CA2005199A1 - Genetically engineered endothelial cells and use thereof - Google Patents

Genetically engineered endothelial cells and use thereof

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Publication number
CA2005199A1
CA2005199A1 CA 2005199 CA2005199A CA2005199A1 CA 2005199 A1 CA2005199 A1 CA 2005199A1 CA 2005199 CA2005199 CA 2005199 CA 2005199 A CA2005199 A CA 2005199A CA 2005199 A1 CA2005199 A1 CA 2005199A1
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CA
Canada
Prior art keywords
endothelial cells
product
cells
gene
genetically engineered
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
CA 2005199
Other languages
French (fr)
Inventor
W. French Anderson
Scott M. Freeman
James A. Zwiebel
J. Anthony Thompson
Una S. Ryan
Philip Kantoff
David Dichek
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
US Department of Energy
University of Miami
Original Assignee
US Department of Energy
University of Miami
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Application filed by US Department of Energy, University of Miami filed Critical US Department of Energy
Publication of CA2005199A1 publication Critical patent/CA2005199A1/en
Abandoned legal-status Critical Current

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86Viral vectors
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61FFILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02Prostheses implantable into the body
    • A61F2/04Hollow or tubular parts of organs, e.g. bladders, tracheae, bronchi or bile ducts
    • A61F2/06Blood vessels
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61FFILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2250/00Special features of prostheses classified in groups A61F2/00 - A61F2/26 or A61F2/82 or A61F9/00 or A61F11/00 or subgroups thereof
    • A61F2250/0058Additional features; Implant or prostheses properties not otherwise provided for
    • A61F2250/0067Means for introducing or releasing pharmaceutical products into the body
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00Reverse transcribing RNA viruses
    • C12N2740/00011Details
    • C12N2740/10011Retroviridae
    • C12N2740/13011Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Abstract

ABSTRACT OF THE DISCLOSURE
Endothelial cells are genetically engineered with a gene for a heterologous protein which is a therapeutic agent. The endothelial cells may be seeded onto a vascular graft and implanted in the vascular system of a mammal to produce the therapeutic agent in vivo.

Description

b ~ 51.,~9 G~N~TICALLY ENGIN~ZR~D ENDOTH~LIAL
OE LLS ~D USE THæ~OF

This invention relates to genetically engineered cells, and to the use thereof. Still more particularly, this invention relates to genetically engineered endothelial cells and the use thereof for e~pressing a therapeutic agent.
There have been numero~s proposal~ with respect to genetically engineering mam~alian cells. In general, retroviruse~ have been employed for introducin~ genetic material into mammalian c~
Thus, there have been proposals to genetically engineer bone marrow and hematopoietic progenitor cell~ by the use of retroviral vectors. In general, there have been drawbacks to the use of -quch cell~, such as the variable ability to express certain gene~
and~or inefficient gene tran~fer. Thus, there have been further propo~als for genetically engineering cells capable of both long term sur~ival and ~table e~pression including cells such a~ fibroblasts, lymphocytes, keratinocytes and hepatocytes for u~e in 8ene therapy.

~c~o~ 9 The present invention is directed to genetically engineered endothelial cells, and the use thereof for expressing a heterologou~ protein. In one embodiment the heterologous protein i9 a therapeutic agen..
According to one aspect of the present invention, there i9 provided endothelial cells which are transformed with at lesst one gene which encode~
for at least one heterologous protein, which is preferably a therapeutic agent.
In accordance with another aspect of the present invention, there is provided a solid support which includes endothelial cells transformed with at least one gene which encodes for at least one heterologous protein, preferably a therapeutic a8ent. In a preferred embodiment, the ~olit support i~ one which i9 compatible with blood and may, for example, be in the form of a blood vessel graft.
In accordance with yet another aspect of the present invention, endothelisl cells which are transformed with at least one gene which encodes for at least one heterologous protein, preferably a therapeutic agent are implanted in a blood vessel.
More particularly, the endothelial cells employed in the present invention are endothelial cells derived from a mammal. The endothelial cells are obtained from a blood vessel. The term "blood ve~sel" as used herein includes veins, arteries and capillaries. Thus, the endothelial cells which are genetically engineered include macrovascular and/or microvascular endothelial cells.
The mammalian cells may be derived from a human or nonhuman mammal. The endothelial cells are preferably deri~ed from a human.
The endothelial cells are tran9formed with st least one gene which encodes for at least one ~6~0~

het~sologous protein which is preferably a therapeutic agent. The cells may be transformed in a manner in which the therapeutic agent is secreted from the transformed cells or may be transformed in a manner in which the therapeutic a~ent remain~ in or on the transformed cells.
The mammalian endothelial cells are transformed with 8 suitable vector or e~pre~sion vehicle which includes a gene for at lesst one therapeutic agent.
The vector includes a promoter for expression in mammalian cells; for example, SV 40, LTR, metallothionein, PGK; CMV; ADA; TK; etc. The vector may also include a ~uitable signal sequence or sequences for secreting the therapeutic agent from the cells. The selection of a suitable promoter is deemed to be within the skill of the art from the teachings herein.
The expression vehicle or vectos is preferably a viral vector and in particular a retroviral vector.
As representative examples of suitable viral vectors which can be modified to include 8 gene for a therapeutic agent, there may b~e mentioned: Harvey Sarcoma virus; ROUS Sarcoma virus, MPSV, Moloney murine leukemia virus, DNA viruses (adenovirus) etc.
Alternatively, the expression vehicle may be in the for~ of a plasmid. The expre~sion vehicle may also be in a form other than a vector; for example, tran~formation may be accomplished by liposome fusion, calcium phosphate or dextran sulfate transfection; electroporation, lipofection, tungsten particles etc. The selaction of a ~uitable vehicle for transformation is deemed to be within the scope of those skilled in the art from the teachings herein.

