WO1994005334A1

WO1994005334A1 - Vascular in vivo expression of macrophage colony stimulating factor

Info

Publication number: WO1994005334A1
Application number: PCT/US1993/008282
Authority: WO
Inventors: John M. Taylor; Robert W. Mahley; Stephen J. Lauer; Tammy K. Ramos
Original assignee: The Regents Of The University Of California
Priority date: 1992-09-02
Filing date: 1993-09-02
Publication date: 1994-03-17
Also published as: AU4845093A

Abstract

Disclosed are the materials and methods enabling the transfection of vascular cells with DNA encoding macrophage colony stimulating factor (MCSF). The transfected cells are transplanted into vascular tissue in vivo such that MCSF is produced as an expression product for local therapeutic effect.

Description

VASCULAR IN VIVO EXPRESSION OF MACROPHAGE COLONY STIMULATING FACTOR

Field of the Invention

The present invention is directed to the medical application of research on the local administration of macrophage colony stimulating factor (MCSF) . The present invention provides local administration of MCSF via vascular in vivo expression of DNA encoding MCSF. The DNA is contained within transfected vascular cells which are then transplanted into vascular tissue and incorporated therein.

Background of the Invention

Macrophage colony stimulating factor (MCSF) is a member of a family of colony stimulating growth factors that are characterized as hematopoietins . Human MCSF has been successfully cloned and expressed, and its characteristics are identified in detail. See, for example Wong, et al . Science 235, 1504 (1987), U.S. Patent 4868119 (1989) , and U.S. Patent 4879227 (1989) , which are hereby expressly incorporated by reference.

Oxidized lipoproteins have been identified in atherosclerotic plaques and lesions in humans. Treatment of endothelial cells with modified low density lipoproteins results in induction of the expression of MCSF as well as other factors. See Rajavashisth, et al . Nature 34 , 254 (1990) . Researchers have observed a decrease in total plasma cholesterol when rabbit models were administered systemically recombinant human MCSF. See Stoudemire, et al . , Blood 77, 750 (1991) . See also U.S. Patent 5021239. Shimano et al . The Journal of Biological Chemistry 265, 12869 (1990) reported that MCSF stimulates the clearance of lipoproteins and reduces plasma cholesterol .

Inaba et al . , The Journal of Biological Chemistry 267, 5693 (1992) , demonstrated expression of c-fms and high affinity binding of MCSF in smooth muscle cells isolated from a rabbit model of arteriosclerosis.

There is no suggestion in the literature to administer MCSF locally, and no prediction of what effects such local administration of MCSF may have.

Summary of the Invention

The present invention provides the unexpected results of research on the local administration of MCSF vascularly. Vascular cells were isolated and transfected with an expression vector that operatively harbored DNA encoding MCSF. Those cells were transplanted vascularly. The resultant tissue at the site of transplantation were thus made capable of providing local administration of MCSF.

The present invention is thus directed to a method of transplantation of mammalian vascular cells. The method comprises isolating vascular cells from a mammal, transfecting them with an expression vector capable of expressing MCSF and transplanting these transfected vascular cells into the vascular tissue of a mammalian host. The site of the transplantation is preferably a site of atherosclerosis or restenosis, and a particularly preferred method would involve autologous transplantation in order to minimize immune response.

The method is useful, for example, in vascular surgery, particularly in arterial vessels, as a means of supplying the therapeutic effects of MCSF locally. The envisioned therapy would include the method of the present invention concomitant with coronary bypass surgery or balloon catheterization of the coronary arteries, or virtually any surgery that would involve resection of vascular walls.

The present invention is further directed to the vascular transplants and to vascular tissue that has been transplanted with vascular cells comprising an expression vector capable of expressing a DNA sequence encoding MCSF.

Further, the present invention is directed to a method for assaying the effects of the local administration of MCSF in proximate vascular tissue. This method would comprise providing or generating a mammal having vascular tissue transplanted with vascular cells that have been transfected with DNA encoding MCSF. The assay would thus provide a means for studying the effect of the local administration of MCSF vascularly by monitoring the deposition and accumulation of cholesterol in the proximate vascular tissue or the production and maintenance of atherosclerotic plaques in the proximate vascular tissue and/or the occurrence and severity of restenosis in the proximate vascular tissue.

