International Journal of Cardiovascular ResearchISSN: 2324-8602

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Research Article, Int J Cardiovasc Res Vol: 2 Issue: 6

Intramyocardial Adenovirus-Mediated VEGF-DΔNΔC Gene Transfer in Patients with No-Option Coronary Artery Disease: Interim Safety Analysis of the Kuopio Angiogenesis Trial 301

Kirsi Muona1, Marja Hedman1, Antti Kivelä1,2, Antti Hedman1, Iiro Hassinen1, Juha Hartikainen1 and Seppo Ylä-Herttuala2,3,4*
1Heart Center, Kuopio University Hospital, Kuopio, Finland
2A.I. Virtanen Institute, University of Eastern Finland, Kuopio, Finland
3Gene Therapy Unit, Kuopio University Hospital, Kuopio, Finland
4Research Unit, Kuopio University Hospital, Kuopio, Finland
Corresponding author : SeppoYlä-Herttuala
A.I.Virtanen Institute, University of Eastern Finland, P.O.BOX 1627, Fi-70211 Kuopio, Finland
Tel: +358 40 355 2075
E-mail: [email protected]
Received: November 06, 2013 Accepted: December 20, 2013 Published: December 25, 2013
Citation: Muona K, Hedman M, Kivelä A, Hedman A, Hassinen I, et al., (2013) Intramyocardial Adenovirus- Mediated VEGF-D?N?C Gene Transfer in Patients with No-Option Coronary Artery Disease: Interim Safety Analysis of the Kuopio Angiogenesis Trial 301. Int J Cardiovasc Res 2:6. doi:10.4172/2324-8602.1000150

Abstract

Intramyocardial Adenovirus-Mediated VEGF-DΔNΔC Gene Transfer in Patients with No-Option Coronary Artery Disease: Interim Safety Analysis

VEGF- mediated gene therapy is a potential new treatment for patients with advanced cardiovascular diseases. However, previous clinical trials have not been able to convincingly demonstrate the efficiency of VEGF gene therapy in humans. Low gene transfer efficiency and insufficient gene expression time could be major contributing factors. To improve these shortcomings we have started a new intramyocardial gene therapy trial with adenoviral (Adv)VEGF-DΔNΔC.

Keywords: VEGF; Gene therapy; Safety; Coronary artery disease; Transseptal puncture

Keywords

VEGF; Gene therapy; Safety; Coronary artery disease; Transseptal puncture

Abbreviations

ACS: Acute coronary syndrome; Adv: Adenovirus; ALP: Alkaline Phosphatase; ALT: Alanine aminotransferase; BET: Bicycle exercise test; CAD: Coronary artery disease; CCU: Cardiac care unit; CMV: Cytomegalovirus; CRP: C-reactive protein; ECG: Electrocardiogram; LDH: Lactate dehydrogynase; MI: Myocardial infarction; PAD: Peripheral artery disease; PSA: Prostate specific antigen; TTE: Transthoracal echocardiography; SAE: Serious adverse event; VEGF: Vascular endothelial growth factor

