CA2256577A1 - Construction and use of genes encoding pathogenic epitopes for treatment of autoimmune disease - Google Patents

Abstract

The present invention relates to the application of genetic engineering to provide a treatment of autoimmune disease. This is achieved preferably through the introduction of one or more recombinant genes encoding self antigens which are the target of an autoimmune response. In particular the invention provides a method of designing and constructing a gene encoding an encephalitogenic epitope of proteolipid protein, and to the in vivo expression of the gene product by a recombinant retroviral vector. The expression and secretion of the encephalitogenic epitope ameliorates the histophathological and clinical characteristics of experimental autoimmune encephalomyelitis (EAE) in the mouse model for multiple sclerosis (MS).

Description

CA 02256577 1998-ll-23 S P E C I F I C A T I Q N

CONSTRUCTION AND USE OF GENES ENCODING PATHOGENIC EPITOPES
FOR TREATMENT OF AUTOIMMUNE DISEASE

~ield of the Invention This invention relates generally to the field of immunotherapy and to the preparation and use of engineered cells having the ability to restore tolerance to self antigens in patients suffering from autoimmune disease. More particularly, this invention relates to the design and construction of a gene encoding an encephalitogenic epitope of proteolipid protein (PLP), to methods of in vilro and in vivo expression of a PLP epitope, to methods of in vivo secretion of a PLP epitope, and to methods of transferring the partial PLP gene to a host to ameliorate the progression of an irnmune response to self antigens derived from myelin proteins.

CA 022~6~77 l998-ll-23 Back~round of the Invention The immune system can respond in two ways when exposed to an antigen. A
positive response leads to differentiation of T and B cells, antibody production and to immunologic memory. A negative response leads to suppression or inactivation of 5 specific Iymphocytes and to tolerance. Tolerance can be defined as the failure of an organism to mount an immune response against a speci~lc antigen. Norrnally, an organism is tolerant of its own antigens.
Autoimmune diseases are thought to result from an uncontrolled immllne response directed against self antigens. In patients with multiple sclerosis (MS), for 10 example, there is evidence that this attack is against the white matter of the ccntral nervous system and more particularly to white matter proteins. Ultimately, the myelin sheath surrounding the axons is destroyed. This can result in paralysis, sensory deficits and visual problems. MS is characterized by a T cell and macrophage infiltrate in the brain. Autoreactive myelin-specific T cells have been isolated from MS patients, 15 although T cells of the same specificity have been detected in norrnal individuals. J.M.
LaSalle et al., J. Tmmllnol. 147:77~-780 (1991), J.M. LaSalle et al., J. Exp. Med.
176:177-186 (1992), J. Correale et al., Neurolo~y.45:1370-1378 (1995). Presently, the myelin proteins thought to be the target of an immune response in MS include myelin basic protein (MBP3, proteolipid protein (PLP), and myelin-oligodendrocyte glycoprotein 20 (MOG). Individuals who do not mount an autoimrnune response to self proteins are thought to have control over these responses and are believed to be "tolerant" of self antigens. The evidence, therefore, that MS is caused by pathogenic T cells is necessarily indirect, but the close resemblance which the ch~r~c~t~ tics of this disease bear to those CA 022~6~77 1998-11-23 of the murine model, experimental autoimmune encephalomyelitis (EAE), suggest that MS is indeed caused by an aberrant immune response mediated by T cells.
~ The EAE mouse model for MS, the subject of intense and fruitful study for several years, displays many of the same histopathological and clinical characteristics as S the relapsing remitting forms of MS. The T Lymphocyte in Experimental Aller~ic Fncephalomyelitis, Ann. Rev. Immunol. 8:579-621 (1990). EAE can be induced in SJL
mice by injection of mouse spinal cord homogenate (MSCH), MBP, PLP, by the injection of synthetic peptides whose sequences correspond to the major encephalitogenic epitopes of myelin basic protein, MBP 84-104, proteolipid protein, PLP l 39-151, or by 10 adoptive transfer of activated CD4+ THI but not TH2 cells specific for encephalitogenic epitopes. The major encephalitogenic epitopes of myelin-derived sequences in EAE, such as MBP, can also activate human T cells of several dirr~ L haplotypes including HLA-DR2. R. Martin, et al., J. Exp. Med. 173: 19-24 (1992). The experimental disease is characterized by a relapsing-remitting course (R-EAE) of neurological dysfunction, 15 perivascular mononuclear infiltration and demyelination. CNS damage is probably mediated by infl~nnm~tory cytokines which can activate additional monocytes and macrophages non-specifically. J.E. Blalock, The Immune System. Our Sixth Sense~ The Immunologist, 2:8-15 (1994).
Although the initial attack in EAE can be in~ cecl by the ~rlmini~tration of either 20 T cells specific for MBP or for PLP, close ex~min~tion of reactivities of T cells in the primary and subsequent relapses demonstrates the presence of T cells which interact with ~ specificities other than the inducing epitopes. This expansion of encephalitogenic epitopes is termed "~letermin~nt spreading". S.D. Miller and W.J. Karpus, Immunolo~y CA 022~6~77 1998-11-23 Todav 15:356-361 (1994), P.V. Lehman, T. Forsthuber, A. Miller, and E.E. Sercarz~
Nature 358-155-157 (1992), H. Jiang, S-I. Zhang and B. Pernis, Science 256:1213-1215 ( 1992). Antigen specific treatment would therefore, be expected to be more effective when ~1ministered early in the course of the disease, before the onset of increasing epitope complexity and eventual non-specific infl~mm~tion.
The goal of imml7nnlogic therapy is to restore tolerance without suppressing theentire immlln~ system which can lead to complications such as infection, hemorrhage, and cancer. Drugs currently used to treat autoimmune ~liccS~ees are non-specificimm-lnosuppressive agents, such as anti-infl~mm~tory agents or drugs which can block cell proliferation or depress proinR~mm~tory cytokines. In general, these agents are effective for limited duration and subject to devastating complications.
It is desirable to suppress the immune system in a more specific way to control the response to self-antigens and theoretically "curc" the disease without down-regulating the entire irnrnune system. Several specific immunot~erapies have been hypothesized and tested in recent years, many of which are impractical or do not work in hllm~n~ For example, high affinity peptides can be synthesized which interact with MHC class II
molecules and prevent the binding of encephalitogenic peptides, thereby preventing the activation of pathogenic T cells. A. Franco et al., The Jmmllnolo~i~t 2:97-102 (1994).
This approach is disadvantageous in that it is difficult to obtain effective concentrations of inhibitor peptides in vivo. G.Y. ~shioka et al., J. ~mmunol. 152:4310-4319. In an alternate strategy, peptides which are analogs of encephalitogenic sequences have been shown to antagonize the T cell receptors of antigen-specific T cells, rendering them unreactive, although the exact mechzlnicm is at present unknown. S.C. Jameson et al., CA 022~6~77 1998-11-23 J. Fxp. Med 177:1541-1S50 (1993), N. Karin et al., J. Exp. Med. 180:2227-2237 (1994), V.K. Kuchroo et al., J. Immunol. 153:3326-3336 (1994). Oral ~mini~tration of myelin has been tested and found to induce a state of immunological unresponsiveness thought to be mediated by the induction of suppressor T cell or of anergy. H.L. Weiner et al., Annu. ~ev. Immunol. 12:809-837 (1994), C.C. Whitacre et al., J. Immunol.. 147:2155-2163 (1991), S.J.Khoury et al., J. Exp. Med. 176:1355-1364 (1992). This treatment has been found to be efficacious for some but not all individuals. H.L. Weiner et al., Science 259:1321-1324 (1993). Thus, it is evident that improvements are needed to treat MS and other autoimmune disorders with an effective, immunospecific approach.

10 Snmm~ry of the Invention The present invention addresses the disadvantages present in the prior art. In general, the invention is based on the discovery that recombinant DNA technology and cell transfer may be employed to restore tolerance to one's own tissues. The present invention provides a means of ~le~ g and constructing a gene, that when expressed 15 and secreted in vivo, can provide a means of halting the progression of an autoimm-lne disease. In filrther aspects the invention provides a method to construct a gene encoding a portion of a CNS protein, insert the gene sequence into a vector and transfect a cell line.
In further aspects, the invention provides a method to construct a gene encoding a portion of a CNS protein, insert the sequence into a retroviral vector, and tr~n~cluce a producer 20 fibroblast cell line to generate supern~t~nt cont~ining the recombinant retrovirus.
Histocompatible fibroblasts are tr~n~d~lçecl with the recombinant retrovirus encoding a CA 022~6~77 1998-ll-23 WO 97/~5144 PCT/US97/10214 portion of the CNS protein and are delivered to animals. These fibroblasts continuously secrete a CNS antigen in vivo but do not themselves produce viral particles.
In accordance with the present invention, we have used synthetic oligonucleotides to construct a gene encoding a portion of the PLP protein, perforrned expression of the S DNA in combination with various expression vectors, and thereby evaluated expression levels of the gene product in vitro and in vivo. After tr~n~d~lçecl histocompatible fibroblasts that secrete the partial PLP protein are transplanted into EAE mice, the disease disappears. The effect is the amelioration of both clinical symptoms and signs and pathological findirlgs.
In a preferred embodiment of the invention, the producer line PA317 is transduced with the PLP retroviral vec~or to generate supernatant cont~ining the recombinant retrovirus. The producer cell line PA317 was developed by Dr. A. Dusty Miller and has been extensively ch~r~ct~ri7.e-1 and approved for human use by the FDA
for other clinical trials, such as for genetic diseases and cancer. Miller and Baltimore, Mol. Cell Psiol. 6:2895-2902 (1986), W.F. Anderson, Science 256:808-813.