The present invention is directed to the foregoing aspects in all of their associated embodiments including all analogous aspects and equivalents. Detailed Description

Preferred Embodiments

Disclosed herein are the methods and results of the research providing the present invention. Having provided that information as a preferred embodiment, it will be well enough understood by those of relevant ordinary skill in the art that one could make modifications to the procedures described consistent with evolving biomedical skill and knowledge. Further, one of relevant ordinary skill having possession of the present information would be capable of extending the present research so as to include analogous processes/results and bioequivalents of the procedures and materials described herein as a preferred embodiment.

For example, the particular rabbit model used herein as an atherosclerotic rabbit model could be extended to other animals, and as it is considered a proper model for the research results provided herein, the results herein with the rabbit model are translatable to humans. Thus, the present invention is properly applicable to therapeutic use in human beings.

Further, the present invention has been demonstrated with polypeptide product (probably processed product from the full length 522 amino acid encoded species) produced from expression of the about 1-1823 bp DNA of Wong et al . , Supra. One of these products has been referred to as the 189 amino acid mature form of human MCSF. One could enablingly apply the present results using other polypeptide sequences encompassed within the sequence (SEQ ID NO: 2) given in Figure 1 hereof. Further, one could use other polypeptides having essential MCSF-like bioactivity. Attention is directed to U.S. Patents 4868119 and 4879227 for details on the identification of such polypeptides and how they could be produced in isolated useful form for use in the present invention.

Other cytokines that modulate the action of MCSF could be used either individually or in a co-transfection with MCSF. These other cytokines could potentiate the action of MCSF to augment or regulate its local physiological action. The use of an interferon could be considered a preferred embodiment of this aspect. The suppression of atherosclerosis by interferon is discussed in Kuo et al . , J. Amer. Coll. Cardiol . 3., 129 (1984) and Wilson et al . , Atherosclerosis 10, 208 (1990) .

A particular vector was employed herein as a vehicle to transfect vascular cells. It will be understood that other suitable vectors could usefully be so employed, and their preparation and use have been well documented in the literature. Attention in this regard is directed for example to Miller et al . Biotechniσues 7, 980 (1989) and Danos et al . Proc . Natl . Acad. Sci. , USA 85, 6460 (1988) , which are hereby expressly incorporated by reference.

The local administration of MCSF in accord with the present invention used sites in vascular tissue that served as models for restenosis and atherosclerosis, thus enabling gene transfer to form the basis of the use of MCSF as a gene therapy protocol in vascular surgery. Such local presence of MCSF enables its direct therapeutic effect, for example, to cause reduction of cholesterol levels at the site as well as reduction overall of plasma cholesterol levels, and consequent atherosclerosis or restenosis.

Thus, the present invention permits a preferred protocol of collecting a patient's venous wall cells prior to a projected vascular surgery, transferring the MCSF DNA sequence to those cells, and transplanting those cells at the site of (projected) vascular surgery.

The current procedure was developed using vascular -smooth muscle cells because these cells grow and proliferate during the development of an atherosclerotic lesion and during restenosis. However, the procedure can be extended to the use of vascular endothelial cells which might offer some advantage in enhancing the therapeutic action of MCSF. Thus, the present results direct the feasibility of transferring MCSF sequences to a site of cardiovascular wall damage for therapeutic purposes .

Brief Description of the Drawings Figure 1 depicts the DNA and amino acid sequence (SEQ ID NOS: 1 and 2) of a family of CSF-1-like growth factors. Human MCSF in its mature form is believed to have a Glu amino terminus following a 32 amino acid presequence (indicated by the (1) above that Glu) and extending at least through amino acid Leu at position 189. The full length species is encoded by the about 1.8 kb DNA sequence as depicted.

Figure 2 depicts the particular recombinant retrovirus vector having a human MCSF cDNA insert that was employed in the present invention.

Figure 3 demonstrates the secretion of authentic human MCSF by the transfected vascular cells.

Figure 4 shows the results of an ELISA assay detecting human MCSF in the media from infected cells. Figure 5 shows the results of the expression of human MCSF in the rabbit carotid artery after transfected smooth muscle cells were seeded therein.

Examples 1. MCSF

Macrophage colony stimulating factor (MCSF, or CSF-1) was identified originally by its ability to stimulate specifically the survival, growth, and differentiation of monocytes and macrophages, including colony formation from bone marrow precursors. The major secreted form of MCSF is a 45 kDa glycoprotein that exists as a homodimer. Sources of MCSF include the placenta and bone marrow stromal cells, as well as oxidized LDL activated endothelial cells, in inducing atherosclerotic lesions, resulting in a rapid stimulation of MCSF expression.

