Name: Large Animal Model for Evaluating the Efficacy of the Gene Therapy in Ischemic Heart (Video) | JoVE Journal
Uploaded: 2021-09-02T14:30:00.000+00:00
Duration: 6 min 7 s
Description: Learn a large animal model for evaluating gene therapy efficacy in ischemic heart disease using transthoracic echocardiography and coronary angiography. This method enables precise preclinical assessment before clinical trials.

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5 minutes

The authors would like to thank Maria Hedman, Tiina Laitinen, Tomi Laitinen, Pekka Poutiainen, Annika Viren, and Severi Sormunen for assistance and permitting 15O-PET imaging at Kuopio University Hospital; and Heikki Karhunen, Minna T&#246;rr&#246;nen, and Riikka Ven&#228;l&#228;inen from National Laboratory Animal Center for their assistance in animal work.
This study is supported by grants from Finnish Academy, ERC, and CardioReGenix EU Horizon 2020 grant.

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
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Administer intramuscular injection of cefuroxime (500 mg) at the beginning of each operation for infection prophylaxis.</li>
	<li>Administer 30 mg of enoxaparin intravenously at the beginning of the ischemia operations and subcutaneously after the operation procedure for thrombosis prevention.</li>
	<li>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 &#181;g/mL at a rate of 10 &#181;g/kg/h. Drug dosages were the same for each pig. Refer to local animal use guidelines for dose administration.</li>
	<li>Anesthetize the animals during all operations. All the operations should be performed in a sterile environment using a sterile technique.</li>
</ol>
2. Transthoracic echocardiography
<ol>
	<li>Perform transthoracic echocardiography before ischemia operation, gene transfer, and euthanasia to evaluate any detectable pericardial fluid and determine the myocardial strain.</li>
	<li>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.</li>
</ol>
3. Endovascular operations under fluoroscopic guidance
<ol>
	<li>Perform left ventricle cine imaging after coronary angiograms before ischemia operation, gene transfer and tissue collection.</li>
	<li>Preparation of the operation
	<ol>
		<li>Prepare for the operations by sedating the pigs with an intramuscular injection of 1.5 mL of atropine and 6 mL of azaperone.</li>
		<li>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 &#181;g/kg/h, respectively. 
		NOTE: The pigs are anesthetized for the entire procedure.</li>
		<li>Support the ventilation by intubation and ventilator and monitor the vital physiological parameters, such as ECG and respiratory parameters.</li>
	</ol>
	</li>
	<li>Introducer sheath placement
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Coronary angiography
	<ol>
		<li>Perform coronary angiography directly before ischemia operation, gene transfer, and tissue collection. The machinery needed for the angiograms is shown in Figure 2.</li>
		<li>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).</li>
	</ol>
	</li>
	<li>Left ventricle cine imaging under rest and dobutamine stress
	<ol>
		<li>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&#160;Single&#160;and&#160;Yes.</li>
		<li>Calculate the ejection fraction by the measurement software of the angiographic workstation. To perform the calculation, select&#160;Ventricular Analysis&#160;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&#39;s method. The ejection fraction measurement is performed during rest and under dobutamine-induced stress.</li>
	</ol>
	</li>
	<li>Stress imaging
	<ol>
		<li>Dose dobutamine intravenously in escalating doses from 10 &#181;g/kg/min to 20 &#181;g/kg/min for the dobutamine-induced stress imaging until the target heart rate of 160 bpm is reached. Then, perform the cine imaging.</li>
	</ol>
	</li>
	<li>Ischemia operation
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Defining the stent size
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Stent placement
	<ol>
		<li>Place a coil to the left coronary artery and glide the bottleneck stent to the LAD, placing it distally to the first diagonal.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
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).
<ol>
	<li>Reference imaging
	<ol>
		<li>Perform computed tomography (CT) scans before rest and stress imaging. Use the CT information for attenuation correction.</li>
	</ol>
	</li>
	<li>15O-labeled radiowater imaging
	<ol>
		<li>Perform rest and stress imaging using an 800 MBq 15H2O bolus.</li>
	</ol>
	</li>
	<li>Stress imaging
	<ol>
		<li>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 &#181;g/kg/min intravenous), as described previously12.</li>
	</ol>
	</li>
</ol>
5. Gene transfer
<ol>
	<li>Electroanatomical mapping
	<ol>
		<li>Proceed to electroanatomical mapping after a coronary angiogram and functional measurements (echocardiography, LV cine imaging).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Finishing the electroanatomical map
	<ol>
		<li>Delete the outlier points to ensure a more reliable electroanatomical map of the left ventricle.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Gene transfer injections
	<ol>
		<li>Introduce an intramyocardial injection catheter to the left ventricle via the femoral sheath under fluoroscopic guidance. Set the injection needle length to 3 mm.</li>
	</ol>
	</li>
	<li>Criteria for intramyocardial injections
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Intramyocardial injections
	<ol>
		<li>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.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>Perfusion fixation of the heart
	<ol>
		<li>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).</li>
	</ol>
	</li>
	<li>Sample collection
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Safety tissue collection
	<ol>
		<li>Harvest samples from remote tissues, such as the lung, liver, kidney, spleen, and ovaries. Take samples into 4% PFA and liquid nitrogen.</li>
	</ol>
	</li>
</ol>
7. Sample storing
<ol>
	<li>Store the samples for staining in 4% PFA for 48 h at 4 &#176;C. 
	NOTE: Replace the PFA daily with a fresh liquid.
	<ol>
		<li>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 &#176;C.</li>
	</ol>
	</li>
</ol>