Introduction

Vascular endothelial growth factor (VEGF)- mediated gene therapy is a new potential option for patients with severe coronary artery disease (CAD) or peripheral artery disease (PAD) that are not suitable for revascularization and remain symptomatic despite optimal medical therapy [1,2]. It is estimated that the number of these patients is constantlyincreasing [3] and therefore new forms of treatment are urgently needed. VEGF-A is the most widely studied member of the VEGF family. However, despite promising initial results in animal and human studies, the efficiency of VEGF-A has not been proven in human trials [4-8]. Major reasons for this might bea low gene transfer efficiency and a short expression time of the transgene. A new member of the VEGF family, the mature short form of VEGF-D is not hypoxia induced like VEGF-A, but it is proteolytically activated and binds mostly to VEGF receptor 2 causing a milder, but longer lasting angiogenic effect [9,10]. A longer expression time seems to be required in order to induce fully stabilized and functioning vessel structures [11].
In preclinical studies AdvVEGF-DΔNΔC has induced angiogenesis and increased myocardial perfusion in porcine heart and induced angiogenesis in a rabbit hindlimbmodel [12,13]. In a study by Byzova et al. VEGF-D gene transfer with adenoviral vector increased angiogenesis and lymphangiogenesis in rat cremastermuscle [14]. Based on preclinical studies, AdvVEGF-DΔNΔC gene therapy is a potential new pro-angiogenic treatment, but until now it has not been used in clinical trials.
A number of theoretical safety issues are associated with VEGF- based gene therapy, such as acceleration of atherosclerosis and tumour growth, immunological responses, tissue oedema and neovascularization of retina. Tissue oedema and transient increase in vessel permeability have been reported to be fairly common side effects of VEGF-A gene therapy especially in the treatment of peripheral artery disease [15-17]. So far, short-term and long-term follow-up of VEGF-A gene therapy patients have not raised any major safety concerns [5,6,18,19]. Short-term adverse events have mainly been restricted to mild transient immune responses, oedema and transient elevation of inflammatory parameters [5,15,17]. In our recent studies VEGF-A mediated gene therapy did neither increase mortality nor the risk of malignancies, diabetes or diabetic retinopathy in CAD and PAD patients in two separate 8- year and 10-year safety follow-up studies [18,19].
Kuopio Angiogenesis Trial 301 (KAT301) is a randomized, single-blinded, placebo-controlled Phase I/II clinical study aiming to assess the periprocedural and short-term safety, feasibility, as well as efficacy of the Intramyocardial AdvVEGF-DΔNΔC gene therapy in no-option CAD patients. In this report we provide an interim safety analysis of the on-going study as required by Regulatory Authorities.

