The European Society of Cardiology
The design and preclinical testing of Ad5FGF-4 to treat chronic myocardial ischaemia
Gene Therapy Department, Berlex Biosciences, Richmond, CA, USA
* G.M. Rubanyi, Gene Therapy Department, Berlex Biosciences, 2600 Hilltop Dr, Richmond CA 98404, USA. Tel.: +1-510-262-7804; fax: +1-510-669-4750
gabor_rubanyi{at}berlex.com
Abstract
Patients with myocardial ischaemia may experience cardiovascular events, sudden death, or constant pain and limited activity despite maximal medical and surgical therapy. Therapeutic angiogenesis is being developed as a novel treatment strategy for patients with myocardial ischaemia. The goal of therapeutic angiogenesis is to stimulate the formation of collateral coronary arteries around obstructed arteries to restore blood flow to ischaemic regions of the heart. It was hypothesized that local production of an angiogenic growth factor would result in increased collateral formation, myocardial perfusion, and wall motion during contraction. A rational development process resulted in the selection of fibroblast growth factor-4 (FGF-4) as the angiogenic growth factor, adenoviral gene transfer as the delivery mechanism and percutaneous intracoronary infusion as the administration technique. Preclinical studies in pigs with myocardial ischaemia caused by ameroid constriction of the left circumflex coronary artery showed that intracoronary injection of Ad5FGF-4 is well tolerated and effective. Intracoronary infusion of Ad5FGF-4 increased myocardial perfusion to control levels and restored normal heart wall motion, whereas injection of a control vector (Ad5LacZ) or an FGF-2 gene without a signal sequence motif did not. Based on these data, clinical trials in patients with myocardial ischaemia were initiated.
Key Words: Myocardial ischaemia • Collateral coronary arteries • Stable angina • Angiogenesis • Fibroblast growth factor • Adenovirus
Introduction
Patients with severe coronary artery disease may not have satisfactory outcomes despite maximal medical therapy and multiple revascularization procedures. The consequences of inadequate therapy may include myocardial infarction and sudden death, or living with anginal pain and physical activity limitations. Although modified medical and surgical procedures continue to be developed, new therapeutic strategies are also being explored.
The goal of therapeutic angiogenesis, a novel treatment alternative, is to stimulate the formation of collateral coronary arteries, which is a natural compensatory mechanism for chronic myocardial ischaemia. Collateral arteries protect the myocardium from ischaemia by forming natural "micro-bypasses" that provide alternative routes for myocardial blood flow (Fig. 1).1 In one study, 96 patients (average age 49 years) with coronary artery disease and varying levels of angiographically visible collateral circulation were chosen prospectively and followed over 15 years.2 Patients with extensive collateral formation were less likely to experience myocardial infarction, ST-segment depression, abnormal exercise tests or heart failure. Most importantly, survival after 10 years was higher with well-formed collaterals.2
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Typically, functional collateral vessels are found in approximately one-fifth to one-quarter of subjects of 60 years of age without coronary artery obstruction.1 These collaterals are able to prevent ECG abnormalities and angina pectoris during a 1-minute balloon-occlusion of a coronary artery.1 Interestingly, collateral formation is often associated with severe coronary artery disease, suggesting that ischaemia stimulates collateral formation.2
With the advent of biomedical technologies that can elucidate the physiology of collateral formation and create tools to safely enhance it, therapeutic angiogenesis has become possible. The rational process of studying the genes involved in angiogenesis, choosing one as a therapeutic agent, and then solving the problems of delivery is anticipated to result in one or more approved clinical therapies in the near future.
This review describes the planning and design processes and preclinical experiments involved in the development of Ad5FGF-4 to stimulate myocardial collateral formation.
Designing an intervention suitable for therapeutic angiogenesis
Therapeutic angiogenesis is designed to enhance collateral formation in patients with severe coronary artery disease, thereby improving blood flow to ischaemic areas of the heart and relieving the short- and long-term effects of myocardial ischaemia. Since none of the existing medical or surgical approaches for treatment of myocardial ischaemia have been shown to facilitate collateral growth, a novel approach was explored.