The actions of MCSF are mediated through its interaction with a high-affinity cell surface receptor encoded by the c-fms proto-oncogene. The MCSF receptor is enriched on mature monocytes and macrophages. While it is not found normally on vascular smooth muscle cells, c-fms is expressed by smooth muscle cells derived from rabbit atherosclerotic lesions.

MCSF is important in the hematopoietic differentiation of mononuclear phagocytes, and it may play a regulatory role in fetal development. It is released at sites of vascular injury and inflammation, where it may complement, potentiate, or antagonize the actions of other cytokines. The clearance of LDL by LDL receptor- dependent and -independent pathways is stimulated by

MCSF in the normal rabbit. Acetyl-LDL uptake by macrophages as well as cholesterol esterification is enhanced by MCSF. In both the normal and the Watanabe Heritable Hyperlipidemic rabbit, MCSF administration results in a decrease in plasma LDL cholesterol .

2. The Rabbit Experimental Model

The rabbit is an animal of choice for investigating the actions and therapeutic effects of MCSF. Upon feeding the rabbit a high-fat, cholesterol-enriched diet, it rapidly develops a hypercholesterolemia that is associated with increased 3-VLDL as well as complex, foam cell-containing atherosclerotic lesions that resemble human lesions. Both lipoprotein metabolism and atherosclerosis have been studied extensively in the rabbit. The rabbit is relatively inexpensive and easy to maintain, and many surgical models have been developed in this animal . The Watanabe Heritable Hyperlipidemic rabbit is a well-characterized model for the human disorder, familial hypercholesterolemia.

3. Gene Delivery System

Several approaches have been developed for the introduction of foreign DNA into primary cells for gene therapy. Direct DNA seeding transfers without a tailored expression vector, such as using lipofection methods, may be employed. For gene transfer, a highly efficient method employs replication-defective retrovirus vectors. The promoter activity of the retrovirus 5' LTR (long terminal repeat) drives the production of retroviral transcripts that are translated normally by the host protein synthetic system. The replication-defective retroviral DNA is transfected into a packaging cell that provides the viral proteins necessary to yield infectious virus vectors. The optimum packaging cells contain the required viral protein genes in separately integrated units to nearly eliminate the possibility of obtaining replication- competent helper virus. The optimum retrovirus vector RNA contains the packaging region to enhance virus production and sequence modifications to minimize recombination events that might yield helper virus . These vectors include cloning sites for cDNA or gene fragments, and they can contain a drug selection marker, such as the neomycin-resistance gene driven by its own separate promoter (i.e. , the SV40 virus early promoter) .

4. MCSF Retrovirus Vector Construction The cDNA for human MCSF was obtained by the method disclosed by Wong et al . , U.S. Patent 4868119 and U.S. Patent 4879227. The patents also identify deposits that are available from the American Type Culture Collection

(ATCC) . The about 1.8 kb cDNA was cloned into a replication-defective retrovirus vector via the

EcoRI/XhoI sites. See Figure 2. The preparation of the vector is described in Miller et al . , BioTechniσues 7_.. 980 (1989) and Danos et al . , Proc. Natl . Acad. Sci, USA 85, 6460 (1988) . Virus packaging cells, ^--CRE and ψ- CRIP were employed.

The thus obtained recombinant retrovirus vector (Fig. 2) was transfected into the ecotropic i/'-CRE cells, and the resultant virus-containing culture medium was used to infect the amphotropic

cells. The latter infection yielded culture media with virus titers >3xl0⁶ cf /ml . This titer was an order or magnitude greater than necessary to obtain effective infection of a broad species range of target cells. Examination of the producer cells by in situ hybridization with a human MCSF probe shows a wide range of virus production in the non-cloned mixed mass culture. An estimated increase of about 10-fold in virus titer could be achieved by cloning the most active producer cells.

An amphotropic MCSF virus-producer cell was characterized further. Culture media from it was used to infect rabbit primary fibroblasts, rabbit primary venous smooth muscle cells, and rabbit primary aortic endothelial cells. Culture media from the infected cells, as well as the virus producer cells were examined by Western immunoblot analysis using a monoclonal antibody to human MCSF with polyacrylamide gels containing SDS. Fig. 3 shows that authentic human MCSF was secreted by each cell type. Culture media from each infected cell type was examined for MCSF biological activity by a mouse bone marrow assay, and colony forming activity in soft agar was detected in each case.

Additional culture media from each cell type was subjected to an enzyme-linked immunosorbant assay

(ELISA) (Fig. 4) that detected human MCSF in the media from infected cells.

