WO2002058718A2 - Use of compositions containing pdgf-bb for promoting angiogenesis - Google Patents

Abstract

Methods of promoting angiogenesis by delivering angiogenic factors are disclosed. The factors can be delivered as a gradient to a localized area of tissue to direct vascular growth.

Description

METHODS AND COMPOSITIONS FOR

PROMOTING ANGIOGENESIS

Background of the Invention This application claims priority to U.S. Provisional Patent Application Serial No.

60/264,457, filed on January 26, 2001, the contents of which are hereby incoporated herein.

Background of the Invention The depletion of oxygen supply to due to obstructed or inadequate blood supply is the common pathological state associated with various tissue ischemias, including myocardial ischemia, ischaemic bowel disease, and peripheral ischemia. The alleviation of tissue ischemia is critically dependent upon angiogenesis, the process by which new capillaries are generated from existing vasculature and tissue. The spontaneous growth of new blood vessels provide collateral circulation in and around an ischemic area, improves blood flow, and alleviates the symptoms caused by the ischemia. Although surgery or angioplasty may help to revascularize ischemic regions in some cases, the extent, complexity and location of the arterial lesions which cause the occlusion often prohibits such treatment. Alternative methods for the treatment of chronic ischemia have focused on the delivery of angiogenic growth factors, over twenty of which are known. To date, modest but significant angiogenesis has been achieved following administration (e.g., by local injection) of exogenous factors to animal models. For example, purified recombinant NEGF-A has been demonstrated to elicit a modest but significant vascularization following injection into ischemic skeletal muscle tissue in a rabbit model of chronic limb ischemia [Takeshita et al, Circulation 90, 228:1994]. In addition, direct injection of vectors containing cDΝA encoding NEGF-A has also been shown to induce a modest stimulation of angiogenesis in ischemia animal models in both skeletal and cardiac muscle [Takeshita et al, Biochemical and Biophysical Research Communications 227, 628:1996; and MacGovern et al., Human Gene Therapy 8, 215: 1997]. However, such local application often results in diffusion of the factors away from the desired site, thus diminishing angiogenic effect. Moreover, it is known that such bolus delivery of angiogenic factors generally results in a local and disorganized hodge-podge of new blood vessels, only a fraction of which (if any) contribute to amelioration of a blockage. An additional limitation associated with current approaches for promoting angiogenesis and effective treatment of ischemia is the inability of stable blood vessels to form by application of single agents using known techniques. In past studies, when vessels were followed for several months after treatment of ischemic animal models, the vast majority of new vessels were observed to regress after the applied growth factor had become depleted. Thus, current approaches for ischemia therapy require repeated applications of factors to maintain newly formed vasculature.

Other related therapy methods attempt to circumvent the need for multiple applications by relying on the transplantation of autologous or non-autologous cells which can produce sustained levels of angiogenic proteins. In one such approach, a subject's endogenous cells are isolated, cultured, and transfected with expression vectors encoding angiogenic proteins. Following in vitro manipulations, these cells are injected back into the patient at the site of tissue ischemia. However, the drawbacks of this approach include the time and effort required to isolate, culture and transfect target cells from each individual patient, as well as difficulties in securing sustained expression of angiogenic proteins. In addition, sub-optimal cell survival and differentiation states of the cells following injection also limit the efficacy and of this approach.

To avoid these problems, cells have been obtained from non-patient (e.g., allogeneic), even non-human, sources and manipulated in the manner described above. However, the modified non-patient or non-human cells are often rejected by the patient's own immune system, making this approach impractical too.

Accordingly, improved therapies for promoting tissue angiogenesis and generating stable vasculature in a safe, reliable, and non-invasive manner are needed to treat tissue ischemia and other related conditions.

Summary of the Invention

The present invention provides novel methods and compositions for promoting angiogenesis to treat a variety of tissue ischemias, including peripheral and myocardial ischemia. Selected angiogenic factors or synergistic combinations of factors, functional analogues of such factors or combinations of factors, or nucleic acids encoding such factors or combinations of factors, are delivered to a localized area of tissue in an amount effective to induce angiogenesis within the area of tissue. The invention further includes improved methods and vehicles for delivering such factors or combinations of factors, functional analogues of such factors or combinations of factors, and nucleic acids encoding such factors or combinations of factors. In one embodiment, the invention provides a method of promoting angiogenesis comprising delivering PDGF-BB to a localized area of tissue in an amount effective to induce angiogenesis within the area of tissue. The PDGF-BB can be delivered either alone or in combination with another angiogenesis-promoting factor, particularly bFGF and/or NEGF-A. The angiogenesis-promoting factor or combination of factors can be administered in the form of a protein composition or an expression plasmid encoding the protein(s). The angiogenesis-promoting factor or combination of factors can also be administered in the form of functional analogues of the factor or combination of factors. For example, anti-idiotypic antibodies of PDGF-BB, NEGF-A and/or bFGF can be administered in accordance with the invention.

When administering the angiogenesis promoting factors of the invention in the form of an expression plasmid, suitable vectors include, but are not limited to, adenoviral vectors, retroviral vectors, adeno-associated viral vectors, RΝA vectors, liposomes, cationic lipids, lentiviral vectors and transposons. In another embodiment, the invention provides a method for promoting angiogenesis by delivering angiogenic factors, such as those described above, to a localized area of tissue using heparin sepharose-containing microcapsules in an amount effective to induce angiogenesis within the area of tissue. The angiogenic factors or expression plasmids encoding the factors are incorporated into the microcapsules as described in the working examples provided below for slow, sustained release into localized areas of tissue.

In a particular embodiment, the microcapsules are made up of uncoated heparin sepharose beads, heparin sepharose beads coated with a single layer of alginate polymer, heparin sepharose beads coated with poly-ethylene glycol (PEG) polymer or heparin sepharose beads coated with alternating layers of alginate and PEG. Typically, the microcapsules range in size from 1-200 microns.

Suitable angiogenic factors for incorporating into the microcapsules include, for example, M-CSF, GM-CSF, NEGF-A, VEGF-B, VEGF-C, NEGF-D, VEGF-E, neuropilin, FGF-1 , FGF-2(bFGF), FGF-3, FGF-4, FGF-5, FGF-6, PDGF-BB, PDGF- AA, Angiopoietin 1, Angiopoietin 2, erythropoietin, BMP-2, BMP-4, BMP-7, TGF- beta, IGF-1, Osteopontin, Pleiotropin, Activin, Endothelin-1, and combinations thereof or an expression vectors encoding such angiogenic factors. The angiogenic factors can be purified from their native sources or produced by recombinant expression.