CA 022~6~77 1998-11-23 Brief Description of the Draw;n~c FIGURE 1 is a map of the partial PLP gene showing the sequence of the gene product and restriction sites.

FIGURE 2 is a map of the GlXSvNa vector illustrating restriction sites and functional 5 features. Figure 2b illustrates the entire DNA sequence of G 1 XSvNa.

FIGURE 3 outlines the method of constructing a GlXSvNa vector cont~ining the PLP
gene insert.

FIGURE 4 shows the level of mRNA expressed in transfected and tr~n~d~lce~l SJL
fibroblast cells as detected by reverse transcriptase PCR. Lane 1 is molecular weight 10 standards, Lane 2 is Negative control from mock transfection, Lane 3 is positive control-PLP-gene plasmid, Lane 4 is cDNA from PBP-transfected SJL fibroblasts, Lane 5 is cDNA from PLP transduced SJL fibroblasts.

FIGURE 5 demonstrates the level of PLP protein in the supernatants of transduced fibroblasts as detected by ELISA.

15 FIGURE 6 demonstrates the level of B-Gal expression in tr~n~ ced fibroblasts.

FIGURE 7 illustrates the clinical scoring system for chronic EAE.

FIGURE 8 illustrates the histological scoring system for EAE.

FIGURE 9 illustrates the clinical ~.ses.~nn~nt of EAE mice treated with retrovirus transduced fibroblasts.

FIGURE lOa shows the pathologic ~ss~s~m~nt of brain and spinal cord of SJL mice S treated with retrovirus tr~n~d~ erl fibroblasts7 and lOb is a summary ofthe pathologic ~se~ment of brain and spinal cord from Days 55-60 through days 90-95.

FIGURE 11 shows the histology of S~L mice with chronic EAE treated with retrovirus transduced fibroblasts.

FIGURE 12 illustrates the results of proliferation assays using EAL mice treated with 10 PLP-expressing fibroblasts.

FIGURE 13 illustrates the results of proliferation assays with and without IL-2 using EAE mice keated with PLP-expressing fibroblasts.

Detailed Description sf the Inv~ntion As indicated above, the present invention relates to the use of engineered cells to 15 restore tolerance to self antigens in patients suffering from autoimmune disease. The engineered cells can be any m~mm~ n cell. As used herein, the term "engineered" is CA 022~6~77 1998-11-23 intended to refer to a cell into which one or more recombinant genes, such as a gene encoding an epitope of a self antigen, has been introduced.
A gene is a deoxyribonucleotide sequence coding for an amino acid sequence.
Recombinantly introduced genes will either be in the form of a synthetic oligonucleotide, a cDNA gene (i.e. they will not contain introns), a copy of a genomic gene sequence, or a hybrid gene which is a fusion of two or more gene sequences. Optionally the gene may be linked to one or more nucleotide sequence capable of directing expression of the gene product. Sequence elements capable of effecting expression of a gene or gene product include but are not limited to promoters, enhancer elements, transcription termination signals, polyadenylation sites, a Kozak box sequence to ensure efficient translation, and leader sequences. Optionally, the gene sequence can include restriction sites to enable the insertion of additional gene sequences. Preferably, the gene will contain a leader sequence to ensure the gene product is synthesi7P~l in the endoplasmic reticulum for later constitutive secretion.
Recomhinzlntly introduced genes carried by the engineered cells can encode one or more epitope, fragment, domain or mini-protein portion of a protein antigen.
Examples of suitable proteins from which an epitope, fragment, domain, or mini-protein may be derived include but are not limited to myelin proteins, acetylcholine receptor, TSH receptor, and collagen.
It is believed that protein self-antigens which are the target of an autoimml-neresponse are highly conserved both among and between species. Thus, although theinvention will primarily be used to treat hllm~nc it can also be used to treat ~nim~lc.
Examples of T cell mediated autoimmune diseases that may be treated using the CA 022~6~77 1998-ll-23 invention include but are not limited to multiple sclerosis, myasthenia gravis, systemic lupus erythem~tosus, psoriasis, juvenile onset diabetes, rheumatoid arthritis, thyroid disease and chronic infl~mm~tor~v demyelin~ting polyneuropathy (CIDP).
Expression vectors are generally deoxyribonucleotide molecules engineered for controlled expression of one or more desired genes. The vectors may comprise one or more nucleotide sequences operably linked to a gene to control expression of the desired gene or genes. There are an abundance of expression vectors available and one skilled in the art could easily select an ~pl~,pliate vector. In addition, standard laboratory m~n~ on genetic engineering provide recombinant DNA methods and methods for m~king and using expression vectors. Optionally, the vector may encode a selectable marker, for exarnple, antibiotic resistance.
The gene can be inserted into the m~mm~ n cell using any gene transfer procedure. Examples of such procedures include but are not limited to, RNA viralmf~ t~-~l gene transfer such as retroviral tr~n~ ction, DNA viral m~ t~l gene transfer, electroporation, calcium phosphate mediated transfection, microinjection or liposome meAi~tefi gene transfer. The type of procedure required to achieve an enginf?ered cell that secretes the desired gene product will depend on the nature and properties of the cell.
The specific technology for introducing such genes into such cells is generally known and well within the skill of the art.
The examples which follow illustrate the design and construction of a portion ofthe PLP gene, in vitro and in vivo expression of the PLP gene product, and the in vivo effects of the PLP gene product.

CA 022~6~77 l998-ll-23 WO 97/45144 PCT/US97/1~)214 The following examples are presented to illustrate the invention, and are not intended to limit the scope thereof.

DESIGN ~ND CONSTRUCTION OF TMF PLP GENE
S In SJL/J mice, the encephalitogenic epitope of PLP comprises amino acids 139-lSl.NT~k~h~hietal.,Cell42:139-148(1985),KSakaietal.,J.Neuro;mm~]nol. 19:21-32 (1988), D.H. Kono et al., J. ~xp. Med. 168:213-227. The vcctor in the presentinvention is designed in order that the gene product encoded by it be constitutively secreted from fibroblasts. Since the complete PLP protein is a hydrophobic transmembrane protein (H-J. Diehl, M Schaich, R-M. B~ incki and W. Stoffel, PNASU.S~A. 83:9807-9811 (1986)), with the encephalitogenic epitope being extracellular, a plasmid encoding amino acids 101-157 and additional amino acids required for secretion was constructed. This sequence is hydrophilic in character.

1. Oli~onucleotide synthesis and constructioll of the P~P pRc/CMV vector Oligonucleotides can be synthesized m~nll~lly, e.g., by the phospho-tri-ester method, as disclosed, for exarnple in R.L. Letsinger, et. al., J. Am chem. Soc. 98:3655 (1967), the disclosure of which is incorporated by reference. Other methods are well known in the art. See also Matteucci and Caruthers, J. ~m. Chem Soc. 103 :3185 (1981), the disclosure of which is incorporated by reference.

CA 022~6~77 1998-ll-23 WO 97/45144 PCT/US97/~0214 Preferably, however, the desired gene se~uence can be made by automated synthesis of individual oligonucleotides at .2~M concentrations. For PLP amino acids 101-157, DNA syntheses were ~ rolllled on a Perkin Elmer/Applied Biosystems Division Model 394 DNA 5ynthesi7~r using cyanoethyl-protected phosphorarnidites. The 5 dimethoxytrityl (DMT~ group was not removed from the 5'hydroxyl group to allow for purification. After normal cleavage from the resin using concentrated arnmonium hydroxide and deprotection at 55~C for 16 hours, the oligonucleotides were purif1ed using oligonucleotide purification cartridges (OPC) according to the manufacturer's instructions (Applied Biosystems Inc.). Five oligonucleotidcs of the following sequences 10 were synthe~i7~1 OLG 1 5' - CGGCGACTACAAGACCACCATCTGCGGCAAGGGCCTGAGCGC
AACGGTAACAGGGGGCCAGAAGGGGAGGGGTTCCAGAGGCCA
ACATCAAGCTCATTCTCTCGAGC-3', OLG2 5' - GAGCTTGATGTTGGCCTCTGGAACCCCTCCCCTTCTGGCCCCCT
GTTACCGTTGCGCTCAGGCCCTTGCCGCAGATGGTGGTCTTGTA
GTCGCCGGGCC-3', OLG3 5' - GGGTGTGTCATTGTTTGGGAAAATGGCTAGGACA,TCCCGACAA
GTTTGTGGGCATCACCTATGCTAGCCTTAAGTAGGATCCTTGAA
TAGGTA-3', ~0 OLG4 5' - AGCTTACCTATTCAAGGATCCTACTTAAGGCTAGCATAGGTGA
TGCCCA-3', and CA 022~6~77 1998-ll-23 OLG5 5'- CAAACTTGTCGGGATGTCCTAGCCATTTTCCCAAACAATGACA
CACCCGCTCGAGAGAAT-3 ' .