Choosing a growth factor
The formation of new blood vessels is a complex process, and a wide variety of genes participate in it, activating endothelial cells, recruiting monocytes, degrading the existing extracellular matrix, stimulating migration and division of smooth muscle cells, or stabilizing nascent vessels.3,4 A partial list of pro-angiogenic molecules involved in this process includes the fibroblast growth factors (FGFs), hepatocyte growth factor (HGF), hypoxia inducible factor (HIF)-1, and vascular endothelial growth factors (VEGF) (Table 1).4,5
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Animal studies show that a single key regulatory factor may be sufficient to trigger therapeutic angiogenesis in ischaemic tissues, which has practical advantages for the design of therapeutic interventions.6 As a consequence, numerous regulatory factors are being tested for their ability to stimulate therapeutic vascular growth as monotherapy (Table 1).4,5 Most investigational therapies that have progressed to clinical trials to date use a form of either VEGF or FGF to stimulate formation of new blood vessels.6
Numerous experimental studies have demonstrated differences between the physiological functions of VEGF and those of FGF (Fig. 2). While VEGF is a survival factor for endothelial cells and stimulates the release of endothelial progenitor cells (EPCs) from the bone marrow, the resultant vessels can be permeable or "leaky",7 or may regress when VEGF levels decrease.6 In contrast, FGF not only stimulates VEGF production but also increases the density of PDGF receptors.3 Additionally, FGF stimulates the proliferation of endothelial cells, smooth muscle cells and fibroblasts.6 The increased cellularity of the FGF-stimulated vessels may promote stability and produce more mature vessels. FGF, but not VEGF, was shown to participate in arteriogenesis, an alternative process of collateral formation.8 Recent studies also demonstrate that in addition to therapeutic angiogenesis, FGF is also involved in cardioprotection,9 which may contribute to its therapeutic benefits.
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Because FGF may produce more stable vessels than VEGF, FGF was chosen as the therapeutic molecule for an angiogenic therapy. However, it is estimated that there are 23 structurally similar forms of FGF. FGF-1, -2, -4 and -5 have been used in angiogenic studies and no differences in efficacy have been found.6
Choosing between protein therapy and gene therapy
Options for administering FGF-4 include injection of purified recombinant (r) protein or delivery of the gene coding for FGF-4. While administration of the purified protein may seem to be the most direct solution, experimental results to date have not been encouraging using protein therapy.
FGF protein can be delivered intravenously, infused into the coronary arteries or injected into the heart muscle. Preclinical testing indicated that myocardial retention of rFGF-2 was poor after intravenous or intracoronary infusion. At 150 min after administration, myocardial retention was 
3% with intravenous administration and between 3% and 5% with intracoronary administration. Essentially the entire dose was cleared from the heart within 24 h.10 Because of poor FGF protein pharmacokinetics (i.e. low retention by the heart), stimulation of myocardial angiogenesis requires high systemic doses of FGF over a long period of time. However, hypotension and tachycardia may result from circulating concentrations of 48 µg/kg or more of FGF-2. Nausea and leukocytosis have also been observed after intracoronary administration of 36 µg/kg rFGF-2.11
In the FGF Initiating RevaScularization Trial (FIRST) intracoronary doses of recombinant FGF-2 protein were reduced below the toxic level, but no convincing efficacy was observed. The FIRST trial was a double-blinded, randomized, controlled clinical trial of 337 patients with stable angina. While the frequency of angina was decreased at 90 days in patients treated with rFGF-2, the difference compared with placebo was not sustained at 180 days. Exercise tolerance was not increased at any time point, but hypotension was observed more often in the rFGF-2-treated patients.12
Injection of a sustained-release preparation of rFGF-2 directly into the heart muscle during coronary artery bypass grafting (CABG) has been proposed as an alternative method for improving FGF protein localization and retention. While this limits the eligible patient population to those undergoing CABG, a small study (
per group) showed that perfusion defects were smaller in patients treated with rFGF-2 and followed for 
32 months.13
While it may be possible to optimize therapeutic angiogenesis with FGF protein, gene therapy provides another option for concentrated, localized, extended FGF delivery. By delivering the gene coding for a protein instead of the protein itself, high amounts of the protein can be locally produced from the DNA continuously over an extended period of time. This approach reduces systemic effects, increases the duration of therapy, and avoids the costs of large-scale protein production. Therefore, a gene therapy approach was chosen to deliver FGF-4 to the myocardium.
