AU9718598A - Genetically modified cells for use in transplantation - Google Patents
Genetically modified cells for use in transplantation Download PDFInfo
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Description
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AUSTRALIA
PATENTS ACT 1990 Ii *1; Name of Applicant: ,tual Inventor: *v Namddress of ApplServicant: ^Oi COMPLETE SPECIFICATION FOR A STANDARD PATENT
ORIGINAL
DIACRIN, INC.
Michael E. EGAN and Scott C. CHAPPEL BALDWIN SHELSTON WATERS MARGARET STREET SYDNEY NSW 2000 Invention Title: GENETICALLY MODIFIED CELLS FOR USE IN
TRANSPLANTATION
Details of Original Application No. 22363195 dated 30 March 1995 The following statement is a full description of this invention including the best method of performing it known to me/us:t i.^ r.A *t& *;fc -i la GENETICALLY MODIFIED CELLS FOR USE IN TRANSPLANTATION Background of the Invention The elucidation of the molecular basis for many inherited disorders together with the molecular isolation of the genes involved in the:: disorders now offer the potential for therapeutic treatments based upon providing a functional gene product to a patient having a defect in that gene product. Gene therapy, in which a gene encoding a functional gene product is introduced into cells of a patient to restore the activity of that gene product in the patient, is now a realistic option for many congenital diseases. Two patients with adenosine 1 0 deaminase deficiency have already been treated for their disease by gene therapy, with Sencouraging results, and a number of other human gene therapy protocols have received J ;approval for limited clinical use. Cystic fibrosis, Duchenne muscular dystrophy and hemophilia are just a few of the inherited diseases which are potentially treatable by gene therapy. Furthermore, gene therapy approaches are being applied to acquired disorders as well, for example by introducing into cells of a patient genes encoding gene products which enhance the responsiveness of the patient's immune system. Novel approaches to treating diseases such as cancer and AIDS are thus also possible by applying the principles of gene therapy. For reviews on gene therapy approaches see Anderson, W.F. (1992) Science 256:808-813; Miller, A.D. (1992) Nature 357:455-460; Friedmann, T. (1989) Science 244:1275-1281; and Coumoyer, et al. (1990) Curr. Opin. Biotech. 1:196-208.
The general approach of gene therapy involves the introduction of exogenous genetic material DNA or RNA) into a cell such that one or more gene products encoded by the introduced genetic material are produced in the cell, for example to restore or enhance a functional activity. Exogenous DNA has been successfully introduced into cells both ex viva in vitro) and in vivo. In recent years, many advances in gene therapy have been reported that address problems relating to the types of gene delivery systems that can be used, the different types of genes which can be introduced into cells and the kinds of cells which can be modified. However, gene therapy is still limited by the need to modify autologous cells. A patient's own cells must be modified because foreign cells, whether they are from the same 30 species (allogeneic) or another species (xenogeneic), are recognized as foreign by the patient's immune system when introduced into the patient and are subsequently rejected.
Thus, in the case of ex vivo gene therapy, cells must first be harvested from the patient, modified in culture and then reintroduced into the patien This procedure is both timeconsuming to complete and invasive forthe patient. While the ability to modify some cells in 35 vivo may overcome sme of theseprobles, certain cell types may not be accessible for modification vivr, or may not be targeted specifically or efficiently modified i viv.
Furermore, the cell modif on procedure performed e v or in vivo, mus be Irepead for each individual patiet i
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a ~19* A a As a means of gene therapy, it would be beneficial to provide a patient with heterologous donor cells that have been modified to express a gene product. This would obviate the need to harvest cells from the patient for genetic modification, thereby reducing both the time and invasiveness of the procedure. In addition, a ready supply of modified donor cells that cc'! be cryopreserved and available for inroduction into one, or multiple, paients could be prepared for use as needed. The ability to use modified heterologous donor cells from either the same species as the patient or from a different species would also expand the source of cells which could be used for gene therapy.
However, transplantation ofheterologous cells allogeneic or xenogeneic cells) into a host elicits an immune response against the cells. Thus, use of modified heterologous cells for therapeutic purposes requires a means by which to avoid immunological rejection o the modified cells by the patient. Current approaches toward inhibiting immune responses against transplanted cells typically involve systemic treatment of the patient, for example with immnosuppressive agents. This has the disadvantage that the patient exhibits nonspecific immunosuppression. Additionally, immunosuppressive drugs are known to have side effects that include an increased susceptibility to infections, renal failure, hypertension and tumor growth. Thus, there is a need for an improved method which allows the use of heterologous donor cells for gene therapy purposes which avoids the detrimental effects of systemic immunosuppressants.
This invention provides a means by which allogeneic cells or xenogeneic cells are used to deliver a gene product to an individual without the need for systemic munsuppresion of the individual. The invention features cells which are modiied to expres a gene product and which have an antigen on the surface of the cell altered to inhibit rejection of the cell when the cell is transplanted into a subject. Prior to alteration, the antigen on the cell surface stimulates an immune response against the cell in the subjecL However, the antigen on the cell surface is altered to inhibit immunological rejection of the cells by the subject. Specifically, the antigen is altered to modify an interaction between the antigen and a heatopoietic cell, preferably a T lymphocyte, in the subject. Since the antigen onthe cell surface is altered prior to transplantation, the recipient subject does not require systemic treatment with an immunosuppressive agent to prevent rejection of the cell. Thusa this invention permits the use of allogeneic or xenogeneic cells as vehiles for delivery of a gne product to a subjeL Moreover this invention allows for the preparation of geneticall modifed cills which can be cryoprsrved and admnistd to a subj when necessmy, thby ircumventing the need to isolate and modify. auologous cells from the subject
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According to a first aspect the present invention provides a cell suitable for transplantation which comprises a recombinant vector comprising a nucleic acid encoding a gene product in a form suitable for expression of the gene product in the cell, the cell having at least one antigen on the cell surface which is capable of stimulating an immune response against the cell in an allogeneic or xenogeneic subject and wherein the .antigen on the cell surface is altered prior to transplantation such that an immune response against the cell is inhibited upon transplantation of the cell into a subject.
According to a second aspect the present invention provides a kit for delivering a H human gene product to a subject comprising: a cell which is modified to express the human gene product and which has an antigen on the cell surface which is capable of stimulating an immune response against -the cell in an allogeneic or xenogeneic subject; and an antibody, or fragment or derivative thereof, which binds to the antigen on the cell surface prior to transplantation such that an immune response to the cell is inhibited upon transplantation of the cell into a subject.
SAccording to a third aspect the present invention provides a kit for delivering a human gene product to a subject comprising: a vector encoding the human gene product in a form suitable for expression of the human gene product in a cell; and an antibody, or fragment or derivative thereof, which binds to an antigen on a cell surface which is capable of stimulating an immune response against the cell in an allogeneic or xenogeneic subject prior to transplantation such that an immune response Sto the cell is inhibited upon transplantation of the cell into a subject.
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s According to a fourth aspect the present invention provides a method for delivering a human gene product to a subject comprising: contacting a cell which has been modified to express the human gene product with at least one molecule which binds to at least one antigen on the cell surface which is capable of stimulating an immune response against the cell in an allogeneic or •xenogeneic subject prior to transplantation such that the antigen is altered and an immune response against the cell is inhibited upon transplantation of the cell into a subject; and administering the cell to the subject.
According to a fifth aspect the present invention provides the use of a cell which has been modified to express a human gene product, wherein the cell has been contacted with at least one molecule which binds to at least one antigen on the cell surface which is capable of stimulating an immune response against the cell in an allogeneic or xenogeneic subject prior to transplantation such that the antigen is altered and an immune response against the cell is inhibited upon transplantation of the cell into a C subject, for the manufacture of a medicament for delivering a human gene product to a subject.
Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise' 'comprising', and the like are to be construed in an S 20 inclusive sense as opposed to an exclusive or exhaustive nse; that is to say, in the sense of "including, but not limited to".
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2c According to the invention, a cell is modified to express a gene product by, for example, introducing into the cell a nucleic acid having a nucleotide sequence which encodes the gene product in a form suitable for expression of the gene product in the cell. In a i 9-4a Xl I ir I :11 J" -faaa~p c~slars~ preferred embodiment, the nucleic acid encoding the gene product is introduced into the cell using a recombinant viral vector, such as an adenoviral vector, an adeno-associated viral vector or a retroviral vector. The gene product can be, for example, a secreted protein, a membrane-bound protein or an intracellular protein. Other gene products include active RNA molecules.
In one embodiment of the invention, an antigen on the surface of the cell is altered by contacting the cell prior to transplantation in vitro) with a molecule which binds to the antigen. A preferred molecule for altering an antigen on the cell is an antibody, or fragment or derivative thereof, such as an F(ab) 2 fragment Alternatively, the molecule is a peptide or 10 derivative thereof a peptide mimetic) which binds the antigen and interferes with an interaction with a hematopoietic cell. In a preferred embodiment, the antigen on the cell surface which is altered is an MHC class I antigen. Other cell surface antigens which can be altered include adhesion molecules such as LFA-l, ICAM-1 and ICAM-2.
In one embodiment of the invention, a non-human cell is modified to express a human gene product. A preferred non-human cell for use in providing a human gene product to a subject is a porcine cell. Cell types which are modified according to the invention include muscle cells, liver cells, neural cells, pancreatic islet cells and hematopoietic cells.
Furthermore, the cell which is modified can be within a tissue or organ.
The modified cells of the invention are administered to subjects to deliver a gene product expressed by the cell to the subject Prior to administering the cell to the subject, one or more antigens on the cell surface are altered, e.g. by contacting the cell in vitro with a molecule which binds to the antigen. Although a cell can be modified to express a gene product in vivo, it is preferred that the cell is modified ex vivo, prior to administering the cell to the subject.
The invention further provides kits for use in delivering a gene product to a subject Swhich include a cell modified to express the gene product and a molecule an antibody, or fragment or derivative thereof) which binds to an antigen on the cell." Alternatively, the kit includes a vector encoding a gene product with which to modify a cell and a molecule i an antibody, or fragment or derivative thereof) which binds to an antigen on the cell surface.
Brief Description of the Draing -Figure 1 depicts the plasmid map of pCMVGH, which contains the gene encoding -I human growth hormone.
Figure 2 depicts a P-galactosidase stain of cultured human myotubes transfected with plasmids pCMVp, which contains a gene encoding p-galactosidase, and pJK2Neo, which displays neomycin resistanc Figme 3 depicts a p-galactosidase stain of human myoblasts transfected with i plasmids pCMVP, which contains a gene encoding pgalactosidase, and pJK2Neo, which displays neomycin resistance, which wue transplanted into rat muscle.
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Figure 4 depicts a rabbit anti-desmin stain of human myoblasts modified with F(ab' fragments of the monoclonal antibody W6/32 and transplanted into cyclosporin-treated mice.