5. Delivery of the MCSF Vector to the

Rabbit Artery Wall by Cell-Seeding a. Collection of venous smooth muscle cells

Specific pathogen free New Zealand White rabbits were used, ranging from 3 to 4 kg. in weight. The rabbit was subjected to halothane anesthesia, the left external jugular vein was exposed and ligated, and about 4 cm was excised. The rabbit was allowed to recover, and used for subsequent seeding of transfect autologous cells to eliminate the immune reaction that would occur with heterologous cells. b. Culture of venous smooth muscle cells

The excised vein was sliced, then incubated briefly in a solution of 0.1% collagenase . The cells were collected in gelatin-coated culture dishes, then incubated until the cells were just confluent. These passage 1 cells were collected by trypsin treatment, split into additional culture dishes, and grown to confluency. These passage 2 cells were essentially homogeneous smooth muscle cells. Endothelial cells, fibroblasts, and other cells are lost in this preparation since the culture conditions provide a selective growth advantage to the smooth muscle cells. c. Transfection of venous smooth muscle cells

The smooth muscle cells were collected by trypsin treatment and split into additional culture dishes, then cultured with conditioned media from the virus producer cells. Infected cells were grown to confluency. The cells were collected, split into additional flasks, then selected by culture in the presence of G418. The neomycin-resistant cells were grown to confluency. d. Seeding of the carotid artery The donor rabbit, after a recovery period of about 4 weeks during which time the smooth muscle cells were grown and transfected, was anesthetized. The left carotid artery was exposed and clamps were placed on the artery about 6 cm. apart from each other. A small incision was made in the artery, into which a balloon catheter was inserted. The balloon was inflated and pulled to strip the intimal surface of the artery; then the balloon was removed. The artery was flushed with cell culture media. Transfected smooth muscle cells suspended in media were added to the artery and incubated briefly. The artery was flushed with fresh media, then the clamps were removed to re-establish blood flow. The rabbit was allowed to recover, and it was maintained for 2 or more weeks. e. Collection of the seeded carotid artery

The donor/recipient rabbit was anesthetized. The seeded artery was excised, retaining about 1 cm. on either side of the seeding site. As a control, the corresponding segment of the opposite carotid artery was collected. The rabbit was then euthanized. f . Delivery of the MCSF vector to the artery wall by direct transfection in situ The carotid artery was exposed as above, but conditioned media from the virus producer cell was transferred into the artery instead of transfected cells. The artery was incubated briefly with the virus- containing media, then it was flushed with fresh media and the rabbit was allowed to recover. g. Analysis of the MCSF transfected carotid artery

The transfected artery segments from three cell-seeded experimental rabbits that had been maintained for 2 weeks following gene transfer were analyzed. MCSF sequences were detected by RNase protection analysis using an antisense hybridization probe for human MCSF.

The results for 2 rabbits are shown in Fig. 5, which includes a comparison to the transfected venous smooth muscle cells in culture prior to seeding the artery (cells from Rabbit A in Fig. 5) . These data show that human MCSF RNA transcripts are detected in the transfected artery segments, whereas control arteries contain no human MCSF. The hybridization probe does not cross-react with rabbit MCSF mRNA.

Concluding Remarks

The foregoing description details specific methods that can be employed to practice the present invention. Having detailed those specific methods initially used to produce the present invention, the art-skilled will well enough know how to devise alternative reliable methods for arriving at the same information and for extending this information to other covered embodiments and related matters.

Thus, however detailed the foregoing may appear in text, it should not be construed as limiting the overall scope hereof; rather, the ambit of the present invention is to be governed only by the lawful construction of the appended claims .

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT : TAYLOR, John M.

MAHLEY, Robert W. LAUER, Stephen J. RAMOS, Tammy K.

(ii) TITLE OF INVENTION: VASCULAR IN VIVO EXPRESSION OF MACROPHAGE COLONY STIMULATING FACTOR

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Walter H. Dreger

(B) STREET: 4 Embarcadero Center, Suite 3400

(C) CITY: San Francisco

(D) STATE: California

(E) COUNTRY : USA

(F) ZIP: 94111

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: PatentIn Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: PCT/US93/

(B) FILING DATE: 02-SEP-1993

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Dreger, Walter H.