Choosing a gene therapy vector
A variety of methods for gene transfer are currently available, ranging from "naked" plasmid DNA to various viral vectors. Gene transfer with plasmid DNA is generally inefficient and produces low levels of protein. Transfer efficiency can be increased somewhat by addition of carrier compounds, such as liposomes. Alternatively, genetically modified viruses have been created that transfer DNA of interest but are not capable of replication. Modified adenovirus, adeno-associated virus, baculovirus, herpes simplex virus, lentivirus, retrovirus and Sendai virus are some of the available options.6 Most clinical trials in gene therapy so far have used a retrovirus, DNA/liposome combination or adenovirus.14
Adenovirus lacking its E1 region is a highly efficient vector for cardiovascular gene transfer. Although it may have lost its ability to replicate, it is capable of high-level transfer of DNA to non-dividing, terminally differentiated cells like those found in the heart. After delivery by Ad5, the DNA does not incorporate into the genome and is gradually lost from the cells over time. Although local inflammation can occur at high concentrations of adenovirus and anti-adenoviral antibodies usually form, the lack of chromosomal integration and transient expression increase vector safety.14 Furthermore, high concentrations of adenovirus can be produced reproducibly and are stable in storage, which is practical for routine use. Thus, an E1-deleted, replication-incompetent adenovirus of serotype 5 (Ad5) was chosen as the vector for transferring the gene for FGF-4 to the myocardium.
Choosing a delivery method
Numerous surgical or catheter-based techniques have been developed that permit access to the heart and that can be adapted for gene transfer. The major approaches used for myocardial gene transfer are epicardial, endocardial and intracoronary delivery (Table 2). Accessing the heart's surface for epicardial techniques requires a thoracotomy, which may be performed during CABG or require a separate surgical procedure. This is an invasive procedure that may result in complications. However, the vector can be specifically injected near ischaemic areas. In contrast, percutaneous endocardial injection is appealing because it is easier for the patient, but finding the ischaemic sites can be challenging. Currently, electromechanical (NOGA) mapping is used to localize ischaemic areas, but this procedure requires specialized catheters, instrumentation and skills.15 Furthermore, direct injection may also cause complications and restrict vector distribution within the myocardium.
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Intracoronary infusion adapts the techniques used in angiography to infuse the vector throughout the coronary vascular tree. This treats all of the coronary arteries with less risk of complications than a myocardial injection. A concern is that not all of the adenovirus may be taken up by the heart. If adenovirus is systemically disseminated, some toxicity may result depending on the dose injected.16 Other possible modes of administering adenovirus include infusion during stenting17 or retrograde infusion into the coronary veins.18 We have chosen percutaneous intracoronary infusion for Ad5FGF-4 delivery.
Experimental results
Preclinical studies on the safety and efficacy of Ad5FGF-4 were conducted in domestic pigs, which have a coronary anatomy similar in size and structure to humans. This model system allowed validation of the delivery method and permitted detailed study of Ad5FGF-4 distribution, toxicity and efficacy.
The experimental system
An ameroid constrictor put around the left circumflex coronary artery was used to simulate myocardial ischaemia in pigs weighing 50 kg each. The ameroid constrictor swells gradually over time, obstructing the coronary arteries after 10 days to induce ischaemia with minimal infarction.19 Natural collateral formation to compensate for the severe blockage is complete within 21 days of the procedure, and remains stable for approximately 4 months, resulting in sufficient blood flow at rest, but not during exertion such as electrical pacing.20 Baseline regional blood flow and function during pacing at 200 beats per minute were measured immediately before and at various times after intracoronary delivery of Ad5FGF-5, Ad5FGF-4, Ad5FGF-2 or Ad5LacZ, which occurred 4–5 weeks after ameroid placement.19
Verification of gene transfer
Polymerase chain reaction (PCR) analysis of the heart muscle of pigs treated with Ad5LacZ, Ad5FGF-519 or Ad5FGF-421 showed Ad5 DNA presence in the heart but not in extracardiac tissues (liver, muscle, eye, etc.) 2 weeks after Ad5 injection in the coronary arteries. Evidence of transgene expression was found in the hearts of pigs treated with Ad5LacZ or Ad5FGF-5.19
The hearts of several pigs treated with Ad5FGF-4 were also examined for evidence of gene transfer. In this experiment, tissue from the left ventricle was removed and homogenized 14 days after delivery of Ad5FGF-4. FGF-4 mRNA was detected by RT-PCR and FGF-4 protein was detected by immunoblot analysis in the heart, but not in the liver or eyes of treated animals (Fig. 3).21
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Taken together, these data indicate that intracoronary delivery of Ad5FGF-4 to the coronary vascular bed of pigs with exertional ischaemia results in adenoviral retention, DNA transfer and gene expression in heart cells, and localized production of the appropriate proteins.