Detailed Description of the Invention This invention provides modified heterologo-s cells and methods for delivering a gene product to an allogeneic or xenogeneic subject without eliciting an immune response against the cell in the subject The invention features a heterologous cell which is modified to express a gene product and which is treated such that an antigen on the cell surface which stimulates an immune response against the cell in an allogeneic or xenogeneic subject is S 10 altered to inhibit rejection of the cell when transplanted into the subject Preferably, the antigen which is altered is an antigen which interacts with a T lymphocyte in an allogeneic or xenogeneic subject. Typically, the heterologous cell is treated to alter the antigen on its surface prior to administering the cell to the subject Thus, it is not necessary to treat the subject systemically with an immunosuppressive agent to prevent rej*ction of the heterologous cell. Rather, following administration of the heterologous cell (which has been altered as described herein), the subject exhibits immunological non-responsiveness specific for the cell. Preferably, the heterologous cell is also modified to express a gene product prior to administering the cell to a subject ex vivo). However, the cell can be modified to express a gene product in vivo, following administration of the cell to the subject S 20 This invention enables gene therapy approaches to be extended to the use allogeneic and xenogeneic cells as donor cells to deliver a gene product to a subject. Since both the procedure to modify a heterologous cell to express a gene product and the procedure to alter an antigen on the surface of the cell can be perfomed ex vivo, prior to administering the cell to a subject, the invention allows for the preparation of genetically modified allogeneic or xenogencic cells which can be cryopreserved and stored until use: When the cells are needed by a subject, the already modified cells canbe thawed, treated to alter an antigen on their (a surface and immediately administered to the subject. Thus, a subject in need of gene therapy can receive immediate treatment, rather than having to wait until his or her own cells can be
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isolated, successfully modified and reintroduced. Additionally, if retreatment is necessary, a second aliquot of already modified cells can easily be thawed, altered and readministered.
Moreover, the ability to use heterologous cells for gene therapy purposes greatly extends the supply of donor cells which can be used in this type of treatment.
Accordingly, this invention provides a heterologous cell which is modified to express a gene product and which has an antigen on the cell surface altered to modify an interaction betwen the antigen anda hematopoictic cell aT lymphocyte). The invention further provides methods for delivering a gene product to a subject by administering cells of the Snvetion. The following subsections des'be in detail 1) the modification of a heterologous ce to ex pre a gen product ad 2) the altatin of an ati on the cell t modify an k interaction between the antigen and a heatopoietic celL 1 I- 1. Modification of a Cell to Express a Gene Product A cell of the invention is "modified to express a gene product". As used herein, the ternn "modified to express a gene product" is intended to mean that the cell is treated in a manner that results ir the production of a gene product by the cell. Preferably, the cell does not express the gene product prior to modification. Altematively, modification of the cell may result in an increased production of a gene product already expressed by the cell or result in production of a gene product an antissee RNA molecule) which decreases production of another, undesirable gene product normally expressed by the cell.
10 In a preferred embodiment, a cell is modified to express a gene product by introducing-genetic material, such as a nucleic acid molecule RNA or, more preferably, DNA) into the cell. The nucleic acid molecule introduced into the cell encodes a gene product to be expressed by the cell- The term "gene product" as used herein is intended to Sinclude proteins, peptidcs and functional RNA molecules. Generally, the gene product encoded by the nucleic acid molecule is the desired gene product to be supplied to a subject.
Alternatively, the encoded gene product is one which induces the expression of the desired gene product by the cell the introduced genetic material encodes a transcription factor which induces the transcription of the gene product to be supplied to the subject).
t he A nucleic acid molecule introduced into a cell is in a form suitable for expression in the cell of the gene product encoded by the nucleic acid. Accordingly, the nucleic acid S molecule includes coding and regulatory sequences required for transcription of a gene (or portion thereof) and, when the gene product is a protein or peptide, translation ofthe gene product encoded by the gene. Regulatory sequences which can be included in the nucleic acid molecule include promoters, enhancers and polyadenylation signals, as well as S 25 sequences necessary for transport of an encoded protein or peptide, for example N-terminal signal sequences for transport of proteins or peptides to the surface of the cell or for secretion.
Nucleotide sequences which regulate expression of a gene product promoter and I enhancer sequences) are selected based upon the type of cell in which the gene product is to be expressed and the desired level of expression of the gene product. For example, a promoter known to confe cell-type specific expression of a gene linked to the promoter can be used. A promoter specific for myoblast gene expression can be linked to a gene of interest to confer muscle-specific expression of that gene product- Muscle-specific regulatory elements which are known in the art include upstream regions from the dystrophin gene (Klamuret aL, (1989) Mot CeL Bo 92396), the creatine kinase gene (Buski and Hauschka, (1989) MoLCell BoL 9:2627) andthetroponin gene (Mar and Ordabl, (1988) Proc NaL Acad Si USA 85:6404). Regulatory lements specific for other cell types are known in the art the albai enancer for liversecif or isu eleents for pancreatic islet cll-specific expressio; various neural cell-specific regulatory elements including neural dyrop i neual enolase and A4 amyloid prmoters).
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-6- Alternatively, a regulatory element which can direct constitutive expression of a gene in a variety of different cell types, such as a viral regulatory element, can be used. Examples of viral promoters commonly used to drive gene expression include those derived from polyoma virus, Adenovirus 2, cytomegalovirus and Simian Virus 40, and retroviral LTRs.
Alternatively, a regulatory element which provides inducible expression of a gene E;ked thereto can be used. The use of an inducible regulatory element an inducible promoter) allows for modulation of the production of the gene product in the cell. Examples of potentially useful inducible regulatory systems for use in eukaryotic cells include hormoneregulated elements see Mader, S. and White, J.H. (1993) Proc. Nail. Acad. Sci. USA 90:5603-5607), synthetic ligand-regulated elements (see, e.g. Spencer, D.M. et al. (1993) Science 262:1019-1024) and ionizing radiation-regulated elements see Manome, Y. et al. (1993) Biochemistry 32:10607-10613; Datta, L et al. (1992) Proc. Natl. Acad Sci USA 89:10149-10153). Additional tissue-specific or inducible regulatory systems which may be developed can also be used in accordance with the invention.
There are a number of techniques known in the art for introducing genetic material into a cell that can be applied to modify a cell of the invention. In one embodiment, the nucleic acid is in the form of a naked nucleic acid molecule. In this situation, the nucleic acid molecule introduced into a cell to be modified consists only of the nucleic acid encoding the gene product and the necessary regulatory elements. Alternatively, the nucleic acid encoding the gene product (including the necessary regulatory elements) is contained within a plasmid vector. Examples of plasmid expression vectors include CDM8 (Seed, Nature 329:840 (1987)) and pMT2PC (Kaufnan, et al, EMBO J. 6:187-195 (1987)). In another embodiment, the nucleic acid molecule to be introduced into a cell is contained within a viral vector. In this situation, the nucleic acid encoding the gene product is inserted into the viral genome (or a partial viral genome). The regulatory elements directing the expression of the gene product can be included with the nucleic acid inserted into the viral genome iCe, linked to the gene inserted into the viral genome) or can be provided by the viral genome itself Examples of methods which can be used to introduce naked nucleic acid into cells and viralmediated transfer of nucleic acid into cells are described separately in the subsections below.
F A Introduction of Naked Nucleic Acid into Cells 1. Transfection mediated by CaPO4: Naked DNA can be introduced into cells by forming a precipitate containing the DNA and calcium phosphate. For example, a HEPES-buffered saline solution can be mixed with a solution containing calcium chloride and DNA to form a preipitate and the preipitis then incubated ih cells. A glycerol or diethyl sulfoxide shock step can be added to increas the amoum of DNA taken up by ctain cells. CO 4 mediated transfection can be used to stably (or transiently) trasfct cells and is only -7applicable to in vitro modification of cells. Protocols for CaPO 4 mediated transfectioncn be found in Crrrent Pratocaok in Molecular Bialogv. Ausubel, F-Nt et al- (eds-) Greene Publishing Associates, (1989), Section 9.1 and in Jleciular Cloning- A Laboratory Manual.
2nd Edition. Samnbrook et al. Cold Spring Harbor Laboratory Press, (1989), Sections 16.32- 1 6AO or other standard laboratory manuals- 2. TransJection mediajed by DE4E-davran: Naked DNA can be introduced into cells by forming a mixture of The DNA and DEAE-dextraII and incubating the mixture with the cells.
A dimethylsulfoxide or chioroquine shock step can be added to increase the amount of DNA uptake. DEAE-dextran transfection is only applicable to in vitro modification of cells and can be used-to introduce DNA transiently into cells but is not preferred for creating stably transfected cells. Thus, this method can be used for short term production of a gene prod uct but is not a method of choice for long-term production of a gene product. Protocols for DEAF-dextran-mediated transfection can be found in Cu=rn Protocols in Molecular BiaIlgyv, Ausubel, F.M- et al. (eds.) Greene Publishing Associates, (1989), Section 9.2 and in Molecular Cloning: A Laboratory Manual- 2nd Edition, Sambrook et L- Cold Spring Harbor Laboratory Press, (1939), Sections 16.41-16.46 or other standard laboratory manuals.
Electroporation: Naked DNA can. also be introduced into cells by incubadig the cells and the DNA together in an appropriate buffer and subjecting the cells to a high-voltage electric pulse. The efficiency with wilich DNA is introduced into -cells by eiectroporation is influenced by the strength of the applied field, the length of the electric pulse, the temperature, the conformation and concentration of the DNA and the ionic composition of the media. Electroporation can be used to stably (or traniently) transfect a wide variety of W 25 cell types and is only applicable to in vitro modification of cells. Protocols for electroporating cells can be found in Current Protocols in MolecuJLgB.ljggg Auspbel, F.M.
et al. (eds.) Greene Publishing Associates, (1989), Section 9.3 and in M6lecular Cloning; A Laborator Manual. 2nd Edition. Sambrook et aL Cold Spring Harbor Laboratory Press, (1989), Sections 16-54-16-55 or othe standard Laboratory manuals.
4. Liposome-mediated transfection Clipofecrion"): Naked DNA can be introduced into cells by mixing the DNA with a liposome'suspension containing cationic lipids. The DNA/liposoine complex is then incubated with cells. Liposome mediated trnsfection can be used to stably (or transiently) transfect cells in culture in vitro. Protocols can be found in Curr=n Protocols in Molecular Rioloo, Ausubel, F.M. et al- (eds.) Greene PublishingJ, Associates, (1989), Section9-4 and other standard laboratoryrnannals. Additionally, gene delivery in vivo has been accomilished using liposomes. See for example Nicolan et al (1987) Met&i Enz, 149:157-176-; Wang and Huang (19g7) Proc- NaiL Acad Sci USA s. i 4 i-t -8- 84:7851-7855; Brigham et al. (1989) Am. J. Med Sci. 298:278; and Gould-Fogerite et al.
(1989) Gene 84:429- 4 3 8.
Direct Injection: Naked DNA can be introduced into cells by directly injecting the DNA into the cells. For an in vitro culture of cells, DNA can be introduced by microinjection.
Since each cell is microinjected individually, this approach is very labor intensive when modifying large numbers of cells. However, a situation wherein microinjection is a method of choice is in the production of transgenic animals (discussed in greater detail below). In this situation, the DNA is stably introduced into a fertilized oocyte which is then allowed to S 10 develop into an animal. The resultant animal contains cells carrying the DNA introduced into the oocyte. Direct injection has also been used to introduce naked DNA into cells in vive (see Acsadi et al. (1991) Nature 332: 815-818; Wolffet al. (1990) Science 247:1465-1468).
SA delivery apparatus a "gene gun") for injecting DNA into cells in vivo can be used.
Such an apparatus is commercially available from BioRad).