The microcapsules are contacted with the localized area of tissue generally by injection or surgical implantation. For example, injection can be performed using a catheter based trans-myocardial injection technology, such as the ΝOGA technology. In yet another embodiment, the present invention provides a method for promoting angiogenesis by contacting a localized area of tissue with a gradient of one or more angiogenic factors or a nucleic acid encoding one or more angiogenic factors, such that directed vascular growth along the gradient is achieved. Such directed vascular growth can be used to achieve interconnection and/or intraconnection of blood vessels (e.g., to circumvent blood flow around a blockage within a blood vessel).

In a particular embodiment, the angiogenic factor or nucleic acid is released in a gradient using a biocompatible material which is contacted with (e.g., implanted within) the localized area of tissue. The angiogenic factor is associated with the biocompatible material (e.g., absorbed onto the biocompatible material) such that it is released onto surrounding tissue. This can be achieved by treating the biocompatible material with the angiogenic factor prior to contact with (e.g., implantation into) a selected area of tissue. The angiogenic factor is then released from the biocompatible material onto the surrounding tissue in a directed gradient determined by the placement of the biocompatible polymer

Suitable biocompatible materials include, for example, polymers or threads which incorporate the angiogenic factor. In a preferred embodiment, the biocompatible material is an absorbable thread, such as polyglyconate monofilament, poliglecaprone 25-(Monocryl), polydiaxonone (PDS II), polyglactin 910, polyglycolic acid, Biodyn glycomer 631, chromic surgical gut or plain surgical gut.

These and other embodiments of the invention are described in the following figure detailed description, examples and figures.

Brief Description of the Figures Figure 1 is a graph comparing levels of angiogenesis in the Matrigel model using a low dose of transduced cells encoding GFP alone (control), NEGF-A, NEGF-C, NEGF-D bFGF or PDGF-BB. C57B1/10 mice were each injected subcutaneously into the abdominal with a low dose of 3 x 10⁵ retrovirally transduced autologous myoblast cells, suspended in 0.4ml of Matrigel. Mice were sacrificed 13 days later and the matrigel pellet and a section of the abdominal muscle adjacent to the pellet was removed. Samples were sectioned and the number of microvessels in the abdominal muscle was quantified by visual inspection of sections under the microscope. Shown is the number of microvessels per 10 high power fields counted. The most potent angiogenic effect was observed with NEGF-A, PDGF-BB and bFGF. Analysis of the dose response curve for PDGF-BB and VEGF-A transduced cells showed that PDGF- BB was more potent than VEGF-A at lower doses. Figure 2 is a graph comparing levels of angiogenesis in the Matrigel model using a high dose of cells transduced to express bFGF, NEGF-A and PDGF-BB. C57B1/10 mice were each injected with a high cell dose of 2 x 10⁶ retrovirally transduced autologous myoblast cells suspended in 0.4ml of Matrigel. Mice were sacrificed 13 days later, the pellets were recovered, sectioned and the number of microvessels counted by visual inspection. Shown are the number of microvessels per 10 high power fields. At this cell dose, PDGF-BB was as potent as either bFGF or NEGF-A at stimulating angiogenesis.

Figure 3 shows photographs of mouse corneas 6 days following the implantation of pellets coated with control saline (A), PDGF-BB (B), NEGF-A (C) or bFGF (D) alone. Bottom panels: Quantification of the angiogenic effect elicited by each factor. Vessel length (E), clock hours (F) and area (G) are shown.

Figure 4 (A) shows photographs of mouse corneas 6 days following the implantation of pellets coated with VEGF-A alone (left panel), bFGF (middle panel) or both factors combined (right panel). (B) shows the quantification of the angiogenic effect elicited by each growth factor in terms of clock hours (left panel), vessel length (middle panel) and area (right panel).

Figure 5 (top panels) shows photographs of mouse corneas 6 days post- transplantation of pellets coated with bFGF alone (left panel) or bFGF combined with PDGF-BB (middle and right panels). Bottom panels show photographs of mouse corneas 6 days post-transplantation of pellets coated with either VEGF-A alone (left panel) or VEGF-A combined with PDGF-BB (right panel).

Figure 6 is a graph comparing the quantification of angiogenesis in the mouse cornea model using PDGF-BB, VEGF-A or bFGF either alone or in combination. Corneal micropockets were created with a cataract knife in the eyes of 8-week old C57B1/6 mice. Into this pocket, aluminum sulfate pellets coated with between 80 and 160ng of recombinant human PDGF-BB, VEGF-A, bFGF or combinations thereof were implanted and mice were monitored daily. A total of 5 mice were transplanted per group. The area of newly grown vessels was assessed 5 days post implantation. Mice implanted with control pellets showed no evidence of angiogenesis. When tested alone, bFGF stimulated the highest level of angiogenesis followed by VEGF-A and PDGF-BB. The level of angiogenesis stimulated by VEGF-A in combination with PDGF-BB was equivalent to that observed for bFGF alone. Unexpectedly, the most potent combination was PDGF-BB and bFGF. Of all combinations tested, PDGF-BB and bFGF together stimulated the greatest level of angiogenesis, significantly greater than that observed for VEGF-A and bFGF.

Figure 7 is a schematic illustration of the experimental strategy to make heparin sepharose/alginate microcapsules. Heparin sepharose beads (Pharmacia: 50- 150 Dm in size) are mixed with a solution of sodium alginate to a final concentration of 200mg/ml. The heparin sepharose/alginate solution is then loaded into a 5ml syringe and slowly injected into a coaxial airflow system constructed at Genetix. The coaxial air flow creates a mist of the heparin sepharose/alginate solution which drops into a 1.5% calcium chloride bath. Once the alginate hits the calcium solution the alginate becomes cross-linked, forming a solid gel capsule roughly in the shape of a sphere. The size of the microcapsules can vary greatly from 50 - 400 Dm. Large microcapsules (greater than 200 Dm in size) are removed from the capsule mixture using a 200 Dm sieve. Once formed the capsules are washed twice in sterile water and stored in buffer composed of 0.9% sodium chloride and ImM calcium chloride. Capsules are loaded with recombinant human PDGF-BB by incubation in binding buffer (0.9% NaCl, ImM CaC12 and 0.05% gelatin) at 4°C overnight (-16 hours) with gentle shaking. The next day the capsules are removed, washed twice in binding buffer and either cultured in vitro to determine the kinetics of PDGF-BB release or injected in vivo to assess angiogenesis. The efficiency of PDGF-BB uptake is quantified by ELISA of the binding buffer following removal of the capsules.