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<li>Ylä-Herttuala, S., Baker, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiovascular+gene+therapy%3A+Past%2C+present%2C+and+future%5BTitle%5D)+AND+%22Molecular+Therapy%22%5BJournal%5D)">Cardiovascular gene therapy: Past, present, and future.</a> Molecular Therapy. 25 (5), 1095-1106 (2017).</li>
<li>Lähteenvuo, J., Ylä-Herttuala, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advances+and+challenges+in+cardiovascular+gene+therapy%5BTitle%5D)+AND+%22Human+Gene+Therapy%22%5BJournal%5D)">Advances and challenges in cardiovascular gene therapy.</a> Human Gene Therapy. 28 (11), 1024-1032 (2017).</li>
<li>Hammond, H. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intracoronary+gene+transfer+of+adenylyl+cyclase+6+in+patients+with+heart+failure%3A+A+randomized+clinical+trial%5BTitle%5D)+AND+%22JAMA+Cardiology%22%5BJournal%5D)">Intracoronary gene transfer of adenylyl cyclase 6 in patients with heart failure: A randomized clinical trial.</a> JAMA Cardiology. 1 (2), 163-171 (2016).</li>
<li>Hartikainen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adenoviral+intramyocardial+VEGF-DDNDC+gene+transfer+increasesmyocardial+perfusion+reserve+in+refractory+angina+patients%3A+A+phase+I%2FIIa+study+with+1-year+follow-up%5BTitle%5D)+AND+%22European+Heart+Journal%22%5BJournal%5D)">Adenoviral intramyocardial VEGF-DDNDC gene transfer increasesmyocardial perfusion reserve in refractory angina patients: A phase I/IIa study with 1-year follow-up.</a> European Heart Journal. 38 (33), 2547-2555 (2017).</li>
<li>Laakkonen, J. P., Ylä-Herttuala, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recent+advancements+in+cardiovascular+gene+therapy+and+vascular+biology%5BTitle%5D)+AND+%22Human+Gene+Therapy%22%5BJournal%5D)">Recent advancements in cardiovascular gene therapy and vascular biology.</a> Human Gene Therapy. 26 (8), 518-524 (2015).</li>
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The authors declare no conflicts of interest.

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 ...

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 diseases6,7. 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 model9,10,11. 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.

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

large animal model for evaluating the efficacy of the gene therapy in ischemic heart

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

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.

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 ...

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.

Coronary artery disease is one of the significant causes of mortality and morbidity worldwide. Despite the progression of current therapeutics, a ...

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Research

JoVE Journal

Medicine

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

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A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation

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Delivery of Cardioactive Therapeutics in a Porcine Myocardial Infarction Model

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Model of Ischemia and Reperfusion Injury in Rabbits

Learn a large animal model for evaluating gene therapy efficacy in ischemic heart disease using transthoracic echocardiography and coronary angiography. This method enables precise preclinical assessment before clinical trials.

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

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