6. Receptor-Mediated DNA Uptake: Naked DNA can also be introduced into cells by complexing the DNA to a cation, such as polylysine, which is coupled to a ligand for a cellsurface receptor (see for example Wu, G. and Wu, C.H. (1988) J. Biol. Chem. 263:14621; Wilson et al. (1992) J. Biol. Chem. 267:963-967; and U.S. Patent No. 5,166,320). Binding of the DNA-ligand complex to the receptor facilitates uptake of the DNA by receptor-mediated endocytosis. Receptors to which a DNA-ligand complex have targeted include the transferrin receptor and the asialoglycoprotein receptor. A DNA-ligand complex linked to adenovirus capsids which naturally disrupt endosomes, thereby releasing material into the cytoplasm can be used to avoid degradation of the complex by intracellular lysosomes (see for example Curiel et al. (1991) Proc. Natl. Acad Sci. USA 88:8850; Cristiano et al. (1993) Proc. Natl.
Acad. ScL USA 90:2122-2126). Receptor-mediated DNA uptake can be used to introduce DNA into cells either in vitro or in vive and, additionally, has the added feature that DNA can be selectively targeted to a particular cell type by use of a ligand which binds to a receptor selectively expressed on a target cell of interest.
Generally, when naked DNA is introduced into cells in culture by one of the transfection techniques described above) only a small fraction of cells (about 1 out of 105) 't typically integrate the transfected DNA into their genomes the DNA is maintained in the cell episomally). Thus, in order to identify cells which have taken up exogenous DNA, it is advantageous to transfect nucleic acid encoding a selectable marker into the cell along with the nucleic acid(s) of interest Prf dsetable marks include those which confer resistance to drugs such as G418, hygromycin and methotrxate. Selectable markers may be bbi oasp t introduced on the same plasmid as the gene(s) of interest or may be introduced on a separate 4- -9- An alternative method for generating a cell that is modified to express a gene product involving introducing naked DNA into cells is to create a transgenic animal which contains cells modified to express the gene product of interest. A transgenic animal is an animal having cells that contain a transgene, wherein the transgene was introduced into the animal or an ancestor of the animal at a prenatal, an embryonic stage. A transgene is a DNA molecule which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. Thus, a transgenic animal expressing a gene product of interest in one or more cell types within the animal can be created, for example, by introducing a nucleic acid encoding the gene product (typically linked to appropriate regulatory elements, such as a tissue-specific enhancer) into the male pronuclei of a fertilized oocyte, by microinjection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Methods for generating transgenic animals, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009 and Hogan, B. i et al., (1986) A Laboratory Manual, Cold Spring Harbor, New York, Cold Spring Harbor Laboratory. A transgenic founder animal can be used to breed more animals carrying the transgene. Cells of the transgenic animal which express a gene product of interest can then be used to deliver the gene product to a subject in accordance with the invention.
Alternatively, an animal containing a gene which has been modified by homologous recombination can be constructed to express a gene product of interest. For example, an endogenous gene carried in the genome of the animal can be altered by homologous recombination (for instance, all or a portion of a gene could be replaced by the human homologue of the gene to "humanize" the gene product encoded by the gene) or an _H 25 endogenous gene can be "knocked out" inactivated by mutation). For example, an endogenous gene in a cell can be knocked out to prevent production of that gene product and then nucleic acid encoding a different (preferred) gene product is introduced into the cell. To I *create an animal with homologously recombined nucleic acid, a vector is prepared which contains the DNA which is to replace or interrupt the endogenous DNA flanked by DNA S 30 homologous to the endogenous DNA (see for example Thomas, K.R and Capecchi, M. R.
(1987) Cell 51:503). The vector is introduced into an embryonal stem cell line by electroporation) and cells which have homologously recombined the DNA are selected (see for example Li, E. et al. (1992) Cell 62915). The selected cells are then injected into a blastocyst of an animal a mouse) to form aggregation chimeras (see for example Bradley, A. in Teraocarcinomas and Embryonic Stem Cells: A Practical Approach, EJ.
Robertson,ed. (IRL,Oxford, 987) pp 113-152). A chimeric embryo can then be implanted Sinto a suitable pseudopregant female foster animl and the embryo brought to term.
Progeny harbouring the homologously recobined in their germ cells can be used to ?breed animals in whch all cells of the animi contain the homologously recombined DNA.
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Cells of the animal containing the homologously recombined DNA which express a gene product Of interest can then be used to deliver the gene product to a subject in accordance with the invention.
B.V ViidMedatdGew& rse A c peerd aproach for introducing nucleic acid encoding a gene product into a cell is y ue f avirl ectr cntining nucleic acid, e.g. a cDNA, encoding the gene product.
infection of cells with a viral vector has the advantage that a large proportion of cells receive the nucleic acid, which can obviate the need for selection of cells which have received the nucleic acid. Additionally, molecules encoded within the viral vector, by &cDNA contained-in the viral vector, are expressed efficiently in cells which have taken up viral vector nucleic acid and viral vector systems can be used either in vitro or in vivo.
1. Refroviruses: Defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D. (1990) Blood 76:271). A recombinant retrovirus can be constructed having a nucleic acid encoding a gene product of interest inserted into the retroviral genorne. Additionally, portions of the retrovirai genome can be removed to render the retrovirus replication defective. The replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Qjnn.BQtQlL MoIlflhjijrjl1,Y Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retrovinises include pLJ, pZIP,,pWE and pEM which are well known to those skilled in the art.
Examples of suitable packaging virus lines include WCrip, XjCre, W~2 and iyAm. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocyt~s, bane marrow cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988).Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natf.
Acad-SSet USA. 85-3014-3018; Armentano et al. (1990) Proc. Nail. Acad Sci. USA 87:6141- 6145; Huber et al. (1991).Proc. Nall. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc.
'Naod. Acad ScL USA 88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van Beusechem et al. (1992) Proc. Naol. Acad' Scd USA 89:7640-7644, K.ay et al. (1992) Human Gene 7therapy31:64-7647; Dai et al(1 9 9 2 Proc. Nall. AcacL Sct. USA 9 9 :10992-1089; H'u i etl. (1993) J: Immnol 15 .41044A1s; U.S. Patent No. 4,868,116; U.S. Patent No.
4.980,286; PCT Applicationi WO 819107136; PCT Application WO.89102468;
PCT
A pplication WO 89105345- end PCT Application.W 2077) Retroviral vectors requr target cec dvision in order for the retroviral genorne (and-foreign nucleic. acid inserted into
A
I
-ii it) to be integrated into the host genome to stably introduce nucleic acid into the cell. Thus, it may be necessary to stimulate replication of the target cell.
2. AdenoviruseS: The genome of an adenovirus can be manipulated such that it encodes and expresses a gene product c!mnterest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See for example Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155.
Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 d1324 or other *s tains; of adenovirus Ad2, Ad3, Ad7.etc.) are well known to those skilled in the art.
Recombinant adenoviruses are advantageous in that they do not require dividing cells to be effective gene delivery vehicles and can be used to infect a wide, variety of cell types, including airway epithelium (Rosenfeld et aL (1992) cited supra), endothelial cells* ~*.(Lemarchand et al. (1992) Proc. Nail. A cad. Sci. USA 89:6482-6486), hepatocytes (H1erz and Gerard (1993) Proc. NatL Acad Sci. USA 90:2812-2816) and muscle cells (Quantin et al.
:(1992) Proc. Nat!. Acad Scl USA 89:2581-2584). Additionally, introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episonial, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et a]l.
cited supra; Haj-Ahmand and Graham (1986) J1 Viral. 57:267). Most replication-defective adenoviral vectors currently in use are deleted for all or parts of the viral El and E3 genes but retain as much as 80;% of the adtinovira1 genetic material.
K Adeno-Associaled Viruses:~ Adqno-a ssociated virus (AAV) is axiaturally occurring defective virus that requires another viU's, such as an aderxovinis or a herpes virus, as a helper virus for efficient replication and a productive, lie yce(oraevwseMizyczka et al.
~.7Curr. Topics in Micro. and Immunol. (1992) 158:97-129). It is also one of the few viruses thatmayinterat it DNAint no-dividing cells, ad exhibits a high fequenicy of stable itgain(see for example Flotte et al. (1992)4n AmJ.Respir. Cell.,Mo!. Biol. 7.349-3 56; Samuliki et al. (1989)J ViraL 63:3822-3828; and Mcdauglin it al. (1989)1 Viral.
62:1963-1973). Vectors containing as little a s'300 base pairs of AAV can be packaged and A J can integne Space foP xgnu N is limited to about 4.5 kb. An AAV vector such as that described in Tratschin et al. (1985) AM,? CO. Biol 5:3251-r3260 can be used to introduce DA inocells. A variety of nucleic acids h~ave been introduced into difeet 11 tpe sigAAV vectors (sve for exaple Hermonat et Jl. 1984 Poc. Kai.-Aa SaL US 81:6466-67;Tasbnc] _(1985) MoL :Cell Biol. 7.272081; Wondisford et al. (19889) Mal EndDCrino 23-;Tratehin efal.(19841.J Vrl 51:611- 619; and Flouc ai al.
(1993). i-Cho-m.269:571-3790).
12- *rr c I *i 9 3r o o *er 91 oi 'a9
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The efficacy of a particular expression vector system and method of introducing nucleic acid into a cell can be assessed by standard approaches routinely used in the art. For example, DNA introduced into a cell can be detected by a filter hybridization technique Southern blotting) and RNA produced by transcription of introduced DNA can be detected, for example, by Northern blotting, RNase protection or reverse transcriptase-polymerase chain reaction (RT-PCR). The gene product can be detected by an appropriate assay, for example by immunological detection of a produced protein such as with a specific antibody, or by a fiuctional assay to detect a functional activity of the gene product, such as an enzymatic assay. If the gene product of interest to be expressed by a cell is not readily 10 assayable, an expression system can first be optimized using a reporter gene linked to the regulatory elements and vector to be used. The reporter gene encodes a gene product which is easily detectable and, thus, can be used to evaluate the efficacy of the system. Standard reporter genes used in the art include genes encoding p-glactosidase, chloramphenicol acetyl transferase, luciferase and human growth hormone.
15 When the method used to introduce nucleic acid into a population of cells results in modification of a large proportion of the cells and efficient expression of the gene product by the cells as is often the case when using a viral expression vector), the modified population of cells may be used without further isolation or subcloning of individual cells within the population. That is, there may be sufficient production of the gene product by the 20 population of cells such that no further cell isolation is needed. Alternatively, it may be desirable to grow a homogenous population of identically mo.dified cells from a single modified cell to isolate cells which efficiently express the gene product. Such a population of uniform cells can be prepared by isolating a single modified cell by limiting dilution cloning followed by expanding the single cell in culture into a clonal population of cells by standard techniques.
C. Other Methods for Modifying a Cell to Exress a Gene Product Alternative to introducing a nucleic acid molecule into a cell to modify the cell to express a gene product, a cell can be modified by inducing or increasing the level of expression of the gene product by a cell. For example, a cell may be capable of expressing a particular gene product but fails todo so without additional treatment of the cell. Similarly, the cell may express insufficient amounts of the gene product for the desired purpose. Thus, an agent which stimulates expression of a gene product canbeused to induce or increase expression of a gene product by the cell.- For example, cells can be contacted with an agent in vitro in a culure medium. The agent which stimulates expression of a gene prouct may function, for istae, by increasing transciption of the gene encoding the product, by -increasing the rate of tranislationorstability post transcriptional modification such as a poly A tail) of n mNA encoding the product or by increasing stability, transport or ~iI! 1- ~Ii i- ~J 1 P~I~Bl~e/B~b~aee~ tT
I
I: -13localization of the gene product. Examples of agents which can be used to induce expression of a gene product include cytokines and growth factors.