Figure 8 is a graph showing that heparin sepharose/alginate capsules bind large amounts of recombinant human PDGF-BB. Shown is the amount of PDGF-BB absorbed by 3000 capsules following incubation with various quantities of growth factor. The amount of PDGF-BB remaining in the binding buffer following incubation with capsules was quantified by ELISA. Three thousand capsules were able to absorb at least 35 μg of PDGF-BB representing 13 ng of PDGF-BB per capsule.

Figure 9 is a graph showing that heparin sepharose/alginate microcapsules provide slow, high level and long term release of bound PDGF-BB in vitro. Ten μg of recombinant human PDGF-BB was incubated with three different types of test samples. The first test sample was composed of non-encapsulated heparin sepharose beads while the second and third groups were composed of alginate encapsulated heparin sepharose beads made using either a 1.2% or a 1.6% alginate solution. Three thousand beads/microcapsules were incubated with PDGF-BB at 4°C overnight with gentle shaking. ELISA analysis of the binding buffer the next day showed absorption of 90% (9μg) of the PDGF by the capsules. Following incubation with PDGF-BB, the beads/microcapsules were washed, resuspended in 5mls of serum free medium and incubated at 37°C. Every 24 hours the medium was changed and the amount of PDGF- BB present in the medium quantified by ELISA. A slow, sustained release of approximately 0.5-3% of the total bound PDGF-BB (representing 125-250ng) was detected each day for a minimum of 14 days, the longest time point tested. Importantly, the proportion of PDGF-BB released per day is equivalent to the amount of PDGF-BB that was estimated to be secreted by muscle cells transduced with the PDGF-BB retro virus in the foregoing Matrigel experiments. The release kinetics for non- encapsulated heparin sepharose beads was better than those observed for the alginate encapsulated heparin sepharose.

Figure 10 is a graph showing that PDGF-BB microcapsules potently stimulate angiogensis in vivo in the stringent Matrigel model. Three thousand microcapsules loaded with lμg or lOμg of PDGF-BB were mixed with 400μl of Matrigel and subcutaneously injected into the abdominal region of C57B1/10 mice. Thirteen days later mice were sacrificed, the pellets and a section of the adjacent abdominal muscle was removed, fixed, sectioned and the number of microvessels quantified by visual inspection of the sections under the microscope. The results showed that the number of microvessels in mice receiving microcapsules loaded with lOmg of PDGF-BB was 2.5- fold greater than that of control mice.

Figure 11 is a graph showing that PDGF-BB microcapsules stimulate angiogenesis in infarcted rat hearts 3 weeks post-injection. Infarcted rat hearts were injected with 1600 microcapsules containing μg (control) or 18μg of PDGF-BB in a volume of 20μl. Three weeks post injection rats were sacrificed, hearts were removed, fixed, sectioned and the number of microvessels within the infarct region quantified by visual inspection under a microscope. Shown is the number of microvessels per 5 high power fields for recipients of control and PDGF-BB microcapsules. Rats injected with PDGF-BB microvessels showed an approximate 2-fold increase in the number of microvessels as compared to control rats.

Figure 12 shows an analysis of cardiac function in rats injected with control vs. PDGF-BB microcapsules following myocardial infarction. Left ventricular pressure (LVP), dP/dT, neg dP/dT and tau were measured prior to sacrifice at 3 weeks post injection. Left ventricular pressure (LNP) is the maximum pressure in the left ventricle during contraction. The dP/dT variable is the first derivative of the pressure wave and is separately viewed for the upstroke (dP/dT) and the downstroke (neg dP/dT). The upstroke (dP/dT) is a measure of contractility and reflects the condition of the muscle independent of the pressure. Neg dP/dT reflects the relaxation of the muscle, which together with the relaxation constant, tau, provides information on the stiffness of the ventricular wall following infarction. A significant improvement in all parameters was detected in rats injected with PDGF-BB microcapsules. Rats injected with PDGF-BB microcapsules showed a 25% increase in left ventricular pressure, a 2-3 fold increase in cardiac contractility/relaxation and a 2.5-3 fold decrease in the relaxation constant tau.

Figure 13 is a graph showing that PDGF-BB and bFGF delivered by slow release microcapsules potently synergize to stimulate angiogensis in vivo in the stringent Matrigel model. Three thousand microcapsules loaded with lμg of bFGF were mixed with 400μl of Matrigel and subcutaneously injected into the abdominal region of C57B1/10 mice. Thirteen days later mice were sacrificed, the pellets and a section of the adjacent abdominal muscle was removed, fixed, sectioned and the number of microvessels quantified by visual inspection of the sections under the microscope. The results showed that the number of microvessels in mice receiving bFGF + PDGF-BB microcapsules was 4-fold greater than that of mice implanted with either growth factor alone.

Figure 14 is a schematic illustration of the structure of various angiogenic expression plasmids. All vectors were constructed using the pCI vector backbone from Promega. All vectors contained the Cytomegalovirus immediate-early enhancer/promoter region, a chimeric intron and the late poly adenylation signal from SV40. The cDNA encoding either human PDGF-BB, NEGF-A or bFGF was inserted into this vector downstream of the chimeric intron. A cDΝA encoding for the mature PDGF-BB protein was cis-linked to the secretory signal from the murine IgGkappa immunoglobulin light chain gene while the NEGF-A cDΝA utilized its endogenous secretory signal. Since endogenous bFGF is not secreted by the usual Golgi pathway and prior groups have had difficulty in obtaining high level secretion of bFGF from cells transduced with the bFGF cDΝA, the bFGF cDΝA was linked in cis to the secretory signal from the human Interleukin-2 cDΝA. The level of angiogenic protein secreted from transiently transfected 293T cells, as assessed by ELISA, is shown to the right. To analyze cardiac function in rats injected with control vs. PDGF-BB expression plasmids following myocardial infarction, test animals are anesthetized and intubated. The chest wall is opened and a myocardial infarct is created by tying off the anterior descending artery. 180μg of control or test expression plasmid is injected into the heart wall in a volume of 20μl. Cardiac function is assessed 3 weeks post injection. Animals are sacrificed, the heart is removed and efficiency of plasmid uptake is assessed by staining with X-gal. The size of the infarct and the extent of angiogenesis is quantified.

