CA2158932A1 - Methods of suppressing autoimmune response - Google Patents

Abstract

Tissue cells of an animal transformed with a recombinant vector construct that (a) expresses a protein or active portion thereof; (b) transcribes an antisense message; or (c) transcribes a ribozyme capable of inhibiting MHC antigen presentation for use in suppressing an autoimmune response are provided. In a related aspect, the cells are transformed with two or more proteins, antisense or ribozymes, or combinations thereof.

Description

W O 9~/06718 2 15 ~ 9 3 2 PCTrUS94/09860 Description METEIODS OF SUPPRESSING AUTOIMMUNE RESPONSE

Technical Field The present invention relates generally to the field of autoimmunity, and more specifically, to methods of supp. ~ss;l1g T-cell recognition of host tissues.

10 Back~round of the Invention Autoimmunity refers to the reaction of the immune system against the body's own tissues, and is characterized by either the production of antibodies or immune effector T-cells that react with host tissue. Several diseases attributed to autoimmunity include multiple sclerosis (MS), rheumatoid arthritis, diabetes and uveitis.
MS is a neurological disorder characterized by recurrent incidence of axon demyelination in the optic nerve, brain, and spinal cord. Although it affects appro~h.lately one million people worldwide, the incidence is higher in populations living above the 37th parallel. This progressive disease may be linked to a defect in the immune system which may cause the self-destruction of myelin sheaths in both the20 central and peripheral nervous systems. MS patients are genetically susceptible to the onset of this disease following infection by a virus co~ inin~e amino acid sequences similar to normal myelin. There is a strong association between MS and the humanleukocyte antigen (HLA) HLA-DR2, providing support for this genetic predisposition.
These patients have Iymphocytes that mistakenly identify host myelin, resulting in an 25 autoimmune response.
MS effects persons under 55 years of age primarily of Western European lineage. Common symptoms include partial loss of vision and problems with speech, balance, and general motor coordination. Symptoms may cease after a few days or weeks but can reoccur after months or years. Eventually relapses lead to increasing 30 disability and weakness. In some instances the symptoms are steadily progressive from their onset and disability develops at a relatively early stage.
Although no known treatment exists to prevent progression of the disorder, corticosteroids, such as prednisone, can hasten recovery from relapse.However, the resulting damage remains unchanged. Intensive immunosuppres~h~e 35 therapy with cyclophosphoamide or azathioprine may aid in arresting the course of chronic progressive active MS, but the possibility of opportunistic infection increases.

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One of the symptoms, spasticity, is treated with the drug Danthrolene, which weakens muscle contraction by interfering with the role of calcium. Although this leads to a decrease in the spasm intensity, tre~tment cannot be a~mini~t~red to patients with poor, espil alOl ~ function or myocardial disease. Another drug, Baclofen, has been 5 the most effective in treating spasticity of spinal origin. Unfortunately, associated side effects include gastrointestin~l disturbances, l~c.cit~lde, fatigue, sedation, uncte~int sc, confusion and h~ cin~tions. While still another drug, Di~7ep~m, modifies spasticity, effective dosages often cause intolerable drowsiness. Alternatively, intrathecal injection of phenol or absolute alcohol has been used to reduce spasticity selectively in one or 10 more ;",po,La~l muscles.
Rheumatoid arthritis is an autoimmune disease characterized by chronic systemic inflammation specifically affecting synovial membranes of the joints. Viral infection in a genetically susceptible individual leads to either a cross reaction between antiviral antibodies and joint tissue antigen, or deviation of Iymphocyte function by 15 incorporation of viral DNA into the cell genome. The pathology of the disease includes chronic synovitis and pannus formation. Synovitis refers to the excessive infl~mm~tion of the synovial membranes surrounding the joints, thereby causing severe pain, while pannus eventually results in fibroid alkalosis by eroding cartilage, bone, lig~ment~, and tendons. The prevalence of rhellm~toid arthritis in the general population is 1%-2%, 20 with female patients outnumbering males 3:1. In general, the age at onset ranges from 20-40 years, although this disease may begin at any age.
The primary objectives for treating rheumatoid arthritis are reduction of infl~mm~tion and pain, preservation of function, and prevention of deformity. Several tre~tments have been used to accomplish these goals including non-steroidal anti-25 inflammatory drugs, anti-malarial drugs, gold salts, corticosteroids, methotrexate, a7athioprine and penicill~mine. Nonsteroidal anti-infl~mm~tory drugs include aspirin, ibuprofen, fenoprofen, naproxen, tolmetin, flurbiprofen, s ~lind~c, meclofen~m~te sodium, piroxicam, diclofenac, and ketoprofen. However, these drugs induce gastric ulceration, perforation, or aggravating infl~mm~tory bowel disease, and in some cases 30 result in renal toxicity.
The anti-malaria drug, Plaquenil (hydroxychloroquine sulfate), is used in patients with mild cases of rheumatoid arthritis because of its low toxicity as compared to nonsteroidal anti-inflammatory drugs. However, only 25% of those treated respond to this drug and in some cases only after 3-6 months of therapy. Side effects include 35 pign,~ retinitis, neuropathologies, and myopathologies of both skeletal and cardiac muscle.