Another type of agent which can be used to induce or increase expression of a gene product by a cell is a transcription factor which upregulates transcription of the gene encoding the product A transcription factor which upregulate~ the expression of a gene encoding a gene product of interest can be provided to a cell, for example, by introducing into the cell a nucleic acid molecule encoding the transcription factor. Thus, this approach represents an alternative type of nucleic acid molecule which can be introduced into the cell (for example by one of the previously discussed methods). In this case, the introduced 10 nucleic acid does not directly encode the gene product of interest but rather causes production S. of the gene product by the cell indirectly by inducing expression of the gene product.
-In yet another method, a cell is modified to express a gene product by coupling the gene product to the cell, preferably to the surface of the cell. For example, a protein can be obtained by purifying the cell from a biological source or expressing the protein recombinantly using standard recombinant DNA technology. The isolated protein can then be coupled to the cell. The terms "coupled" or "coupling" refer to a chemical, enzymatic or other means by binding to an antibody on the surface of the cell or genetic engineering of linkages) by which a gene product can be linked to a cell such that the gene product is in a form suitable for delivering the gene product to a subject. For example, a protein can be chemically crosslinked to a cell surface using commercially available crosslinking reagents (Pierce, Rockford IL). Other approaches to coupling a gene product to a cell include the use of a bispecific antibodywhich binds both the gene product and a cell-surface molecule on the cell or modification ofthe gene product to include a lipophilic tail by inositol phosphate linkage) which can insert into a cell membrane.
SII Alteration of an Antigen an the Cell SIn addition to modification ofa cell to express a gene product, this invention involves Saltering an antigen on the cellsurface to reduce the immunogenicityof the cell and thereby Sinibit rejectidn of the cell when transplanted into an allogeneic orxenogeneic subject. In an unalteredstate, the antig on the cellsurface stiulates an immune response against the cell (also referred to herein asthe donor cell) when the cell is administered to a subject (also referred to herein as the recipient or host). By altering theantige the normalmm ologica recognition of the dnor celi by the imunesystem cellsof the recipient is disrupted and additionally, "abnoral" immunological recogtin of this altered form theanigecan c i:p:c lead to donor cl ccon t in the recipient Thus, aheration of an antigen on the donor cell prito administering cell to a recipient interfees with the iilphaseof recognition of th donor cell by the cells of the hoss immune system a B eie:-hsfii Imm'- MM -sm 7 subsequet to admstraon of the cell. Furthermore, alteration of the antigen may induce immunological nonresponsiveness ortolerance, thereby preventing the induction of the c 7-7-7=
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-14effector phases of an immune response cytotoxic T cell generation, antibody production etc.) which are ultimately responsible for rejection of foreign cells in a normal immune response. As used herein, the term "altered" encompasses changes that are made to a donor cell antigen which reduce the immunogenicity of the antigen to thereby interfere with immunological recognition of the antigen by the recipient's immune system. Preferably, immunological nonresponsiveness to the donor cells in the recipient subject is generated as a result of alteration of the antigen. The term altered is not intended to include complete elimination of the antigen on the donor cell since delivery of an inappropriate or insufficient signal to the host's immune cells T lymphocytes) may be necessary to achieve 10 immunological nonresponsiveness.
Antigens to be altered according to this invention include antigens on a donor cell which can interact with a hematopoietic cell in an allogeneic or xenogcneic recipient and thereby stimulate a specific immune response against the donor cell in the recipient The interaction between the antigen and the hematopoietic cell may be an indirect interaction 15 mediated by soluble factors which induce a response in the hematopoietic cell) or, more preferably, is a direct interaction between the antigen and a molecule present on the surface of the hematopoietic cell. As used herein, the term hematopoietic cell is intended to include T lymphocytes, B lymphocytes, monocytes and other antigen presenting cells. Preferably, the antigen to be altered is one which interacts with a T lymphocyte in the recipient the 20 antigen normally binds to a receptor on the surface of a T lymphocyte).
In a preferred embodiment, the antigen on the donor cell to be altered is an MHC .class I antigen. MHC class I antigens are present on almost all cell types. In a normal immune response, self MHC molecules function to present antigenic peptides to a T cell S receptor (TCR) on the surface of self T lymphocytes. In immune recognition of allogeneic or xenogeneic cells, foreign MHC antigens (most likely together with a peptide bound thereto) on donor cells are ecognized by the T cell receptor on host T cells to thereby elicit an immune response. MHC class I antigens on a donor cell are altered to interfere with their S- recognition by T cells in an allogeneic or xenogeneic host a portion of the MHC class I S antigenwhich is normallyrecognized the T cellreceptor is blocked or "asked" such that nornal recognitio of 2 e MHC class I antigen fails to occur). Additionally, an altered form C of arLMn C cies I antgen which is exposed to host T cells available for presentation to S. the host Tcell receptor) may deliver an inappropriate or insufficient signal to the host T cell Such tAat, rather than stimulating an imune response againsthe allogeneic or xenogeneic Scell, donorellsecific T cell non-espnsivenss is induc.ed For example, itis known that T cells which receive an inapropriate or insufficient sigal through their T cell receptor :y ding to an HC antigen in the absece of a costimumAtory signal, such as that provided by B7) co eanergic rater than acivated and renain ref i actory to restiulation for long pods of e (see fo examplDale t al. Pro NaL Aca S. USA 78:5096- S5100; .esslauer etal. (1986) Er. mm io. 16:1289-1295; Gimmi, et a. (1991) Proc.
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i- Nail. Acad Sci. USA 88: 6575-6579; Linsley et al. (1991) J. Exp. Med 173:721-730; Koulova et al. (1991). Exp. Med 173:759-762; Razi-Wolf, et al. (1992) Proc. Natl. Acad Sci USA 89:4210-4214).
Alternative to MHC class I antigens, the antigen to be altered on a donor cell can be n.MHC class II antigen. Similar to MHC class I antigens, MHC class II antigens function to present antigenic peptides to a T cell receptor on T lymphocytes. However, MHC class II antigens are present on a limited number of cell types (primarily B cells, macrophages, dendritic cells, Langerhans cells and thymic epithelial cells). In addition to or alternative to MHC antigens, other antigens on a donor cell which interact with molecules on host T cells 10 and which are known to be involved in immunological rejection of allogeneic or xenogeneic •cells can be altered. Other donor cell antigens known to interact with host T cells and contribute to rejection of a donor cell include molecules which function to increase the avidity of an interaction between a donor cell and a host T cell. Due to this property, these molecules are typically referred to as adhesion molecules (although they may serve other 15 functions in addition to increasing the adhesion between a donor cell and a host T cell).
Examples of preferred adhesion molecules which can be altered according to the invention include LFA-3 and ICAM-1. These molecules are ligands for the CD2 and LFA-1 receptors, respectively, on T cells. By altering an adhesion molecule on the donor cell, (such as LFA-3, ICAM-1 or a similarly functioning molecule), the ability of the host's T cells to bind to and 20 interact with the donor cell is reduced. Both LFA-3 and ICAM-1 are found on endothelial C •cells within blood vessels in transplanted organs such as kidney and heart. Altering these a 'antigens may facilitate transplantation of any vascularized implant, by altering recognition of S.those antigens by CD2+ and LFA-I+ host T-lymphocytes.
The presence of MHC molecules or adhesion molecules such as LFA-3, ICAM-1 etc. on a particular donor cell can be assessed by standard procedures kowvn in the art. For Sexample, the donor cell can be reacted with alabeled antibody directed against the molecule Sto be detected (e.g.,MHC molecule, ICAM-1, LFA-1 etc.) and the association of the labeled antibody with the cell can be measured by a suitable technique immunohistochemistry, flow cytometryetc A preferred method for altering an antigen on a donor cell to inhibit an immune response against the cell is to contact the cell with molecule which binds to the antigen on the cell suface. It is preferred that thecell be c actewith the molecule which binds to the antigen to be altered prior to administering the cell toa recipient the cell is contacted with the mnolecule in vitroj For example, the cel ca be inncubated with the molecule whichbinds the antigenuder conditionswhich dlo bidig ofthe molecule to the antigen and t und molecule can be rnmooved (sch as described in the Exapls below .i ::admni;ta in- Folhe onoft ifi ceaie a to recipi ithe molcule remains bound to the igen on thf cell for a sufficient time to munologic ognition by h1s cellsh and induce on-responsiveness inthe recipmen a -16- Os sc *5 S S Preferably, the molecule for binding to an antigen on a donor cell is an antibody, or fragment or derivative thereof which retains the ability to bind to the antigen. For use in therapeutic applications, it is necessary that the antibody which binds the antigen to be altered be unable to fix complement, thus preventing donor cell lysis. Antibody complement fixation can be prevented by deletion of an Fe portion of an antibody, by using an antibody isotype which is not capable of fixing complement, or, less preferably, by using a complement fixing antibody in conjunction with a drug which inhibits complement fixation. Alternatively, amino acid residues within the Fc region of an antibody which are important for activating complement (see Tan et al. (1990) Proc. Natl. Acad Sci. USA 87:162-166; Duncan and 10 Winter (1988) Nature 332: 738-740) can be mutated to reduce or eliminate the complementactivating ability of an intact antibody. Likewise, amino acids residues within the Fc region of an antibody which are necessary for binding of the Fc region to Fc receptors (see e.g.
Canfield, S.M. and SL. Morrison (1991) J. Exp. Med. 173:1483-1491; and Lund, J. etal.
(1991) J Im munoL 147:2657-2662) can also be mutated to reduce or eliminate Fc receptor binding if an intact antibody is to be used.
A preferred antibody fragment for altering an antigen is an F(ab)2 fiagment.
Antibodies can be fragmented using conventional techniques. For example, the Fc portion of an antibody can be removed by treating an intact antibody with pepsin, thereby generating an F(ab2 fragment. In a standard procedure for generating F(ab)2 fragments, intact antibodies are incubated with immobilized pepsin and the digested antibody mixture is applied to an immobilized protein A column. The free Fc portion binds to the column while the F(ab) 2 fragments passes through the column. The F(ab) 2 fragments can be fuher purified by HPLC or FPLC. F(ab')2 fragments can be treated to reduce disulfide bridges to produce Fab' fragments.
An antibody, or fragment or derivative thereof, to be used to alter an antigen can be derived from polyclonal antisera containing antibodies reactive with a number otepitopes on an antigen. Preferably, the antibody is amonoclonal antibody diret against the antigen.
P olyclonal andmonoclonal antibodies can be prepared by standard techniques known in the art. For example, a mammal, amouse, hamster, or rabbit) can be immunized with the antigen or with acell.which expresses the antigen on thecellsurface) to elicitan antibody response against the antigen in the mammal. Alternatively, tissue or a whole organ S which expresses the antigen can be used to elicit antibodies. The progress of immunization can b monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immumoassay can be used with the antigen to assess the levels of antibodies. Following immunizatin, ans can b obtained and, if desed, polyclonal antibodies isolated from the scra. To produce monoclonal antibodies. antibody producing cells (lymphocytes) can be havested from an immunized animal nd fused with mycloa cells by standard somaticell fusion procedues thus immortalizing these cells and yielding hybridomacells. Such tehniques are well known in the art. For example, the hybrido tecnique originally tehi m k :n-ntha x~ d
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a. a c( developed by Kohler and Milstein ((1975) Nature 256:495-497) as well as other techniques such as the human B-cell hybridoma technique (Kozbar et al., (1983) Immuol. Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al.
(1985) Monoclonal Antibodies in Cancer Therapy, Alien R. Bliss, Inc., pages 77-96) can be used. Hybridoma cells can be scr:eed immunocherrically for production of antibodies specifically reactive with the antigen and monoclonal antibodies isolated.
