Pigs (n = 14) were randomly divided into three groups receiving 1 × 1013 vg of either AAV2-VEGF-B186 (n = 7), or AAV2-GFP (n = 7). Pigs were followed up until 1 month. One AAV2-VEGF-B186 pig did not reach this time point since it died prematurely. Another group of animals (n = 7) treated with 1 × 1013 vg of AAV2-VEGF-B186 (n = 3) or AAV2-GFP (n = 4) was followed up until 6-month time point. The study protocol is detailed in Fig. 1. All experiments were performed using female domestic pigs and were approved by the Animal Experiment Board in Finland. The animals were 3 months old and weighed ~30 kg at the beginning of the experiments. Sample size was determined by using resource equation method. The animals were randomized into the groups before the gene transfer and the investigators remained blinded until the data was analyzed.
Thirteen animals were followed up until 1-month time point, and seven animals until 6-month time point. PET imaging was performed one day prior to the gene transfer and sacrification. Angiographic measurements and blood sampling were performed at the time of angiographic procedures and sacrification.
AAV2 vectors
AAV2 vectors were prepared by National Virus Vector Laboratory and Kuopio Center for Gene and Cell Therapy. Briefly, the production of the vectors was based on HEK293 cell transfection using pAAV2 vector plasmids and pDG2 helper plasmid (Plasmid Factory) complexed with polyethyleneimine (PEIpro, Polyplus transfection). Affinity chromatography was used for AAV2 vector purification [21]. AAV2 preparations were tested for sterility, mycoplasma, infectivity, and functionality.
Bottleneck stent model
To induce chronic myocardial ischemia, a bottleneck stent was placed in the proximal part of the anterior descending branch of the left coronary artery 14 days before the gene transfer, as described in our previous studies [22].
Medication
Animals received daily 200 mg of amiodarone and 2.5 mg of bisoprolol perorally to prevent fatal ventricular arrhythmias. The medication started 1 week before the ischemia operation and continued daily until the end of the follow-up. Loading doses of clopidogrel (300 mg, peroral) and acetylsalicylic acid (300 mg, peroral) were administered 1 day before the ischemia operation to prevent in-stent thrombosis after the bottleneck stent placement. Also, 30 mg enoxaparin was administered intravenously at the beginning of the ischemia operations and subcutaneously after the operation. To prevent ventricular arrhythmias during the ischemia operation, 100 mg of lidocaine and 2.5 mL MgSO4 were administered intravenously. As infection prophylaxis, cefuroxime (500 mg, intramuscular) was administered at the beginning of each operation.
Before the operations, pigs were sedated with an intramuscular injection of 1.5 mL atropine and 6 mL of azaperone. After the initial sedation, animals were put under general propofol and fentanyl anesthesia with doses of 15 mg/kg/h and 10 µg/kg/h.
To reduce inflammatory responses to the vector, a daily peroral 40 mg dose of prednisolone was given to all animals for 3 weeks, beginning 3 days before the gene transfer procedure. The treatment was then continued with 20 mg prednisolone daily. Simultaneously, 40 mg of peroral pantoprazole was administered daily to prevent possible ulcus formation caused by the high prednisolone dose.
Cardiac output measurement
Cardiac output (l/min) was measured by left ventricular cine imaging using fluoroscopic imaging at rest and under dobutamine-induced stress at increasing infusion rates. Upon reaching the target heart rate of 160 bpm, the infusion rate was kept constant during fluoroscopic imaging. The cardiac output was calculated by using the software of the angiographic station (GE Innova 3100IQ Pro, NY, USA) and Simpson’s rule. Cardiac output was then normalized to the weight of the animal at the moment of imaging.
Gene transfer
For the gene transfer procedures, an intramyocardial injection catheter, MyoStar® (Biosense Webster, a Johnson & Johnson company, Diamond Bar, CA, USA), was introduced to the left ventricle via a femoral sheath. Under fluoroscopic guidance (GE Innova 3100IQ Pro, NY, USA) and using the NOGA® mapping system (Biologics Delivery Systems, a Johnson & Johnson company, Irwindale, CA, USA), an electroanatomical map of the left ventricle was acquired. Using this map as a guide, 1 × 1013 vg divided into ten injections (300 μl each) were injected into the hypokinetic but still viable areas of the left ventricle. For viability, a unipolar voltage over 5 mV was used as a criterion. For hypokinesia, a local linear shortening (LLS) at least below 12% but preferably below 6% was selected [23]. The injection needle length was set for 3 mm. An injection duration was 30 s, and the injection needle was kept inside the myocardium for an additional five seconds before retraction to prevent backflow into the ventricle.