In employing a retrovlral vector a~ the expre~ion vehicle for transforming endothelial cells, steps should be taken to eliminate and/or minimize the chances for replication of the virus.
Various procedures are known in the art for providing helper cells which produce viral vector particles which are essentially free of replicating virus.
Thus, for e~ample, ~arkowitz, et al., "A Safe Packaging Line for Gene Transfer: Separating Viral Genes on Two Different Plasmidq," Journal of Viroloxy, Vol. 62, No. 4, P8q- 1120-1124 (April 1988); Watanabe, et al., "Construction of a Helper Cell Line for Avian Reticuloendotheliosi3 Virus Cloning Vectoss, "Molecular and Cellular Biolo~Y, Vol. 3, No. 12, p8s- 2241-2249 (Dec. 1983); Danos, et al., "Safe and Efficient Generation of Recombinant Retroviruses with Amphotropic and Ecotropic Host Ranges, "Proc. Natl. Acsd. Sci. Vol. 85, pgs.
6460-6464 (Sept. 1988); and Bosselman, et al., "Replication-Defective Chimeric Helper Proviruses and Factors Affecting Generation of Competent Virus:
Expression of Moloney Murine LeuXemia Virus Structural Genes via the Metallothionein Promoter, "Molecular and Cellular Biolo~, Vol. 7, No. 5, pg~.
1797-1806 (May 1987) di~clo~e procedures for producing a helper cell which minimize~ the chances for producing a viral particle which includes replicating virus. This procedure and other procedures may be employed for genetically engineering with endothelial cells by use of a retrovirsl vector.
The endothelisl cells which are to be genetically engineered in accordance with the present invention may be derived from a mam~al, and as hereinabove indicated, quch endothelial cells msy be )51~39 obtainet from an appropriate blood ve~el, ~uch a~ an artery, vein or capillary. The proc~dure for obtaining ~uch endothelial cells from the blood vessel of a mammsl are generally ~nown in the art and a representative procedure i~ disclo~ed in the E~amples.
She inventi~n will be further described with respect to endothelial cells genetically engineered with a gene for a therapeutic sgent; however, the scope of the invention is not to be limited thereby.
For example, the endothelial cells may be genetically engineered with a gene for a protein which is not a therapeutic aBent; for example, a marker protein, such as beta-galactosidase.
The endothelial cells are genetlcally engineered to include a gene for a therapeutic agent by the use of an appropriate vector, with the vector preferably being a retroviral vector. A representative proceture for genetically engineering endothelial cells by the u~e of a retroviral vector is described in the e~amples, and such general procedure and others may be employed for int~roducing other genes into mammalian endothelial cells. Thus, as described in the E~amples, the procedure basically involves introduction of an appropriate promoter and DNA for the de8ired therapeutic agent into an appropriate retroviral vector. In addition to the promoter and the gene for the therapeutic agent, other material may be included in the vector such as a selection gene; for example a neomycin resistance gene; a sequence for enhancing expression, etc.
The appropriate vector now containing a gene for at least one desired therapeutic agent i9 employed for transducing mammalian entothelial cells by procedures generally available in the art.

X~53~9~

-B-In accordance with an aspect of the present invention, genetically en8ineered endothelial c~lls and in particular those genetlcally engineered with at least on~ gene for at least one therapeutic a8ent may be supported on a solid support. The solid support is preferably one which ls biocompatible with blood whereby the solid support including the genetically engineered endothellal cells may be placed in communication with the blood system of a patient. Thus, for example, the solid support msy be employed in an e~tracorporeal device or implanted in a blood vessel (the term implant in a blood vessel includes a by-psss or a shunt for a blood vessel).
The implantation may take the form of a blood vecsel graft (the term graft includes a shunt or bypass).
It is to be understood, however, that the solid ~upport may take a variety of forms, such as pads, strips, gels, etc. and is not limited to grafts.
The genetically engineered mammalian endothelial cells, which include a gene for a therapeutic agent, may be implsnted in a blood vessel of a mammal. The mammalian endothelial cells wh~ich are genetically engineered in accordance with the present invention are derived from a mammal, and the transformed endothelial cell~ are implanted in a blood vessel of a mammal of the ~ame species. In a preferred embotiment~ the genetically engineered mammalian endothelial cells are implanted in the blood vessel of a host from which the cells were originally derived; i.e., autologous cells are employed. Thus in a preferred embodiment, endothelial cells are derived from a blood vessel of a patient, genetically engineered to include a gene for at lea~t one therapeutic agent and the genetically engineered cells are implanted in a blood ves~el of the patient . . X~)0~199 fron which they were derived. In this manner, autologous genetically engineered endothelial cells are employed for in vivo production of a therapeutic agent for treatment of a patient, i.e., gene therapy.
It is to be understood that the genetically engineered endothelial cells may be implanted in a blood ves~el on a solid support or implanted directly onto a blood vessel (without the use of a ~olid support). It is also to be understood that the endothelial cells may be placed on a solid support in an e~tracorporeal device in communication with the blood sy~tem.
In accordance with a preferred embodiment, the genetically engineered mammalian cells are implanted in 8 blood vessel by providing a blood vessel graft which includes the genetically engineered endothelial cell3. The graft now including genetically en8ineered endothelial cells may be inserted intc a blood vessel of a ho~t. Thus, in accordance with one aspect of the present invention, there i9 providet a blood vessel graft which includes genetically engineered endothelial cells which are suitable for use in a mammalian host, which may be a human or nonhuman mammal.
The blood vessel graft may be any one of a wide variety of vascular grafts and such grafts may be of various sizes. The graft may be used in a vein, an artery, or a capillary. The selection of appropriate grafts is deemed to be within the scope of those skilled in the art from the teaching~ herein.
Although in most caQes a synthetic vascular graft is preferred, it is possible within the spirit and scope of the present invention to provide a blood ves~el derived from a host with genetically engineered endothelial cells and then graft such blood ves~el 3~ 9 bac~ into the host. Thus the term ~raft i~clude~
nstural and synthetic graft 9 .
The graft may be provided with genetically engineered endothelial cells in accordance with the present invention by ~eeding the genetically engineered endothelial cells onto a ~uitable blood vessel graft. Represèntstive graft and procedure iq disclosed in the Examples. The present invention is not limited to such grafts and procedures. Other grafts and procedures for qeeding endothelial cells onto the graft are known in the art ant may be used in the present invention. For e~ample, Herring et al Eds. Endothelial Seeding in Vascular Surgery (Grune &
Stratton, Inc. Orlando 1987); Ziller et al Eds.
Endothelialization of Vasculsr Grafts, 1st European Workshop~ on Advanced Technologies in Yascular Surgery, Vienna, Nov. 5-6 ~Karger, Basel, 1986). As representative graft materials, there may be mentioned polye~ters (for example DACRON); expanded polytetrafluroethylene (Gore-Tex); polyurethane~;
coated polyurethanes; such as a silicone coated polyurethane msnufactured by C~orvita corporation in Miami, Florida; tubular slotted stainle~s ~teel stents (Johnson and Johnson) which are coated with a substrate to permit adhesion of the endothelial cells to the stents; natural blood vessels, etc.
The graft, now including geneticslly engineered endothelial cells, may then be inserted into a blood vessel of 8 host. The procedures for placing 8 graft in an appropriate blood vessel are generally known in the art, and such procedures are spplicable to the present invention.
Alternatively, endothelial cells may be removed from a blood vessel of 8 patient, genetically 19C~