Another method of generating specific antibodies, or antibody fragments, reactive with an antigen is by use of expression libraries encoding immunoglobulin genes, or portions thereof, expressed in bacteria which can be screened with the antigen (or a portion thereof).
For example, complete Fab fragments, VH regions, FV regions and single chain antibodies can be expressed in bacteria using phage expression libraries. See for example Ward et al., (1989) Nature 341:544-546; Huse et al., (1989) Science 246:1275-1281; and MeCafferty et al. (1990) Nature 348:552-554. Alternatively, a SCID-hu mouse can be used to produce antibodies, or fragments thereof (available from Genpharm). Antibodies of the appropriate binding specificity which are made by these techniques can be used to alter an antigen on a donor cell.
An antibody, or fragment thereof, produced in a non-human subject can be recognized to varying degrees as foreign when the antibody is administered to a human subject wiei a donor cell with an antibody bound thereto is administered to a human subject), 20 resulting in an immune response against the antibody in the subject. One approach for minimizing or eliminating this problem is to produce chimeric or humanized antibody derivatives, i.e, antibody molecules comprising portions which are derived from non-human Santibodies and portions which are derived from human antibodies. Chimeric antibody molecules can include, for example, an antigen binding domain from an antibody of a mouse, rat, or other species, with human constant regions. A variety of approaches for making chimeric antibodies have been described. See, for example, Morrison et al., Proc. Nal. Acad Sci. US.A. 81, 6851 (1985); Takeda et al., Nature 314,452 (1985), Cabilly et al., U.S. Patent No. 4,816,567; Boss et al, U.S. PatentNo. 4,816,397; Tanaguchi et al, European Patent Publication EP171496; European Patent Publication 0173494, United Kingdom Patent GB 2177096B. For use in therapeutic applications, it is preferred that an antibody used to alter a donor cell antigen not contain an Fc portion. Thus, a humanized F(ab)2 fragment in which parts of the variable region of the antibody, especially the conserved framework regions of the antigen-binding domain, are of human origin and only the hyprvariable regions are of non-human origin is a preferred antibody derivative. Such altered immunoglobulin molecules can be produced by any of several techniques known in the art, Teng et a, Proc. NalL Acad Sci 80, 7308-7312 (1983); Kozbor et al, Immunology Today, 4, 7279 (1983); Olsson et al., Meth. EnymoL, 92, 3-16(1982)), and are preferably produced taccoing tthem ing ofPCT Publication W092f06193 orEP 0239400. Humanized -i oaaPlr~ssaRI ~l.i a -n i- :a ae-
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antibodies can be commercially produced by, for example, Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain.
Each of the cell surface antigens to be altered, the MHC class I antigens, MHC class II antigens, LFA-3 and ICAM-1 is well-characterized and antibodies reactive with these antigens are commercially available. For example, an antibody reactive with human MHC class I antigens an anti-HLA class I antibody),W6/32, is available from the American Type Culture Collection (ATCC HB 95). This antibody was raised against human tonsillar lymphocyte membranes and binds to HLA-A, HLA-B and HLA-C (Barnstable, CJ. et al.
(1978) Cell 14:9-20). Another anti-MHC class I antibody which can be used is PT85 (see 10 Davis, W.C. et al. (1984) Hybridoma Technology in Agricultural and Vetrinary Research NJ. Stemn nd H.R. Gamble, eds., Rownman and Allenheld Publishers, Totowa, NJ, pl21; commercially available from Veterinary Medicine Research Development, Pullman WA).
This antibody was raised against swine leukocyte antigens (SLA) and binds to class I antigens from several different species pig, human, mouse, goat). An anti-ICAM-1 antibody can be obtained from AMAC, Inc., Maine. Hybridoma cells producing anti-LFA-3 antibodies can be obtained from the American Type Culture Collection, Rockville, Maryland.
A suitable antibody, or fragment or derivative thereof, for use in the invention can be identified based upon its ability to inhibit the immunological rejection of allogeneic or xenogeneic cells using a protocol such as that described in the Examples. Briefly, an antibody (or antibody fragment) to be tested is incubated for a short period of time minutes at room temperature) with cells or tissue to be transplanted and any unbound antibody is washed away. The cells or tissue are then transplanted into a recipient animal The ability of the antibody pretreament to inhibit or prevent rejection of the transplanted cells or tissue is then determinedby monitoring for rejection of the cells or tissue compared to untreated controls.
It is preferred that an antibody, or fragment or derivative thereof, which isused to alter an antigen have aaffinity for bindingto the antigen ofatleast 10-7 M. The affinity of an antibody or other molecule for binding to an an antigen can be determined by conventional techniques (see Masan, D.W. and Williams, A.F (1980) Biochem. J. 187:1-10). Briefly, the antibody to be tested i! labeled with 1125 and incubated with cells expressing the antigen at increasing concentrations until equilibrium is reached. Data are plotted graphically as [bound antibody]/[free antibody] versus [bound antibody] and the slopeofthe line is equal to the kD (Scatchard analysis).
Other molecules which bind to an antigen on a donor cell and produce a functionally similar result as anbodies, or fragments or derivatives thero other molecules which intefere with the interaction of the antigenvit aematopoietic cell and induce immunological nomesponsivcness) can be used to alter the antigen on the donor cell, One su- h molel is a soluble form of aigand for an antigen a receptor) on the donor cell S ich can be used to alter the antige on the donor cell. For example, a soluble form of CD2 S(L comprising the xtracellular domain of CD2 without the transmembrane or cytoplasmic 4- s r
IT-
9*l I 4* 9.54 p 5.* domain) can be used to alter LFA-3 on the donor cell by binding to LFA-3 on donor cells in a manner analogous to an antibody. Alternatively, a soluble form of LFA-l can be used to alter ICAM-l on the donor cell. A soluble form of a ligand can be- made by standard recombinant DNA procedures 1 using a recombinant expression vector containing DNA encoding the ligand encompassing an extmzcellular domain lacking DNA cnzoding the transmembrane and cytoplasmnic domains). The recombinant expression vector encoding the extraceliular domain of the ligand can be introduced into host cells to produce a soluble ligand, which can then be isolated- Soluble ligands of use have a binding affinity for the receptor on the donor cell sufficient to remain bound to the receptor to interfere with immunological recognition 10 and induce non-responsiveness when the cell. is administered to a recipient preferably, the affinity for binding of the soluble ligand to the receptor is at least about 10- M).
Additionally, the soluble ligand can be in the form of a fusion protein comprising the receptor binding portion of the Jigand fused to another protein or portion of a protein. For example, an immunoglobulin fusion protein which includes an extracellular domain, or functional portion of CD2 or LFA-l finked to an immnmglobulin heavy chain constant region the hinge, CH2 and CH3 regions of a human iinmuneglobulin such as IgGl) can be used.
Immunoglobulin ffusion proteins can be prepared, for example, according to the teachings of Capon, DJ. et al. (1989) Nature 3,1-:525-531I and U.S. Patent No. 5,116,964 to Capon and Lasky.
20 Another type of molecule which can be used to alter an MHC antigen and MHC class I antig en) is a peptide which binds to the MOHC ant igen and interferes with the interaction of the MHC antigen with a T lymphocyte. In one embodiment, the soluble peptide mimics a region of the T cell receptor which contacts the MHC antigen. This peptide can be used to interfere, with the interaction of the intact T cell receptor (on a T lymphocyte) with the MHC antigen Such a peptide binds to a region of the- MHC molecule which is specifically recognized by a portion of-the T cell receptor the alphajl or alpha- 2 loop of an MHC class I antigen), thereby altering the MHC class I antigen and inhibiting recognition ofthe antigen by the T cell receptor. In another embodiment~ the soluble peptide mimics a region of a T cell surface molecule which contacts the MUC antigen, such as a reinof the CD8 molecule whicti contacts an MHC class I antigen or a region of a CD4 molecule which contacts an MHC class HI antigen. For example, a peptide which binds to a region of the alpha-3 loop of an MHC class I antigen can be used to inhibit binding toCD& to the antigen thereby inhibiting recognition of the antigen byT cells- T cell receptor-derived peptides have beeni used to inbibit.MHC class I-restricted immune responses (see eg., Clabergt; C. et aL (1993) Trwzqp[TgIProc 25.477-478) and prolong allogeneic skin graft suvvlin vM'owhen injected sbutnouI into the recipient (see oss, JL.A et at.
(1993) Pro. NaiL Aca Sci US 90:9872916).
An antigen on a donor clfiurthi cain be altered by using-two ornxor molecules vihbind to the samne or drfint antigens. For cxample~jwo6 different antibodies with
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K 7 specificity fbr two different epitopes on the same antigen can be used two different anti- MHC class I antibodies can be used in combination). Alternatively, two different types of molecules which bind to the same antigen can be used an anti-MHC class I antibody and an MHC class I-binding peptide). A preferred combination ofanti-MHC class I antibodies which can be used with human donor cells is the W6/32 antibody and the antibody or F(ab)2 fragments thereof Another anti-MHC class I antibody which can be used is the monocional antibody 9-3 generated at Diacrin, Inc. 9-3 reacts with porcine MHC class I. The epitope for the monoclonal antibody 9-3 has been shown to be on the alpha-3 domain of MHC class I (the alpha-3 domain of porcine MHC class I is known-see, Satz, MLJ. et 10 al. (1985) J. ImmunoL 135:2167-2175) and is separate from the epitope for the monoclonal antibody When the donor cell to be administered to a subject bears more than one S" hematopoictic cell-interactive antigen, two or more treatments can be used together. For example, two antibodies, each directed against a different antigen an anti-MHC class I S 15 antibody and an anti-ICAM-1 antibody) can be used in combination or two different types of molecules, each binding to a different antigen, can be used an anti-ICAM-l antibody and an MHC class I-binding peptide). Alternatively, a polyclonal antisera generated against the entire donor cell or tissue containing donor cells can be used, following removal of the Fc region, to alter multiple cell surface antigens of the donor cells.
