAD before gene transfer. Please click here to download this Video.

Video 3: Left anterior descending artery diameter measurement. Please click here to download this Video.

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 methods and imaging is crucial so that the results are comparable between the timepoints and animals. Second, the samples must be collected from the exact gene transfer area and processed successfully to perform further analyses. Also, this protocol requires profound familiarity with angiographic procedures and different imaging modalities. For example, coronary angiogram and viral injections to a beating heart require extensive training as well as performing correct transthoracic echocardiography. Nevertheless, these imaging modalities measure myocardial function and perfusion to provide essential information for further studies.

The cardiovascular system of a pig resembles the human one due to its anatomical and physiological similarities, and therefore, pigs are often used to model cardiovascular disease mechanics and procedures. However, the follow-up time is restricted to approximately 6 months due to the rapid growth of the animal. After 6 months, the handling of the animal becomes challenging, and the imaging quality deteriorates.

Also, pigs are considerably resistant to atherosclerosis, making diet-induced atherosclerosis complicated to model in pigs¹⁷. However, chronic ischemia models have been developed to mimic the original disease. The significant advantage of the bottleneck stent ischemia model used in this protocol is that the gradual occlusion of the stent represents coronary artery disease better than sudden occlusion. Compared to the ameroid constrictor model, this method is less invasive. Secondly, percutaneous bottleneck stent placement is a quick procedure to perform. Utilizing the electroanatomical mapping system enables targeting the gene transfer to the hibernating myocardium, not to the infarct area, which is a possible outcome when ultrasound guidance is used to target the injections. However, the downside of the electroanatomical mapping is the length of the procedure. Furthermore, since the pig heart is highly sensitive to ventricular arrhythmias, mapping can induce ventricular fibrillation during the mapping procedure. However, these arrhythmias are easily defibrillated.

The endpoints used in this large animal model identify those used in clinical trials, enhancing the transition to the clinics. In addition, these methods are applicable for large animal studies evaluating the efficacy of myocardial gene therapy with different follow-up times and other adjunct endpoints in addition to the ones described in this model. This protocol has been standardized after a vast experience of large animal experiments. In the future, this protocol applies to evaluating the safety and efficacy of myocardial gene therapy before translation to the clinics.

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