eng~n~er~d and returned to a blood vessel of the patient, without u~e of an implantable ~olid support.
The endothelial cells, which sre genetically engineered with an appropriate therapeutic agent, may be genetically engineered in a manner such that the therapeutic agent i9 secreted into the blood, whereby ~uch therapeutic agent may e~ert its effect upon cells and tissues either in the immediate vicinity or in more di~tal locations. Alternatively, the therapeutic agent may not be secreted from the cell~, and exert its effect within or on the genetically engineered endothelial cells upon substances that diffuse into the cell. Thus, for example, adenosine deaminase (ADA) may function within the cell to inactivate adenosine, a toxic metabolite that accumulates in severe combined immunodeficiency ~yndrome; phenylalanine hytroxylase may function within a cell to inactivate phenylalanine, a to~ic metabolite in phenylketonuria, etc.
As hereinabove indicated, the endothelial cells are transformed with a gene for at least one heterolo~ou~ protein, prefersbly a therapeutic agent.
The term therapeutic agent is u~ed in its broadest sen~e and means any agent or material which has a beneficial effect on the ho~t. The therapeutic agent may be in the form of one or more protein~. As repre~entative examples, there may be mentioned:
CD-4; Factor VIII, Factor IX, von Willebrand Factor, TPA; urokins~e; hirudin; the interferon~; tumor necrosis factor, the interleukins, hematopoietic growth factors (G-CSF, GM-CSF, IL3 erythropoietin), antibodies, glucocerebrosidsse; ADA; phenylalanlne hydroxyla~e, human growth hormone, in~ulin, etc. The selection of a suitable gene is deemed to be within ~t~05~9~

the 5cope ~f those skilled in t:he art from the teachings herein.
In u~ing the genetlcally engineered endothellal cell~, it iq possible to employ a mi~ture of endothelial cells which includes endothelial cells genetically enBineered with a gene for a first therapeutic agent and endothelisl cells genetically engineered with a gene for a second therapeutic agent. It is al~o poqsible to transform individual endothelial cells with more than one gene.
The genetically engineered endothelial cells may be implsnted in a blood vessel alone or in combination with other genetlcally engineered endothelial cell or with other genetically engineered cells, such as smooth muscle cells, fibroblasts, glial cells, keratinocytes, etc.
The use of genetically engineered endothelial cellq permits a therapeutic a8ent to be introduced directly into the blood. As a result of the location of the endothelial cell~ in immediate contact with the circulating blood, the survival and delivery of a therapeutic agent is facilitat~ed.
The genetically engineered endothelial cells (by selection of high producing clonal populations and/or the use of vectors with enhanced expression) may be employed to produce, in vivo, therapeutically effective amounts of a desired therapeutic aBent for treatlng a patient. In determining the number of cells to be implanted, factors such as the half life of the therapeutic agent; volume of the va~cular system; production rate of the therapeutic agent by the cells; and the desired dosage level are con~idered. The selection of such vectors and cells is dependent on the therapeutic agent and i9 deemed 2~05~99 to be w$thin the scope of those skilled in the art from the teachings herein.
The drawing i9 a schematic illustration of vectors used in the present invention.
The following Examples further illustrate the present invention; however, the scope ~f the invention i9 not to be limited thereby. In the Examples, unless otherwise specified, restriction enzyme digests, ligations, transformations, etc. may be performed as described in Molecular Clonin~, A
Laboratory Manual by Maniatis et 81.
E~ample 1 A. To construct the pG2N retroviral vector of the drawing u~ed to genetically engineer endothelial cells to produce rat growth hormone, an SV40 promoted neomycin resistance gene and a rst growth hormone cDNA were placed into the pB2 retroviral vector (Laboratory of Molecular Hematology, NI~). A growth hormone cDNA was obtained by digesting the plasmid RGH-l (Nature 270, 494 (1977)) with Xho I and Mae III
restriction endonucleases (~oehringer Mannheim Biochemicals). This rat growth.hormone cDNA was eletrophoretically i~olated out of an agaorse gel and purified via binding/elution to glass beads, Geneclean (BI0 101, LaJolla, California). This growth hormone cDNA was then blunted using the large fra~ment of DNA polymerase (Klenow) (New England Biolabs) and nucleotide triphosphates 8S recommended by the manufacturer. This fragment was then purified with Geneclean.
The B2 vector was constructed in order to replace the NeoR gene in N2, ~M.A. Eglitis, P.
Kantoff, E. Gilboa, W.F. Anderson, Science 230, 1395 (1985); D. Armentano et al., J. Virol, 61, 1647 (1987) and shown in the draw$ng] with a multiple ~)0~)19~