Alternative to binding a molecule an antibody) to an antigen on a donor cell to inhibit immunological rejection of the cell, the antigen on the donor cell can be altered by other means. For example, the antigen can be directly altered mutated) such that it can no longer interactnormally with a hematopoietic cell aT lymphocyte) in an allogeneic Sor xenogencic recipient and induces immunological non-responsiveness to the donor cell in the recipienL For example, a mutated form of a class I MHC antigen or adhesion molecule LFA-3 or ICAM-1) which does not contribute to T cell activation but rather delivers an inappropriate or insufficient signal to aT cell upon binding to a recept on the T cell can be created by mutagenesis and selection. A nucleic acid encoding the mutated form of the P antigen can then be inserted into the genome of a non-human animal, either as a transgene or by homologous recombination (to replace the endogenous gene encoding the wild-type antigen). Cells from the non-human animal which express the mutated form of the antigen can then be modified to express a gene product of interest according to one ofthe procedures described earlier. The modified cell expressing the gene product of interest and the mutated altered) form ofthe antigeancan then be used as a donor cell to deliver a gene product to n allogeneic or xenogeneic recipient SAltenatively, an antigen on the donor cell can be altered by downmodulating or altering its level of expression on the surface ofthe donor cell such that the interaction Sbetween. .e antigen and a recipient beratopoii c cell is modified. By dereasing the level Sof surface expressionof one or re antigns on the donor cell, the avidity of the interacion r -21 a amisA between the donor cell and the hernatopOietiC call T lymphocyte) can be reduced. The level of surface expression of an antigen on the donor cell can be downinodulted by inhibiting the traiscripti~n, translation or transport of the antigen to the call surface. Agents whir-h decrease surface expressionl of the antigen can be contacted with the donor cell. For exainp!, a number of oncogeflic viruses have been, demonsUtatd to dcrease MHC class expesson n ifeced ell (se rvers et (19S0) 17711 Syrap. on Aging in Cancer, 175180; k=e et al- (1988) Br. J1 Cancer 57:374-7) In addition,ithsbefodtaths effect on MHC Ciess I expresion c' be achieved using fragments of viral genomes, in addition to intact viru-s. For example, trasfection of cultured kidney cells with fiagments of atlenovirus causes elimination of surface MHC class .antigeicu expression (hsie l (198) J 16:2l5-2l Forpuro~ 0 fdecreasiflg MIIC class I expression on the surfaces of donor cells, viral fragments which are niOn-infectlOus are preferable to whole viruses. a r e Alternatively, the level of anantigen an the donor cell surace can be altere by capping the antigen_ Capping is a term referring to the use of antibodies to cause aggregation and inactivation of surface antigens. To induce capping, a tissue is contacted with a first antibody specific for an antigen to be altered, to allow formation of antigen-antibody imimune complexes. Subsequently, the tissue is contacted with a second antibody which forms immune complexes with the first antibody. As a result of treatmenIt with the second antibody, the first antibody is aggregated to form a cap at a single location on the cell surface. The technique of capping is well knownt and has been described, in Taylor et al. (1971), Nat.
Me~w lBoL 233:225-227; and Santiso et al. (1986), Blood, 67:343-349. To alter NfflC class I antigens, donor cells are incubated with a ffist antibody WUt32 antibody, antibody) reactive with MHC class!I molecules, followed by incubation with a second antibody reactive with the donor species, goat anti-mou11-;e anitibody, to sut in aggregation.
TI GntialYM~i~e ~1swih AltrdSufteAien a% Donor Celsf& Thisinventin prodes a means for modif in a variety of cell Types to express a gene product and for reducing the inmunogenidty of such cells in an allogeneic- or -xcnogeneiC host. Depending on the type of cell to be modified, a gene expression systeM (er. vetrt prpraergltr elements) is selected to allow expression of a gene prodUct in thiat particular CClU tpe. One or more appropriatemlele)isaoslctdo bind to antigen(s) o n the cell .surface to, alter the antigen (cg. one or more antz.class
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antbd which bind the MUC class I antigens on that particular Cl type). Cells which cart be modified and aluend according to thr ento inld liver cells hepaICet1s).
Muscle cells myoblasts MYOCYtes, ytue) neural Cell, panmCratic islet cells and beniaopoIctiecells. The- use of hepatCYte for teeXPressium of a particular gene prodii
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0*fl S S 4 :~t allows for the production of proteins that require a specific co- or post-translational modification, such as vitamin K-dependent y-carboxylation many of the blood clotting factors require this modification for biological activity). Myoblasts have the advantage that injected myoblasts fuse with existing muscle fibers in a recipient (see Partridge, et al.
(1989) Nature 337:176; and Karpati et al. (1989)Am. J Pathology 135-27). Hematopoietic stem cells are advantageous in that they continue to divide and repopulate a number of cell types (see Chang and Johnson (1989) Int. J. Cell Cloning 7:264; Williams (1990) Hum.
Gene Ther. 1:229; Karlsson et al. (1985) Proc. Nail. Acad Sci. USA 82:158; and Bodine et al.
(1989) Proc. Nal. Acad. Sci. USA 86:8897). Alternatively, mature hematopoietic cells, such 10 as lymphocytes or monocytes, can be used. A gene product can be continuously produced by modifying a cell which continues to divide a stem cell, such as a hematopoietic stem cell) or a gene product can be produced in a limited amounts by modifying a differentiated cell which does not divide a myotubes or a neural cell). Recombinant retroviral vectors are suitable for modifying dividing cells but are not suitable for modifying non-dividing cells.
The modified cells can be contained within a tissue or whole organ. For example, a tissue or organ can be modified to express a gene product by infecting the tissue or organ with a recombinant virus retrovirus, adenovirus, adeno-associated virus etc.). One or more antigens on the tissue or organ can be altered by contacting the tissue or organ with a molecule which binds the antigen. For example, an organ can be perfused with a solution 20 containing the molecule an antibody) using conventional organ perfusion methods.
This invention further allows cells to be modified to express a variety of gene products and thus allows many different types of gene products to be delivered to a subject.
For example, the gene product can be a secreted protein. In this situation, the modified donor cell secretes the gene product in the subject, either locally or systemically. Non-limiting examples of secreted gene products of therapeutic interest which a cell can be modified to Sexpress include glucocerebrosidase, -glucouronidase, al-antitrypsin, phenylalanine hydroxylase, tyrosine hydroxylase, omithine transcarbamylase, arginosuccinate synthetase, UDP-glucuronysyl transferae, apoAl, TNF, soluble TNF receptor, human growth hormone, insulin, erhropoietin, anti-angiogenesis factors and interleukins. For example, the secreted protein can replace a missing function in a subject insulinin a diabetic subject) or can stimulate a response in a subject TNF or IL-2 can be produced in a tumor-bearing subject to stimulate an immune response against the tumor in the subject). Alteatively, the Sgene product can be a membrane-bound protein. In thiscase, the gene product remains associated with the membrane f the difed donorcell and fnctions for example, by binding a solublesubstance in a host binding of LDL cholesterol by an LDL receptor) or by bindingto aothr brane-bounptein (e.g a reeptor) on cells ofthe host to Stgger a sil within te ipient cells. Nn-itig les of membrane-bound gene products which a cell can be modfiedto express include the LDL receptor,CF d CD Alternatively, thegene product can be an intracellular protein. The intracellular protein 1.
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3' .4 Bi AI 1 -23within modified donor cells can be introduced into cells of a recipient by fusion of the donor cells to recipient cells fusion of modified myoblasts or myocytes with muscle cells within the recipient, to deliver dystrophin). An intracellular protein can also function by acting upon substances within a recipient that are taken up by the modified cell to detoxify substances within the recipient) Non-limiting examples of intracellular proteins which a cell can be modified to express include dystrophin, P-globin and adenosine deaminase.
In one embodiment, the cell to be modified is a non-human cell and the gene product is a human gene product Human gene products have been expressed in non-human cells (see A 10 Dai, Y. et al. (1992) Proc. Nafl. Acad. Sci. USA 89:10892-10895; Arnentano, et al.
(1990) Proc.-Natl. Acad. Sci. USA 87:6141-6145; van Beusechem, V.W. etal. (1992) Proc.
Nail. Acad Sci. USA 89:7640-7644). The human gene product expressed in a non-human I.'cell can be the human version of a gene product typically expressed by that cell type, e.g., human insulin can be expressed in non-human islet cells or human Factor IX can be 15 expressed in non-human hepatocytes (see, e.g. Armentano, et al. (1990) Proc. Nail. Acad Sci USA 87:6141-6145). Altemratively, the human gene product expressed in the non-human cell can be a gene product which is not normally expressed by that cell type. For example, human growth hormone can be expressed in non-human myoblasts or tyrosine hydroxylase can be *expressedin non-human myoblasts (see e.g. Jiao, S. etal. (1993) Narure 362:450-453 and Dal, Y. et al. (1992)Proc. Nail Acad Sdc. USA 89:10892-10895 for examples of the expression ina cell of a gene product not normally expressed by that cell type). A preferred non-human cell for use in the methods of this invention is a porcine cell. Genetically inbred stiains of pigs miniature pigs), which have organs of approximately equivaleit size to humans and have well characterized MHC antigens, are available in the art.
Alteration of one or more antigens on the surface of a cell prior to transplantation reduces the immunogenicity of the cell such that rejection of the cell by an allogeneic or xenogenm recipient is.inhibited following tansplantation. Accordinglythe invention provides a method for reducing the immunogenicity of a cell which is modified to express a gene product in whichthe cellis contacted, prior to transplantation in vitro), with at
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least one molecule which bindsto at least one antigen on the cell surface. The antigen to be altered stimulates an imune response against the cell in an allogeneic or xenogeneic subject.
Thus, alteration of the antigen on the cell surface inhibits rejection of the cell whentransplanted into a subject. It is preferred that the cell is contacted in vitro with a molecule.
antibody, or fragment or derivative thereof, such as an F(ab)2 fragment) which binds to the antigen on the cell suface but does not activate complement or induce lysis of the cell.
re the antigen on the cell surface which is alteredis anMHC class I antigen.
P ferably- ih6 iintion on the c I e -su, -hici A modified cell of the invention is used to delivera gene-produc to a subject by administeng the cellto ubject. The term "subjec" is inteded to include mammals, preferably humans, in which an immune response is elicited against allogenin or xenogene ;-is~idd~: 1M.- -24a r-.
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91 a cells. A cell can be administered to a subject by any appropriate route which results in delivery of the gene product to a desired location in the subject For example, cells can be administered intravenously, subcutaneously, intramuscularly, intracerebrally, subcapsular under the kidney capsule) or intraperitoneally. The cells can be administered in a S physiologically compatible carrier, such as a buffered saline solution. When cells are within a tissue or organ, the tissue or organ can be transplanted into a suitable location in the subject by conventional techniques.
It is preferable that a cell is mo ified to express a gene product prior to administering e. the cell to a subject modified ex vivo).- It is also preferable that the cell is modified to express the gene product prior to altering an antigen on the cell surface. However, a cell can b e modified to express a gene product ex vivo after the antigen has been altered, so long as the modification method does not disrupt an association between the antigen on the cell surface and the molecule an antibody) which is bound to the antigen. Furthermore, a N" cell can be modified to express a gene product in vivo following alteration of a surface S 15 antigen(s) ex vivo and administration to a subject. In vivo methods for genetically modifying a cell using retroviral or adenoviral vectors) are known in the art. These embodiments are encompassed by the invention.
The methods of the invention for delivering a gene product to a subject can further comprise additional treatments which inhibit rejection of the transplanted cells by the subject.
For example, an immunosuppressive agent a drug) can be administered to the subject at a dose and for a period of time sufficient to induce tolerance to the transplanted cells in the S• subject. A preferred immunsuppressive agent for administration to a subject is cyclosporin k A. Other immunsuppressive:agents which can be used include FK506 and RS-61443. Such immunosuppressive agents can be used in conjunction with a steroid glucocorticoids 25 such as prednisone, methylprednisolone and dexamethasone) or chemotherapeutic agent S-zathioprine and cyclosphosphamide), or both. Alternatively, an agent which depletes or S inhibits T cell activity in the subject can be administered to the subjeef For example, an antibody which binds to a surface antigen on T cell inthe subject can be used to deplete T cells within the subject. Preferred surface antigens to which aT cell-depleting antibody can S 30 bind include CD3,-CD2, CD4 and CDS. Other antibodies which can be used to inhibit T cell activityin a subje in eincludeantibodies against IL-2 or other T cell growth factors and antibodies against the IL-2 recepto oother T cell growth factor receptors.