Detailed Description of the Invention

While growth promoting factors have been described as an angiogenic agents, the efficacy of such proteins as a therapeutic agents for the treatment of peripheral and/or myocardial ischemia has not yet been demonstrated. The present invention provides, for the first time, methods for promoting angiogenesis and treating such ischemia using particular angiogenic factors, such as PDGF-BB, as well as particular combinations of factors (e.g., combinations which include PDGF-BB). The present invention also provides improved methods for delivering the factors to increase their efficacy, for example, by enabling directed and/or controlled release of the angiogenic factors onto surrounding tissue (e.g., ischemic myocardium).

ANGIOGENIC FACTORS

As used herein, the term "angiogenic factor" refers to any known protein factor capable of promoting growth of new blood vessels from existing vasculature ("angiogenesis"). Suitable angiogenic factors for use in the invention include, for example, PDGF-BB, PDGF-AA, M-CSF, GM-CSF, NEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, neuropilin, FGF-1 , FGF-2(bFGF), FGF-3, FGF-4, FGF-5, FGF-6, Angiopoietin 1, Angiopoietin 2, erythropoietin, BMP-2, BMP-4, BMP-7, TGF-beta, IGF-1, Osteopontin, Pleiotropin, Activin, Endothelin-1 and combinations thereof. The term "angiogenic factor" also refers to functional analogues of the above- mentioned factors. Such functional analogues include, for example, functional peptides or portions of the factors. Functional analogues also include anti-idiotypic antibodies which bind to the receptors of the factors and, thus, mimic the activity of the factors in promoting angiogenesis. Methods for generating such anti-idiotypic antibodies are well known in the art and are described, for example, in WO 97/23510, the contents of which are incorporated by reference herein.

Antigens have specific epitopes to which certain antibodies will bind. The region of the antibody that specifically interacts with the epitopes on the antigen is called the antigen combining site. The antigen combining site is composed of a collection of idiotopes which are unique sequences on the antibody that specifically interact with the epitopes on the antigen. The specific collection of idiotopes that interact with the epitopes on the antigen is defined as an antibody's "idiotype". Accordingly, an anti- idiotype antibody is an antibody that is directed against the antigen combining site of the first antibody. Anti-idiotype antibodies combine with those specific sequences and may resemble or act as the epitope to which the first antibody reacts. For example, one can have an antibody that binds to specific epitopes on bFGF. One can then make a second antibody (an anti-idiotype antibody) that specifically interacts with antigen combining site of the first antibody. This anti-idiotype antibody may then mimic the biological activity of bFGF itself by binding to the bFGF receptor and activating it.

The use of anti-idiotype antibodies (as growth factor analogues) in the methods of the present invention can be advantageous in that many growth factors have a very short half life and therefore most of the factor that is given to a patient is not utilized. In contrast, antibodies have much greater half lives and therefore their potency is maintained for a greater length of time in vivo. In addition the levels of growth factors that are required to achieve a biological effect can, in certain instances, produce adverse reactions such as toxicity and hypotension. Since lower levels of anti-idiotype antibodies may be required to produce the same biological effect these adverse side effects may be prevented.

Angiogenic factors used in the present invention can be purified from their native sources or produced by recombinant expression and subsequently administered to patients as a protein composition. Alternatively, the factors can be administered in the form of an expression plasmid encoding the factors, as is described in further detail below. The construction of suitable expression plasmids is well known in the art. Particular angiogenic expression plasmids for use in the invention are shown in Figure 14 and are described in Example 1. Suitable vectors for constructing expression plasmids include, for example, adenoviral vectors, retroviral vectors, adeno-associated viral vectors, RNA vectors, liposomes, cationic lipids, lentiviral vectors and transposons. Accordingly, in one embodiment, the present invention provides novel methods and compositions for promoting angiogenesis to promote angiogenesis and to treat a variety of tissue ischemias. Selected angiogenic factors or synergistic combinations of factors, functional analogues of such factors or combinations of factors, or nucleic acids encoding such factors or combinations of factors, are delivered to a localized area of tissue in an amount effective to induce angiogenesis within the area of tissue.

In a preferred embodiment, the invention provides a method of promoting angiogenesis comprising delivering PDGF-BB to a localized area of tissue in an amount effective to induce angiogenesis within the area of tissue. The PDGF-BB can be delivered either alone or in combination with another angiogenesis-promoting factor. Particularly preferred combinations include PDGF-BB combined with bFGF and/or VEGF-A. DELIVERY VEHICLES FOR ANGIOGENIC FACTORS

I. In another embodiment, the present invention provides a means for delivering angiogenic factors to a localized area of tissue in a controlled, sustained fashion. One problem in using purified (e.g., recombinant) angiogenic proteins to stimulate angiogenesis for the treatment of myocardial and peripheral tissue ischemia can be the short half-life of the protein upon injection in vivo. This problem is addressed by way of the present invention using slow-release heparin sepharose-containing microcapsules in an amount effective to induce angiogenesis within the area of tissue. One property that many angiogenic factors share is the ability to bind to heparin, a highly sulfated glycosaminoglycan that plays a role in anti-coagulation in vivo. This property is exploited in making the heparin sepharose containing microcapsules of the present invention which bind angiogenic factors and provide slow release of the factors when contacted (e.g., implanted) with localized tissue. The experimental process for making the microcapsules is described in detail in Example 3 and shown schematically in Figure 7.

In one embodiment, the microcapsules are injected or surgically implanted into localized areas of tissue. In another embodiment, the microcapsules are delivered to localized areas of tissue using the NOGA system (Biosense). NOGA is a 3 dimensional catheter based transmyocardial injection system. A catheter is inserted into a major vein/artery and is snaked up into either of the ventricals of the heart. The end of the catheter contains a needle and a space in which therapeutic agents can be inserted and subsequently injected intra-myocardially into the damaged areas of the heart muscle. This delivery method is much easier and safer for the patient and is much more efficacious than other methods of delivery including intracoronary injections or placing therapeutic agents against the wall of the heart. Due to the nature of the system, only therapeutic agents which can be physically delivered through a 25-27 gauge needle can be used. Thus macrocapsules described in the prior art cannot be used with such a system, whereas microcapsules of the present invention can be used with the system. The microcapsules are composed of single heparin sepharose beads which optionally can be coated with a thin layer of alginate polymer. In a particular embodiment of the invention, the microcapsules are made up of uncoated heparin sepharose beads, heparin sepharose beads coated with a single layer of alginate polymer, heparin sepharose beads coated with poly-ethylene glycol (PEG) polymer or heparin sepharose beads coated with alternating layers of alginate and PEG.