cloning ~lte. N2 was first digested with Eco RI, thereby releasing both the 5' and 3' LTRs with the ad~oining MoMLV flanXing sequences. The 3' LTR
fra8ment was ligated into th^ EcoRI ~ite of the plasmid GEM4 (Promega Biotech). The 5' LTR fra8ment with its flanking gag sequence was then digested with Cls I, Hind III linkers were sdded, and the fragment wa~ inserted into the-Hind III ~ite of pGEM4.
The pB2 ~ector wa3 digested with the HincII
restriction endonuclease (New England ~iolab~), and pho~phstased using calf alkaline phosphatase.
(Boehringer Mannheim Biochemicals). The pB2 plaYmid W85 then purified with Geneclean. The pB2 vector and the rat growth hormone cDNA were then ligated using T4 ligase (New England Biolabs). The ligation was then transformed into competent DH5 bacteris (Bethe~
da Research Labs). Colonie~ were then screened for a growth hormone cDNA containing vector. The new vector was called pG2. pG2 was then digested with BamHI (New England Biolabs), purified with Geneclean (Bio 101), and blunt ended with the Klenow fragment (New England Biolabs). A 340 ~sse pair SV40 promoted neomycin resistance gene fragment wa~ isolated from the pSV2CAT plasmid (ATCC accession number 37155) by di8e~ting with PvuII and HindIlI (New England Biolsb~). This fragment wa~ i~olated by agarose gel electrophoresis ant purified with Geneclean. The SV40-neomycin resistance fragment W8~ then ligated using T4 ligase (New England Biolsbs) with pG2 and tran~formed into DH5 competent bacteria per the manufacturers in~truction (B~L). Colonies were screened and the resulting pla9mid con~truct was called pG2N.

5~9 The SA~ vector shown ln th~ drawing was obtained as described in Proc. Na~l. Acad. Sci. USA 83:6563 (1986).
The recombinant vectors (N2,SAX, G2N) used in the study were each separately transfected into the currently available retroviral vector packaging cell lines, including the amphotropic packaging lines, PA12 (Science 225:630 (19~4) and PA317 (Mol. Cell.
Biol 6:2895 (1986), and the ecotropic line, P~i2 (Cell 33:153 (1983). These lines were developed in order to allow the production of helper virus-free retroviral vector particle~.
Aortic endothelial cells were obtained from New Zealand White rabbits (2-5 kilograms) by methods described previously for obtaining endothelial cell~
from bovine pulmonary artery (U.S. Ryan, M. Mortara, C. Whitaker, Tissue ~ Cell 12, 619 (1980)). The rabbit was anesthetized (1 ml sodium pentobarbital) and the aorta was removed and placed in Hanks buffered saline containing 3X antibiotics. The vessel wa~ slit longitudinally and the luminal surface was ~craped with a #}1 scalpel blade taking care to scrape each area only once. The initial isolates were grown in Ryan Red medium [Ryan et al J.
Tissue Cult. Method~, 10:3 (1986)], purified by selection of endothelial "islands" and pas~aged with a rubber policeman. Passsged cells were grown in Rrimaria 25 cm2 flasks.
A confluent 100 mm ti9sue culture dish (Costar) was harvested with a cell scraper. Following the dispersal of the cells by titurating 10-20 times with a 5 ml pipet, the cells were plated into 2-100 mm tissue culture dishes with 8 ml Ryan Red medium.
After an overni~ht incubation, the medium was removed snd 5 ml retroviral Yector supernatant was added with )5~3'9 Polybrene at a final concentrstion of 8 ug/ml. After a 2 hour incubation an additional 5 ml of Ryan Red was added to the di~h. The cells were incubated overni~ht and the medium was replaced with 8 ml of Ryan Red. After another overnight incubation G418 was added to a final concentrstion of 200 ug/ml. The cells were the fed every 3-4 d~ys with Ryan Red containing 200 u6/ml G418. The cell were sub3equently maintained in Ryan Red without G418.
G2N-infected RAEC that had been selected in G418-containing growth medium were harvested with a rubber policçmsn from 2 confluent T75 flssks and su~pended in 5 ml of Ryan Red. The cell su~pension was titurated 6-7X with a 6 cc syringe and a 23 gauge needle. The cells were pelleted and resuspended in 1.25ml of Ryan Red. A vascular clamp was attached to one of a lO cm 2 4 mm (inner diameter) Corvita grsft (Cor~ita Corp., Miami, FL) which is a silicone coated polyurethane graft. The cell suspenslon was vortexed and introduced into the open end of the graft with a 3 cc syrin~e and 20 gsuge needle. A second vascular clamp was attached to the ope~ end and the graft was placed into a 50 ml conical tube filled with Ryan Red medium. The conical tube was capped, wrapped in parafilm, and placed into a roller bottle. The roller bottle was rotated overnight at 37 degrees Centrigrade. The ne~t day the clamps were removed and the 8raft was placed into a 500 ml bottle containing 150 ml of Ryan Red. The bottle was placed in an incubator equilibrated with 5% C02, at 37 degrees C. It remained in the incubator and was periodically rotated for the next nine days. At that time the graft was transferred 1nto a T75 flask, fed with fresh medium and periodically sampled for rst growth hormone produc~ion over the ne~t four weeks.