Materials and Methods

Patient selection and screening
Fifteen patients (Table 1) with CAD not eligible for coronary angioplasty or bypass operation (“no-option patients”) due to diffuse coronary stenosis, small coronary vessels, repeated revascularization or too high risk for the operation meeting the study criteria (Table 2) were enrolled in the study.
Table 1: Baseline characteristics of the patients.
Table 2: Inclusion and exclusion criteria. CCS=Canadian Cardiovascular Society; LV=left ventricular; TTE=transthoracal echocardiography; DM=diabetes mellitus; EF=ejection fraction; CO=Cardiac output; BMI=Body mass index.
Patients with no evident contraindications were invited for a screening visit. Their suitability for the study was evaluated by clinical examination, laboratory tests, electrocardiogram (ECG), transthoracal echocardiography (TTE) bicycle exercise test (BET) and chest x-ray. Patients with type 2 diabetes mellitus underwent ophthalmological examination to exclude pre-existing retinopathy. Concomitant diseases and medication were reported. An informed consent was signed by each patient. The study was approved by the local Ethics Committee and the Finnish Medicines Agency.
Randomization
The patients were randomized 4:1 to the treatment and control groups. The randomization codes were prepared prior to the start of the study. The randomization codes were given in successive order as each patient was recruited in the study. The randomization codes were covered so that only the operator performing the injections knew the code. The patients and other personnel were blinded for the study drug.
Vector
First generation, replication-deficient E1-E3- deleted adenoviruses (serotype 5) were produced in HEK 293 cells by using GMP production methods (reference to product masterfile (PMFVD- 08-001)). Adenoviral vectors were manufactured and provided by Ark Therapeutics Oy and the details of the methods are described elsewhere [20].
Gene
Mature form of human AdvVEGF-DΔNΔC under a cytomegalovirus (CMV) promoter was injected into the target area of the mapped, ischemic myocardium. This VEGF-DΔNΔCcDNA lacks N-and C-terminal propeptides and contains only a central VEGF-D homology domain. An escalating dose of AdvVEGF-DΔNΔC was used for the first fifteen patients. The first four of five patients received the lowest titer of 1x109vpu or placebo, the next five patients received a titer of 1x1010 vpu or placebo and the last five patients 1x1011vpu or placebo. The best tolerated dose will be used for the following study patients.
Transseptal puncture and endocardial electromechanical mapping with or without gene injection
Endocardial gene transfer was performed in the cardiac catheter laboratory under fluoroscopic guidance. A 8.0 F introducer sheath was inserted into the right femoral vein. Transseptal puncture was performed with a transseptal needle 8.5 FrAgilis™NxT steerable Introducer catheter (St Jude Medical™, St.Paul, Minnesota) under fluoroscopic guidance. After the transseptal puncture unfractionated heparin was given to maintain ACT between 300-350 s. An electroanatomical-mapping and injection catheter (NOGA©, Cordis Corp., Johnson & Johnson company, Miami Lakes, Florida) was introduced into the left ventricle via the transseptal catheter. Left ventricular endocardium was mapped spot-by-spot to detect the areas of ischemia and reduced contraction to evaluate the optimal sites fort the gene injections (Figure 1). After the mapping the NOGA© catheter was used to inject the solution of AdvVEGF-DΔNΔC to ten different sites of ischemic myocardium (Figure 2). The depth of the injections was 5-6 mm and the injections were given 5 to 10 mm apart from each other (200μl per injection).
Figure 1: Electroanatomical mapping of the left ventricle in a 69-year old patient. Left panels show unipolar voltage maps and right panels show left ventricular contractility maps (local linear shortening). Injection sites are defined as red dots on the posterolateral wall.
Figure 2: X-ray image of a NOGA mapping and injection catheter (black arrow) introduced via a transseptal guiding catheter (white arrow). * Denotes a temporary pacemaker lead in the right ventricle.
Safety follow-up
Safety protocol: The patients were admitted to the hospital ward one day prior to the procedure. ECG, vital signs and baseline laboratory parameters were evaluated. On the day of the procedure blood gas analysis and vital signs including blood pressure, heart rate and body temperature, were taken at the time of the gene transfer. ECG, vital signs and laboratory assessment were controlled 4 h after the procedure. Patients were monitored at Cardiac Care Unit (CCU) until the first postoperative morning and if no signs of adverse reactions were detected they were transferred to a cardiology bed ward. If no complications occurred, the patients were discharged on the second postoperative day.
Laboratory and ECG assessments were repeated on the days 6 and 14. At 90 days patients were invited for a 3-month follow-up visit. Clinical signs, laboratory parameters and ECG were assessed during the visit. To detect potential pericardial effusion, TTE was performed to all patients. In case of no contraindications, patients also underwent a magnetic resonance imaging (MRI) at rest and during adenosine infusion (Figure 3).
Figure 3: A. 2-D echocardiography of a 74- year-old patient. An apical 3-chamber projection for the evaluation of the left ventricle wall motion and pericardial space. B. An ECG-gated cardiac MRI of the same patient. A long axis 4-chamber view.
All adverse events were reported regardless of their causality to the study drug or the procedure. They were classified as either serious or non-serious based on strictly objective definitions by European Commission Enterprise and Industry Directorate [21]. The data was monitored according to the Good Clinical Practice (GCP) guidelines.
Analysis of the results
The results are presented in a descriptive form and no statistical analysis was performed due to the small number of the patients in the study groups.