Using, for example, the coaxial air flow technology shown in Figure 7 and described in Example 1, the microcapsules can also be made small enough for use in the NOGA delivery system. For example, the microcapsules typically range from 1-200 microns in size. Moreover, they are able to absorb large quantities of angiogenic factors, such as FGF-2, VEGF-A and PDGF-BB, and slowly release the factors over extended periods of time at levels which are able to stimulate the growth of new blood vessels in vivo.

II. In another embodiment, the present invention provides a means for delivering angiogenic factors to a localized area of tissue using a gradient of one or more angiogenic factors or a nucleic acid encoding one or more angiogenic factors, such that directed vascular growth along the gradient is achieved. Angiogenic factors, such as those described in the preceding examples, work by providing a gradient of angiogenic factor that stimulates the chemotaxis and proliferation of endothelial cells, and their supporting cells towards the source of the factor. Regulating the growth of new vessels from existing vasculature (angiogenesis) that effectively bypass an arterial lesion requires strict spatial and temporal control. Accordingly, by forming a directed gradient of angiogenic factors in accordance with the present invention, interconnection and/or intraconnection of blood vessels (e.g., to circumvent blood flow around a blockage within a blood vessel) can be achieved.

In a particular embodiment, the angiogenic factor (or a nucleic acid encoding the factor) is released in a gradient using a biocompatible material which contains the factor such that the factor is released onto surrounding tissue when the biocompatible material is contacted with (e.g., implanted within) the tissue. This can be achieved by treating the biocompatible material with the angiogenic factor prior to contact with (e.g., implantation into) a selected area of tissue in a manner which allows for release of the factor from the material in vivo. The biocompatible material is then contacted with (e.g., implanted into) a localized area of tissue in a configuration which provides a directed gradient of the angiogenic factor once it is released from the material.

Suitable biocompatible materials for use in the invention include, for example, polymers or threads which incorporate the angiogenic factor. In a preferred embodiment, the biocompatible material is an absorbable thread, such as polyglyconate monofilament, poliglecaprone 25-(Monocryl), polydiaxonone (PDS II), polyglactin 910, polyglycolic acid, Biodyn glycomer 631, chromic surgical gut or plain surgical gut. The biocompatible material can be coated with one or several angiogenic factors to allow delivery of growth factor or growth factor combinations that provide the optimal angiogenic stimulus. The biocompatible material can also be engineered to release certain growth factors at specific rates and at specific times that may help mimic the natural angiogenic process more closely. The invention is further illustrated by the following examples which should not be construed as limiting.

EQUIVALENTS

Although the invention has been described with reference to its preferred embodiments, other embodiments can achieve the same results. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments described herein. Such equivalents are considered to be within the scope of this invention and are encompassed by the following claims.

INCORPORATION BY REFERENCE

The contents of all references and patents cited herein are hereby incorporated by reference in their entirety.

EXAMPLES

The following examples were perfomed to demonstrate and quantify the angiogenic potential of PDGF-BB alone and in combination with other angiogenic factors. A variety of assays were used, including the stringent Matrigel assay, the mouse cornea assay, and the ischemic rat heart assay, all as described in detail below.

EXAMPLE 1 - Construction of Retroviral Vectors Containing Angiogenic cDNAs , Analysis of Virus Titer and Assessment of Stable Gene Transfer

In order to provide stable, high-level delivery of PDGF-BB in the Matrigel model, primary myoblasts from C57B1/10 mice were transduced with retroviral vectors encoding human PDGF-BB. To compare the angiogenic potential of PDGF-BB to other known angiogenic agents, retroviral vectors encoding human VEGF-A165, VEGF-C, VEGF-A, VEGF-D, PDGF-BB, or bFGF also were constructed and tested. All vectors are shown schematically in Figure 14. Since VEGF-C, VEGF-D and PDGF-BB cDNAs encode proteins which are inactive (or less active in the case of PDGF-BB) in their non- processed form, vectors containing cDNAs encoding the mature forms of the aforementioned proteins linked to the powerful secretory signal from the murine IgG kappa immunoglobulin gene were constructed.

All vectors also contained the gene encoding the green fluorescent protein (GFP) to enable the fast efficient and non-toxic selection of transduced target cells by fluorescence activated cell sorting (FACS). FACS sorting is used to isolate the brightest 10% of GFP positive retrovirally transduced cells. Since both GFP and the angiogenic cDNA are translated from the same mRNA molecule, this ensures that the sorted cells also express high levels of the angiogenic protein. A strong correlation between the fraction of top GFP expressing cells and the amount of angiogenic protein secreted by the sorted cells exists. This result, in combination with data from Southern blot analysis of transduced cells which showed 3-6 proviral copies per genome in sorted GFP positive cells, indicates that the levels of angiogenic protein production and secretion are at their maximal level using this system.

All vectors were tested for stability of gene transfer and virus titer. Vectors demonstrated a virus titer ranging from approximately 5 x 10⁵ - 1.2 x 10⁶ infectious virus particles per ml and all vectors showed stable transfer of the angiogenic cDNA to target primary skeletal muscle myoblasts from C57B1/10 mice. To enable the easy localization and quantification of transduced myoblasts following injection in vivo, all myoblast samples were also marked by infection with a retroviral vector encoding a β- galactosidase/neomycin (β-GEO) resistance fusion gene.

I. Characterization of the Protein Expression and Secretion Properties of

Retrovirally Transduced C57B1/10 Myoblasts

High-level expression and secretion of the encoded angiogenic proteins from transduced myoblast cells was demonstrated by either Western blot or ELISA analysis of supernatants from virally transduced myoblasts.