rGH continued to be ~ecreted into the tissue culture medium at a rate of appro~imately 1000 ng/106 cell~/day for at least 4 weeks after seeding the graft a~ follows.

Cell o~ ~raft Rat growth hormone6 6 ~ 10 production (ng/lOE
(cells/cm2) cells/24 hours) Dsy 13 930 Day 32 1060 B. Rabbit endothelial cells were sl~o tran~fected with the vector SAX by the hereinabove described procedure and quch tranfected cells were found to expres~ human ADA.
C. The CD4 containing plasmld (pT4B, a gift of Richard Axel of College of Physicians and Surgeonq Columbia University, New YorX, New York) was digeqted with the rs~triction endonucleasas Eco RI and Bam HI
New England Biolabs, Beverly MA) to release the CD4 gene which was isolated by agaro~e gel electrophoresis ~ollowed by purification via binding/elution to glass beads (using the geneclean product, BIO 101, La Jolls CA in the manner recommended by the msnufacturer). The CD4 fragment was li~ated (usin~ T4 DNA ligase as recommended by the suppller, New England Biolabs) into Eco RI plus Bam HI cut Bluescript cloning vector (~tratagene Co.
La Jolla CA). The ligation was then transformed into competent DH5 alpha bacteris (Bethesda Research Labs, Gaithersburg MD) and white colonies were isolated and screened for proper insert size to yield the plasmid pCDW. To produce a suitable plasmid ba~ed e~pres~ion vector for the CD4 gene; the plasmid SV2neo (obtained form American Type Culture Collection, Rockville MD~
was digested with Hind 3 plus ~pa I, and a ~ynthetic -lB-polylin~er sequence from the pUC-13 vector (Pharamicia, Piscataway NJ) was inserted (via T4 DNA
ligase) in place of the neo 8ene of pSV2neo. This ligation wss transformed into DH5 bscteria (Bethesda Research Labs) and colonie3 screened for the presence of restriction enzyme sites unique to the polylinker to yield the vector pSVPL. The pSVPL e~pression vector wss further modified by the insertion of an Xho I linker (conditions and reagents supplied by, New England Biolabs) into the Pvu II site on the 5' side of the SV40 early region promoter to produce pSVPLX.
The pCDW and pSVPLX pla~mids were digested with enzymes Hind 3 plus ~ba I (New England Biolabs) and their DNAs isolated (using the Gene Clesn product) following agarose gel electrophoresis. Ligation of the CD4 fra8ment into the pSVPL~ vector wa~ performed and colonies were screenet to yield pSVCDW in which the SV40 viru~ early region promoter is used to drive the expression of the complete CD4 8ene product. The next step wss to produce a form of the CD4 gene such that it would be exported from the cell as an extracellular product.
The production of a soluble form of CD4 was accomplished by the use of a specially designed oligonucleotide adaptor to produce 8 mutant form of the CD4 gene. This adaptor has the unique property that when inserted into the Nhe I site of the CD4 gene it produces the preci~e premature termination of the CD4 protein amino acid sequence while re8enerating the Nhe I site and creating a new Hpa I
site. This oligonucleotide adaptor (~ynthesized by Midland certified resgent Co.) was produced by annealing two phosphorylatet oligonucleotides; 1) 5' CTAGCITGAGTGAGIT 3', 2) AACTCACTCAAG and then this ~0051~9 product wa~ llgated into the ~lte of pSVCDW. The ligation reaction was then cleaved with Hpa I and then ~ho I linkers were added (New England Biolabs).
The linker resction wss terminated by heating at 65C
for 15 min. and then sub~ected to digestion with Xho I restriction endonuclease (New England Biolab~).
This reaction was then subJected to agarose gel electrophoresi~ and the fragment containing the SV40-CD4 adsptor i~olated (Geneclean). The retroviral vector N2 was prepared to accept the SV40-CD4-adaptor fragment by digestion with Xho I and treatment wlth Calf intestinal phosphstase (Boehringer Mannheim, Indianpoli~ IN). The ligation of CD4 expres~ion cas~ette wa9 performed with an insert to vector ratio of 5:1 and then transformed into DH5 competent bacteria (Bethesda Research Labs).
Constructs were analyzed by restriction endonuclease digestion to screen for orientation and then grown up in large scale. The construct where the SV40 virus promoter i~ in the same orientation as the viral LTR
promoters is known as SSC while the construction in the reverse or reverse orientation is called SCSC.
The SSC vector is packaged into PA317 cell line as described by Miller et al supra. to provide PA 317 cells capable of producing soluble CD4 protein.
The SSC vector packaged PA 317 cells were used to transduce rabbit endothelial cells as hereinabove described.
The transduced endothelial cells were found to express soluble CD-4.
D. Collsgen sponges containing sdsorbed HBGF-I
were 9urgically implantet in the abdominal cavity of a rat near the li~er (Science 241, 1349 (1988).
Seven (7) to ten (10) day8 post implantation, sponges were surgically removet and dige~tet 30 to 60 min. at i~C~05~'~9 37C with a solution of colla6enase in phosphate buffered ~aline (1 mg/ml) using a tissue culture incubator (5% C02). Released cells were collected by centrifugation (10 min., 1000 RPM, 20C) and washed once with phosphste buffered saline (PBS) and pelleted by centrifu6stion. Cell~ were resuspended with 30 ml of media contsining:
Ml99 media (Gibco) ECGF (crude brain e~tract ) 7.2mg Hepsrin (Up~ohn) 750 units 20% conditioned cellular media collected a~
supernatant from confluent dishes (48 hr.) of either bovine sortic or human umbllicsl vein endothelial cells 10% Fetal Calf serum (Hyclone) 3000 units Peniclllan G (Biofluids) 3000 units streptomycin sulfate (Biofluids) and plated for 16 hour~ on 100 mm tissue culture disk coated with fibronectin (human) using lug/cm2.
Plated cells were washed with P~S three times and fed 15ml of previously mentioned media. Media was changed every 2 days for the duration of the procedures .
Selected rat endothelial cells were tran~duced with the N-7, SA~, G2N and SSC vectors by the following procedure:
1. 2 X 106 microendothelial cells (monolayer 80% confluent) 2. 2 X 106 viral supernatant 3. Polybrene (8ug/ml) -Combine 1, 2, 3 in 5ml total volume for 2-3 hours at 37C (5~ C02).
-Add 20ml of ti~sue culture media for 16 hrs. at 37C
(5% C02).