wo 95/06718 2 1 5 ~ 9 3 ~ PCT/US~ D(I

In some patients, gold salts have been shown to retard bone erosions ~csoci~ted with rhe..m~toid arthritis. Thirty-two percent of these patients experience toxic side effects similar to heavy metal poisoning. The toxicity "lanil~ls itself as dermatitis, sto.llaL;lis, neutropenia, nephritis and nitritiod reactions.
Corticosteroids are generally used for their imme~i~te anti-infl~mm~tory effect without altering the natural progression of rhe.lm~toid arthritis. Unfortunately, they mask the underlying disease, and therefo. e increase the tçn-lency of the patient and physician to neglect general supportive l,e~ e~-~, physical therapy and orthopedic measures.
Methotrexate is given to patients with severe rhel-m~toid arthritis who fail to respond to nonsteroidal anti-infl~rnm~tory drugs and gold salts. This drug produces beneficial effects within t~,vo to four weeks as conlpared to gold salts which take two to six months. However, acsociated disadvantages include gastric irritation, stomatitis, pneumonitis, fibrosis, and cirrhosis. Therefore, liver biopsies are performed periodically to monitor treated individuals.
Azathioprine, like methotrexate, is effective for the treatment of severe rheum~toid arthritis. However, its use is restricted because of the potential for severe toxicity, including leukopenia, thrombocytopenia, and immunosuppression complicated by opportunistic im^ection.
Penicill~mine is also effective for treating severe rhe~ toid arthritis.
However, up to one-half of the patients experience some side effects, such as oral ulcers, loss of taste, fever, rash, thrombocytopenia, leukopenia and aplastic anemia. Other immune complex di.ce~ces appear to be in-iuced by the drug including myasthenia gravis, systemic lupus erythematosus, polyrnyositis, and Goodpasture's syndrome.
Diabetes mellitus is a disease characterized by an absence of circul~ting insulin, elevated plasma glucagon, and destruction of pancreatic B cells resulting in a disordered metabolism and hyperglycemia. The disease is most often treated by insulin injection, which prevents ketosis, reduces hyperglucagonemia, and decreases the elevated blood glucose level. Two major classifications of this disease are type I or insulin-dependent diabetes mellitus (IDDM) and type II or non-insulin-dependent diabetes mellitus (NIDDM). IDDM, occurs most commonly in children. Twenty percent of all diabetics in Scandinavia suffer from IDDM decreasing to 13% in southern Europe, 8% in the United States, and less than 1% in Japan and China.
Certain HLA have been associated with IDDM. HLA-DR3 and ~A-DR4 are present in 95% of patients as compared to 45%-50% in controls. In addition, antigen HLA-DQw3.2 is present in DR4 patients with IDDM, while the protective gene WO 9S/06718 PCT/US94/(19860 2158~a~ 4 HLA-DQ3. 1 is found predo~,una~Lly in DR4 controls. Anti-insulin and circul~ting islet cell antibodies have been detected in 85% of pqtientc in the first few weeks following the onset of diabetes. Considering these immllne characteristics, IDDM is believed to result from an infectious or toxic environment~l insult to pancledlic B cells of individuals 5 whose immune system is genetic~lly predisposed to develop an autoimmune response against .-tered pancreatic B cell antigens. Factors that effect B cell function include damage caused by viruses su~,h as mumps or coxsackie B4 virus, toxic chemical agents or cytotoxins, and antibodies released from sen.citi7ed immllnocytes. Sust~ined hyperglycemia causes osmotic diuresis, res..ltin~ in increased urination. This lowered 10 plasma volume produces fi;,,.;ness and weakness that is further exacerbated by potassium loss and catabolism of muscle protein. Acute stages of ketoacidosis exacerbates the dehydration and hyperosmolarity by producing anorexia, nausea and vomiting. This condition interferes with oral fluid replacement and as serum osmolarity increases, the patient experiences impaired consciousness eventually progressing to 1 5 coma.
Type II diabetes occurs predominantly in adults. MDDM is not associated with HLA markers, is non-ketotic, lacks islet cell antibodies, and does not require insulin therapy to sustain life. Although the cause of this form of diabetes is unclear, an unknown primary genetic factor is implicated which is aggravated by 20 enh~ncPrs of insulin t ~ict~nce such as aging and abdominal visceral obesity. Genetic infl~ences have been ~urther supported by epidemiological data showing that when one monozygotic twin over forty years of age develops diabetes, the second will develop the disease within the same year.
In the United States over 90% of all diabetics are type II. These patients 25 are initially asymptomatic, although chronic skin infections are common. An e~ ted 7 million people in the United States suffer from diabetes of which 560,000 have type I
diabetes. Treatment generally requires controlling dietary caloric intake to m~int~in weight. Other tre~tmentc involve the use of hypoglycemic drugs such as sulfonylureas.
Due to the limited duration in the system, these drugs must be continuously ~rimini.stered 30 to insulinopenic diab~ics to improve insulin release.
Drugs from the biguanides class were in- iuced in the 1950's but were discontinued in the United States because of their lmplication in lactic acidosis.
Metformin has been used in France since 1957 and is currently awaiting FDA approval.
This drug is used in conjunction with sulfonylureas and has many side effects including 35 anorexia, nausea, vomiting, abdominal discomfort and diarrhea.