Results

Serious adverse events (SAE)
Baseline characteristics of the patients are presented in Table 1. In total, seven SAEs were reported during the 3-month follow-up period (Table 3). One patient in the treatment group died 10 days after the procedure. Autopsy revealed that acute myocardial infarction (MI) was the cause of the death Table 4. In addition, one patient in the control group and one patient in the treatment group experienced acute MI or acute coronary syndrome (ACS). One of these patients had MI two times during the follow-up. Both of the MIs occurred after the discharge.
Table 3: Procedural and acute phase safety.na = not available.
Table 4: Serious adverse events (SAE). *Both MIs occurred in the same patient.
One patient from the control group presented with fever, cough and elevated inflammatory parameters on the day of the gene transfer. Pneumonia was suspected based on symptoms and chest x-rays, but the diagnosis remained uncertain. Antibiotic treatment was started and the symptoms resolved within 5 days. The patient was discharged on the sixth postoperative day.
One control patient had an iatrogenic pericardial puncture during the procedure. The patient was followed at the CCU for 2 days before transfer to the bed ward. TTE was performed on both days and no signs of pericardial tamponation were detected. No specific treatment was needed. The patient underwent successful procedure three weeks later.
Pyelonephritis was diagnosed in one patient in the treatment group. The infection was most likely related to the use of a urinary tract catheter. Symptoms occurred after the discharge and resolved with oral antibiotic treatment. No hospitalization was required.
Arrhythmias and pericardial effusion
None of the patients had serious arrhythmias, such as ventricular or atrial tachycardias or ventricular fibrillation. No sinus arrests or II or III degree AV-blocks were detected during the procedure or hospitalization. During the follow-up there were no changes in rhythm compared to the baseline rhythm.
In TTE performed on the second postoperative day mild pericardial effusion (3-6mm) was detected in two patients in the treatment group. Control TTE was not required for one of the patients and the other one showed no longer signs of effusion at the one-week control TTE. Another patient from the same group was shown to have a mild pericardial effusion at the 3-month follow-up visit. However, this was not seen in the MRI. The size of the effusion was considered minimal and no additional treatment or controls were required.
Laboratory assessment
Results of the laboratory assessment are presented in Table 5. Mild elevation in CRP-level was detected in all patients during hospitalization regardless whether they were treated with gene transfer or not. No such changes were seen in blood leukocyte levels. At 3-month control visit one patient in the treatment group presented with elevated PSA not seen in the baseline visit (3.07-9.17 μg/l). Prostate biopsies were taken, but these showed no signs of malignancy or dysplasia. No elevation in the liver function parameters was found (ALT, ALP, LDH) in any of the patients. Three patients in the treatment group had slightly elevated adenovirus antibody titers at the two weeks’ time point, but this elevation was considered clinically non-significant.
Table 5: Laboratory assessement. *During hospitalization, ** after discharge until day 90. CRP indicates C-reactive protein,proBNP N-terminal pro-brain natriuretic peptide, ALT alanine aminotransaminase, ALP alkaline phosphatase, LDH lactate dehydrogenase, PSA prostate specific antigen and AVAb adenovirus antibody.†Taken on the admission day.†Taken on the screening day.