~05~'3~

-Aspirnte off media (virus contsining), add fresh culture media.
-After 48-96 hours add G418 (800ug/ml~ and culture media.
-Select for one to two week~ changing media every two dsys.
Cells tran~duced with N7 e~pressed neomycin, those tran~duced with SAX expressed ADA; tho~e transduced with G2N e~pressed rat growth hormone; and those transduced with SSC e~pressed sCD4.
The rat endothelial cells transduced with G2N
expressed rat growth hormone in vitro as follows:
106 cells produce 3.0 Y8 after 24 hrs.; 6 ,ug after 48 hr~.; and 9.0 ,ug after 72 hours.
E~ample 2 A. Endothelial cells were harvested from segments of adult sheep jugular vein, carotid artery, and femoral vein using the method of Jaffee et al.
J.Clin.Invest., 52:2745-2756 (1973). A total of four vessels from three sheep were used. Identification of har~ested endothelial cells was confirmed by their cobblestone structure and by confirmed by their cobbleston~ structure and by binding of the fluorescent ligand DiI-Acetyl-LDL J.Cell.Biol, 99:2034-2040 (1984)). (Biomedicsl Technologies, Stoughton, Massschusetts). Cells were cultured on fibronectin (Collaborative Research, Bedford, Ma~sachusetts) coated plastic cul~ure dishe~ (1.0 ,ug/cm2 in M-199 Biofluids, Rock~ille, Maryland) with 20% fetal calf serum (Hyclone Laboratories, Logan, Utah), 100 U/ml penicillin, 100 ~g/ml streptomycin, and 0.25 ~ug/ml amphotericin B (Biofluids). Cells were pas3aged using trypsin-EDTA (Biofluid~
digestion. Remo~al of ~heep vessels was done according to protocols appro~ed by the snimsl use ~O~S~3!3 committee of the Nationsl Heart, Lung, and ~lood Institute .
B. A murine ecotropic psckaglng line capable of tr~nsmitting the ~-galactosida~e-containing "BAG"
vector (Proc. Nat~.Acad Sci, 84:156-160 (1987) wa~
provided by Constance Cepko (Harvard University, CambridKe, Massachusetts). Supernstant from this packaging line was used to generate an amphotropic psckaging line from PA-317 cells. A human t-PA cDNA
(in plasmid pPA34'f) (J.Biol. Chem, 260 t:ll223-11230(1985)) was provided by Sandra Degan (University of Cincinnati, Cincinnati, Ohio). This t-PA cDNA was u~ed, through several subcloning ateps, to construct a t-PA contalning retroviral vector, ~2NSt analogous in construction to the SA~ vector.
The corre~ponding pla~mid, based on the B2 plasmid, (Science), 343:220-222 (1989)) W8S transfected into GPE-86 cells, (J.Virol., 62:1120-1124 (1988)) and supernatant from these cells, thereby, generating amphotropic packaging clones capable of transmitting the t-PA gene. Endothelial cell3 were transduced by incubation for 2 hours with supernatant-containing virions with the retrovirsl vector, along with 8 ~g/ml G-418 for at least 16 days. Duplicate cultures of cells from each vessel harvest were transduced simultaneously with either the t-PA- or B-galactosidase-containing retroviral vector and, then, cultured, passaged, and selected using identical procedures. In thls manner, the t-PA- and B-galactosidase-transduced cells served as controls for one another in experiments involving either B-galactosidase activity or t-PA secretion.
C. Tubular slotted stainlesa steel 1.6-mm diameter stents (Circulation, 76:IV-27 (1987)) (Johnson and John~on Interventional Systems, Warren, New Jer~ey) 3~ 3 were cut at the articulstion, and each half was seeded with endothelial cells, u~ing a modification of the method of Van der Gei~en et al. (J.Intervent.
Cardiol, 1:109-120 (1988)). A totsl of 10 stent segments were qeeded. Endothelial cells will not grow on bsre metal, and therefore the application of a substrate is necessary before cell ~eeding. A
fibronectin costing is used in vitro to allow endothelial cell adhesion to the 3tent~. Stents were submerged in 100 ~g/ml human fibronectin for 15 minutes at 37 and, then, transferred to polypropylene tubes containing a suspension of 6-lOX104 endothelial cells in 0.8 ml culture medium.
The tubes were plsced in a 37 incubator containing 5% C2 and rotated 180 every 10 minute~ for 2 hours, after which the 3tents and cell suspension were placed in well~ of plastic tissue-culture dishes and additional culture medium added. Covera~e of the stent surfaces was monitored both by phase-contrast microscopy and by incubatlon of the stents for 4 hours in medium containing DiI-Acetyl-LDL followed by fluorescence microscopy.
D. The presence of the B-galactosidase gene product was determined by staining with 5-Bromo-4-chloro-3-indolyl-B-D-galactopyranoside (~-Gal) EMB0 J., 5:3133-3142 (1986)) of cells either on tissue-culture dishes or in situ on the stents.
Levels of human t-PA were determined by enzyme-linked immunosorbent assay (ELISA) on tissue culture supernatants using a commercially available kit (Thromb. Res., 41:527-535 (1986)). (American Diagnostica, New York, New York). Supernatant to be assayed was collected above confluent monolayers in 35-mm dishes, 48 hours after addition of 2 ml fresh medium. For measurement of t-PA secret~on from a 1'3~ `