Sacrification
Animals were sacrificed with intravenous KCl injection under general anesthesia. The heart was perfused with PBS (Dulbecco’s Phosphate Buffered Saline). Tissue samples were collected from the gene transfer site in the heart, and safety tissue samples were collected from lung, liver, spleen, kidney, ovary, brain, retina, and both proximal (thoracic cavity) and distal (femoral plexus) lymph nodes. Samples were fixed in 4% paraformaldehyde (PFA, pH 7.2) for 48 h at 4 °C and then placed in 15% sucrose for at least 48 h before embedding.
Capillary analysis
Mean capillary area (%) was measured from CD31-immunostained (1:100, AF806; R&D) sections of the pig myocardium [9]. All measurements were done with Fiji software in a blinded manner from 25 randomly selected fields at ×200 magnification from five sections of each pig.
Transgene mRNA expression and AAV2-vector biodistribution
To evaluate transgene mRNA expression in the myocardium, three samples from the gene transfer area from four VEGF-B186 and four GFP animals were analyzed, in addition to control samples from the posterior wall of the left ventricle. Half of the analyzed animals from both groups were sacrificed at a 1-month time point and the rest at a 6-month time point. Also, transgene mRNA was quantified from the extracardiac samples of the four animals from the VEGF-B186 group. VEGF-B186 group was chosen since the higher mRNA expressions in the heart.
Tissue RNA was extracted using TRI Reagent® (Life Technologies) and treated with DNAse (DNA-Free™, Life Technologies), according to the manufacturer’s instructions. cDNA was synthesized from 1 µg of total RNA using RevertAid Reverse Transcriptase (Thermo Scientific) and Random Hexamer Primer (Thermo Scientific), and the levels of VEGF-B186 and GFP cDNA were measured by qPCR using VEGF-B186 and GFP specific Taqman-based assays (Integrated DNA Technologies). The expression of VEGF-B186 and GFP in the samples was normalized to the expression of the housekeeping gene HPRT (Applied Biosystems; ss03388274 m1 HPRT1). Samples were considered negative for transgene mRNA expression if no amplification was seen after 40 cycles.
To study the biodistribution of the AAV2 vector, we analyzed the vector genome copy numbers from different tissues. The tissue DNA was extracted using NucleoSpin® DNA RapidLyse kit (Machery-Nagel), according to the manufacturer’s instructions. The total amount of AAV2 vector was quantitated using AAV2 ITR specific PrimeTime® qPCR assay (Integrated DNA Technologies) and TaqMan Fast Advanced Master Mix (Applied Biosystems) and measured with StepOnePlus™ Real-Time PCR instrument (Applied Biosystems). Samples were considered negative for AAV2 ITR if no amplification was seen after 40 cycles.
Radiowater perfusion imaging
Rest and stress [15O]H2O PET/CT scans were performed using a Siemens Biograph mCT scanner (Siemens Healthcare, Erlangen, Germany). Computed tomography (CT) scans were performed at rest, and stress imaging and CT information were used for attenuation correction. An on-site cyclotron (PETtrace 860, GE Healthcare, UK) and radiowater generator (Hidex Oy, Finland) produced [15O]H2O bolus. Rest and stress imaging was performed using an 800 MBq [15O]H2O bolus. The dynamic acquisition included frames of 14 × 5, 3 × 10, 3 × 20, and 4 × 30 s (total duration 280 s). After suitable decay of 12 min, stress imaging was performed with a further 800 MBq [15O]H2O bolus. The dynamic acquisition was performed during adenosine-induced hyperemia. Images were reconstructed on a 128 × 128 matrix using the ordered subsets expectation maximization (OSEM) iterative algorithm (2 iterations, 21 subsets, zoom 2, Gaussian 6 mm post-filter).
Myocardial perfusion reserve analysis
Regional myocardial perfusion (mL/g/min) was measured using Carimas 2 software (Turku PET Center, Turku, Finland; http://www.turkupetcentre.fi/carimas). Gene transfer area was selected as a region of interest (ROI) by comparing the PET image to the NOGA map from the ischemia operation [24]. The blood perfusion of the gene transfer area was normalized to the area of maximal perfusion of each heart both at rest and at stress. Myocardial perfusion reserve (MPR) was calculated as the ratio of the perfusion at stress to rest.
[18F]FDG-PET imaging
The pigs received intravenous [18F]FDG injections of 363 ± 15 MBq. To induce euglycemic hyperinsulinemic clamp, intravenous boluses of insulin (10 IU) and glucose (1 g/kg) were administered to the animals. Imaging was performed 60 min after [18F]FDG injection with the Siemens Biograph mCT PET/CT scanner (Siemens Healthcare, Erlangen, Germany). CT scan was used for attenuation correction. PET acquisition was performed in one bed position using an acquisition time of 10 min. The images were reconstructed with ordered subset expectation maximization (OSEM) algorithm using 2 iterations and 21 subsets (matrix size was 256 × 256, Gaussian 3 mm post-filter).