Each purified oligonucleotide was dried under vacuum, washed with 1 ml of sterile double distilled water and then concentrated to dryness under vacuum (Speed vac 5 evaporator; Savant Inc.). BOpM of each oligomer was kin~ecl at 37~C for I hour by u~ nding in 56.6,u1 of lX kinase buffer (Polynucleotide Kinase Buffer, Boehringer Mannheim, Indianapolis, IN) cont~ining 10 units of polynucleotide kinase (Boehringer MarLnheim) and 1 00,uM of ATP. The individual oligonucleotides were combined in the presence of 2X SSC (0.03M Sodium Citrate, pH 7.0, and 0.3M NaCl) in a PCR tube with 10 their respective complementary oligomer partners for ~nn~Tin~. Each annealed set measured 200,u1 in volume. Oligomer OLG 1 was annealed with OLG2, and oligomers OLG4 and OLG5 were annealed with OLG3. Annealing was perforrned in a Perkin-Elmer 9600 Thermocycler, programmed as follows: 1)99.9~ for 2 nnin~tec7 and 2) 99.9~
to 4~ in 15 minutes. During the temperature descent to 4~C, when the thermocycler 15 t~ peld~ reached 37~C, the solution cont~ining the oligomer duplex OLGl and OLG2 was combined with the solution contz-ining the oligomers OLG3, OLG4, and OLG5. The descent cycle was then continued until it reached 22~C. Subsequently, S units (5,ul) of T4 ligase (Boehringer Mannheim, Tn~ n~l~olis, IN) and 45,u1 of manufacturer's 10X T4 D~A ligation buffer (Boehinger Marmheim, Tnc~i~n~polis, IN) was added, and ligation 20 proceeded overnight at 10 ~C.
The ligated DNA was precipitated with 2 volumes of 100% ethanol and incubated at -70~C for I hour. The precipitate was centrifuged for 30 minutes at 17000 x g at 4~C.

CA 022~6~77 1998-ll-23 The supernatant was discarded and pellet was washed with 1 ml of 70% ethanol andcentrifuged for 10 minutes at 17000 x g at 4 ~C. The DNA pellet was dried under vacuum (Speed vac evaporator; Savant Inc.) and resuspended in 45,u1 sterile double distilled water.
DNA of the correct molecular weight was isolated by electrophoresis. 5,ul of 1 0x loading buffer (6.25g Ficoll and 0.93g Disodium EDTA/25ml 10% SDS, Orange G, Xylene Cyanole, and Bromophenol Blue) was added to the sample and loaded onto a 14.5 cm x 16cm x 0.15mm urea/acrylamide gel (7M urea/8% acrylamide with 1.1% Bis).
TBE (89mM Tris, 89mM Boric acid, and 2mM EDTA pH8.0) was used as both gel and electrophoresis buffer. The sample was electrophoresed at 35mA until the Orange G dye line had migr~t~?d within 1 cm of the bottom of the gel. The acrylamide gel was washed twice with water for S miml~t-s After the last wash, the gel was incubated for 3 minl]te~
in a 500 ml solution cont~inin~ 10ul of 10mg/ml of ethidium bromide, and vi~ li7f ~1 under a W-light source. The band corresponding to the ligated DNA was excised and cut into small pieces for electroelution in an IBI electroelutor ~ Lus (Model UEA:
International Biotechnologies Inc., New EIaven, CT).
For electroelution, the salt trap of the apparatus was filled with 125,ul of 7M
sodiurn acetate/bromophenol blue dye solution. The buffer chamber was filled with 1/2X
TBE. The sarnple was electroeluted for I hour at 85V. After removing the eluted DNA, the sarnple well was washed with 1/2X TBE and combined with the initial eluate. Thc eluted DNA was then l,rt;.;i~iLa~d overnight at -70~C with 2 volumes of 100% ethanol.
The precipitate was pelleted, washed as previously described, and resuspended in I Sul of sterile double distilled water.

CA 022~6~77 1998-ll-23 Preceding the ligation of the eluted partial PLP gene to the pRc/CMV vector (Invitrogen, San Diego, CA), the p~c/CMV vector construct was cut with the restriction endonucleases Apa I and Hind III according to the Manufacturer's instructions (Boehringer Mannheim, Tn~ n~polis, IN). The resuspended PLP gene construct was then added to a 5~1 mixture containing 0.3,ug of pRc/CMV cut vector (2~1), 1 unit T4 ligase (l,ul) (Boehringer Mannheim, Tnclizln~polis, IN), and 2,u1 of Manufacturer's lOX
T4 DNA ligation buffer (Boehringer Mannheim, Tn~i~n~polis, IN). The ligated vector was then transformed into the competent cell line AG1.
Tla~ lation proceeded by combining the ligation mixture with the AG1 cells and incubating it on ice for 20 minutes. The cell/vector mixture was then incubated at 42~ for 2 minutes and plated overnight onto a Luria Broth agar (LB; BiolO1, Vista, CA) plate, supplemented with 80 mg/ml of ampicillin (Sigma, St. Louis MO). Colonies were screened for the correct sequence vector by first isolating the plasmid DNA and then sequencing the DNA.
To isolate the plasmid, a commercially available plasmid purification kit, Wizard Minipreps (Promega, Madison, WI) was used. Colonies were picked from the LB/Amp plates and grown for 3.5 hours in 5 ml of LB medium (BIO 101, Vista, CA) supplt-m~nted with 80mg/ml of ampicillin (Sigma, St. Louis, MO). 3 ml of the medium was centrifuged at 17000 x g at room temperature, for 1 minute to pellet the cells.
Isolation ofthe plasmid proceeded according to the h/l~nl-f~tnrer's instructions. l,L~g of the isolated DNA was used for sequencing.
The oligonucleotide sequence can be checked by methods well known in the art, such as that described by Sanger et. al..PNAS U.S.A. 70:1209 (1973) or by the Maxam-CA 022~6~77 1998-11-23 Gilbert method, Meth. Fn7ymolo~y 65:499 (1977), the disclosures of both of which are incorporated herein by reference. Preferably, the plasmid can be sequenced using an automated DNA sequencer. For the PLP pRc/CM~ construct, the plasmid was sequenced using automated fluorescent DNA sequencing procedures (Perkin S Elmer/Applied Biosystems Inc, Foster City, CA) using the following primers:
GATTTAGGTGACACTATAG and TAATACGACT(~ACTATAGGG. These primers primed off the vector, which flanked the Kozak and "stop" site of the total construct.
Figure 1 shows a map of the partial PLP gene showing the sequence of the gene product and restriction sites. At the 5' end of the construct we had previously inserted a 10 hydrophobic leader sequence from the MHC class I Ld gene to enable the gene product to be synth~i7P.1 in the endoplasmic reticulum (ER) for later constitutive secretion. Linsk et al. J.Exp Med. 164:794-813 ~199~) In addition, a Iysine codon at the 3' end was added to ensure that the protein could not be retained in membrane. A Kozak box was included in the construct to ensure efficient translation. Restriction sites Afl II and 15 BarnHI were included in the construct to allow for insertion of further epitopes.

ITR~ FXPRESSION OF THF PLP PROTFIN
The following experiments were performed in order to demonstrate that the PLP
vector encodes a protein which is constitutively secreted. Specifically, the mRNA levels 20 of PLP were evaluated in SJL fibroblast cells transfected with the pRc/CMV-PLP vector, and mRNA and protein levels of PLP were evaluated in SJL fibroblast cells transfected with the pGlPLPSvNa vector.

.

CA 022~6~77 1998-11-23 1. F~tabli~hm~nt of Fibroblast Cultures Syngeneic fibroblasts (derived from SJL mice) were obtained from Dr. G.
Dveskler (Uniformed Services University, Bethesda, MD) and expanded at 3 7 ~
incubation using DMEM growth medium, supplemented with 5% ghlt:~mine and 10%
5 ~CS. The cells were harvested and frozen at 1 x 107 cells per vial, and aliquots were quality control tested for mycoplasma, sterility and viability.