seeded stent, the stent wa~ transEerred to a new well containing fresh medium ant, then, began a timed collection of culture medium. Harvested supernatant was centrifuged at 15,000g for 15 mlnutes to remove cellular debris, made 0.01% with Tween-80, and frozen at -70C until sssayed. The rate of t-PA secretion in nanogrsms per 106 cells per 24 hours was calculsted using a confluent cell density of 3 ~ 104 cells per cm2 of tissue culture plastic (data not shown) .
E. Seeded stents were incubated in medium containing DiI-Acetyl-LDL for 4 hours before e~pansion. The stents were v~sualized by fluorescence microscopy to confirm endothelial coverage, snd, then, manually placed over 8 deflated 3.0-mm dismeter coronsry angioplssty balloon catheter (Scimed Life Systems, Maple Grove, Minnesota). After balloon inflation to 4-6 atmosphere~, resulting in complete ~tent e~pansion, the bslloon wss deflated and the ~tents were removed fro~ the catheter~ and, agsin, viewed by fluorescence micro~copy.
F. Transduced sheep endothe~ial cells retained their cobblestone structure and their ability to bind the fluorescent ligand DiI-Acetyl-LDL. No difference in structure was detectable between those cells that had been transduced with the B-gslactosidase vector snd those thst were transduced with t-PA vector.
G. Only cells in cultures trsnsduced with the B-galsctosidase gene e~hibited deep blue cytoplasm on staining with X-Gal. After G-418 selection, most of the B-galctosidase-transduced cells stained deep blue with X-Gal.
H. Endothelial cells from all four vessel~, when transduced with the t-PA vector, secreted immunoreactive t-PA. Rates of t-PA secretion (mesn X~05~9 -a3-SD of duplicste tissue culture wells, e~pres~ed as ng/lO4 cells/24 hours) were femoral vein, 370 ~ 8;
carotid artery, 660 + 240; ~ugular vein l, 230 ~ 6;
~ugular vein 2, 200 ~ 18. t-~'A production by the ~-galacto~idase- transduced cell~ was below the lower limit of ~ensitivity of the as3ay (i.e., less than 5 ng/lO4 cells/24 hours) in all of the supernatant~
testet.
I. Fluorescence microscopy of si~ of the seeded stents confirmed complete co~erage of the visible stent surfaces. When eight stents seeded with either B-galactosidase- or t-PA-transduced endothelial cells were stained with X-Gal, the stents covered with B-galactosidase- carrying celLi turned blue, whereas the stents covered with t-PA-secreting cells did not.
Measurement of human t-PA levels from the cell culture medicum surrounding the stents confirmed that t-PA was being ~ecreted only by the t-PA-transduced endothelial cells. Three stents seeded with t-PA-transduced endothelial cells, secreted 6.3, 4.8, and 2.6 n8 t-PA/24 hours. t-PA secretion by the B-galactosidase-transduced cel~l~ on each of three stents, if present, was below the limit of detection of the assay. To check the internal consistency of our results, the measured t-PA secretion from each of three lines of transduced cells was used both before and after they were seeded onto stents to calculate the surface area of the stent~. This calculation i~
based on the assumption that the density of the cells and the rate of t-PA secretion do not change when the cells are on the stents. A stent surface area (mean i SD) of 48 i 19 mm was calculated, not significantly different fsom the manufacturer's value of 42 mm2 (personal communication, John90n and Johnson Int2rventionsl Systems, Warren, New Jersey).

