WO1999048537A1

WO1999048537A1 - Use of scatter factor to enhance angiogenesis

Info

Publication number: WO1999048537A1
Application number: PCT/US1999/006452
Authority: WO
Inventors: Itzhak D. Goldberg; Eliot M. Rosen
Original assignee: Long Island Jewish Medical Center
Priority date: 1998-03-26
Filing date: 1999-03-26
Publication date: 1999-09-30
Also published as: JP2002507584A; CA2326053A1; MXPA00009440A; EP1066061A1; AU3202999A; EP1066061A4

Abstract

This invention relates to a method of enhancing wound healing and to a method of enhancing organ transplantation utilizing scatter factor, either alone or in combination with a growth factor.

Description

USE OF SCATTER FACTOR TO ENHANCE ANGIOGENESIS

Statement of Government Interest This invention was made with government support under NIH Grant No. CA50516. As such, the government has certain rights in the invention.

Field of the Invention This invention relates to a method of enhancing wound healing and to a method of enhancing organ transplantation comprising the administration of scatter factor to promote angiogenesis .

Background of the Invention Scatter factor has previously been described as a cytokine which is secreted by fibroblasts (see Stoker et al., J. Cell Sci . , Vol. 77, pp. 209-223 (1985) and Stoker et al., Nature (London), Vol. 327, pp. 238-242 (1987)) and by vascular smooth muscle cells (see Rosen et al . , In Vi tro Cell Dev. Biol . , Vol. 25, pp. 163-173 (1989)). Scatter factor has been shown to disperse cohesive epithelial colonies and stimulate cell motility. In addition, scatter factor has been shown to be identical to hepatocyte growth factor (HGF) (see eidner et al . , Proc. Nat ' 2 . Acad. Sci . USA, Vol. 88, pp. 7001-7005 (1991) and Bhargava et al . , Cell Growth Differ. , Vol. 3, pp. 11-20

(1992) ) , which is an independently characterized serum mitogen (see Miyazawa et al . , Biochem . Biophys . Res .

Coromun . , Vol. 169, pp. 967-973 (1989) and Νakamura et al . ^'

Nature (London) , Vol. 342, pp. 440-443 (1989)). Scatter factor induces kidney epithelial cells in a collagen - 2 - matrix to form branching networks of tubules, suggesting that it can also act as a morphogen (see Montesano et al . , Cell , Vol. 67, pp. 901-908 (1991)).

Scatter factor (HGF) is a basic heparin-binding glycoprotein consisting of a heavy (58 kDa) and a light (31 kDa) subunit . It has 38% amino acid sequence identity with the proenzyme plasminogen (see Nakamura et al . , Nature (London ) , Vol. 342, pp. 440-443 (1989)) and is thus related to the blood coagulation family of proteases. Its receptor in epithelium has been identified as the c-met protooncogene product, a transmembrane tyrosine kinase (see Bottaro et al . , Science, Vol. 251, pp. 802-804 (1991) and Νaldini et al . , Oncogene, Vol. 6, pp. 501-504 (1991)). Scatter factor has been found to stimulate endothelial chemotactic and random migration in Boyden chambers (see Rosen et al . , Proc . Soc . Exp . Biol . Med . , Vol. 195, pp. 34-43 (1990)); migration from carrier beads to flat surfaces (see Rosen et al . , Proc . Soc . Exp . Biol . Med. , Vol. 195, pp. 34-43 (1990)); formation of capillary-like tubes (see Rosen et al . , Cell Motili ty Factors , (Birkhauser, Basel) pp. 76-88 (1991)) and DΝA synthesis (see Rubin et al . , Proc . Nat ' l . Acad . Sci . USA, Vol. 88, pp. 415-419 (1991)). In addition, preliminary studies have suggested that scatter factor induces endothelial secretion of plasminogen activators (see Rosen et al., Cell Motili ty Factors, (Birkhauser, Basel) pp. 76-88 (1991) ) .

The term "angiogenesis" , as used herein, refers to the formation of blood vessels. Specifically, angiogenesis is a multistep process in which endothelial cells focally degrade and invade through their own - 3 - basement membrane, migrate through interstitial stroma toward an angiogenic stimulus, proliferate proximal to the migrating tip, organize into blood vessels, and reattach to newly synthesized basement membrane (see Folkman et al . , Adv. Cancer Res . , Vol. 43, pp. 175-203 (1985)). These processes are controlled by soluble factors and by the extracellular matrix (see Ingber et al . , Cell , Vol. 58, pp. 803-805 (1985) ) .