2. ~etrovir~l Const~uct~
A recombinant retroviral vector in which exogenous genes are inserted into a 10 retroviral vector was constructed. The cloning strategy was to construct a pGlXSvNa vector (W. French Anderson, University of Southern California) cont:~ining the PLP
insert from pRc/CMV-PLP. The pGlXSvNa vector, like most retroviral vectors used in preclinical and clinical trials, is derived from the Moloney murine leukemia retrovirus (Mo-MLV). Rosenberg et al., N Fr~. J. Med. 323:570-578 (1990), Culver et al., Science 256:1550-1552 (1992). The GlXSvNa vector is a 5865 bp vector whose map, functional features and complete DNA sequence are shown in Figures 2a and 2b. Figure 3 illustrates the procedure for constructing the pGlPl,PSvNa vector. F~enti~lly, the pRc/CMV-PLP vector was digested with BstEII/HindIII and PLP encoding fragment was isolated by gel electrophoresis. After electroelution, ~IindIII/NotI adapters (Stratagene, 20 La Jolla, CA) were ligated into the ~indIII site of the eluted fragment. A NotI digestion was performed to generate NotI ends. A NotI digest was perforrned on pGlXSvNa and the 5865 bp fragment was isolated, electroeluted, and a CIAP (Calf intestine ~Ik~lin~
phosphatase trezltment) was performed on the fragment ends. The NotI site of the insert CA 022~6~77 1998-11-23 was ligated into the NotI site of the vector. BstEII ends of the insert and NotI site of the vector were Klenowed. A blunt end ligation is performed to close the vector. HB 101 cells were transformed with ligation mix and restriction analysis was performed to determine which vectors contain insert and the insert orientation. The recombinant 5 retroviruses are non-replicating and incapable of producing infectious virus.

3. Retroviral vector supern~tsnt To prepare supernatant co~ PLP-recombinant retrovirus, the PLP-tr~n~luçe~ retroviral p~ ging cell line PA3 17 was grown in 4 ml of ~ u~,idle culture 10 medium in a T25 flask (Corning, Cambridge, MA). Retroviral vector supernatant is produced by harvesting the cell culture medium when cells were 80-90% confluent, and stored in 1 ml aliquots at -70C ~.

The following tests were performed on the PLP cell line and/or the vector supernatants:
(1~ The viral titer is determined using 3T3 cells. Viral ple~)~dLions with titers 15 greater than 5 X 104 colony forming units/ml are used.
(2) Sterility of the producer cell line and the supernatant is assured by testing for aerobic and anaerobic bacteria, fungus and mycoplasma.

The PLP-vector p~ ~dlions from PA3 17 can be extensively tested to assure that no ~letect~ble replication competent virus is present. This is particularly relevant to the 20 embodiment of the invention wherein the invention is used to treat hum~n.~ Tests on both the viral supernatant and on the tr~n~c~uçed fibroblasts can be performed to CA 022~6~77 1998-11-23 determine if there is replication competent virus present. The following tests can be performed on the producer cell line and/or the viral supt?rn~t~nt s (1) The viral titer is determined using 3T3 cells. Viral piepaldlions with titers greater than 5 X 104 colony forming units/ml are used.
(2) Southern blots are run on the producer cell line to detect the partial PLP
genc.
(3) PLP production by the producer cell line is measured and should be significantly above baseline control values, as determined by ELISA assay.
(4) Sterility of the producer cell line and the supernatant is assured by testing for aerobic and anaerobic bacteria, fungus and mycoplasma.
(S) Viral testing is performed including: MAP test, LCM virus, thymic agent, S + L-assay for ecotropic virus, S + L assay for xenotropic virus, S + L-assay for amphotropic virus and 3T3 amplification.
(6) Electron microscopy is performed to assure the absence of adventitious 1 5 agents.
Following the introduction of the gene into fibroblasts, the following tests areperformed on the fibroblasts prior to ~r~mini~tration to patients.
(1 ) Cell viability is greater than 70% as tested by trypan blue dye exclusion.
(2) Cytologic analysis is performed on over 200 cells prior to infusion to assure that tumor cells are absent.
(3) Sterility is assured by testing for aerobic and anaerobic bacteria, fungus and mycoplasma.
(4) S + L-assay including 3T3 amplification must be negative.

CA 022~6~77 1998-11-23 (5) PCR assay for the absence of 4070A envelope gene must be negative.

(6) Reverse transcriptase assay must be negative.

(7) Southern blots run on the tr~nc~ efl fibroblasts to assure that intact provirus is present.

(8) PLP protein assay to assure the production of PLP protein.

4. Tr~n~fection of fibroblasts Prior to the transfection of the SJ~ fibroblasts, highly purified PLP-pRc/CMV
vector was isolated from the transformed AG1 cells. Large scale purification was performed by using a commercially available kit and CsCl gradient b~n~ling Initial 10 purification was accomplished using a Wizard Megaprep Kit (Promega, Madison, WI).
A lOOOml culture oftransformed AG1 cells, grown overnight in LBtAmp at 37~C, was pelleted and the plasmid DNA isolated according to the Manufacturer's instructions. The isolated DNA, which was suspended in 3 ml of TE buffer (I OmM Tris-~ICI, pH 7.4, and 1 mM disodium EDTA, pH, 8.0) was further processed by CsCI gradient banding. A
15 modified C~SCI banding of the DNA was performed based on procedures found in "Current Protocols in Molecular Biology, Vol 1 " (Greenc Publishing Associates and Wiley-Interscience) .
After the DNA band was extracted from the ultracentrifuge tubes, ethidium bromide was removed from the sample by washing it with 3 volumes of SSC saturated 20 isopropanol. The wash was repeated until the aqueous layer appeared clear. CsCl was removed by precipitation. 2 volumes of 0.2M NaCl/TE and 2 volumes of 100% ethanol (relative to the combined total volume of DNA solution and 0.2M NaCI/TE) were added CA 022~6~77 1998-ll-23 to the sample, mixed and placed on ice for 10 minl7tes. The precipitated DNA was pelleted by centrifugation at 10000 x g for 10 minutes at 4~C The pellet was washed ~ with cold 70% ethanol, recentrifuged at 10000 x g for 10 minutes at 4~C, and dried under vacuum (Speed vac evaporator; Savant Inc.). The puri~led DNA was resuspended with 5 double-distilled sterile water and utilized in the transfection process.
Test SJL fibroblasts were transfected using LipofectAMINE Reagent (Life Technologies Inc./Gibco BRL) according to the manufacturer's instructions. Control SJL
fibroblasts underwent the same procedure without the presence of a DNA construct. 3,ug of CsCl purified PLP-pRc/CMV plasmid and 25,ul of Lipofectamine were used for transfection. Approximately 3 ~ 105 SJL cells, seeded overnight into 25cm 2culture flasks (Corning Costar Corp., Cambridge, MA.) and grown at 37~ with 5% CO2 ~n 5ml of DMEM culture medium (Dulbecco's Modificd Eagle's Medium (Irvine Scientific, Santa Ana, CA), supplemented with 5% glutamine, 10% Fetal Calf Serum, 25 Units/ml of penicillin G sodium, and 25,ug/ml of streptomycin sulfate, were washed with 3ml serum free HL- 1 medium (Hycor Biomedical Inc., Irvine, CA). ~fter the DNA/lipofect~min(~. complexes were incubated with cells for 6 hours at 37~ with 5% Co2 1 ml of DMEM was added to the flasks. The flasks were incubated overnight at 37~ with 5% CO2 The medium was replaced with 5ml of fresh DMEM the next morning. 36 hours after the end of the transfection period, the medium was replaced with Sml of DMEM cn~ " ~ g 900,~cg of G418 (Life Technologies Inc./Gibco BRL)/ml of medium.
The test cells were grown in the presence of 900,ug of G418 of medium until all the control cells had died, and no more cell death could be observed in the test sample flask.

CA 022~6~77 1998-ll-23 The G418 concentration was then reduced to 600,ug/ml of culture medium for duration of cell culturing procedures.

5. Transduction of Fibroblasts Retroviral constructs cont~ining a neo-selectable marker together with either the S PLP gene or the Lac-z gene were used to tr~n~ ce fibroblasts. Transduction with the retrovirus was performed on healthy cells (90% viable, as determined by trypan blue staining). 2 X 106 cells were plated in 0.5 ml DMEM-I0 media (DMEM media supplemented with 10% fetal calf serum, 2 mM L-glnt~rnin~, 50 U/ml penicillin G, 50 mg/ml streptomycin in one well of a 24-well plate (Falcon, Franklin Lakes, NJ). Cells 10 were placed in the incubator and allowed to settle (37~, 5% C~2) After cells had settled, I ml of retroviral supernatant and polybrene (Sigma, St. Louis, MO) (final concentration I 0,ug/ml) was added to the well. Cells were incubated as above for 2.5 hours without ~hslkin~. After 2.5 hours, cells were transferred to a T25 flask and DMEM-10 media was added to a total volume of 8 ml. Selection media (culture media comprising DMEM- 10 supplemented with 900 ,ug/ml G418 (Gibco, Grand Island, NY) was added on the third day after tr~n~ .tion. The G4 18 concentration was then reduced to 600,ug/ml of culture medium for the duration of cell culturing procedures.