WO 9~/06718 2 1 S 8 9 3 PCT/US94/09860 s Insulin injection is used for type I and non-obese type II diabetes.
However, subcutaneous injections cannot reproduce physiological patterns of intraportal insulin secretions. The.~ore, tre~tmPnt requires con~a..l monitoring of blood glucose levels followed by insulin injections over the patient's lifetime.
Uveitis is an intraocular i~ .f~ tory disease present in the anterior or posterior segment of the eye or equally distributed between the two. It is categorized as acute or chronic and granulomatous or nongranulomatous.
Anterior uveitis is characterized by infl~mm~tory cells within the aqueous humor. In granulomatous anterior uveitis, large p.ecip;lates and iris nodules cause blurred vision and infl~mm~tion. Diseases producing granulomatous anterior uveitis include sarcoidosis, tuberculosis, syphilis, toxopl~mocis, Vogt-Koyanagi-Harada syndrome and sympathetic ophth~tmi~, In non-granulomatous anterior uveitis, the precipitates are smaller and lack iris nodules. This causes unilateral pain, redness, photophobia and loss of vision.
In severe non-granulomatous anterior uveitis, fibrin is present within the anterior chamber. Systemic disorders associated with acute non-granulomatous anterior uveitis are the HLA-B27-related conditions sacroilitis, ankyloing spondylitis, Reiter's syndrome, psoriasis, ulcerative colitis and Chron's disease. Other infections that may cause non-granulomatous anterior uvieitis are herpes simplex and herpes zoster.
In posterior uveitis, there are cells in the vitreous humor and infl~mm~tory lesions in the retina or choroid. Visual loss may be due to vitreous haze, opacities, inflammatory lesions involving the macula, macular edema, retinal vein occlusion or associated optic neuropathy. Autoimmune retinal vasculitis and parsplanitis are conditions that produce posterior uveitis.
There are limited treatments for uveitis. Anterior uveitis may be treated with topical corticosteroids, while posterior uveitis requires systemic corticosteroid therapy. Posterior uveitis may also be treated through systemic immunosuppression with azathioprine or cyclosporine. However, high dosage use can result in hepatotoxicity and nephrotoxicity. Other side effects include renal dysfunction, tremor, 30 hirsutism, hypertension and gum hyperplasia.
Consequently, there is a need in the art for improved methods of suppressing the autoimmune response without the side effects or disadvantages associated with previously described methods. The present invention fulfills these needs and further provides other related advantages.