S Another aspect ofthe invention pertains to a kit for ~se in delivering a gene product to a subject. In one embodiment, thekit includes a cell which is modified to express a gene 35 product; the cell having an antigen n the surfae which stimulates an immuneresponse Sagains the cell in anallogeneic orxengeneic subjectThe kitfurther incudes a molecule e.g, an antibody, or fragment or derivative thereof) which bindsto the antigen on the cel Ssurfic. In another eibodiment, th kit includes a vector encoding a gene product in a form suitable for expression of the gen product in a cell n a molecule an antibody, or i i o
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fragment or derivative thereof) which binds to the antigen on the cell surface. In this embodiment, the kit can optionally include a cell which has the antigen to be altered on the cell surface. When cells are included in the kit genetically modified cells or cells to be genetically modified), the cells can be cryopreserved in liquid nitrogen or on dry ice) and thawed before use. The molecule an antibody, or fragment or derivative thereof) which binds to an antigen on a cell can be provided in the kit in a physiologically acceptable carrier, such as a buffered saline solution. A preferred molecule for binding to an antigen, such as an MHC class I antigen on a cell is a F(ab')2 fragment. The components of the kit can be supplied in appropriate containers tubes, vials) for each component cells, antibodies, vectors) and each component supplied within an appropriate holder container). A kit of the invention can also include instructions for use of the kit to deliver a gene product to a subject This invention is further illustrated by the following Examples which should not be construed as limiting. The contents of all references and published patents and patent applications cited throughout the application are hereby incorporated by reference.
EXAMPLES
a.
595 EXAMPLE I: PRODUCTION OF GENETICALLY MODIFIED HUMAN 20 MYOBLASTS AND TRANSPLANTATION OF THE MODIFIED MYOBLASTS INTO MICE Satellite myoblasts were isolated from a frozen biopsy of human muscle following Sminute digestions in trypsin/collagenase/bovine serum albumin mix at 370C. Released cells from each digestion were seeded in 100 mm plates in the following growth medium (GM): MCDB 120 (JRH Biosciences, Lenexa, KS) epidermal growth factor dexamethasone+ fetal bovine serum. Primary cultures were re-fed once with GM before being.
S trypsinized for electroporation ten days after digestion.
Cell harvests from digestions 3 through 10 were combined and washed twice with ice Scold Hepes buffered saline pH 7.0, counted and resuspended at a final concentration of 2.6 x 10 5 cells/ 0.8 mlof Hepes buffered saline for placement in an 0.4 m gap width S. elecroporation cuvette Cells were incubated on ice for 10 minutes with 20 pg of Scal d igested pCMV plasmid (Clontch, Palo Alto, CA aplasmid which containsthe gene S encoding P-galactosidase, and 2 pg of Nsil digested pkJ2Neo(Dinsmore, JH. and Solomon, F. (1993) Neuroprorocols 2:19-23), which displays neomycin resistance, or pCMVGH, a 'plasmid containing a gene encoding human growth hormone and which also displays neoniycin resistance. pCMVGH was constructed as follows: plasmidp0GH (Selden, R.F. et oRIind cloneid aL (1986) Mo Cell. Bol 31733179) was cut with BaHI and EcoR and cloned into plasmid pcDNA3 (Clontech, Palo Alto, -CA) previo.usly cut with the same enzymes. The resultant plasmid, pCMVGH, which is shown in Figure 1, contains the gene encoding human St- g9. 2: j i;; -26growth hormone. The pCMVGH plasmid was then linearized with Scal in preparation for introduction into human myoblasts via electroporation. Electroporation was performed with a BioRad electroporation device with capacitance extender at 240V, 500jF. After electroporation, cells were left to recover for 10 minutes at room temperature and seeded into four 6-well plates at 6.5 x 104 cells per well (assuming no cell death during electroporation), and cultured in GM 800ggG418/ml for 11 days to select for stable transfectants. Cloning efficiency was 0.01 0.02%. ElectroporAtion was determined to be more efficient for transfection than either lipofection (BRL) or calcium phosphate-mediated transfection (Rosenthal, N. (1987) Meth. Enzymo. 52:704-720).
10 For determing expression in vitro, cells transfected with pCMVP and pJK2Neo were fixed in 0.05% glutaraidehyde, rinsed three times (5 minutes each rinse) in PBS, stained with X-GAL for3-24.hours (NaHPO 4 -8mM, NaHzPO 4 -20mM, MgC 2 -1.3mM, X-GAL- Img/ml, K 3 Fe(CN) 6 -3mM, K 4 Fe(CN) 6 -3mM in d1 2 0) according to standard protocols.
Clones after G418 selection often showed sporadic expression of P-gal when stained with X- GAL substrate mix. However, expression increased with consecutive passages and was at 100% upon fusion of cells (Figure For determining expression of human growth hormone in vitro, medium from cells transfected with pCMVGH was sampled and measured for 2. human growth hormone content using a growth hormone radioimmunoassay from Nichols Labs, San Jan Capistrano, CA. Human growth hormone was produced at 800-1800 ngI106 cells/hour.
For transplantation experiments, nude mice were anesthetized by intraperitoneal administration of Avertin (250 mg/kg body weight) and a flank incision was made to expose the kidney. A small incision was made on the kidney and a small fire polished glass rod was inserted between the kidney epithelium and the kidney tissue to create a space for cells to be ransplanted. Prior to transplantation, 106 cells were spun down in an Eppendorf pipette tip, then were placed under the kidney capsule together witha piece of sponge blocking the tip.
This type of transplant can be easily localized even with a bare eye. The skin incision was then closed with a wound clip and the animal transferred to a cage for recovery.
Mice transplanted with myoblasts were sacrificed thirty days after transplantation and 0 Ahen the regin of muscle which received the transplant is dissected and placed in glutaraldehyde. After fixation, tissue was rinsed three times (five minutes each rinse) in PBS and then frozen. The frozen tissue was later thawed, sectioned and stained with X-GAL for 24-48h (X-GAL solution wa the same as above except for increased K 3 Fe(CN) and d
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4 Fe(CN) 6 concentration to 50mM). -gal expression was detected 30 days after Uansplantation.
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.0 0 *0 0 7 0t 0.0 jO 0 EXAMPLE I: TRANSPLANTATION OF GENETICALLY MODIFIED HUMAN MYOBLASTS/MYOTUBES INTO CYCLOSPORIN- TREATED RATS Satellite myoblasts were isolated from human muscle, cultured and transfected as described in Fample I. l-gal and human growth hormone expression in these cells were also measured as described in Example I. P-gal expression was measured after 34 days in vitro (Figure This was the latest time point examined and does not reflect a limit on the length of time in which expression is expected.
Expression of hGH in stable transfectants in vitro was maintained over 5 passages up to 14 days in culture. Clones were never cultured till senescence to assay for GH but expression is stable in cultures from different frozen passages. In myoblasts, hGH was expressed at 200-500 ng/10 6 /hour; in myotubes, hGH was expressed at 800- 1500ng/106/hour.
The Lewis rats used for the transplantation experiment were obtained from Charles River, Wilmington, MA. Rat tibialis anterior muscle was damaged by injection of pivacaine and hyaluronidase three days prior to transplantation. One day prior to transplantation and daily thereafter, the rats were treated with cyclosporin (10-15mg/kg). Human myoblasts stably transfected with p-gal or hGH expression vectors as described above were transplanted as myoblasts or induced to form myotubes then transplanted to the damaged site in recipient muscle. Two weeks after transplantation the muscle was removed for histological analysis to detect 3-gal expression. Rat serum was also sampled to measure circulating hGH levels at days 3 and 7 post transplantation. Production of human growth hormone in the rats, as a result of expression of human growth hormone by the introducedmodified cells, is monitored by detecting the presence of human growth hormone in th circulationfthe rats. Aliquots of blood from.the rats were collected periodically, from the tail vein, and hGH present within the blood sample was detected using a growth hormone radioimmunassay from Nichols Labs, San Juan Capistrano, CA.
S-gal expression was measured 14 days after transplantation in viv (Figure This was the latest time point examined and does not reflect a limit on the length of time in which expression is expected.
n vivo detection of human growth hormone production was observed to last for three days. Aftermyoblast and myotube injection into normal Lewis rat TA muscle, a much reduced number of cells were detected with ahuman specificprobe 14days post_ implantatin. Tus, the inability to detct GHexpression ps three days post- 35 transplantation would appear to be due to poorcell survival rather than a los- of H expression from the transgcne.
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I~ i:i \-r i-i; li j; :1 i:: -r -I~e~a~e~P~s~pr~BIBBII~-- -s~a :r 1 EXAMPLE II: TRANSPLANTATION OF MODIFIED HUMAN MIYOTUBES INTO CYCLOSPORIN TREATED MICE
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Human rayotubes were modified by incubation with purified F(ab')2 fragments of the monoclonal antibody W6132 (F(abh) fragments were were generated using :he Immunopure F(ab%) preparation kit sold by Pierce Chemical Company, Rockford, illiois) at a concentrationl of 10 ILg of antibody fragment for approximately 5 x 106 cells in 500 p11 of PBS for one hour on ice with intermittent shaking. Alter the incubation, the treated myotubte.
were washed, spun down once and resuspended at transplant concentration in PBS and then immediately transplanted (5 x 106 nuclei per mouse) into the kidney capsules, as described in Example I, of four experimental Balb/c mice. Two of these mice -were treated with cyclosporin (20.mg/kg) beginning one day prior to transplantation and continued daily thereafter. The other two mice were masked with F(ab 2 fragments of the monoclonal antibody W6/32 and not subject to cyclosporin treatment. As a positive control, untreated mryotubes (5 x 106 nuclei per mouse) were transplanted under Ithe kidney capsules of two nude mice. As a negative control, untreated myotubes (5 x 1 06 nuclei per mouse) were transplanited under the kidney capsule of two normal Balbfc mice.
The mice kidneys were removed 30 days after transplantation, fixed in 4% paraforinaldehyde for 24 hours, and stained with rabbit anti-desmin from BioGenex Labs, -San Ramnon, CA (Figure Human myotubes were detected in all experimental kidneys days after transplantation including the kidneys from the negative control. The negative control, however, showed evidence of ongoing rejection of -the myotubes,- vacublated ares with-obvious cell necrosis. Evidence of ongoing rejection was not observed in the test mice (mice transplanted with myotubes and subject to cyclosporin treatmrent, mice transplanted with F(ab12 modified myotubes, and nude mice transplanted with myotubes).
S.S
ci EXAMPLE TV'. PRODUCTION AND TRANSPLANTATION OF GENETICALLY MODIFIED PORCINE MYOBLASTS SUITABLE FOR TRANSPLANTATION An expresson vecztor containing a genie encoding human growth hormone is introduced into porcine myoblasts to create: a mhodified cell which expresses human growth hormone. -The rondfled porcine cell is then treated with an anti-MHC class I antibody F(ab')2-fragment thereby altein pocieMU las Intign fi.e.. SLA anigens) mI oa cel. Thie genetically Modified cells with altered MHC class; I antigens are then administered to a mouse to demonstrate the deliveryof a human gene product to a subject uting a cell ~~~Nucleic9acd enoin te gene for hizmn gwh hormone (hGH) is cloned into h comrciall~ available plasinid exsinvctrpDA .Nucecai eiicding h humnh growth hormone geme can be obtained by anstadard Procedure based upon the At.' -29reported nucleotide Isequence of the gene (Goeddel et al. (1979) Nature21:4)foex pl either by designing PCR pimr based upon the genesqic adapfygafamnto DNA encompass-16g the coding region of-the gene by PCR or by screening a cDNA or genoic NA lbiry ithprimers based upon the gene sequence. Nucleic acid encoding S unrowth DNA aei clndit h cinker of pCDNAIII to create a vector (phGH) containing the hiargrowth hormone gene under the tran~scriptional control of the cytomegaloviruS jrninoter. The vector also contains a bacterial selectable marker (amnpicillin resistance) and aManimalian selectable marker (neomycin resistance).