6. mR:NA expression analysis mRNA isolation was performed using aseptic techniques, RNAse free supplies, 20 and DEPC (Diethylpyrocarbonate) treated solutions. 4 X I o6 experimental and control SJL cells were washed twice with cold Phosphate-buffered saline, resuspendcd in 200,u1 CA 022~6~77 1998-11-23 cell Iysis mix ~lOmM TRIS pH 7.5, 0.15M NaCI, 1.5mM MgCI2, 0.65% NP 40), vortexed, and centrifuged at 17000 x g at 4~ for 5 minutes. The supernatant was transferred to a tube containing 200,~11 of urea mix (7M urea, 1% SDS, 0.35M NaCI, lOmM EDTA, and lOmM Tris-HCL, pH 7.5) and 400,u1 of phenol:chloroform;isoamyl alcohol (25:24:1). The solution was vortexed and centrifuged for 1 minute at 17000 x g. This procedure was repeated twice using the aqueous layer and then transferred to a tube cont~inin~ 400,u1 of phenol and washed as before. The aqueous layer was kansferred again to another tube, and precipitated with 1 ml of 100% ethanol overnight at -20~C. The precipitated RNA was washed with 1 ml 70% ethanol. After the ethanol was discarded, the pellet was dried under vacuum. 1,ug ofthe RNA was used for RT-PCR analysis.
RT-PCR was p~;lro~ ed using a commercially available kit, GeneAmp RNA PCR
Kit (Perkin Elmer/ABI) according to the Manufacturer's instructions. The following primers were used to amplify the cDNA: 5'-GCGACTACAAGACCACCATCT-3' and 5'-TAAGGCTAGCATAGGTGATG-3'. The PCR products were electrophoresed on a 1.5% agarose (SeaKem GTG; FMC)/TAE gel with 1,ul of lOmg/ml of ethidium bromide/ml of agarose solution. The gel was electrophoresed using TAE buffer at a constant 40mA. Eleckophoresis was continued until the molecular weight marker bands had separated adequately enough, to verify the PCR products' approximate molecular size. The DNA band of interest was then excised and gel purified, using the commercially available MERmaid Kit (Bio 101, Vista, CA), according to the Manufacturer's instructions. The purified DNA was then sequenced by automated Fluorescent DNA sequencing procedures (Perkin Elmer/ABI, Foster City, CA).

CA 022~6~77 1998-ll-23 Figure 4 iS an agarose gel showing PLP-specific RT-PCR products. The data illustrates that mRN~ is present in both PLP-tr~n~duced and PLP-transfected cells. The correlation between mRNA and secreted protein remains to be determined since peptide concentration does not necessarily correspond to the level OrmRNA.

5 7. Protein Expression Analysis The in vitro qualitative expression of tl~e proteins encoded by the PLP gene was detected immllnologically by ELISA. Undiluted supernatants from cultures of fibroblasts k~nccl~lce~l with the PLP gene were tested. Wells of 96 microtiter plate were coated with the supernatants. Primary anti-PLP-antibody 4E10 139-151, from Dr. M. Lees (Harvard), is specific for PLP 139-151 and was added to wells as undiluted hybridoma supernatant followed by horseradish peroxidase (HRP~-conjugated goat anti-mouse secondary antibody in a concentration of 1:500. The plate was developed and analyzed at 490 nm on a microplate reader. Figure 5 illustrates the results of ELISA assays on transduced fibroblast supern~t~nt~ Samples 1 and 2 were PLP (amino acids 139-151) and HIV
15 gpl20 peptides used at a concentration of 5ug/ml. This experiment illustrates that the tr~n~cl~lced PLP-transduced fibroblasts do produce and secrete the partial PLP protein.

IN VIVO FFFECTS OF THE PLP PROTFTN
Critical to the success of this invention in the embodiment of this exarnple is the 20 ability to deliver genetically manipulated fibroblasts to patients so that the cells survive CA 022=,6=,77 1998-ll-23 in sufficient numbers and for long periods of time, in order that continuous secreted antigen may be provided to the patient.
- To assess the fate of transplanted transduced fibroblasts, SJL fibroblasts tr~ncfl~lcecl with retrovirus encoding B-galactosidase were injected subcutaneously S between the shoulders of SJL mice. All mice were female mice of the SJL strain between 6-8 weeks old and were obtained from Jackson Labs. Animals were housed and maintained according to NIH guidelines (National Research Council, 1986). These fibroblasts survived in large numbers after 60 days. Fibroblasts injected into the footpad or intramuscularly could not be detected at eight days.

10 1. In Yivo fatc B-~al tr~ncduced cellc The activity of the B-Galactosidase marker was evaluated using two groups of eight normal mice. Two mice were injected subcutaneously on the back, two mice were injected intramuscularly and two mice were injected in the footpad with Lac-Z
tr~n.c(l~lced cells. One animal was injected with fibroblasts tr~nccl~ ed with neo-marker 15 only, and the last mouse was injected with untr;m.ccll-~ecl fibroblasts. ~fter harvesting and washing, the different cell lineages were suspended in a concentration of 107 cells in .2 ml of Hank's PBS and slowly injected using a 25 gauge needle at different sites.
Animals were sacrificed at 10 and 15 days post treZltn~ent and injection sites were submitted to histochemical study. Pieces of tissue were fixcd in 4% paraforrnaldehyde 20 for one hour, washed in PBS three times and then kept in 8.4% acrylamide solution overnight. The next morning tissues were embedded in acrylamide which after hardening were cut and frozen. The frozen sections were done in 1 Oum by cryostat and stained with CA 02256577 1998-ll-23 WO 97/4~;144 PCT/US97/10214 1 ml of 5-Bromo-4-chloro-3-indolyl-B-d-galactopyranoside (X-Gal) in PBS. The X-Gal was dissolved in DMSO at 40mg/ml and then addcd to the reaction mixture. Incubation was for 14-18 h at 37~. Figure 6 illustrates B-Gal expression in transduced fibroblasts 60 days in vivo. There was no evidence of an infl~mm~Jory response, suggesting that the retrovirus used to transduce syngeneic fibroblasts, does not evoke an immune response or rejection process.

2. Effect of PLP in no~nal SJL mice Another important aspect of this invention in the embodiment of this example is determ;ning whether transduced l~lbroblasts secreting PLP actually produce EAE in normal z~nim~ To test this, 107 PLP-secreting SJL fibroblasts were injected into 12 normal SJL mice. Six ~nim~l.c had fibroblasts placed subcutaneously and six animals had fibroblasts injected intraperitoneally. Animals were sacrificed at day 16 and showed no evidence of infl~mm~tory disease or EAE. Figure 7 illustrates the clinical scoring system for chronic EAE. Y-A Lu et al., Mol. Immllnol.. 28:623-630 (1991), J. Williamson et al., J. I~euro;mmnnol. 32:199-207 (1991). In the EAE model for multiple sclerosis, using spinal cord homogenates plus adjuvant, infl~mm~tion in the CNS can be seen by day 1~.
In this study, normal ~nim~l~ injected with PLP-secreting SJL fibroblasts did not show any signs of clinical disease even at day 60. In addition, the ~nimAl~ did not show any histologic evidence of infl~mm~tion in the CNS at day 60. Figure 8 illustrates the histological scoring system for EAE. J. Governman et al., Cell 72:551-560 (1993).

CA 022~6~77 1998-ll-23 WO 97/45144 PCT/US97tlO214 3. Clini~ nfl histolo~ical assessment of acute EAE Inice treated with retrovirus tr~ncduced fibroblasts.

- Six week SJL mice were infected with mouse spinal cord homogenate (MSCH) in complete Freund's Adjuvant (CFA) and with MSCH in incomplete Freund's Adjuvant IFA, seven days later. J. ~mmnnol. 144:909-915 (1990). The initial EAE attack was observed on days 14-18, with full recovery by 21. Ninety-five percent of animals showed clinical evidence of an acute attack and these were given either 107 PLP secreting SJL
fibroblasts or control fibroblasts on day 21. Animals not showing clinical disease were elimin~t~-rl from the experiment. Figure 9 illustrates the clinical z~ccec~m~nt of EAE mice treated with retrovirus tr:~n.c~nce~l fibroblasts. Animals receiving the PLP secreting fibroblasts had a marked reduction of clinical signs and had dramatic reduction in infl~mm~tory cells, particularly in the brain. Figure 10a illustrates the pathologic assessment of brain and spinal cord of SJL mice treated with retrovirus transduced fibroblasts. Figure 1 Ob is a summary of the pathologic assessment of brain and spinal cord from days 55-60 and 90-95. Histological ~csçssment of EAE Grades in Brain and Spinal Cord were performed following the ~ ~dtion of hematoxylin and eosin stained sections.