Because proteases, such as plasminogen activators (the endothelial secretion of which is induced by scatter factor) are required during the early stages of angiogenesis, and since endothelial cell migration, proliferation and capillary tube formation occur during angiogenesis, the inventors hypothesized that scatter factor might enhance angiogenic activity in vivo . In addition, it is desirable to enhance angiogenic activity so that wound healing and organ transplantation can be enhanced.

It is therefore an object of this invention to provide a method of enhancing angiogenic activity.

It is a further object of this invention to provide a method of enhancing wound healing.

It is a still further object of this invention to provide a method of enhancing organ transplantation.

Summary of the Invention This invention is directed to a method of promoting angiogenesis by administration of scatter factor. The promotion of angiogenesis can be used for promoting wound healing and treating various conditions where the - 4 - promotion of angiogenesis is desirable, including, but not limited to, ischemia.

Brief Description of the Drawings The above brief description, as well as further objects and features of the present invention, will be more fully understood by reference to the following detailed description of the presently preferred, albeit illustrative, embodiments of the present invention when taken in conjunction with the accompanying drawing wherein:

Figures 1A-1C. Figures 1A-1C show the effects of SF/HGF gene transfer on rat myocardium. Figure 1A shows immunostaining with monoclonal antibodies directed against human SF/HGF in myocardial tissue 5 days following infarction and gene transfection. Figure IB and 1C show myocardial tissue from SF/HGF-treated and control animals, respectively, treated with anti -endothelial CD-34 antibody and stained using peroxidase-based immunohistochemistry .

Detailed Description of the Invention The present invention is directed to a method of promoting angiogenesis in a tissue or subject by administering scatter factor to a subject in need of angiogenesis promotion. Specifically, the method provided by the present invention involves the administration of scatter factor to promote angiogenesis in various tissues to promote wound healing,

The administration of scatter factor may be effected by administration of the protein itself or administration - 5 - of a nucleic acid encoding scatter factor by the use of standard DNA techniques .

Scatter factor protein may be administered to a tissue or subject topically or by intravenous, intramuscular, intradermal, subcutaneous or intraperitoneal injection. Scatter factor protein is administered in amounts sufficient to promote angiogenesis in a subject, which is in the amount of about .1-1000 ng/kg body weight . Scatter factor protein may be administered as the wild type scatter factor protein, or analogues thereof, and may be produced synthetically or recombinantly, or may be isolated from native cells. As used herein, "analogue" means functional variants of the wild type protein, and includes scatter factor protein isolated from mammalian sources other than human, such as mouse, as well as functional variants thereof.

A nucleic acid sequence encoding scatter factor administered to a mammal may be genomic DNA or cDNA. The nucleic acid sequence may be administered using a number of procedures known to one skilled in the art, such as electroporation, DEAE Dextran, monocationic liposome fusion, polycationic liposome fusion, protoplast fusion, DNA coated microprojectile bombardment, by creation of an in vivo electrical field, injection with recombinant replication-defective viruses, homologous recombination, and naked DNA transfer. It is to be appreciated by one skilled in the art that any of the above methods of DNA transfer may be combined. A nucleic acid encoding scatter factor may also be administered to a mammal using gene therapy, i.e. by the - 6 - administration of a recombinant vector containing a nucleic acid sequence encoding scatter factor. The nucleic acid sequence may be, for example, genomic DNA or cDNA. The recombinant vector containing nucleic acid encoding scatter factor may be administered to a mammal using any number of procedures known to one skilled in the art, including, but not limited to, electroporation, DEAE Dextran transfection, calcium phosphate transfection, cationic liposome fusion, protoplast fusion, by creation of an in vivo electrical field, DNA coated microprojectile bombardment, injection with recombinant replication- defective viruses, homologous recombination, gene therapy, and naked DNA transfer. It is to be appreciated by one skilled in the art that any of the above methods of nucleic acid transfer may be combined. Accordingly, a cell, such as a stem cell or a tumor cell which expresses scatter factor introduced therein through viral transduction, homologous recombination, or transfection is also provided by the present invention. This cell may then be administered to a subject to promote angiogenesis.