(B) REGISTRATION NUMBER: 24,190

(C) REFERENCE/DOCKET NUMBER: FP-57328/WHD

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (415) 781-1989

(B) TELEFAX: (415) 398-3249

(2) INFORMATION FOR SEQ ID NO: 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2237 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE: (A) NAME/KEY: CDS

(B) LOCATION: 146..1807

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 :

CCTGGGTCCT CTCGGCGCCA GAGCCGCTCT CCGCATCCCA GGACAGCGGT GCGGCCCTC 60

GCCGGGGCGC CCACTCCGCA GCAGCCAGCG AGCGAGCGAG CGAGCGAGGG CGGCCGACG 120

GCCCGGCCGG GACCCAGCTG CCCGT ATG ACC GCG CCG GGC GCC GCC GGG CGC 172

Met Thr Ala Pro Gly Ala Ala Gly Arg 1 5

TGC CCT CCC ACG ACA TGG CTG GGC TCC CTG CTG TTG TTG GTC TGT CTC

220

Cys Pro Pro Thr Thr Trp Leu Gly Ser Leu Leu Leu Leu Val Cys Leu 10 15 20 25

CTG GCG AGC AGG AGT ATC ACC GAG GAG GTG TCG GAG TAC TGT AGC CAC

268 Leu Ala Ser Arg Ser lie Thr Glu Glu Val Ser Glu Tyr Cys Ser His

30 35 40

ATG ATT GGG AGT GGA CAC CTG CAG TCT CTG CAG CGG CTG ATT GAC AGT

316 Met lie Gly Ser Gly His Leu Gin Ser Leu Gin Arg Leu lie Asp Ser 45 50 55

CAG ATG GAG ACC TCG TGC CAA ATT ACA TTT GAG TTT GTA GAC CAG GAA

364

Gin Met Glu Thr Ser Cys Gin He Thr Phe Glu Phe Val Asp Gin Glu 60 65 70

CAG TTG AAA GAT CCA GTG TGC TAC CTT AAG AAG GCA TTT CTC CTG GTA

412 Gin Leu Lys Asp Pro Val Cys Tyr Leu Lys Lys Ala Phe Leu Leu Val 75 80 85

CAA GAC ATA ATG GAG GAC ACC ATG CGC TTC AGA GAT AAC ACC CCC AAT

460 Gin Asp He Met Glu Asp Thr Met Arg Phe Arg Asp Asn Thr Pro Asn 90 95 100 105

GCC ATC GCC ATT GTG CAG CTG CAG GAA CTC TCT TTG AGG CTG AAG AGC

508 Ala He Ala He Val Gin Leu Gin Glu Leu Ser Leu Arg Leu Lys Ser

110 115 120

TGC TTC ACC AAG GAT TAT GAA GAG CAT GAC AAG GCC TGC GTC CGA ACT

556 Cys Phe Thr Lys Asp Tyr Glu Glu His Asp Lys Ala Cys Val Arg Thr 125 130 135 TTC TAT GAG ACA CCT CTC CAG TTG CTG GAG AAG GTC AAG AAT GTC TTT