4. Clinir~l and histological asse.c.cment of chronic EAE miçe treated with retrovirus k~ncduced fibroblasts.
150 mice were inoculated with MSCH in CFA. A second immlmi7~tion was given 7 days later. A.M. Brown and D.E. McFarlin, Laboratory Invest. 45:278-284 (1981~. On day +14 to 16, 1 13 animals developed clinical disease lasting 3-4 days. These positive ~nim~l~ were separated for subsequent experiments and had their first relapse on CA 022~6~77 1998-11-23 WO 97/45144 PCTtUS97/10214 day +55 to 60, with 100 animals becoming sick. These were again separated and on day +137, 67 had a relapse. Eight days after relapse, animals were each transplanted with 107 i~lbroblasts and then sacrificed 18 to 23 days la~er. Four different types of fibroblasts were used, those tr~n.~ ce~l with retrovirus encoding PLP, encoding B-galactosidase and 5 encoding neo-selectable marker as well as untransduced cells. Figure 11 shows the histology of SJL mice with chronic EAE treated with retrovirus tr~n~ çe~ fibroblasts.
There were no animals receiving PLP secreting fibroblasts with 2+ to 3+ infl~mm~tion~

5. Peripheral immune status of treated mice v. control E~F mice.
Spleen cells from our EAE control mice and from four EAE mice which had been 10 treated with fibroblasts expressing the PLP protein were used in proliferation assays, in which they were incubated with 40~1M PLP peptide 139-151 or 4011M HIV gpl20 peptide 308-322 for 4 days and then pulsed with 3H-thymidine for 24 hours.
Briefly, animals were sacrificed by CO~ asphyxiation. Spleen cells were dispersed to single cell suspensions in RPMI 1640 by passing through a size 60 mesh, and washed once before being cultured (8 x 105 per well) in 0.2 ml of HL-1 medium (Hycor Biomedical, Irvine, CA), supplemented with 2mM glutamine, l OOU/ml penicillin, l OO,ug streptomycin either alone or with 40,uM of peptide in 96-well tissue culture plates for 4 days at 37~C with 5% CO2. PLP peptide 140-151 and MBP peptide 89-101 were used for antigen-specific proliferation while HIV gp 120 peptide 308-322 was used as negative 20 control. Where indicated, some wells also contained 1 OU/ml of recombinant mouse IL-2 (Boehringer Mannheim, Indianapolis, IN). During the last 18-24 h of culture, each well was pulsed with 1,uCi of 31~1-thymidine (ICN, Irvine, CA), harvested onto 'Xtal Scint' CA 022~6~77 1998-11-23 WO 97/45144 PCT/US97/1~214 glass fiber filters (Beckman, Fullerton, CA) and counted using a Beckman L~6000 Scintillation counter. Thymidine incorporation values (experimental counts per minute -- background counts per minute) were calculated and represent means of triplicate cultures ~ standard deviation.
The results are shown in Figure 12 and suggest that PLP specific proliferative responses are reduced significantly in EAE mice which have received PLP expressing fibroblasts.
Figure 13 illuskates the same experiment as in Figure 12 but with the addition of mouse IL-2 (lOU/ml) for 5 days. These results illustrate that the mech~ni~m by which the PLP specific proliferative responses are reduced significantly may suggest the possibility of deletion of T cells rather than anergy because these Iymphocytes do not respond to IL-2.
Although the mechanism by which the present invention acts to restore tolerance in individuals suffering from T-cell mediated autoimmlme disease is not entirelyunderstood, the benefits of the treatment are clearly advantageous over alternative treatments. The method is a genetic approach to immunospecifically silence pathogenic T-cell responses and does not down-regulate the entire immune system. In the case where an individual with a T-cell mediated autoimmune disease exhibits pathogenic T-cells of multiple specificities, the invention may easily be adapted to target those specificities. For example, DNA encoding multiple self-antigenic epitopes may beinkoduced into the patient' s cells. The invention is also advantageous in that the reagents can easily be made or obtained in sufficient quantity to carry out the invention.

CA 022~6~77 1998-11-23 The present invention is not to be limited in scope by the exemplified embodiments disclosed herein which are intended as illustrations of single aspects of the invention, and clones, DNA or amino acid sequences which are functionally equivalent are within the scope of the invention. Various modifications of the invention, in addition 5 to those shown and described herein, will become al~cuclll to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.
Various publications are cited herein that are hereby incorporated by reference in their entireties.

WO97145144 PCT~S97/10214 S~OUF.NC~ TlIsTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Weiner, Leslie P.
McMillan, Minnie (ii) TITLE OF INVENTION: Construction and Use o~ Genes Encoding Pathogenic Epitopes For Treatment of Autoimmune Disease (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Kaye, Scholer, Fierman, Hays &
Handler LLP
(B) STREET: 1999 Avenue o~ the Stars (C) CITY: Los Angeles (D) STATE: California (E) COUNTRY: USA
(F) ZIP: 90067 (V) COM~U'l'~K READABLE FORM:
(A) MEDIUM TYPE: Diskette, 3.50 inch (B) COMPUTER: IBM
(C) OPERATING SYSTEM: Windows 3.1 (D) SOFTWARE: Wordper~ect 6.1 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 08/654,737 (B) FILING DATE: 29-MAY-1996 (C) CLASSIFICATION: Unknown (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Thomson, William E.
(B) REGISTRATION NUMBER: 20,719 (C) REFERENCE/DOCKET NUMBER: USC-002 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (310) 788-1050 (B) TELEFAX: (310) 788-1200 (2) INFORMATION FOR SEQ ID NO: 1 (i) SE~U~:~ CHARACTERISTICS:
(A) LENGTH: 317 base pairs (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: Genomic DNA

CA 022~6~77 l998-ll-23 WO97/45144 PCT~S97/10214 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: l GATGGTGACC GGAGATCTGC CGCCACCATG
GGGGCGATGG CTCCGCGCAC GCTGCTCCTG
CTGCTGGCGG CCGCCCTGGC CCCGACTCAG
ACCCGCGCGG GGCCCGGCGA CTACAAGACC
ACCATCTGCG GCAAGGGCCT GAGCGCAACG
GTAACAGGGG GCCAGAAGGG GAGGGGTTCC
AGAGGCCAAC ATCAAGCTCA TT~lLlCGAG
CGG~l~l~lC ATTGTTTGGG AAAATGGCTA
GGACATCCCG ACAAGTTTG TGGGCATCAC
CTATGCTAGC CTTAAGTAGG ATCCTTGAAT
AGGTAAGTTG CTAGCCC

(2) INFORMATION FOR SEQ ID NO: 2 ~i) SEQu~N~ CHARACTERISTICS:
(A) LENGT~: 5865 base pairs (~3) TYPE: Nucleic Acid (C) STRAN~ : Double (D) TOPOLOGY: Circular (ii) MOLECULE TYPE: Vector DNA
(xi) ~Qu~: DESCRIPTION: SEQ ID NO: 2 121 TGAATACCAA ACAGGATATC lVlG~lAAGC G~ll~lGCC CCGGCTCAGG GCCAAGAACA
181 GATGAGACAG CTGAGTGATG GGCCAAACAG GATA'l'~'l'~'l'G GTAAGCAGTT CCTGCCCCGG

361 TAACCAATCA GTTCGCTTCT CG~ll~l~l~ CGCGCGCTTC CG~l~l~A GCTCAATAAA
421 AGAGCCCACA ACCCCTCACT CGGCGCGCCA GTCTTCCGAT AGACTGCGTC GCC~'1'AC
481 CCGTATTCCC AATAAAGCCT CTTG~l~lll~ GCATCCGAAT CGTG~ CG CTGTTCCTTG

661 AGCAACTTAT ~'l'~'l'~'l'~'L~'l' CCGA~ll~lLl~ AGTGTCTATG TTTGATGTTA TGCGCCTGCG721 TCTGTACTAG TTAGCTAACT AG~l~l~lAT CTGGCGGACC CGTGGTGGAA CTGACGAGTT
781 CTGAACACCC GGCCGCAACC CTGGGAGACG TCCCAGGGAC TTTGGGGGCC ~'~ ll~lGG
841 CCCGACCTGA GGAAGGGAGT CGATGTGGAA TCCGACCCCG TCAGGATATG ~ ~lGGT
901 AGGAGACGAG AACCTAAAAC AGTTCCCGCC TCCGTCTGAA lllllGCTTT CG~lll~AA
961 CCGAAGCCGC G~l~ll~l'~ TGCTGCAGCG CTGCAGCATC ~l~l~l~'l'~'l''l' ~'l'~'l'~'l'~'l'CT
1021 GA~l~l~l-ll CTGTATTTGT CTGAAAATTA GGGCCAGACT GTTACCACTC CCTTAAGTTT