Angiogenic Factor Amount of Protein Secreted (ng/1 x 10⁶ cells/24 hours)

VEGF-A 88

VEGF-C 790

VEGF-D 580

PDGF-BB 56 bFGF 20

EXAMPLE 2 - Quantification of Angiogenic Stimulation by Transduced Myoblasts

Using the Stringent Matrigel Assay

The potency of the angiogenic proteins secreted from the transduced myoblasts described in Example 1 was assessed in vivo using the stringent Matrigel assay. In brief, 3 x 10⁵ - 2 x 10⁶ transduced myoblasts were suspended in Matrigel and injected subcutaneously into the dorsal abdominal region of C57B1/10 mice. The Matrigel pellets, in addition to a section of abdominal muscle adjacent to the Matrigel pellet, were recovered 13 days post-injection. Following harvesting of the Matrigel pellet and the adjacent abdominal muscle 13 days post-injection, Matrigel pellets were stained with X- gal and analyzed for the presence of blue, retrovirally transduced myoblasts. In addition, the number of microvessels in the adjacent abdominal muscle was quantified by visual inspection.

A significant (p< 0.05) angiogenic response was observed for VEGF-A, VEGF- C, PDGF-BB, and bFGF (see Figures 1 and 2). Transplantation transduced myoblasts secreting the aforementioned growth factors resulted in approximately a 4-5-fold increase in the number of microvessels observed. The most potent angiogenic response was observed for PDGF-BB, followed by VEGF-A and bFGF (Figures 1 and 2).

EXAMPLE 3 - Assessment of the Angiogenic Potential of PDGF-BB using the Mouse Cornea Model

The potential of factors, such as PDGF-BB, as angiogenic agents was also investigated using the mouse cornea model. Corneal micropockets were created with a cataract knife in the eye of 8-week old C57B1/6 mice. Into this pocket, a 0.34mm X 0.34mm sucrose aluminum sulfate pellet coated with hydron polymer containing 160ng of recombinant human PDGF-BB, 160ng of human VEGF-A, or 80 ng of human bFGF was implanted and mice were monitored daily. While those mice implanted with control pellets showed no evidence of angiogenesis, all mice receiving PDGF-BB coated pellets showed evidence of potent angiogenesis (see Figure 3). Thus, recombinant PDGF-BB protein can potently stimulate the growth of new vessels in both the mouse Matrigel and corneal assays. In contrast to the results obtained using the Matrigel assay, PDGF-BB by itself, although inducing a clear and potent angiogenic response, was less potent than either VEGF-A or bFGF (Figure 3). The differences observed for the Matrigel and mouse corneas models could be explained by the production of additional endogenous angiogenic factors by transduced myoblasts that synergize more readily with PDGF-BB compared to VEGF-A. Alternatively, the levels of recombinant VEGF-A and PDGF- BB produced in vivo may differ to the levels produced in vitro. Unexpectedly, the combination of bFGF and PDGF-BB proved to be, by far, the most potent combination (Figure 5), producing an angiogenic effect many times greater than the any factor alone (Figure 3) or bFGF and VEGF-A combined (Figure 4). Moreover, the level of this synergistic effect appeared to be specific to PDGF-BB and bFGF since PDGF-BB combined with VEGF-A did not elicit nearly as potent an effect (see Figure 6). Therefore, the most potent combination of angiogenic factors observed was PDGF-BB and bFGF. EXAMPLE 4 - Therapeutic Effect of PDGF-BB Delivered Via Slow Release Microcapsules for the Treatment of Myocardial Ischemia

The following studies were performed to assess the ability of PDGF-BB to promote angiogenesis in vivo in the stringent Matrigel model and to improve cardiac function in ischemic rat models having myocardial infarction, using a slow release delivery system employing heparin-sepharose/alginate microcapsules. This delivery approach was based upon the ability of certain factors, such as bFGF and PDGF-BB, to bind strongly to heparin molecules both in vitro and in vivo. Sepharose beads coated with heparin (approximately 50-150μm in size) were purchased from Pharmacia. The beads were sterilized using UV irradiation and mixed with a 1.6% solution of alginate polymer. This polymer is able to form gels through chemical cross-linking with multivalent cations such as calcium. The procedure for making heparin-sepharose/alginate capsules, shown in Figure 7, was as follows. Sterilized heparin-sepharose beads were mixed with a 1.6% alginate solution and the mixture was loaded into a 5ml syringe. The mixture was then extruded through a needle and a mist of heparin sepharose/alginate, produced using a coaxial air flow system, dropped into a wash bath of 1.5% calcium chloride solution. Once the alginate hit the calcium solution, the alginate became cross-linked, forming a solid gel capsule in the shape of a sphere. Once formed, the capsules were forced through a 250μm sieve, washed twice in sterile water and stored in buffer composed of 0.9% sodium chloride and ImM calcium chloride. Visual analysis of the capsules under the microscope showed that the vast majority of microcapsules were composed of individual heparin sepharose beads coated with a thin layer of alginate. Heparin-sepharose/alginate microcapsules were incubated overnight at 4 degrees

Celcius in binding buffer composed of 0.9% sodium chloride, ImM calcium chloride, 0.05% gelatin and lOμg of recombinant PDGF-BB for 16 hours. The next day, the binding buffer was removed from the microcapsules and analyzed by ELISA to quantify the amount of PDGF-BB absorbed by the capsules. In a typical experiment, approximately 75-90% of the PDGF-BB protein is absorbed by the microcapsules (see Figure 8). Next, the heparin-sepharose/alginate microcapsules were washed twice in fresh binding buffer and either placed in vitro to assess release kinetics or injected into the myocardium of rats that had undergone surgically induced myocardial infarction. To assess the release kinetics of the bound PDGF-BB in vitro, three thousand heparin-sepharose/alginate microcapsules or three thousand non-alginate encapsulated heparin sepharose beads containing 9μg of bound PDGF-BB were placed in serum free medium and incubated at 37 degrees Celcius. Every 24 hours the medium was changed and the amount of PDGF-BB present in the medium quantified by ELISA. The results showed a slow, sustained release of approximately 0.5-3% of the total bound PDGF-BB for a minimum of 14 days, the longest time point analyzed (see Figure 9). Importantly, the proportion of PDGF-BB released per day was equivalent to the amount of PDGF-BB that we estimated to be secreted by muscle cells transduced with the PDGF-BB retrovirus in the Matrigel experiments described in Example 1.