Quantification of the [18F]FDG uptake in the myocardium was performed as previously described [25]. Briefly, the myocardial contours from the [15O]water images were copied to the co-registered [18F]FDG images. The polar maps of [18F]FDG uptake expressed as standardized uptake value (SUV) in the LV myocardium were generated using matching image orientation and sampling points. ROI defining the ischemic area was copied from the [15O]water polar maps to measure [18F]FDG uptake in this region. The mean SUVs were determined from the gene therapy area and the ischemic area defined as resting myocardial blood flow <67% of the remote area [26]. The apical segment 17 was excluded from the analysis.
Detection of anti-AAV2 IgG antibodies
Serum anti-AAV2 antibodies were quantified by enzyme-linked immunosorbent assay (ELISA). Recombinant AAV2 particles were diluted in PBS (pH 7.4), and 100 μl was added to the wells in a 96-well Nunc MaxiSorp plate (Thermo Fisher Scientific, Waltham, USA) and incubated at 4 °C overnight. PBS without AAV2 was used as a no coating control. On the following day, the plate was washed four times with PBS, and 150 μl of blocking solution (5% BSA in PBS) was added to each well and incubated at 37 °C for 2 h. The plate was again washed four times with PBS, and 100 μl of pig serum diluted from 1:100 to 1:24300 was added and incubated at 37 °C for 90 min. After four washes, 100 μl of anti-pig IgG peroxidase antibody (Sigma-Aldrich, St. Louis, USA) was added and incubated at 37 °C for 1 h. After washing, 100 μl of tetramethylbenzidine (TMB) substrate solution (Sigma-Aldrich) was added and incubated for 20 min at room temperature. 100 μl of Stop reagent for TMB substrate (Sigma-Aldrich) was added, and absorbances were read at 450 and 650 nm.
Neutralizing anti-AAV2 antibody assay
On day 1, 96-well plates were seeded with 1.5 × 104 293T cells per well. On day 2, Compound C (Sigma-Aldrich) (ref. [27]) was added to the wells in a final concentration of 5 µM in serum-free DMEM and incubated for 1 h at 37 °C and 5 % CO2. Serum samples were heat-inactivated at 56 °C for 30 min. Serum, subjected to threefold serial dilutions from 1:3 to 1:19683 with fetal bovine serum, was incubated with AAV2 expressing murine secreted alkaline phosphatase (muSEAP) at 37 °C for 1 h; this mixture was then added to a culture well pre-treated with Compound C, and incubated at 37 °C and 5% CO2 for 24 h. Pig serum dilution series without AAV2-muSEAP was used as a negative control as well as DMEM only, and AAV2-muSEAP without the serum was used as a positive control. On day 3, the culture medium was collected, and muSEAP expression was measured with a Phospha-Light chemiluminescent reporter gene assay system (Applied Biosystems, Foster City, USA). The neutralizing titer was reported as the highest serum dilution that inhibited the AAV2-muSEAP transduction by ≥50% compared with the positive control without serum.
Isolation of peripheral blood mononuclear cells
To investigate a possible T-cell response towards AAV2, four additional animals received an intramyocardial AAV2 gene transfer, and fresh blood samples were collected before and 1 month after the gene transfer. Peripheral blood mononuclear cells (PBMC) were isolated from blood samples by using Ficoll-Paque PLUS (Sigma-Aldrich) and SepMate tubes (STEMCELL, Vancouver, Canada) by the instructions of the manufacturer. Isolated PBMC were resuspended in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine.
IFNgamma ELISpot assay
The number of AAV2-specific interferon-gamma-producing PBMC was determined using Porcine IFNgamma ELISpot kit (R&D). Briefly, 200,000 fresh isolated mononuclear cells were seeded per well and incubated with AAV2-mock (1.05 × 1011 vg/ml or 0.50 × 1011 vg/ml) at 37 °C and 5% CO2 for 48 h. Unstimulated PBMC were used as a negative control as well as cell cultures with media only. PBMC stimulated with phytohemagglutinin (PHA) were used as a positive control. Spots were counted manually by an optical microscope.
Statistical analysis
Unpaired t test was used for statistical analysis of capillary analysis data, [15O]H2O-PET, and [18F]FDG-PET data. The cardiac output data were analyzed by using ANOVA following the Bonferroni post-test.