The recombinant vector may comprise a nucleic acid of or corresponding to at least a portion of the genome of a virus, where this portion is capable of directing the expression of a nucleic sequence encoding scatter factor, operably linked to the viral nucleic acid and capable of being expressed as a functional gene product in the subject mammal. The recombinant vectors may be derived from a variety of viral nucleic acids known to one skilled in the art, e.g. the genomes of HSV, adenovirus, adeno- associated virus, Semiliki Forest virus, vaccinia virus, and other viruses, including RNA and DNA viruses. - 7 -

The recombinant vectors may also contain a nucleotide sequence encoding suitable regulatory elements so as to effect expression of the vector construct in a suitable host cell. As used herein, "expression" refers to the ability of the vector to transcribe the inserted DNA sequence into mRNA so that synthesis of the protein encoded by the inserted nucleic acid can occur. Those skilled in the art will appreciate that a variety of enhancers and promoters are suitable for use in the constructs of the invention, and that the constructs will contain the necessary start, termination, and control sequences for proper transcription and processing of the nucleic acid sequence encoding scatter factor when the recombinant vector construct is introduced into a mammal. Vectors suitable for the expression of the nucleic sequence encoding scatter factor are well known to one skilled in the art and include pMEX, pRSX24 (provided by Dr. George Vande oude, Frederick Cancer Center, Frederick, Maryland) , pSV2neo (Clonetech) , pET-3d (Novagen) , pProEx-1 (Life Technologies) , pFastBac 1 (Life

Technologies) , pSFV (Life Technologies) , pcDNA II

(Invitrogen) , pSL301 (Invitrogen) , pSE280 (Invitrogen) , pSE380 (Invitrogen), pSE420 (Invitrogen), pTrcHis A,B,C

(Invitrogen), pRSET A,B,C (Invitrogen), pYES2 (Invitrogen) , pAC360 (Invitrogen) , pVL1392 and pVll392

(Invitrogen) , pCDM8 (Invitrogen) , pcDNA I (Invitrogen) , pcDNA I (amp) (Invitrogen), pZeoSV (Invitrogen), pcDNA 3

(Invitrogen) , pRc/CMV (Invitrogen) , pRc/RSV (Invitrogen) , pREP4 (Invitrogen) , pREP7 (Invitrogen) , pREP8 (Invitrogen) , pREP9 (Invitrogen) , pREPIO (Invitrogen) , - 8 - pCEP4 (Invitrogen), pEBVHis (Invitrogen), and λPopδ . Other vectors would be apparent to one skilled in the art. Suitable promoters include, but are not limited to, constitutive promoters, tissue specific promoters, and inducible promoters. Expression of the nucleic acid sequence encoding scatter factor may be controlled and affected by the particular vector into which the nucleic acid sequence has been introduced. Some eukaryotic vectors have been engineered so that they are capable of expressing inserted nucleic acids to high levels within the target cell. Such vectors utilize one of a number of powerful promoters to direct the high level of expression. Eukaryotic vectors use promoter-enhancer sequences of viral genes, especially those of tumor viruses. A particular embodiment of the invention provides for regulation of expression of the nucleic acid sequence encoding scatter factor using inducible promoters. Non- limiting examples of inducible promoters include, but are not limited to, metallothionine promoters and mouse mammary tumor virus promoters. Depending on the vector, expression of the nucleic acid sequence encoding scatter factor would be induced in the mammal by the addition of a specific compound at a certain point in the growth cycle of the cells of the mammal. Other examples of promoters and enhancers effective for use in the recombinant vectors include, but are not limited to, CMV (cytomegalovirus) , SV40 (simian virus 40) , HSV (herpes simplex virus) , EBV (epstein-barr virus) , retroviral, adenoviral promoters and enhancers, and tumor cell specific promoters and enhancers. - 9 -

It is within the confines of the invention that scatter factor may be administered in combination with a growth factor to promote angiogenesis, including, but not limited to TGF-α, FGF and PDGF . Scatter factor, in the form of a protein, nucleic acid, or a recombinant vector containing nucleic acid encoding scatter factor, may be administered to a subject prior to, simultaneously with or subsequent to administration of a growth factor. For the purposes of gene transfer into a tissue or subject, a recombinant vector containing nucleic acid encoding scatter factor may be combined with a sterile aqueous solution which is preferably isotonic with the blood of the recipient . Such formulations may be prepared by suspending the recombinant vector in water containing physiologically compatible substances such as sodium chloride, glycine, and the like, and having buffered pH compatible with physiological conditions to produce an aqueous solution, and rendering such solution sterile. In a preferred embodiment of the invention, the recombinant vector is combined with a 20-25% sucrose in saline solution in preparation for introduction into a mammal.

The amounts of nucleic acid encoding scatter factor, or nucleic acid encoding scatter factor contained in a vector are administered in amounts sufficient to promote angiogenesis in a subject. However, the exact dosage will depend on such factors as the purpose of administration, the mode of administration, and the efficacy of the composition, as well as the individual pharmacokinetic parameters of the subject. Such therapies may be - 10 - administered as often as necessary and for the period of time as judged necessary by one of skill in the art.