604 Phe Tyr Glu Thr Pro Leu Gin Leu Leu Glu Lys Val Lys Asn Val Phe 140 145 150

AAT GAA ACA AAG AAT CTC CTT GAC AAG GAC TGG AAT ATT TTC AGC AAG

652 Asn Glu Thr Lys Asn Leu Leu Asp Lys Asp Trp Asn He Phe Ser Lys

155 160 165

AAC TGC AAC AAC AGC TTT GCT GAA TGC TCC AGC CAA GAT GTG GTG ACC

700 Asn Cys Asn Asn Ser Phe Ala Glu Cys Ser Ser Gin Asp Val Val Thr 170 175 180 185

AAG CCT GAT TGC AAC TGC CTG TAC CCC AAA GCC ATC CCT AGC AGT GAC

748 Lys Pro Asp Cys Asn Cys Leu Tyr Pro Lys Ala He Pro Ser Ser Asp

190 195 200

CCG GCC TCT GTC TCC CCT CAT CAG CCC CTC GCC CCC TCC ATG GCC CCT

796 Pro Ala Ser Val Ser Pro His Gin Pro Leu Ala Pro Ser Met Ala Pro 205 210 215

GTG GCT GGC TTG ACC TGG GAG GAC TCT GAG GGA ACT GAG GGC AGC TCC

844 Val Ala Gly Leu Thr Trp Glu Asp Ser Glu Gly Thr Glu Gly Ser Ser 220 225 230

CTC TTG CCT GGT GAG CAG CCC CTG CAC ACA GTG GAT CCA GGC AGT GCC

892 Leu Leu Pro Gly Glu Gin Pro Leu His Thr Val Asp Pro Gly Ser Ala

235 240 245

AAG CAG CGG CCA CCC AGG AGC ACC TGC CAG AGC TTT GAG CCG CCA GAG

940 Lys Gin Arg Pro Pro Arg Ser Thr Cys Gin Ser Phe Glu Pro Pro Glu 250 255 260 265

ACC CCA GTT GTC AAG GAC AGC ACC ATC GGT GGC TCA CCA CAG CCT CGC

988 Thr Pro Val Val Lys Asp Ser Thr He Gly Gly Ser Pro Gin Pro Arg

270 275 280

CCC TCT GTC GGG GCC TTC AAC CCC GGG ATG GAG GAT ATT CTT GAC TCT

1036 Pro Ser Val Gly Ala Phe Asn Pro Gly Met Glu Asp He Leu Asp Ser 285 290 295

GCA ATG GGC ACT AAT TGG GTC CCA GAA GAA GCC TCT GGA GAG GCC AGT

1084 Ala Met Gly Thr Asn Trp Val Pro Glu Glu Ala Ser Gly Glu Ala Ser 300 305 310 GAG ATT CCC GTA CCC CAA GGG ACA GAG CTT TCC CCC TCC AGG CCA GGA

1132 Glu He Pro Val Pro Gin Gly Thr Glu Leu Ser Pro Ser Arg Pro Gly 315 320 325

GGG GGC AGC ATG CAG ACA GAG CCC GCC AGA CCC AGC AAC TTC CTC TCA

1180 Gly Gly Ser Met Gin Thr Glu Pro Ala Arg Pro Ser Asn Phe Leu Ser 330 335 340 345

GCA TCT TCT CCA CTC CCT GCA TCA GCA AAG GGC CAA CAG CCG GCA GAT

1228 Ala Ser Ser Pro Leu Pro Ala Ser Ala Lys Gly Gin Gin Pro Ala Asp

350 355 360

GTA ACT GGT ACA GCC TTG CCC AGG GTG GGC CCC GTG AGG CCC ACT GGC

1276 Val Thr Gly Thr Ala Leu Pro Arg Val Gly Pro Val Arg Pro Thr Gly 365 370 375

CAG GAC TGG AAT CAC ACC CCC CAG AAG ACA GAC CAT CCA TCT GCC CTG

1324 Gin Asp Trp Asn His Thr Pro Gin Lys Thr Asp His Pro Ser Ala Leu 380 385 390

CTC AGA GAC CCC CCG GAG CCA GGC TCT CCC AGG ATC TCA TCA CTG CGC

1372 Leu Arg Asp Pro Pro Glu Pro Gly Ser Pro Arg He Ser Ser Leu Arg 395 400 405

CCC CAG GGC CTC AGC AAC CCC TCC ACC CTC TCT GCT CAG CCA CAG CTT

1420 Pro Gin Gly Leu Ser Asn Pro Ser Thr Leu Ser Ala Gin Pro Gin Leu 410 415 420 425

TCC AGA AGC CAC TCC TCG GGC AGC GTG CTG CCC CTT GGG GAG CTG GAG

1468 Ser Arg Ser His Ser Ser Gly Ser Val Leu Pro Leu Gly Glu' Leu Glu

430 435 440

GGC AGG AGG AGC ACC AGG GAT CGG AGG AGC CCC GCA GAG CCA GAA GGA

1516 Gly Arg Arg Ser Thr Arg Asp Arg Arg Ser Pro Ala Glu Pro Glu Gly 445 450 455

GGA CCA GCA AGT GAA GGG GCA GCC AGG CCC CTG CCC CGT TTT AAC TCC

1564 Gly Pro Ala Ser Glu Gly Ala Ala Arg Pro Leu Pro Arg Phe Asn Ser 460 465 470

GTT CCT TTG ACT GAC ACA GGC CAT GAG AGG CAG TCC GAG GGA TCC TCC

1612 Val Pro Leu Thr Asp Thr Gly His Glu Arg Gin Ser Glu Gly Ser Ser 475 480 485 AGC CCG CAG CTC CAG GAG TCT GTC TTC CAC CTG CTG GTG CCC AGT GTC