WO 9~il06718 PCT/US94/09860 ~5~v~ 6 Summary of the Invention The present invention provides methods for inhibiting MHC antigen presenlation in order to suppress T-cell recognition of host tissues. Within one aspect of the present invention, methods are provided for supples~;ng the a~toimm~ne response 5 within an animal, comprising transforming tissue cells of an animal with a leco..-binalll vector construct that eApresses a protein or an active portion of a protein capable of inhibiting MHC antigen prese..l~t;on, such that an autoimml-ne response against the cells is suppressed. Within one embodiment of the present invention, the recombil1al1t vector construct directs the tA~"e~ion of a protein capable of binding ~2-microglobulin, such 10 as H301. In another embodiment, the leco--.binanL vector construct directs the e,~,res~ion of a protein capable of binding the MHC class I heavy chain moleculeintraGellul~rly, such as E3/19K.
Within another aspect of the invention, a method is provided for suppressil1g an autoimmune response within an animal, comprising transforming tissue 15 cells of an animal with a .eco..,bi"ant vector construct that transcribes an ~nti~n~e message capable of inhibiting MHC antigen presentation, such that an autoirnmuneresponse against the cells is suppressed. Within various embodiments of the present invention, the recGl,.binanl vector construct transcribes an ~nticence message which binds a conserved region of ~IC class I heavy chain llàns~,l;pLS, ~2-microglobulin 20 transcript, or the PSFl transporter protein transcript.
Within still another aspect of the invention, a method is provided for supple~sin~ an autoimmune response within an animal, comprising transforming tissue cells of an animal with a recombinant vector construct that transcribes a ribozyme capable of inhibiting MHC antigen presentation, such that an autoimmune response25 against the cells is suppressed. Within various embo-liments of the present invention, the leco",binant vector construct transcribes a ribozyme that cleaves a conserved region of MHC class I heavy chain t~an~c~ 2-microglobulin l~ansc~ t~ or the PSF1 transporter protein transcript.
Within yet another aspect of the invention, a method is provided for 30 suppress;ng an autoimmune response within an animal, comprising transforming tissue cells of an animal with a multivai~nt recombinant vector construct that expresses a protein or active portion of a protein capable of inhibiting MHC antigen presentation, and an ~nti~ense message or ribozyme capable of inhibiting MHC antigen presentation, such that an autoimmune response against the cells is suppressed. Within a related 35 aspect of the invention, the multivalent recombinant vector construct directs the ,A~,.es~ion of an antisense message and a ribozyme capable of inhibiting MHC antigen wo 95/06718 21 ~ 8 9 3 2PCTluss4los86n pl~sP.~ ;on, such that an autoimmune response to the cells is suppressed. Withinanother related aspect of the present invention, the multivalent reconlbh~anl vector construct directs the c,~ ession of two or more proteins or active portions of proteins, two or more ~nticçnce messages, or two or more ribozymes capable of inhibiting MHC
5 antigen presentation, such that an autoimm~.ne response to the cells is suppressed.
Within various embodiments of the invention, the multivalent reuJ...binanl vector construct expresses or transcribes at least two of the following in any co..~binalion: a protein or active portion of the proteins E3/19K or H301, an ~nticçnse message that binds the transcript of a conserved region of MHC class I heavy 10 chains, ~2-microglobulin or PSFl transporter protein, or a ribozyme that cleaves the transcript of a conserved region of the MHC class I heavy ch`ains, ~2-microglobulin or PSFl transporter protein.
Within preferred embodiments, the recombinant vector construct is a recombinant viral vector construct. Within a particularly p.ere.led embodiment, the 15 recobinalll vector construct is a recobinaL retroviral vector construct. Within other embodiments, the recombinant vector construct is carried by a virus selected from the group consisting of togaviridae, picornaviridae, poxviridae, adenoviridae, parvoviridae, herpesviridae, paramyxoviridae and coronaviridae.
Within the methods briefly ~iccnssed above, suitable tissue cells of an animal include myelin nerve sheath cells, synovial membrane cells, pancreatic islet cells, hepatocytes and keratocytes. Within a preferred embodiment of the present invention~
the animal cells are transformed in vivo by direct injection of a recombinant vector construct.
These and other aspects of the present invention will become evident upon reference to the following detailed description.

Detailed Description of the Invention Prior to setting forth the invention, it may be helpful to an underst~nding thereof to set forth definitions of certain terms that will be used hereinafter."Autoimmune response" as used herein refers to a condition - characterized by a specific humoral or cell-mediated imm~lne response against con.ctituents of the body's own tissue. Within the context of the present invention, - "suppression" of the autoimmune response refers to interference with MHC antigen presentation, such that an immune response is diminiched or prevented.
"Transformin~" tissue cells refers to the transduction or transfection of tissue cells by any of a variety of means recognized by those skilled in the art, such that WO 9~i/06718 PCT/US94/09860 2~-58g~lz 8 the trar.~ro""cd tissue cell c ~I"esses additional polynucleotides as cor,.paled to a tissue cell pnor to the transforming event.
"Reco~"binanl vector construct" or "vector construct" refers to an assembly which is capable of c~yles~ g sequences or genes of interest. The vector S construct must include promoter elern~nts and may include a signal that directs polyadenylation. In addition, the vector construct preferably includçs a sequence which, when transcribed, is operably linked to the sequences or genes of interest and acts as a translation initiation sequence. Preferably, the vector construct include~ a selectable marker such as neomycin, thymidine kinase, hygromycin, phleomycin, histidinol, or 10 dihydrofolate reductase (DHFR), as well as one or more restriction sites and a translation terrnination sequence. In addition, if the vector construct is used to make a retroviral particle, the vector construct must include a retroviral Fac~gin~ signal and LTRs approp-iate to the retrovirus used, provided these are not already present. The vector construct can also be used in combination with other viral vectors or inserted 15 physically into cells or tissues as described below. As noted above, the vector construct incllldes a sequence that encodes a protein or active portion of the protein, ~ntiSence message or ribozyme. Such sequences are designed to inhibit MHC antigen presentation, in order to suppress an autoimmune response of class I restricted T-cells against transformed tissues cells.
In general, the recombinant vector constructs described herein are prepared by selecting a plasmid with a strong promoter, and approp~iate restriction sites for insertion of DNA sequences of interest downstream from the promoter. As noted above, the vector construct may have a gene encoding antibiotic resistance for selection as well as termination and polyadenylation signals. Additional elements may include 25 enh~ncçrs and introns with functional splice donor and acceptor sites.
The construction of multivalent recombinant vector constructs may require two promoters when two proteins are being cApressed, because one promoter may not ensure adequate levels of gene expression of the second gene. In particular, where the vector construct eA~,Ie~ses an anti.~ence message or ribozyme, a second 30 promoter may not be necçss~ry. Within certain embodimentc, an internal ribosome binding site (IRBS) or herpes simplex virus thymidine kinase (HSVTK) promoter isplaced in conjunction with the second gene of interest in order to boost the levels of gene eA~ression of the second gene. Briefly, with respect to IRBS, the upslle~alll untr~n~l~ted region of the immunoglobulin heavy chain binding protein has been shown 35 to support the intemal engagement of a bicistronic message ~Jacejak etal., Nature 353:90, 1991). This sequence is small, appro~ ately 300 base pairs, and may readily be 215~`~32:

incorporated into a vector in order to express multiple genes from a multi-cistronic message whose cistrons begin with this sequence.
Where the reco~ u~anl vector construct is carried by a virus, such constructs are prepared by inserting sequences of a virus cont~inin~ the promoter, 5 splicing, and polyadenylation signals into pl~cmids con~ -g the desired gene of interest using methods well known in the art. The ~co"~binan~ viral vector cor.~ .g the gene of interest can replicate to high copy number after tr~n~duction into the target tissue cells.
Subsequent to preparation ofthe ,eco.nbinan~ vector construct, it may be 10 prt;re,~ble to assess the ability of vector transformed cells to down regulate MHC
presentation. In general, such assessments may be performed by Western blot, FACS
analysis, or by other methods recognized by those skilled in the art.
Within pref~. . ed embodiments, the l eco-.-binant vector construct is carried by a retrovirus. Retroviruses are RNA viruses with a single positive strand 15 geno".c which in general, are nonlytic. Upon infection, the retrovirus reverse transcribes its RNA into DNA, forming a provirus which is inserted into the host cell genome.
P,q~a,a~ion of retroviral constructs for use in the present invention is described in greater detail in an application entitled "Recombinant Retroviruses" (U. S . S.N.
07/586,603, filed September 21, 1990) herein incorporated by reference. The retroviral 20 genome can be divided conceptually into two parts. The "trans-acting" portion consists of the region coding for viral structural proteins, including the group specific antigen (gag) gene for synthesis of the core coat proteins; the pol gene for the synthesis of the reverse transcriptase and integrase enzymes; and the envelope (env) gene for thesynthesis of envelope glycoproteins. The "cis-acting" portion consists of regions of the 25 genome that is finally packaged into the viral particle. These regions include the p~cL~gin~ signal, long terminal repeats (LTR) with promoters and polyadenylation sites, and two start sites for DNA replication. The internal or "trans-acting" part of the cloned provirus is replaced by the gene of interest to create a "vector construct". When the vector construct is placed into a cell where viral p~c~ging proteins are present (see 30 U.S.S.N. 07/800,921), the transcribed RNA will be packaged as a viral particle which, in turn, will bud off from the cell. These particles are used to transduce tissue cells, allowing the vector construct to integrate into the cell genome. Although the vector construct express its gene product, the virus carrying it is replication defective because the trans-acting portion of the viral genome is absent. Various assays may be utilized in 35 order to detect the presence of any replication competent infectious retrovirus. One plefe"ed assay is the extended S+L- assay described in Example 9. Preferred retroviral z~58~ ~o vectors are murine leukemia amphotropic or xenotropic or VsVg pseudotype vectors(see WO 92/14829; incol~o~ted herein bv reference).
Recombinant vector constructs may also be developed and utilized with a variety of viral carriers incl.ldin~, for ~Y~mple, poliovirus (Evans et al., Nature 339:385, 5 1989, and Sabin etal., J. of Biol. Standardization 1:115, 1973) (ATCC VR-58);
rhinovirus (Arnold et al., J. Cell. Biochem. L401, 1990) (ATCC VR-1110); pox viruses, such as canary pox virus or vaccinia virus (Fisher-Hoch et al., PNAS 86:317, 1989;
Flexner et al., Ann. N.Y. Acad. Sci. 569:86, 1989; Flexner et al., Vaccine 8:17, 1990;
U.S. 4,603,112 and U.S. 4,769,330; WO 89/01973) (ATCC VR-111; ATCC VR-2010);
10 SV40 (Mulligan et al., Nature 277:108, 1979) (ATCC VR-305), (Madzak et al., J. Gen.
Vir. 73:1533, 1992); influçn7~ virus (Luytjes etal., Cell 59:1107, 1989; McMicheal et al., The New En~land Journal of Medicine 309:13, 1983; and Yap et al., Nature273:238, 1978) (ATCC VR-797); adenovirus (Berkner, et, al., Biotechniques 6:616,1988, and Rosenfeld et al., Science 252:431, 1991) (ATCC VR-1); parvovirus such as 15 adeno-associated virus (Sam~.lc~i et al., J. Vir. 63:3822, 1989, and Mendelson et al., Virolo~y 166:154, 1988) (ATCC VR-645); herpes simplex virus (Kit et al., Adv. Exp.