Non-limiting examples of tissues into which nucleic acid encoding scatter factor may be introduced to promote angiogensis include fibrous, endothelial, epithelial, vesicular, cardiac, cerebrovascular, muscular, vascular, transplanted, and wounded tissues.

Transplanted tissues are, for example, heart, kidney, lung, liver and ocular tissues. The tissues into which nucleic acid encoding scatter factor may be introduced to promote angiogensis include those associated with diseases or conditions selected from the group consisting of ischemia, circulatory disorders, vascular disorders, myocardial ischemic disorders, myocardial disease, pericardial disease or congenital heart disease. Non-limiting examples of ischemia are myocardial ischemia, cerebrovascular ischemia and veno- occlusive disorder. An example of myocardial ischemia is coronary artery disease. In further embodiments of the invention, scatter factor is used to enhance wound healing, organ regeneration, and organ transplantation, including the transplantation of artificial organs. In addition, scatter factor can be used to accelerate endothelial cell coverage of vascular grafts in order to prevent graft failure due to reocclusion, and to enhance skin grafting. Further, antibodies to scatter factor can be used to treat tumors and to prevent tumor growth.

The present invention is described in the following Examples which are set forth to aid in the understanding of the invention, and should not be construed to limit in - 11 - any way the scope of the invention as defined in the claims which follow thereafter.

Experimental Details Preparation of pRSX24 Plasmid. The plasmid pRSX24 was constructed by ligating full-length 2.3 kb SF cDNA into the BamHI-Kpnl site of the pMEX vector, and was provided by Dr. George Vande Woude (Frederick Cancer Center, Frederick, MD) . Expression of Scatter Factor in normal ischemic tissue . In order to ascertain potential expression of HGF using the plasmid PRSX24 on normal ischemic tissue, Sprague-Dawley rats weighing 210-300 were anesthetized, intubated and placed on a positive-pressure respirator. The left coronary artery was ligated 3-4 millimeters from its origin to produce myocardial infarction, and at the same time, the apices of the heart were injected with 40 micrograms of the PRSX24 (HSF) plasmid.

Expression analysis was performed using antihuman HGF monoclonal antibodies by analyzing cross-sectional sections of the apex of rat hearts using immunochemistry techniques. Five days following injection of the plasmid, positive staining was seen in the myocardium which supports expression of HSF in the tissue. Effects of SF/HGF Gene Transfer on Rat Myocardium.

In order to determine the effect of SF/HGF gene transfer on rat myocardium, male Sprague-Dawley rats were treated to induce myocardial ischemia by ligation of the left coronary artery, and 40 μg of control or SF/HGF plasmid was injected into the apex of the heart. Figure 1A shows immunostaining with monoclonal antibodies directed against - 12 - human SF/HGF in myocardial tissue 5 days following infarction and gene transfection; staining patterns were significantly more dense in group treated with SF/HGF plasmid than controls (not shown) . Figures IB and 1C show myocardial tissue from SF/HGF-treated and control animals, respectively, treated with anti-endothelial CD-34 antibody (Becton Dickenson) and stained using peroxidase-based immunohistochemistry (Ventana Medical Systems) ; increased vascularity is seen in the myocardium of SF/HGF-treated animals 20 days following surgery and transfection.

Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of various aspects of the invention. Thus, it is to be understood that numerous modifications may be made in the illustrative embodiments and other arrangements may be devised without departing from the spirit and scope of the invention.

Claims

- 13 -hat is claimed is:

1. A method for promoting angiogenesis in a tissue comprising introducing nucleic acid encoding scatter factor to the tissue so that the scatter factor is expressed in an amount effective to promote angiogenesis in the tissue.

2. The method of Claim 1, wherein the nucleic acid is introduced by a method selected from the group consisting of electroporation, DEAE Dextran transfection, calcium phosphate transfection, cationic liposome fusion, protoplast fusion, by creation of an in vivo electrical field, DNA coated microprojectile bombardment, injection with recombinant replication-defective viruses, homologous recombination, gene therapy, and naked DNA transfer.

3. The method of Claim 1, wherein the tissue is selected from the group consisting of fibrous, endothelial, epithelial, vesicular, cardiac, cerebrovascular, muscular, vascular, transplanted, or wounded .

4. The method of Claim 3, wherein the tissue is associated with diseases or conditions selected from the group consisting of ischemia, circulatory disorders, vascular disorders, myocardial ischemic disorders, myocardial disease, pericardial disease or congenital heart disease. - 14 -

5. The method of Claim 4, wherein the ischemia is myocardial ischemia, cerebrovascular ischemia, or veno- occlusive disease.

6. The method of Claim 5, wherein the myocardial ischemia is coronary artery disease.