CA 022~6~77 1998-11-23 WO 97/45144 PCT~S97/10214 1321 TGACCCCCCT CCCTGGGTCA AGCLL'1"1"1'L'1' ACACCCTAAG CCTCCGCCTC CTCTTCCTCC

1441 TC QGCCCTC A~''1'L'L''1''1'CTC TAGGCGCCGG AATTCGCGGC CGCTACGTAG TCGACTCGCT

1741 AA'1''1"1"1''1"1''1''1' ATTTATGCAG AGGCCGAGGC CGCCTCGGCC TCTGAGCTAT TCCAGAGTA
1801 GTGAGGAGGC '1"1''1''1''1''1'GGAG GCCTAGGCTT TTGCAAAAAG CTCGAAGATC AATTCCGATC
1861 TGATCAAGAG ACAGGATGAG GATL'~'1"1"1'L'G CATGATTGAA CAAGATGGAT TGCACGCAGG

1981 CTGCTCTGAT GCCGL'LG'1'L'1' TCCGGCTGTC AGCGCAGGGG CGCCCGGTTC '1"1''1''1"1'L-'1'CAA

2341 CGGTLl l~lL GATCAGGATG ATCTGGACGA AGAGCATCAG GGGCTCGCGC QGCCGAACT

2521 CCGGCTGGGT GTGGCGGACC GCTATCAGGA CATAGCGTTG GCTACCC~1~ ATATTGCTGA
2581 AGAGCTTGGC GGCGAATGGG CTGACCGCTT CL'1'LL'1'GCTT TACGGTATCG CCGCTCCCGA
2641 TTCGCAGCGC ATCGCCTTCT ATCGCCTTCT TGACGAGTTC TTCTGAGCGG GACTL'1'GGG~

2881 TGGAACAGCT GAATATGGGC CAAACAGGAT A'1'L'LL-'1'~GTA AGCAGTTCCT GCCCCGGCTC

3001 TTCCTGCCCC GGCTCAGGGC CA~GAACAGA TGGTCCCCAG ATGCGGTCCA GCCCTCAGCA
3061 GTTTCTAGAG AACCATCAGA '1'L'1''1''1'CCAGG GTGCCCCAAG GACCTGAAAT GACL~L~ GC3121 CTTATTTGAA CTAACCAATC AGTTCGCTTC TCGCTTCTGT TCGCGCGCTT CTGCTCCCCG

3241 CGCCCGGGTA CCLL~1L~1~ATC CA~TAAACCC TCTTG QGTT GCATCCGACT '1~1~L1L1CG
3301 L~ CCTTG GGAGGGTCTC CTCTGAGTGA TTGACTACCC GTCAGCGGGG GTL'1"1"1'LATT
3361 TGGGGGCTCG TCCGGGATCG GGAGACCCCT GCCCAGGGAC CACCGACCCA CCA~LG~AG
3421 GTAAGCTGGC TGCCTCGCGC L-'1"1"1'CL-L'1'~A TGACGGTGAA AACCTCTGAC ACATGCAGCT
3481 CCCGGAGACG GTCACAGCTT GTCTGTAAGC GGATGCCGGG AGCAGACAAG CL'L~'1'CAGGG
3541 CGCGTCAGCG GGTGTTGGCG G~'1'L-'1'LGGGG CGCAGCCATG ACCCAGTCAC GTAGCGATAG

CA 022~6~77 1998-11-23 WO97/4S144 PCT~S97/10214 3841 TGAGCAAAAG GCCAGCA~AA GGCCAGGAAC CGTAAAAAGG CCGCGTTGCT GGC'~lllllC

4021 C~l~llC~A CCCTGCCGCT TACCGGATAC CTGTCCGCCT TTCTCCCTTC GGGAAGCGTG

4141 CTGGGCTGTG TGCACGAACC CC~'ll'CAG CCCGACCGCT GCGCCTTATC CGGTAACTAT
4201 C~l~ll~AGT CCAACCCGGT AAGACACGAC TTATCGCCAC TGGCAGCAGC CACTGGTAAC

4321 TACGGCTACA CTAGAAGGAC AGTA'll"l'~'l' ATCTGCGCTC TGCTGAAGCC AGTTACCTTC

4441 'lLl~lll~CA AGCAGCAGAT TACGCGCAGA AAAAAAGGAT CTCAAGAAGA l~lll~ATC
4501 lll-l~lACGG G~l~l~ACGC TCAGTGGAAC GAAAACTCAC GTTAAGGGAT TTTGGTCATG
4561 AGATTATCAA AAAGGATCTT CACCTAGATC ~llllAAATT AAAAATGAAG TTTTA~ATCA
4621 ATCTAAAGTA TATATGAGTA AA~ll~'l~l' GACAGTTACC AATGCTTAAT QGTGAGGCA
4681 CCTATCTCAG CGAT~l~l~l ATTTCGTTCA TCCATAGTTG CCTGACTCCC C~l~l~lAG

4861 AGAAGTGGTC CTGCAACTTT ATCCGCCTCC ATCCAGTCTA TTAA'll~ll~ CCGGGAAGCT

4981 GTGGTGTCAC GCTC~l~ll TGGTATGGCT TCATTCAGCT CCGGTTCCCA ACGATCAAGG
5041 CGAGTTACAT GATcccrrAT ~ll~l~CAAA AAAGCGGTTA GCTCCTTCGG lC~l~GATC
5101 GTTGTCAGAA GTAAGTTGGC CGCA~l~ll'A TCACTCATGG TTATGGCAGC ACTGCATAAT
5161 TCTCTTACTG TCATGCCATC CGTAAGATGC llLl~l~l~A CTGGTGAGTA CTCAACCA~G
5221 TCAll~l~AG AATAGTGTAT GCGGCGACCG AGTTGCTCTT GCCCGGCGTC AAcAr~r~AT

5341 CGAAAACTCT CAAGGATCTT ACCG~'l'~'l''l~ AGATCCAGTT CGATGTAACC CA~l-~l~CA

5461 AGGCAAAATG CCGCAAAAAA GGrAATAA~G GCGACACGGA AAl~l'l~AAT ACTCATACTC
5521 TT~lllllC AATATTATTG AAGCATTTAT CAGGGTTATT ~l~l~aTGAG CGGATACATA
5581 TTTGAATGTA TTTAGAA~AAA TAAACAAATA GGGGTTCCGC GCACATTTCC CCGAAAAGTG

5761 GTCCCCCTCA CA~lCC~AAA TTCGCGGGCT TCTGL~l~ll AGACCACTCT ACCCTATTCC

Claims

What is claimed is:
1. A method of treating a patient for a T-cell mediated autoimmune disease comprising:
introducing DNA comprising a sequence encoding one or more antigenic proteins into the cells of said patient, said cells expressing in said patient a therapeutically effective amount of said antigenic protein or proteins to restore T-cell tolerance to said patient.

2. The process of claim 1 wherein said patient is human.

3. The process of claim 2 wherein said cells are fibroblast cells.

4. The process of claim 3 wherein said fibroblast cells are histocompatible.

5. The process of claim 2 wherein said DNA encodes an amino acid sequence derived from a nervous system protein.

6. The process of claim 2 wherein the disease is multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, juvenile onset diabetes, Sjogren's disease, thyroid disease, myasthenia gravis, or chronic inflammatory demyelinating polyneuropathy (CIDP).

7. The method of claim 1 wherein the disease is multiple selerosis.

8. A method of treating a human for a T-cell mediated auto immune disease comprising:
introducing DNA comprising a sequence encoding one or more amino acid sequence derived from a self-antigenic protein into the cells of said human, said cells expressing in said human a therapeutically effective amount of said self-antigenic protein or proteins to restore T-cell tolerance to said human.

9. A method of treating a human for multiple sclerosis comprising:
introducing DNA comprising a sequence encoding one or more amino acid sequence derived from a nervous system protein into the cells of said human, said cells secreting in said human a therapeutically effective amount of said self-antigenic protein or proteins to restore T-cell tolerance to said human.

10. A method of treating a patient for a T-cell mediated autoimmune disease comprising:
introducing mammalian cells into a patient, said sells having been treated in vitro to insert therein a DNA segment encoding one or more antigenic protein, said mammalian cells expressing in vivo in said patient a therapeutically effective amount of said antigenic protein or proteins to restore T-cell tolerance to said patient.

11. The process of claim 10 wherein said patient is human.

12. The process of claim 11 wherein said cells are fibroblast cells.

13. The process of claim 12 wherein said fibroblast cells are histocompatible.

14. The process of claim 11 wherein said disease is multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, juvenile onset diabetes, Sjogren's disease, thyroid disease, or myasthenia gravis or chronic inflammatory demyelinating polyneuropathy (CIDP).