Med. Biol. 215:219, 1989) (ATCC VR-977; ATCC VR-260); HIV (EPO 386,882, B~lc~ cllçr et al., J. Vir. 66:2731, 1992); measles virus (EPO 440,219) (ATCC VR-24); Sindbis virus (Xiong et al., Science 234: 1188, 1989) (ATCC VR-68); and 20 coronavirus (Hamre et al., Proc. Soc. Exp Biol. Med. 121:190, 1966) (ATCC VR-740).
It will be evident to those in the art that the viral carriers noted above may need to be modified to express proteins, antisen~e messages or ribozymes capable of inhibiting MHC antigen presenla~ion.
Once a vector construct has been prepared, it may be atiminictered to a 25 warm-blooded animal in order to transform tissue cells through a variety of routes, inc~ ing in vivo by direct injection. More specifically, naked DNA or a recombinant vector construct co.~ g a sequence that codes for a protein or active portion of a protein, an ~nticence message or ribozyme sequence capable of inhibiting MHC antigen presentation, can be directly injected into the interstitial space of tissues including 30 muscle, brain, liver, skin, synovial membrane cells, pancreatic islet cells, and keratocytes (see WO 90/11092). Other representative examples of in vivo ~minictration of vector constructs include transfection by various physical methods, such as lipofection (Felgner etal., PNAS 84:7413, 1989); microprojectile bombardment (Williams etal., PNAS
88:2726, 1991); liposomes (Wang etal., PNAS 84:7851, 1987); calcium phosphate 35 (Dubensky et al., PNAS 81:7529, 1984); DNA ligand complexes (Wu et al., J. of Biol.
Chem. 264:1698S, 1989; Cotten etal., PNAS 89:6094, 1992). As noted above, the Il 215~32 vector construct may be carried by a virus such as vaccinia, Sindbis, or corona. Further, methods for a.lminictering a vector construct via a retroviral vector by direct injection are described in greater detail in an application entitled "Recombinant Retroviruses"
(U.S.S.N. 07/586,603) herein incorporated by reference.
S As ~liccussed above, the present invention provides methods and compositions suitable for inhibiting MHC antigen prestlllaLion in order to suppress the autoimmune response of the host. Briefly, CTL are specific~11y activated by the display of peptides in the context of self MHC molecules along with accessory molecules such as CD8, inexcellular adhesion molecule -1 (ICAM-l), ICAM-2, (Singer, Science 255:
1671, 1992; RAO, Crit.Rev.Immunol. 10: 495, 1991 leukocyte functional antigen-l (LFA-1) (Altmann et al., Nature 338:521, 1989), the B7/BBl molecule (Freeman et al., J. Immunol. 143: 2714. 1989), LFA-3, or other cell adhesion molecules. Antigenicpeptide presentation in association with MHC class I molecules leads to CTL activation.
Transfer and stable integration of specific sequences capable of eA~,ressing products expected to inhibit MHC antigen presen~alion block activation of T-cells, such as CD8+
CTL, and therefore suppress the autoimmune response. A standard CTL assay is used to detect this response, as described in detail in Example 13. Components of the antigen presentation pathway include the 45Kd MHC class I heavy chain, ~2-microglobulin,processin~ enzymes such as proteases, accessory molecules, chaparones and transporter proteins such as PSF1.
Within one aspect of the present invention, vector constructs are provided which direct the expression of a protein or active portion of a protein capable of inhibiting ~IC antigen presentation. Within the present invention, an "activeportion" of a protein is that fragment of the protein which must be retained for biological activity. Such fragments or active domains can be readily identified by systematically removing nucleotide sequences from the protein sequence, transforming target cells with the resultin~ recombinant vector construct, and detel.,fining MHC class I presentation on the surface of cells using FACS analysis or other immunological assays, such as a CTL assay. These fragments are particularly useful when the size of the sequenceencoding the entire protein exceeds the capacity of the viral carrier. Alternatively, the active domain of the ~vIHC antigen p~esentaLion inhibitor protein can be enzymatically digested and the active portion purified by biochemical methods. For example, a monoclonal antibody that blocks the active portion of the protein can be used to isolate and purify the active portion of the cleaved protein. (Harlow et al., Antibodies: A
Laboratory Manual, Cold Springs Harbor, 1988).