Apro2;jimly prepared phGH plasmid (eg. cesium chloride purified and linearized) is introduced into Porcine myoblasts by electroporation. In a general procedure, approximnate y l x io7 cells are suspended in 0.5 ml of icc-cold electroporation buffer (Possible electoporatioli buffers include: PBS without Ca 2 orM 2 ;H ESbfee slin andtisue ultr medium without fetal calf sem. eg., RPMI) and PIGH DNA (aproimaely1-1 isaddd. he NA~eIIsuspension is placed in an eleCtrOPOration cuvutt.is and incubated on ice for 5-710 minutes. The cuvette is then placed inan elecropoatio aparatus; commercially available from BioRad) and pulsed at a desired voltage and capacitance setting. The voltage and capacitance conditions for most efficient eectroporation are determined empirically (for example by testing avreyo odtos determining the percentage of cell death with each condition and selecting a condition that achieves approximately 50 cell death) but typical conditions are-bten20t 0 with500 to 1000g 1capacitace After the cellsre shocte ctlsOee inicub ice for 10 minutes and then washed and plated in an approriat culur ed a ntboi To ftlect cells which have incorporated-the introduced phGH plidteatboi -0418 is added to the culur media three days aft elciroporation and the Cells =r refed On alternate days with 0418-containig media. The concentration of.G418 used for efficientselection .of cells is determined empirically (for -example, by determining the dosage of 041 S -which is neededto kill mock transfected cells )btpcay is "nh .ag of00aigmit l Selective cell death of cells which hive not taken up the hGH plasmid occurs V bgnnn buto aS fe hesatofdu eection and by day 30 surviving cells arm considered to be selected. Wnividual clones of calls are then ioae n xaddit Specific cell lines and analyzdfrepeso ftegn rdc.
-ExPresion of human gr-owth hormone by te iansfected cells is dectected usinga comercaly aaiabl to-steradioinmmun6asa (RLA frrn Nichols Institute Diagnostics) vWipure hGki standards for comlparisonto determine the levelo G 35 expessin b thecels. Hmangrowth hormone produced by.1the cells iS secreted into the culture medium, thereby allowing assesment of hGH expression by samplig an alqoto the culture medfia for the presence of hH ie RIA Thbus, -t he pmqil#uction, of hGH by the cells can be continuouslY nitared by, repeatedly sampling the cult=r media 'over time-
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A stably tranfected cell line which produces maximal expression of the introduced hGH DNA is chosen by screening different clones that have been drug selected for hGH production. Such a cel line can then be prepared for transplantation into a xenogeneic recipient animal by treating the cells with an F(ab')2 fragment of the PT85 anti-MHC class I antibody (commercially available from Veterinary Medicine Research Development, Pullman WA) which binds to SLA class I antigens. An F(abj 2 fiagment is prepared from intact antibodies as follows: Purified PT85 at 20 mg/ml is incubated with immobilized pepsin for 4 hours at 370 C in a pH 4.7 digestion buffer in a shaking water bath. The crude digest is removed fom the pepsin and immediately neutralized with a pH 7.0 binding buffer. The crude digest is applied to an immobilized protein A column and the eluate containing the F(ab)2 fragments is collected. The F(ab) 2 fragments are dialyzed against PBS for 24 hours using 50,000 MW cutoff dialysis tubing to remove any contaminating Fc fragments.
CHAPS
is added to the dialysis bag at a concentration of 10 mM. The completeness of the F(ab2 digest is monitored by silver staining of 15 SDS gels. Final purification ofthe fragments 15 is achieved by FPLC using a Superose 12 column (Pharmacia, Upsala, Sweden).
Cells modified to express hGH are prepared for transplantation by incubating the cells with the purified F(ab)2 fragments at a concentration of 1 mg of antibody for approximately SIx 106 cells for 30 minutes at room temperature. After the incubation, the treated cells are washed once in Hank's buffer containing 2% fetal calf seum and then immediately transplanted into Balb/c mice by syringe.injection at an appropriate site into the muscle of the hindleg). Production of human growth hormone in the mice, as a result of expression of human growth hormone by the introduced modified cells, is monitored by detecting the presence of human growth hormone in the circulation ofthe mice. An aliquot of blood from the mouse is collected periodically, for example from the tail vein, and hGH present within the blood sample is detected using the radiomunassay described previously.
t p n aTtV A LIT S u' l so' h Those s ed in the art will recognize, orbe able to ascertain using no more than routine exprimentation,many equivalents the fc ebodiments ofthe invention described herein. Such equivalents are intended to be encompassed by the following claims.
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Claims (23)
1. A cell suitable for transplantation which comprises a recombinant vector comprising a nucleic acid encoding a gene product in a form suitable for expression of the gene product in the cell, the cell having at least one antigen on the cell surface which is capable of stimulating an immune response against the cell in an allogeneic or xenogeneic subject and wherein the antigen on the cell surface is altered prior to "transplantation such that an immune response against the cell is inhibited upon Stransplantation of the cell into a subject.
2. A cell of claim 1, wherein the gene product is a secreted protein. to 3. A cell of claim 1, wherein the gene product is a membrane-bound protein.
4. A cell of claim 3, wherein the membrane-bound protein is a cell surface receptor. A cell of claim 1, wherein the gene product is an intracellular protein.
6. A cell of any one of claims 1 to 5, which is a muscle cell.
7. A cell of any one of claims I to 5, which is a liver cell.
8. A cellof any one of claims 1 to 5, which is a neural cell.
9. A cell of any one of claims 1 to 5, which is a pancreatic islet cell. A cell ofanyone of claims I to 5,which is a hematopoietic cell.- s i.11. A cell of any one of claims 1 to 10, wherein the antigen is altered to modify an interaction between the antigen and T lymphocyte in an allogeneic or xenogeneic subject -32-
12. A cell of any one of claims 1 to 1 wherein the recombinant vector is an adenovirus or an adeno-associated virus.
13. A cell of any one of claims 1 to 11, wherein the recombinant vector is a retrovirus.
14. A cell of any one of claims 1 to 11, wherein the antigen on the cell surface which is altered is an MHC class I antigen.
15. A cell of claim 14, which is contacted prior to transplantation with at least one S anti-MHC class I antibody, or fragment or derivative thereof, which binds to the MHC class I antigen but does not activate complement or induce lysis of the cell.
16. A cell of claim 15, wherein the anti-MHC class I antibody is an anti-MHC class I F(ab')2 fragment
17. A cell of claim 16, which is contacted with a F(ab') fragment of a monoclonal antibody W6/32 or a F(ab') fragment of a monoclonal antibody PT85 or F(ab')2 fragments of both W6/32 and
18. A cell of claim 14, which is contacted prior to transplantation with at least one peptide which binds to an MHC class I antigen. S19. A kit for delivering a human gene product to a subject comprising: a cellwhich is modified to express the human gene product and which has an antigen on the cell surfacewhich is capable of stimulatingan immune response against thecell in an allogeneicor xenogeneic subject; and- S- 0 an antibody, or fragment or derivative thereofwhich binds to the antigen o the cell surface prior to transpantation sch that an immune response to the cell is inhibited upon tranlantationof the cell into a subject. v i: I! .j i- r -33- 4* 4 4- 0 44 304~ 4**A 4> S. 11*4 *4 A kit of claim 19, wherein the antibody, or fragment or derivative thereof, is a fragment ofthe antibody.
21. A kit of claim 19 or claim 20, wherein the antigen is an MHC class I antigen.
22. A kit for delivering a human gene product to a subject comprising: a vector encoding the human gene product in a form suitable for expression of the human gene product in a cell; and an antibody, or fragment or derivative thereof, which binds to an antigen on a cell surface which is capable of stimulating an immune response against the cell in an allogeneic or xenogeneic subject prior to transplantation such that an immune response to the cell is inhibited upon transplantation of the cell into a subject-
23. A kit of claim 22 further comprising a non-human cell which has an antigen on the cell surface which is capable of stimulating an immune response against the cell in an allogeneic or xenogeneic subject, wherein the antibody, or fragment or derivative thereof binds to the antigen.
24. A method for delivering a human gene product to a subject comprising: contacting aceU which has been modified to express the hunan gene product with at least one molecule whicbinds to at least one antigenon the cll surface which is capableof stimulating an immune response against the cell in an allogeneic or xenogeneic subject prior to transplantation such iat the antigen is altered and an immune response againstthe cell is inhibited upontransplantation of the cell into a subject; and (b)-administeriig the ce tothe subect I; V. C:; i r ^S t 4 -34 A method of claim 24, wherein the antigen on the cell surface which is altered is an MH4C class I antigen.v
26. A method of claim 25, wherein the cell is contacted prior to transplantation with at least one anti-M-C class f antibody, or firannent or derivative thereof.- Vhich bindAs to the _MLIC class I antigen but does not activate compleiint or induce I.sis of the cell.- _27. A method of claim 26. wherein the anti-IMC class I antibody is an anti-MHC class I F(ab' )2 fr-agment 2- Use of a cell which has been modified to express a hufm gene product- wherein the cell has been contacted with at least one molecula which binds to at least one antigen, zo on the cell surface which is capable of stimulatie an imunre response agpinst the cell in an allogeneic or xeno rneic subject ~or ttan1aaioschatheaignis altered and an immune response .against the cell is inhibited Upontranspantatiori ofte cell into a subject. fior the mannficture of a me-dicament for deliver-iniz a hiuman genec product to a subj~ct_
29.- Use of claimr 28, whe4rein the antigen on dle cell surface which is altered is an_ MHC class I antigen- Use of claim 29, whe-rei the c ell is ,contaucted prior to transplant~itiont with at le ast one anti-MHC class I antibody, or fragment or derivative theof whchbinds to the- MHC class I antigen but does not activate complement or induce lysis oftecI 'Use of claim 30. wherein the anti-vlHC class- I antibody is an an~ti-M}IC class I F(ab!'2 fragment 32 A Ielacrigt claim 1, and substantially as herein described wihrf ene o any one ofthexpls A-
33. A kit according to claim 19 or claim 22, and suabstatially as herein described with reference to any one of the examples.
34. A method according to claim 24, and substantially as herein described with reference any one of the examples-
35. Use according to claim 28 and substatially as herein described with reference to any one of the examples. DATED this 17thi day of December 1998 DIACRIN, Inc. Anomey PAUL G HARRSON-J Feflow 1astit of Pacent Atton2My Of Auztrliz of BALDWIN SHELSTON WATERS ~q S A. .0 S 44* >4 p. I p 44 4 .4 54 I. U 4' 4 e (V t~~
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US22140494A | 1994-03-31 | 1994-03-31 | |
US221404 | 1994-03-31 | ||
AU22363/95A AU2236395A (en) | 1994-03-31 | 1995-03-30 | Genetically modified cells for use in transplantation |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU22363/95A Division AU2236395A (en) | 1994-03-31 | 1995-03-30 | Genetically modified cells for use in transplantation |
Publications (1)
Publication Number | Publication Date |
---|---|
AU9718598A true AU9718598A (en) | 1999-03-25 |
Family
ID=25618557
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU97185/98A Abandoned AU9718598A (en) | 1994-03-31 | 1998-12-17 | Genetically modified cells for use in transplantation |
Country Status (1)
Country | Link |
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AU (1) | AU9718598A (en) |
-
1998
- 1998-12-17 AU AU97185/98A patent/AU9718598A/en not_active Abandoned
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