X0051'39 J. ~our stent~ covered with DlI-Acetyl-LDL- stained endothelial cells were expanded using balloon catheters and immediately viewed with a fluorescene micso~cope. Near-complete retention of the cell~ on the e~terior surfaces of all four stents was confirmed. X-Gal stainin8 of stents was confirmed.
X-Gal staining of stents carrylng B-galactosidase-transduced cells permitted evaluatlon of cellular retention on all ~urfaces after balloon inflation.
The stents were viewed with a dlssecting microscope, and cellulsr retention on all surfaces was estimated.
A total of ~ight expanded stent~ were observed after X-Gal staining, four covered with B-galacto~idase-transduced endothelial cells. Much of the interior lumen surface of the stents was free of cells after balloon in1ation but that the cellular layer on the exterior and lateral ~tent-strut surfaces was largely intact.
Although the ~cope of the present invention is not intended to be limited to any theoretical reasoning, it is believed that an intravascular stent seeded with endothelial cells ~as hereinabove described may produce a local thrombolytic environment in vivo. High level secretion of t-PA
ad~acent to a forming clot may permit t-PA to be concentrated through the high affinity binding of t-P~ to fibrin. (Thorsen, et al., Thromb D.
Haemorrh, 28:65-74 (1972)). In this manner, fibrinolytic acti~ity would be directed to microthrombi beginning to form on the ~tent surface or do~nstream, thus prevent~ng the formation of occlusive thrombi. It has been demonstrated (Hergreuter, et al., Plast. Reconstr. Sur~., 81:418-424 (1988~) in a rabbit model that locally administered t-PA could abort thrombus formation on a ~O~a~99 -as-hi6hly thrombogenic inverted artery. Intravascular stents are far lass thrombogenic than is an inverted vessel, and it is possible that localized delivery of nanogram quantitie9 of t-PA will re~ult in qufficient thrombolytic activity to prevent stent-related thrombotic events.
It has also been t'neorized that the implantation of genetically en8ineered endothelial cells on stent surfaces offers a potential means of preventing intimal hyperplasia because implanted endothelial cells would be in direct contact with the intima and could be engineered to secrete proteins capable of inhibiting intimsl growth.
Numerous modifications snd variations of the present invention are possible in light of the above teachings; therefore, within the scope of the appended claims, the invention may be practiced otherwise than as particularly described.

Claims (39)

1. A product, comprising:
endothelial cells genetically engineered with at least one gene for at least one heterologous protein.
2. The product of claim 1 wherein the cells are human endothelial cells.
3. The product of claim 1 wherein the endothelial cells are microvascular endothelial cells.
4. The product of claim 1 wherein the endothelial cells are microvascular endothelial cells.
5. The product of claim 1 wherein the endothelial cells are genetically engineered with a retroviral vector including a gene for a heterologous protein.
6. The product of claim 5 wherein the heterologous protein is a therapeutic agent.
7. The product of claim 1 wherein the endothelial cells are mammalian endothelial cells.
8. The product of claim 1 wherein the heterologous protein is secretable from the cells.
9. A product comprising:
a solid support, said solid support including endothelial cells genetically engineered with at least one gene for at least one heterologous protein.
10. The product of claim 9 wherein the solid support is compatible with blood.
11. The product of claim 10 wherein the solid support is a vascular graft.
12. The product of claim 11 wherein the graft is a synthetic graft.
13. The product of claim 10 wherein the solid support is a tubular slotted stainless steel intravascular stent, said stent being coated with a substrate for permitting adhesion of said endothelial cells to said stent.
14. The product of claim 11 wherein the cells are human endothelial cells.
15. The product of claim 11 wherein the endothelial cells are microvascular endothelial cells.
16. The product of claim 11 wherein the endothelial cells are microvasculsr endothelial cells.
17. The product of claim 11 wherein the endothelial cells are genetically engineered with a retroviral sector including 8 gene for a heterologous protein.
18. The product of claim 12 wherein the heterologous protein is a therapeutic agent.
19. The product of claim 18 wherein the heterologous protein is secretable from the cells.
20. A process for gene therapy, comprising:
implanting in a blood vessel of a host endothelial cells genetically engineered with at least one gene for a heterologous protein which is a therapeutic agent for the host.
21. The process of claim 20 wherein said endothelial cells are implanted by implanting in a blood vessel of the host a biocompatible solid support containing genetically engineered endothelial cells.
22. The process of claim 21 wherein the host is a human and the genetically engineered endothelial cells are human endothelial cells.
23. The process of claim 22 wherein the endothelial cells are genetically engineered with a retroviral vector including 8 gene for the therapeutic agent.
24. The process of claim 21 wherein the host is a human patient and the endothelial cells are autologous endothelial cells.
25. The process of claim 24 wherein the solid support is a vascular graft.
26. The process of claim 25 wherein the endothelial cells are genetically engineered with 8 retroviral vector including a gene for the therapeutic agent.
27. The process of claim 26 wherein the therapeutic agent is secretable from the cells.
28. The process of claim 20 wherein said endothelial cells are comprised of a first portion genetically engineered to express 8 first therapeutic agent and a second portion genetically engineered to express a second therapeutic agent different from the first therapeutic agent.
29. The product of claim 7 wherein said gene encodes for soluble CD-4.
30. The product of claim 7 wherein said gene encodes for ADA.
31. The product of claim 7 wherein said gene encodes for TPA.
32. The product of claim 10 wherein the gene encodes for a member selected from the group consisting of soluble CD-4, Factor VIII, factor IX, von Willebrand Factor, TPA, urokinase, hirudin, the interferont, tumor necrosis factor, the interleukins, hematopoietic growth factors, antibodies, glucocerebrosidase, ADA, phenylalsnine hydroxylase, human growth hormone, and insulin.
33. The process of claim 24 wherein the gene encodes for a member selected from the group consisting of soluble CD-4, Factor VIII, Factor IX, von Willebrand Factor, TPA, urokinsse, hirudin, the interferons, tumor necrosis factor, the interleukins, hematopoietic growth factors, antibodies, glucocerebrosidase, ADA, phenylalanine hydroxylsse, human growth hormone, insulin and erythropoietin.
34. The product of claim 10 wherein the gene encodes for soluble CD-4.
35. The product of claim 10 wherein the gene encodes for ADA.
36. The product of claim 10 wherein the gene encodes for TPA.
37. The process of claim 33 wherein the gene encodes for soluble CD-4.
38. The process of claim 33 wherein the gene encodes for ADA.
39. The process of claim 33 wherein the gene encodes for TPA.
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