2~93~ 12 Within one embodiment, the recombinant vector construct directs the eAp-~ssion of a protein or active portion of a protein that binds to newly syntheci~Pd MHC class I molecules intracellul~rly. This binding prevents migration of the MHC
class I molecule from the endoplasmic reticulum, reslllting in the inhibition of terminal S glycosylation. This blocks transport of these molecules to the cell surface and prevents cell recognition and Iysis by CTL. For inct~nc~, one of the products of the E3 gene may be used to inhibit transport of MHC class I molecules to the surface of the ll~n~ro~ ed cell. More specifically, E3 encodes a l9kD l,~s--.~...b-~ne glycoprotein, E3tl9K, transcribed from the E3 region of the adenovirus 2 genome. Within the context of the 10 present invention, an animal is injected directly with a reco...bh~al-~ vector construct co~ ining the E3/19K sequence, which upon expression produces the E3/19K protein.
The E3/19K protein inhibits the surface eApres~ion of an MHC class I surface molecules, and the cells transformed by the vector construct evade an immune response. The construction of a le~resentali~e recombinant vector construct in this regard is presented 15 in Example 7.
Within another embodiment of the present invention, the recombinant vector construct directs the ,~p.~ssion of a protein or an active portion of a protein capable of binding ,~2-microglobulin. Transport of MHC class I molecules to the cell surface for antigen pres~..lalion requires association with ~2-microglobulin. Thus, 20 proteins that bind ~2-microglobulin and inhibit its association with MHC class I
indirectly inhibit MHC class I antigen presentation. Suitable proteins include the H301 gene product. Briefly, the H301 gene, obtained from the human cytomegalovirus (CMV) encodes a glycoprotein with sequence homology to the ~2-microglobulin binding site on the heavy chain of the MHC class I molecule (Browne et al., Nature 347:770, 25 1990). H301 binds to ~2-microglobulin, preventing the maturation of MHC class I
molec~lle~, and renders transformed cells unrecognizable by cytotoxic T-cells, thus evading MHC class I restricted immune surveillance.
Other proteins, not fliscl~ssed above, that function to inhibit or down-regulate MHC class I antigen presentation may also be identified and utilized within the 30 context of the present invention. In order to identify such proteins, in particular those derived from m~rnm~ pathogens (and, in turn, active portions thereof), a recombinant vector construct that expresses a protein or an active portion thereof suspected of being capable of inhibiting MHC class I antigen presentation is transformed into a tester cell line, such as BC. The tester cell lines with and without the sequence encoding the 35 c~n-lid~te protein are compared to stimul~tors and/or targets in the CTL assay. A