Safety and toxicology studies
When the heart and liver tissues from the pigs treated with Ad5FGF-4 were examined microscopically, no evidence of inflammation or necrosis was found up to the highest dose tested (1.6x1012 viral particles [vp] per pig). No evidence of inappropriate angiogenesis or any other product-related toxicity was observed in a good laboratory practices (GLP)-toxicology study in non-ischaemic pigs receiving Ad5FGF-4 either in the coronary artery (1010–1012 vp/animal) or systemically (1012 vp/animal) up to 90 days after treatment. Systemic administration of Ad5FGF-4 to male mice showed no evidence of germ-cell transfection.22
Efficacy studies
The extent of angiogenesis was initially measured after Ad5FGF-5 treatment by counting the number of capillaries surrounding each muscle fibre in heart tissue.19 A significant 30% increase in capillary density was observed in the endocardial wall of both ischaemic and non-ischaemic regions, reflecting new vessel formation.
Myocardial perfusion in treated hearts was measured using a contrast medium detectable by echocardiography. The relative amount of contrast medium detected in the left ventricle increased to control (non-ischaemic) levels 14 days after treatment with either Ad5FGF-5 or Ad5FGF-4 (Fig. 4)19,21,23 indicating that blood flow was increased in the ischaemic areas. Perfusion was increased at Ad5FGF-4 doses ranging from 1010 to 1.6x1012 vp but not at 109 vp.21 Although angiogenesis was not measured after Ad5FGF-4 treatment, the similarity between FGF-5 and FGF-4 on improving perfusion suggests that, similar to FGF-5, FGF-4 also stimulates coronary angiogenesis. Direct proof for Ad5FGF-4-induced angio/arteriogenesis was provided by a separate study in which delivery of Ad5FGF-4 to ischaemic rabbit hind-limb increased the number of capillaries (angiogenesis) and angiographically visible collaterals (arteriogenesis).24
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Similar to the perfusion deficit, the pacing-induced, wall-thickening deficit was also eliminated 2 weeks after Ad5FGF-519 or Ad5FGF-4 injection (Fig. 5).21,23 The dose response for the functional improvement resembled that of the perfusion changes, with no response at 109 vp Ad5FGF-4 but improved wall motion from 1010 to 1.6x1012 vp.21 Improved perfusion and function were maintained for the duration of the experiment (12 weeks), suggesting that newly formed vessels were stable for at least that time period.21
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The role of signal peptide
In designing an adenovirus vector carrying an angiogenic growth factor gene for myocardial therapeutic angiogenesis, the presence or absence of a signal peptide (needed for effective secretion of proteins from cells) is an important issue. To examine the role of signal peptide, we compared the effect of intracoronary delivery of Ad5 vectors (5x1011 vp) expressing a mutant form of FGF-2, either with a signal peptide (FGF-2LI [+sp]) or without (FGF-2LI [–sp]). In the pig ameroid model of stress-induced myocardial ischaemia, blood flow to the ischaemic region was improved only in animals that received the vector with the transgene containing the signal peptide.21 These studies indicated that the presence of signal peptide (such as found in the Ad5FGF-4 construct) may reduce the dose of Ad5 required for a therapeutic effect, thereby providing an improved safety margin when used in patients.
Conclusion
Research to date suggests that angiogenic gene therapy for myocardial ischaemia may be a viable therapeutic option for patients with ischaemic heart disease. Genes that stimulate angiogenesis are known; vectors for transferring the therapeutic genes to the vicinity of ischaemic tissue are available; and the necessary technologies to make, grow and store the therapeutic vector exist. Furthermore, routine percutaneous techniques can be used to access the coronary vasculature.
Testing in a pig model of ischaemia provided promising results, supporting both the safety and efficacy of therapeutic angiogenesis with Ad5FGF-4. The results to date indicate that a single intracoronary injection of Ad5FGF-4 can effectively transfect the myocardium, produce FGF-4 protein exclusively in the heart for several weeks, and improve both myocardial perfusion and function. Based on these data, human studies examining the effects of therapeutic angiogenesis with Ad5FGF-4 in patients with myocardial ischaemia were initiated.
Footnotes
All authors from Berlex/Schering are employees of Schering/Berlex. The AGENT Trials were funded by Schering. Preclinical studies with Ad5FGF-4 were also funded by Schering.
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