15. The process of claim 10 wherein said disease is multiple sclerosis.

16. The process of claim 10 wherein said DNA segment has been inserted into said cells in vitro by a recombinant vector.

17. The process of claim 10 wherein said DNA segment has been inserted into said cells in vitro by a viral vector.

18. The process of claim 17 wherein said viral vector is a retroviral vector.

19. The process of claim 10 wherein said DNA segment encodes a protein comprising an amino acid sequence derived from a nervous system self-antigenic protein.

20. The process of claim 11 wherein said DNA segment encodes a protein comprising an amino acid sequence derived from a nervous system self-antigenic protein.

21. The process of claim 14 wherein said DNA segment encodes a protein comprising an amino acid sequence derived from a self-antigenic protein.

22. The process of claim 12 wherein said DNA segment encodes a protein comprising an amino acid sequence derived from a nervous system protein.

23. The process of claim 13 wherein said DNA segment encodes a protein comprising an amino acid sequence derived from a nervous system protein.

24. The process of claim 15 wherein said DNA segment encodes a protein comprising an amino acid sequence derived from a nervous system protein.

25. The process of claim 16 wherein said DNA segment encodes a protein comprising an amino acid sequence derived from a nervous system protein.

25. The process of claim 17 wherein the DNA segment encodes a protein comprising an amino acid sequence derived from a nervous system protein.

26. The process of claim 18 wherein the DNA segment encodes a protein comprising an amino acid sequence derived from a nervous system protein.

28. The process of claim 10 wherein said DNA segment encodes a protein comprising an amino acid sequence derived from a nervous system protein selected from the group consisting of myelin basic protein, proteolipid protein, and myelin-oligodendrocyte glycoprotein.

29. The process of claim 11 wherein said DNA segment encodes a protein comprising an amino acid sequence derived from a nervous system protein selected from the group consisting of myelin basic protein, proteolipid protein, and myelin-oligodendrocyte glycoprotein.

30. The process of claim 12 wherein said DNA segment encodes a protein comprising an amino acid sequence derived from a nervous system protein selected from the group consisting of myelin basic protein, proteolipid protein, and myelin-oligodendrocyte glycoprotein.

31. The process of claim 13 wherein said DNA segment encodes a protein comprising an amino acid sequence derived from a nervous system protein selected from the group consisting of myelin basic protein, proteolipid protein,and myelin-oligodendrocyte glycoprotein.

32. The process of claim 15 wherein said DNA segment encodes a protein comprising an amino acid sequence derived from a nervous system protein selected from the group consisting of myelin basic protein, proteolipid protein, and myelin-oligodendrocyte glycoprotein.

33. The process of claim 16 wherein said DNA segment encodes a protein comprising an amino acid sequence derived from a nervous system protein selected from the group consisting of myelin basic protein, proteolipid protein, and myelin-oligodendrocyte glycoprotein.

34. The process of claim 17 wherein said DNA segment encodes a protein comprising an amino acid sequence derived from a nervous system protein selected from the group consisting of myelin basic protein, proteolipid protein, and myelin-oligodendrocyte glycoprotein.

35. The process of claim 18 wherein said DNA segment encodes a protein comprising an amino acid sequence derived from a nervous system protein selected from the group consisting of myelin basic protein, proteolipid protein, and myelin-oligodendrocyte glycoprotein.

36. The process of claim 10 wherein said DNA segment encodes a protein comprising an encephalitogenic epitope selected from the group consisting of encephalitogenic epitopes of myelin basic protein, encephalitogenic epitopes of myelin-oligodendrocyte glycoprotein, and encephalitogenic epitopes of proteolipid protein.

37. The process of claim 11 wherein said DNA segment encodes a protein comprising an encephalitogenic epitope selected from the group consisting of encephalitogenic epitopes of myelin basic protein, encephalitogenic epitopes of myelin-oligodendrocyte glycoprotein, and encephalitogenic epitopes of proteolipid protein.

38. The process of claim 12 wherein said DNA segment encodes a protein comprising an encephalitogenic epitope selected from the group consisting of encephalitogenic epitopes of myelin basic protein, encephalitogenic epitopes of myelin-oligodendrocyte glycoprotein, and encephalitogenic epitopes of proteolipid protein.

39. The process of claim 13 wherein said DNA segment encodes a protein comprising an encephalitogenic epitope selected from the group consisting of encephalitogenic epitopes of myelin basic protein, encephalitogenic epitopes of myelin-oligodendrocyte glycoprotein, and encephalitogenic epitopes of proteolipid protein.

40. The process of claim 15 wherein said DNA segment encodes an protein comprising an encephalitogenic epitope selected from the group consisting of encephalitogenic epitopes of myelin basic protein, encephalitogenic epitopes of myelin-oligodendrocyte glycoprotein, and encephalitogenic epitopes of proteolipid protein.

41. The process of claim 16 wherein said DNA segment encodes an protein comprising an encephalitogenic epitope selected from the group consisting of encephalitogenic epitopes of myelin basic protein, encephalitogenic epitopes of myelin-oligodendrocyte glycoprotein, and encephalitogenic epitopes of proteolipid protein.

42. The process of claim 17 wherein said DNA segment encodes a protein comprising an encephalitogenic epitope selected from the group consisting of encephalitogenic epitopes of myelin basic protein, encephalitogenic epitopes of myelin-oligodendrocyte glycoprotein, and encephalitogenic epitopes of proteolipid protein.

43. The process of claim 18 wherein said DNA segment encodes a protein comprising an encephalitogenic epitope selected from the group consisting of encephalitogenic epitopes of myelin basic protein, encephalitogenic epitopes of myelin-oligodendrocyte glycoprotein, and encephalitogenic epitopes of proteolipid protein.

44. The process of any one of claims 19-43 wherein said DNA segment additionally comprises a hydrophobic leader sequence, said hydrophobic leader sequence enabling the gene product to be synthesized in an endoplasmic reticulum for later constitutive secretion.

45. The process of any one of claims 19-43 wherein said DNA segment further comprises a Kozak box, said Kozak box permitting efficient translation of an mRNA transcribed from said DNA segment.

46. The process of any one of claims 19-43 wherein said DNA segment further comprises a codon corresponding to a charged amino acid at the 3' end to ensure that the protein is not retained in membrane.

47. The process of any one of claims 19-43 wherein said DNA segment further comprises one or more restriction sites to permit insertion of additional gene sequences.

48. The process of any one of claims 19-43 wherein said DNA sequence encodes amino acids 101-157 of proteolipid protein.

49. A method of treating a human patient for multiple sclerosis comprising:
introducing mammalian cells into said human patient, said mammalian cells having been treated in vitro to insert therein a DNA segment encoding one or more encephalitogenic epitope derived from nervous system protein, said mammalian cells expressing in vivo in said human patient a therapeutically effective amount of said encephalitogenic epitope or epitopes to restore T-cell tolerance to said human patient.

50. The method of claim 49 wherein said mammalian cells are fibroblasts cells.

51. The method of claim 50 wherein said mammalian fibroblast cells are histocompatible.

52. A method of treating a human patient for multiple sclerosis comprising:
introducing histocompatible fibroblast cells into said human patient, said histocompatible fibroblast cells having been treated in vitro to insert therein a DNA segment encoding amino acids 101-157 of proteolipid protein, said DNA
segment introduced into said histocompatible fibroblasts cells in vitro by a recombinant retroviral vector, said DNA sequence comprising a hydrophobic leader sequence whereby said leader sequence enables said amino acids 101-157 of proteolipid protein to be synthesized in the endoplasmic reticulum of said histocompatible fibroblast cells for later constitutive secretion, said DNA
segment further comprising a Kozak box permitting efficient translation of mRNA transcribed from said DNA segment, said DNA segment further comprising a codon corresponding to a charged amino acid at the 3' end to ensure that the protein is not retained in membrane, said DNA segment further comprising one or more restriction sites to permit insertion of additional gene sequences, whereby the gene product or gene products of said DNA segment is expressed in said human in a therapeutically effective amount to restore T-cell tolerance to said human.

53. An engineered cell comprising a gene encoding one or more antigenic protein which can be expressed, wherein said gene has been introduced into the cell by means of a recombinant vector.

54. An engineered cell comprising a gene encoding one or more antigenic protein which can be secreted, wherein said gene has been introduced into the cell by means of a recombinant vector.

55. An engineered cell comprising a gene encoding one or more encephalitogenic epitope which can be expressed, wherein said gene has been introduced into the cell by means of a recombinant vector.

56. An engineered cell comprising a gene encoding one or more encephalitogenic epitope which can be secreted, wherein said gene has been introduced into the cell by means of a recombinant vector.

57. Any one of claims 53-56 wherein said recombinant vector is a retroviral vector.

58. The cell of claim 56 wherein said gene comprises the sequence encoding amino acids 101-157 of proteolipid protein.