1660 Ser Pro Gin Leu Gin Glu Ser Val Phe His Leu Leu Val Pro Ser Val 490 495 500 505

ATC CTG GTC TTG CTG GCT GTC GGA GGC CTC TTG TTC TAC AGG TGG AGG

1708 He Leu Val Leu Leu Ala Val Gly Gly Leu Leu Phe Tyr Arg Trp Arg

510 515 520

CGG CGG AGC CAT CAA GAG CCT CAG AGA GCG GAT TCT CCC TTG GAG CAA

1756 Arg Arg Ser His Gin Glu Pro Gin Arg Ala Asp Ser Pro Leu Glu Gin 525 530 535

CCA GAG GGC AGC CCC CTG ACT CAG GAT GAC AGA CAG GTG GAA CTG CCA

1804 Pro Glu Gly Ser Pro Leu Thr Gin Asp Asp Arg Gin Val Glu Leu Pro 540 545 550

GTG TAGAGGGAAT TCTAAGCTGG ACGCACAGAA CAGTCTCTCC GTGGGAGGAG

1857 Val

ACATTATGGG GCGTCCACCA CCACCCCTCC CTGGCCATCC TCCTGGAATG TGGTCTGCC 1917

TCCACCAGAG CTCCTGCCTG CCAGGACTGG ACCAGAGCAG CCAGGCTGGG GCCCCTCTG 1977

CTCAACCCGC AGACCCTTGA CTGAATGAGA GAGGCCAGAG GATGCTCCCC ATGCTGCCA 2037

TATTTATTGT GAGCCCTGGA GGCTCCCATG TGCTTGAGGA AGGCTGGTGA GCCCGGCTC 2097

GGACCCTCTT CCCTCAGGGG CTGCACCCTC CTCTCACTCC CTTCCATGCC GGAACCCAG 2157

CCAGGGACCC ACCGGCCTGT GGTTTGTGGG AAAGCAGGGT GGACGCTGAG GAGTGAAAG 2217

ACCCTGCACC CAGAGGGCCT 2237

(2) INFORMATION FOR SEQ ID NO:2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 554 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 :

Met Thr Ala Pro Gly Ala Ala Gly Arg Cys Pro Pro Thr Thr Trp Leu 1 5 10 15

Gly Ser Leu Leu Leu Leu Val Cys Leu Leu Ala Ser Arg Ser He Thr 20 25 30

Glu Glu Val Ser Glu Tyr Cys Ser His Met He Gly Ser Gly His Leu 35 40 45

Gin Ser Leu Gin Arg Leu He Asp Ser Gin Met Glu Thr Ser Cys Gin 50 55 60

He Thr Phe Glu Phe Val Asp Gin Glu Gin Leu Lys Asp Pro Val Cys 65 70 75 80

Tyr Leu Lys Lys Ala Phe Leu Leu Val Gin Asp He Met Glu Asp Thr

85 90 95

Met Arg Phe Arg Asp Asn Thr Pro Asn Ala He Ala He Val Gin Leu 100 105 110

Gin Glu Leu Ser Leu Arg Leu Lys Ser Cys Phe Thr Lys Asp Tyr Glu 115 120 125

Glu His Asp Lys Ala Cys Val Arg Thr Phe Tyr Glu Thr Pro Leu Gin 130 135 140

Leu Leu Glu Lys Val Lys Asn Val Phe Asn Glu Thr Lys Asn Leu Leu 145 150 155 160

Asp Lys Asp Trp Asn He Phe Ser Lys Asn Cys Asn Asn Ser Phe Ala

165 170 175

Glu Cys Ser Ser Gin Asp Val Val Thr Lys Pro Asp Cys Asn Cys Leu 180 185 190

Tyr Pro Lys Ala He Pro Ser Ser Asp Pro Ala Ser Val Ser Pro His 195 200 205

Gin Pro Leu Ala Pro Ser Met Ala Pro Val Ala Gly Leu Thr Trp Glu 210 215 220

Asp Ser Glu Gly Thr Glu Gly Ser Ser Leu Leu Pro Gly Glu Gin Pro 225 230 235 240

Leu His Thr Val Asp Pro Gly Ser Ala Lys Gin Arg Pro Pro Arg Ser

245 250 255

Thr Cys Gin Ser Phe Glu Pro Pro Glu Thr Pro Val Val Lys Asp Ser 260 265 270

Thr He Gly Gly Ser Pro Gin Pro Arg Pro Ser Val Gly Ala Phe Asn 275 280 285 Pro Gly Met Glu Asp He Leu Asp Ser Ala Met Gly Thr Asn Trp Val 290 295 300