Discussion

This study is the first to use Intramyocardial AdvVEGF-DΔNΔC gene transfer in a clinical trial. The short form VEGF-DΔNΔC has a longer expression time compared to VEGF-A and therefore has potentially a better efficiency to form new collateral vessels, improve myocardial perfusion and relieve symptoms of CAD [9,13,14]. The primary aim of this interim analysis was to evaluate the perioperative and short-term safety of the AdvVEGF-DΔNΔC gene therapy in patients with severe CAD. The main KAT 301 trial will include further patients and the main interest will be in the efficacy and safety analyses of the treatment.
One of the safety issues related to the VEGF gene therapy is associated to tissue oedema caused by increased vascular permeability. This could be harmful when occurring in myocardium or pericardium. Other short-term safety concerns are mainly related to immunological and inflammatory responses. Also, the gene transfer procedure itself using transseptal puncture and intramyocardial injections could theoretically lead to complications.
Periprocedural safety
The gene transfer procedure itself was feasible and well tolerated. No signs of arrhythmias, chest pain or hypotension were registered during the gene transfer. One perioperative SAE occurred in the form of pericardial puncture which, however, did not lead to clinical consequences. A successful procedure was performed three weeks later.
Oedema
VEGF-A induces increased permeability at the endothelial level. When a vessel is exposed to VEGF-A, the endothelial cell junctions are loosened through a specific signalling pathway. This leads to increased permeability, which enables the sprouting of new vessels from the existing ones and is thus an essential component of angiogenesis [22]. A similar increase after the use of AdvVEGFDΔNΔC is also possible. Therefore, all study patients were followed by TTE during hospitalization and at 3-month visit after the gene delivery procedure for the detection of possible pericardial effusion or structural abnormalities. In addition, cardiac MRI was performed. Non-significant pericardial effusion was detected in three patients in the treatment group. No additional procedures were required and the effusion was spontaneously resolved in all of the patients.
Inflammation and immunological findings
Adenovirus was chosen as a vector for this trial because of its high efficiency. In the previous studies replication-deficient adenoviruses have increased the risk for immune system activation compared to i.e. plasmid vectors. However, the immunological reactions reported have remained mild and transient [5,23].
Slightly elevated CRP levels were detected in all patients shortly after the procedure. This suggests that the elevation is related to the procedure itself rather than the adenoviral vector or the transgene. In addition, leukocyte levels remained normal indicating CRP elevation to be a consequence of tissue manipulation. Adenoviral antibody titers were elevated in three patients at day 14, but this elevation was not considered clinically significant and no other indicators of infection were detected in these patients. One suspected pneumonia case was detected in the control group. The symptoms occurred on the day of the procedure and thus the relationship to the procedure is unlikely. Also, urinary tract infections are common complications after prolonged use of urinary tract catheters and therefore the catheter was the most likely cause of the pyelonephritis.
Other
No clinically significant changes were reported in haemoglobin, leukocyte count, thrombocytes or liver parameters. One patient in the treatment group had a significant elevation in PSA at 3 months visit. However, the biopsies showed no signs of malignancy. VEGF-A has not been shown to increase the incidence of cancer or to accelerate tumour growth in adenovirus-based clinical gene therapy trials [18,19], although it is known to be over expressed in malignant tumours [24]. In this interim study the follow-up time was not long enough to evaluate the long-term risk of malignancies and other slowly progressing diseases.
In this study one death occurred 10 days after the procedure due to myocardial infarction. The patient was a 67 year-old male with diffuse CAD and multiple concomitant diseases. In addition, two patients encountered a myocardial infarction from both groups. All study patients had a severe advanced CAD along with multiple concomitant diseases and therefore acute coronary events were anticipated.
All patients recruited in the first part of the trial were male. Male gender is known to be a risk factor for earlier onset of CAD [25], which might be why male patients meeting all the criteria were easier to find for the trial. Many of the patients had had CAD diagnosis for several decades and the disease had slowly progressed to advanced stage. These no-option patients are also sometimes considered as the “survivors” of CAD. However, population size in this study is not big enough to make definite conclusions about the gender distribution of the end stage CAD patients.
Dose
The patients received an escalating dose of gene and based on the safety and tolerability analysis of the first 15 patients, the highest 1x1011vpvirus dose would be the recommended dose for the remaining study patients. It has been suggested that improvement in myocardial perfusion is dose dependent [26].
In conclusion, this interim safety analysis suggests that AdvVEGF-DΔNΔC mediated intramyocardial gene therapy by using the transseptal puncture manoeuvre is feasible and well tolerated. However, the final results of the KAT301 will eventually give a more thorough understanding of the safety aspects of the AdvVEGF-DΔNΔC gene therapy as well as the efficacy of the treatment.

Acknowledgments

This study was supported by grants from Finnish Academy, European Research Council (FutureGenes250050), Finnish Foundation for Cardiovascular Research, Kuopio University Hospital (EVO grant), and UEF Spearhead Program. We also thank Ark Therapeutics Oy for providing AdvVEGF-DΔNΔC for the study.

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