The ability of the heparin-sepharose/alginate microcapsules to stimulate angiogenesis in vivo was assessed using the stringent Matrigel assay. Three thousand microcapsules loaded with 1 μg or 1 Oμg of PDGF-BB were mixed with 400μl of Matrigel and subcutaneously injected into the abdominal region of C57B1/10 mice. Thirteen days later mice were sacrificed, the pellets and a section of the adjacent abdominal muscle was removed, fixed, sectioned and the number of microvessels quantified by visual inspection of the sections under the microscope. As shown in Figure 10, the number of microvessels in mice receiving microcapsules loaded with lOmg of PDGF-BB was 2.5-fold greater than that of control mice.

In addition, as shown in Figure 13, PDGF-BB and bFGF delivered by slow release microcapsules synergize to stimulate angiogensis in vivo in the stringent Matrigel model. Three thousand microcapsules loaded with lμg of bFGF were mixed with 400μl of Matrigel and subcutaneously injected into the abdominal region of

C57B1/10 mice. Thirteen days later mice were sacrificed, the pellets and a section of the adjacent abdominal muscle was removed, fixed, sectioned and the number of microvessels quantified by visual inspection of the sections under the microscope. Figure 13 shows that the number of microvessels in mice receiving bFGF + PDGF-BB microcapsules was 4-fold greater than that of mice implanted with either growth factor alone.

PDGF-BB microcapsules were also tested for their ability to stimulate angiogenesis in infarcted rat hearts 3 weeks post-injection. Infarcted rat hearts were injected with 1600 microcapsules containing μg (control) or 18μg of PDGF-BB in a volume of 20μl. Three weeks post injection rats were sacrificed, hearts were removed, fixed, sectioned and the number of microvessels within the infarct region quantified by visual inspection under a microscope (i.e., number of microvessels per 5 high power fields for recipients of control and PDGF-BB microcapsules). As shown in Figure 11, rats injected with PDGF-BB microvessels showed an approximate 2-fold increase in the number of microvessels as compared to control rats. The ability of the heparin-sepharose/alginate microcapsules to stimulate angiogenesis in vivo was also assessed using the ischemic rat heart model as follows. Adult male rats were anesthetized, intubated and ventilated with a Harvard respirator. Under sterile conditions, a left lateral thoractomy was performed. The heart was exposed and the left descending coronary artery was ligated with a 8-0 Prolene suture.

Immediately after infarction each heart was injected twice intramyocaridally with lOμl of a buffer suspension containing approximately 800 heparin sepharose/alginate microcapsules with 9μg of absorbed recombinant human PDGF-BB protein. Thus, a total of 1600 microcapsules containing 18μg of human PDGF-BB protein were injected into each rat heart. The lungs were then inflated and the wound was closed in layers. Three weeks later, cardiac function was assessed using a variety of parameters including left ventricular pressure (LVP), dP/dT (a measure of cardiac contractility), negative dP/dT (a measure of relaxation of the cardiac muscle) and tau (the relaxation constant) (see Figure 12). In rats injected with PDGF-BB microcapsules, a 25% increase in left ventricular pressure was detected (see Figure 12). Moreover, cardiac contractility /relaxation increased 2.5 - 3 -fold while the relaxation constant, tau, was decreased by approximately 3 -fold (Figure 12). Thus, a significant improvement in all parameters was detected in rats injected with PDGF-BB microcapsules.

EXAMPLE 5 - Directed Delivery of Angiogenic Factors Using Biocompatible Threads

Regulating the growth of new vessels from existing vasculature (angiogenesis) that effectively bypass an arterial lesion requires strict spatial and temporal control.

Angiogenic factors, such as those described in the preceding examples, work by providing a gradient of angiogenic factor that stimulates the chemotaxis and proliferation of endothelial cells, and their supporting cells towards the source of the factor.

Today, most efforts to stimulate the growth of new vessels involve the injection of proteins into or near affected areas. Such injections can result in the spread of the angiogenic factor over a large area, greatly diminishing their efficacy, and can dilute the angiogenic stimulus over a large region resulting in an unorganized hodge-podge of new vessels that do not provide any therapeutic benefit.

To solve this problem, biocompatible absorbable threads containing angiogenic factors can be employed to provide a small, highly localized and orderly gradient of angiogenic factors in the appropriate and crucial areas. This enables the creation of a

"molecular road map" that directs the growth of new vessels from around the site of the arterial occlusion to join again at a point below or downstream of the blockage. To achieve this, absorbable surgical threads can be coated with the appropriate angiogenic factors (e.g., PDGF-BB, FGF-2, VEGF-A and PDGF-B). Such threads can then be surgically placed at the site of arterial occlusion such that they provide a clear spatial direction and gradient of angiogenic factor(s) to direct the generation of new vessels around the block.

The biocompatible threads can be coated with one or several angiogenic factors to allow delivery of growth factor or growth factor combinations that provide the optimal angiogenic stimulus. Moreover, such threads can be engineered to release certain growth factors at specific rates and at specific times that may help mimic the natural angiogenic process more closely.

EXAMPLE 6 — Treatment of Ischemia Using Angiogenic Expression Plasmids

Figure 14 shows a variety of expression plasmids encoding angiogenic factors that can be administered directly to localized areas of tissue to promote angiogenesis. To show that the plasmids encode biologically active PDGF-BB protein, supernatant from 293T cells transiently transfected with the PDGF-BB expression plasmid was added to NIH 3T3 cells growing under serum free conditions. Seventy two hours later cells were trypsinized, spun down and counted using a hemocytometer. The results showed that the PDGF-BB supernatant specifically and potently induced the proliferation of NIH3T3 cells.

To analyze cardiac function following administration of expression plasmids in vivo, test animals (e.g., rats) can be injected with control vs. angiogenic plasmids (e.g., PDGF-BB expression plasmids) following myocardial infarction. To achieve this, test animals can be anesthetized and intubated. The chest wall is opened and a myocardial infarct is created by tying off the anterior descending artery. 180μg of control or test expression plasmid is injected into the heart wall in a volume of 20μl. Cardiac function is assessed 3 weeks post injection. Animals are sacrificed, the heart is removed and efficiency of plasmid uptake is assessed by staining with X-gal. The size of the infarct and the extent of angiogenesis is quantified.

Claims

What is claimed is:

1. A method of promoting angiogenesis comprising delivering PDGF-BB to a localized area of tissue in an amount effective to induce angiogenesis within the area of tissue.