Pro Glu Glu Ala Ser Gly Glu Ala Ser Glu He Pro Val Pro Gin Gly 305 310 315 320

Thr Glu Leu Ser Pro Ser Arg Pro Gly Gly Gly Ser Met Gin Thr Glu

325 330 335

Pro Ala Arg Pro Ser Asn Phe Leu Ser Ala Ser Ser Pro Leu Pro Ala 340 345 350

Ser Ala Lys Gly Gin Gin Pro Ala Asp Val Thr Gly Thr Ala Leu Pro 355 360 365

Arg Val Gly Pro Val Arg Pro Thr Gly Gin Asp Trp Asn His Thr Pro 370 375 380

Gin Lys Thr Asp His Pro Ser Ala Leu Leu Arg Asp Pro Pro Glu Pro 385 390 395 400

Gly Ser Pro Arg He Ser Ser Leu Arg Pro Gin Gly Leu Ser Asn Pro

405 410 415

Ser Thr Leu Ser Ala Gin Pro Gin Leu Ser Arg Ser His Ser Ser Gly 420 425 430

Ser Val Leu Pro Leu Gly Glu Leu Glu Gly Arg Arg Ser Thr Arg Asp 435 440 445

Arg Arg Ser Pro Ala Glu Pro Glu Gly Gly Pro Ala Ser Glu Gly Ala 450 455 460

Ala Arg Pro Leu Pro Arg Phe Asn Ser Val Pro Leu Thr Asp Thr Gly 465 470 475 480

His Glu Arg Gin Ser Glu Gly Ser Ser Ser Pro Gin Leu Gin Glu Ser

485 490 495

Val Phe His Leu Leu Val Pro Ser Val He Leu Val Leu Leu Ala Val 500 505 510

Gly Gly Leu Leu Phe Tyr Arg Trp Arg Arg Arg Ser His Gin Glu Pro 515 520 525

Gin Arg Ala Asp Ser Pro Leu Glu Gin Pro Glu Gly Ser Pro Leu Thr 530 535 540

Gin Asp Asp Arg Gin Val Glu Leu Pro Val 545 550

Claims

1. A method for transplantation of mammalian vascular cells comprising: a) isolating a population of vascular cells from the vascular system of a mammal, b) transfecting said population of vascular cells with an expression vector that expresses an inserted DNA sequence encoding macrophage colony stimulating factor, c) transplanting said transfected vascular cells into the vascular tissue of a mammalian host.

2. The method of Claim 1 wherein said population of vascular cells comprises vascular smooth muscle cells .

3. The method of Claim 1 wherein the transfected vascular cells are transplanted into the carotid artery of the mammalian host.

4. The method of Claim 1 wherein said population of vascular cells is transfected using a retroviral gene transfer vector.

5. The method of Claim 1 wherein said inserted DNA sequence encodes human macrophage colony stimulating factor having the amino acid sequence 1 to 189 as set forth in Figure 1 (SEQ ID NO: 2) hereof.

6. The method of Claim 1 wherein said mammalian host is a rabbit.

7. The method of Claim 1 or 5 wherein said vascular tissue comprises a site of atherosclerosis or restenosis.

8. Mammalian vascular transplant cells comprising transplanted vascular cells comprising an expression vector that expresses an inserted DNA sequence encoding macrophage colony stimulating factor.

9. The mammalian vascular tissue of Claim 8 wherein said transfected cells are vascular smooth muscle cells.

10. The mammalian vascular tissue of Claim 8 wherein said DNA sequence encodes human macrophage colony stimulating factor having the amino acid sequence 1 to 189 as set forth in Figure 1 (SEQ ID NO: 2) hereof.

11. The mammalian vascular tissue of Claim 8 or 10 comprising a site of atherosclerosis or restenosis.

12. A method for the assay of the effects of local administration of macrophage colony stimulating factor in proximate vascular tissue comprising: a) providing a mammal having transplanted vascular tissue comprising vascular cells transfected with an expression vector that expresses DNA encoding macrophage colony stimulating factor locally proximate said vascular cells and, b) monitoring the effect of the resultant localized administration of said macrophage colony stimulating factor provided by the transplanted, transfected vascular cells on at least one of: 1) the deposition and accumulation of cholesterol in vascular tissue proximate said vascular cells,

2) the production and maintenance of atherosclerotic plaques in vascular tissue proximate said vascular cells and

3) the occurrence and severity of restenosis in the vascular tissue proximate said vascular cells.

13. The method according to Claim 12 wherein said transplanted vascular tissue comprises a site of atherosclerosis or restenosis.

14. The method according to Claim 12 or 13 wherein said DNA encodes human macrophage colony stimulating factor having the amino acid sequence 1 to 189 as set forth in Figure 1 (SEQ ID NO: 2) hereof.