2. The method of claim 1, wherein the PDGF-BB is delivered in combination with one or more other angiogenesis-promoting factors.

3. The method of claim 2, wherein said one or more other angiogenesis- promoting factors is selected from the group consisting of PDGF-AA, M-CSF, GM- CSF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, neuropilin, FGF-1 , FGF- 2(bFGF), FGF-3, FGF-4, FGF-5, FGF-6, Angiopoietin 1, Angiopoietin 2, erythropoietin, BMP-2, BMP-4, BMP-7, TGF-beta, IGF- 1 , Osteopontin, Pleiotropin, Activin, Endothelin-1 and combinations thereof.

4. The method of claim 1 or 2, wherein the angiogenesis-promoting factor(s) is administered locally in the form of a protein composition.

5. The method of claim 1 or 2, wherein the angiogenesis-promoting factor(s) is delivered in association with a polymer.

The method of claim 5, wherein the polymer comprises a matrix.

7. The method of claim 6, wherein the matrix is selected from the group consisting of heparin sepharose/alginate, chitosan/tricalcium phosphate sponge, poly- lactide-glycolide sponge, polylactide glycolic mesh, methyl cellulose, polysulfone, extrudable ethylene vinyl acetate, alginate/poly-L-lysine/alginate and agarose/poly-L- lysine/alginate.

8. The method of claim 1 or 2, wherein the angiogenesis-promoting factor(s) is delivered by expression from isolated DNA encoding the factor following delivery of the DNA to the localized area of tissue.

9. The method of claim 8, wherein the isolated DNA is contained within a vector.

10. The method of claim 8, wherein the DNA is delivered in an adenoviral vector, retroviral vector, adeno-associated viral vector, RNA vector, liposome, cationic lipid, lentiviral vector, AAV or transposon.

11. The method of any one of claims 1-10, wherein the induction of angiogenesis is used to treat ischemia.

12. A method for promoting angiogenesis comprising contacting a localized area of tissue with heparin sepharose-containing microcapsules in an amount effective to induce angiogenesis within the area of tissue.

13. The method of claim 12, wherein the microcapsules comprise heparin sepharose in a form selected from the group consisting of heparin sepharose beads, heparin sepharose beads coated with a single layer of alginate polymer, heparin sepharose coated with poly-ethylene glycol (PEG) polymer, and heparin sepharose beads coated with alternating layers of alginate and PEG.

14. The method of claim 12, wherein the microcapsules range in size from 1- 250 microns.

15. The method of claim 13 , wherein the heparin sepharose beads encapsulate an angiogenic factor selected from the group consisting of M-CSF, GM- CSF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, neuropilin, FGF-1 , FGF- 2(bFGF), FGF-3, FGF-4, FGF-5, FGF-6, PDGF-BB, PDGF-AA, Angiopoietin 1,

Angiopoietin 2, erythropoietin, BMP-2, BMP-4, BMP-7, TGF-beta, IGF-1, Osteopontin, Pleiotropin, Activin, Endothelin-1 and combinations thereof, or an expression vector encoding said angiogenic factor.

16. The method of claim 15, wherein the angiogenic factor is purified from its native source or produced by recombinant expression.

17. The method of any one of claims 12-16, wherein the microcapsules are contacted with the localized area of tissue by injection or surgical implantation.

18. The method of claim 17, wherein the injection is performed using a catheter based trans-myocardial injection technology (ie. NOGA).

19. The method of claim 15, wherein the angiogenic factor is control released from the microcapsule into the localized area of tissue.

20. The method of any one of claims 12-19, wherein the induction of angiogenesis is used to treat ischemia.

21. A method for promoting angiogenesis comprising contacting a localized area of tissue with a gradient of one or more angiogenic factors or a nucleic acid encoding one or more angiogenic factors, such that directed vascular growth along the gradient is achieved.

22. The method of claim 21 , wherein the directed vascular growth results in interconnection of blood vessels.

23. The method of claim 21 , wherein the directed vascular growth results in intraconnection of blood vessels.

24. The method of claim 21 , wherein the directed vascular growth circumvents blood flow around a blockage within a blood vessel.

25. The method of claim 21, wherein the angiogenic factor or nucleic acid is released in a gradient from a biocompatible material contacted with the localized area of tissue.

26. The method of claim 25, wherein the biocompatible material is a polymer or thread which incorporates the angiogenic factor.

27. The method of claim 26, wherein the biocompatible material comprises an absorbable thread.

28. The method of claim 27, wherein the thread comprises a material selected from the group consisting of polyglyconate monofilament, poliglecaprone 25- (Monocryl), polydiaxonone (PDS II), polyglactin 910, polyglycolic acid, Biodyn gly comer 631 , chromic surgical gut and plain surgical gut.

29. The method of claim 25, wherein the biocompatible material is implanted into the localized area of tissue.

30. The method of claim 21 , wherein the nucleic acid is contained in an 5 adenoviral vector, retroviral vector, adeno-associated viral vector, RNA vector, liposome, cationic lipid, lentiviral vector, AAV or transposon.

31. The method of claim 21 , wherein the one or more angiogenic factors is selected from the group consisting of M-CSF, GM-CSF, VEGF-A, VEGF-B, VEGF-C,

10 VEGF-D, VEGF-E, neuropilin, FGF-1, FGF-2(bFGF), FGF-3, FGF-4, FGF-5, FGF-6, PDGF-BB, PDGF-AA, Angiopoietin 1, Angiopoietin 2, erythropoietin, BMP-2, BMP-4, BMP-7, TGF-beta, IGF-1, Osteopontin, Pleiotropin, Activin, Endothelin-1 and combinations thereof.

15 32. The method of any one of claims 21-31, wherein the induction of angiogenesis is used to treat ischemia.

33. A method for promoting angiogenesis comprising: applying one or more angiogenic factors, or a nucleic acid encoding one or more 20 angiogenic factors, to a biocompatible material to form a treated material; and contacting the treated material with a localized area of tissue, such that the angiogenic factor or nucleic acid is released into the surrounding tissue in a directed gradient.

25.

34. The method of claim 33, wherein the biocompatible material is an absorbable thread.

35. The method of claim 34, wherein the thread is surgically implanted into the localized area of tissue.

30

36. The method of claim 33, wherein the one or more angiogenic factors includes PDGF-BB.