WO 95/06718 2 ~ ~ S g ~ 2 PCT/US94tO9860 decrease in cell Iysis corresponding to the transformed tester cell indicates that the c2n-1id~te protein is capable of inhibiting ~IC presentation.
An alternative method to determine down-regulation of MHC class I
surface eA~,ession is by FACS analysis. More speçific~lly, cell lines are transformed 5 with a recombinant vector construct encoding the candidate protein. After drugselection and expansion, the cells are analyzed by FACS for ~ffIC class I e,~i)ression and cG",paled to that of non-transformed cells. A decrease in cell surface cAI,ression of MHC class I indicates that the c~n~lid~te protein is capable of inhibiting MHC
pre~se~ t;on (see, for inct~ncP, Example 12).
Within another aspect of the present invention, methods are provided for su,~)pressing an autoimmune response within an animal by transforming tissue cells of an animal with a recombinant vector construct which transcribes an antisense message capable of inhibiting MHC class I antigen presentation. Briefly, oligonucleotides with nucleotide sequences complementary to the protein coding or "sense" sequence are15 termed "antisense". ~nti~ence RNA sequences function as regulators of gene expression by hybridizing to comple",e"~a~ mRNA sequences and a,le~Ling translation (Mizunoet al., PNAS 81:1966, 1984; Heywood et al., Nucleic Acids Res. 14:6771, 1986).
~nticen.ce molecules comprising the entire sequence of the target transcript or any part thereof can be synthesized (Ferretti et al., PNAS 83:599, 1986), placed into vector 20 constructs, and effectively introduced into cells to inhibit gene tAI~ression ( Izant et al., Cell 36:1007, 1984). In addition, the synthesis of antisence RNA (asRNA) from DNA
cloned in inverted orientation offers stability over time while constitutive asRNA
CAIJI ession does not interfere with normal cell function.
Within one embodiment of the present invention, the recombinant vector 25 construct transcribes an antisense message capable of binding a conserved region of the MHC class I transcript, thereby inhibiting cell surface expression and MHC class I
antigen presentation. One may identify such conserved regions through computer-~c.cisted comparison of sequences represçntin~ di~Tele"t classes of MHC genes (for example, HLA A, B and C), available within DNA sequence d~t~h~nkc (e.g., Genbank).
30 Conserved sequences are identified through computer-ac.cicted alignment for homology - of the nucleotide sequences. The conser~ed region is a sequence having less than 50%
mismatch, preferably less than 20% mismatch, per 100 base pairs between ~IC class I
genotypes.
Within another embodiment of the present invention, the recombinant J5 vector construct transcribes an ~ntisence message responsible for binding to ,~2-microglobulin transcript. This binding prevents translation of the ~2-microglobulin protein and thereby inhibits proper assembly of the MHC class I molecule complexnecesc~.y for cell surface cA~les~;on. Within a prefc:.-ed embodiment, the nucleotide sequence for ~2-microglobulin is cloned into a vector construct in the reverse orientation. The proper ~nticçnce orientation may be determined by restriction enzyme 5 analysis.
Within still another embodiment, the recoll.binant vector construct ll~nsc.il~es an ~nticence m.ocs~ge responsible for binding PSF1 transcript, a peptide transporter protein. Since this protein is necçss~ry for the efficient assembly of MHC
class I molecules, an ~ntisence to PSF1 l.ansc.ipt blocks the transport of processed 10 antigenic peptide fragments to the endoplasmic reticulum (ER) prior to association with the ,~2-microglobulin and MHC class I molecular complex. Within a prefe~ I ed embodiment7 the nucleotide sequence for PSFl is prepared and inserted in reverseorientation into the vector construct and determined by restriction enzyme analysis.
As ~iica~ssed above, the sequences of other proteins involved in antigen 15 presentation may also be identified, and used to design a reco~..bh1a~ vector construct capable of transcribing an ~ntisence message that inhibits MHC antigen plesenLalion.
More specifically, the nucleotide sequence of the gene encoding the protein is e~mined, and the identified sequence is used to synthesi7e an appropriate anticence message. It is preferable to use a sequence complimentary to a portion upstream or close to the start 20 sequence of the target message. This allows the antisense sequence to bind to the mRNA preventing translation of a significant portion of the protein. Examples of such molecules are ICAM-l, ICAM-2, LFA-l, LFA-3, and B7/BBl. Down-regulation of MHC class I expression or antigen presentation may be assayed by FACS analysis or CTL assay, respectively, as described in Examples 13 and 15 or other means as 25 described above for proteins capable of inhibiting MHC class I presentation.
Within another aspect of the present invention, a method is provided for suppressing an autoimmune response within an animal by tr~nsducing selected cells of the animal with a recombinant vector construct which transcribes a ribozyme responsible for the enzymatic cleavage of a component involved in MHC antigen presenl~ion.
30 Briefly, ribozymes are RNA molecules with enzymatic activity used to digest other RNA
molecules. They consist of short RNA molecules pocsçcsing highly conserved sequence-specific cleavage domains flanked by regions which allow accurate positioning of the enzyrne relative to the potential cleavage site in the desired target molecule. They provide highly fleAible tools in inhibiting the eA~l~ssion and activation of specific genes 35 (Haseloffet al., Nature 334:585, 1988). Custom ribozymes can be designed, provided that the transcribed sequences of the gene are known. Specifically, a ribozyme may be W O 95/06718 PCT~US94/09860 2 1 ~ ~ 9 ? 2 designed by first choosing the particular target RNA sequence and ~tt~ching compliment~ry sequences to the beginning and end of the ribozyme coding sequence.
This ribozyme producing gene unit can then be inserted into a reco,nbinanl vector construct and used to transform tissue cells. Upon e,.l"ession, the target gene is 5 neutralized by complim~nt~ry binding and cleavage, guaranteeing pe"~anenl inactivation. In addition, because of their enzymatic activity, ribozymes are capable of destroying more than one target.
Within one embodiment, vector constructs conî~ 'g specific ribozymes are used to cleave the l,~nsc,iyt of a conserved region of the MHC class I heavy chain 10 molecule in order to inhibit antigen present~tion. Within another embodiment of the present invention, the recombinant vector const~uct transcribes a ribozyme responsible for the enzyrnatic cleavage of the ~2-microglobulin ll~ns~liyt. Specifically, a ribozyme with fl~nking regions complimentary to a sequence of the ~2-microglobulin message cleaves the transcript, thereby preventing protein translation and proper assembly of the 15 MHC class I molecule complex. This inhibits transport of the MHC class I complex to the cell surface, thereby preventing antigen presentation.
Within still another embodiment of the present invention, the recombinant vector construct transcribes a ribozyme responsible for the enzymatic cleavage of the PSFl transcript, thereby suppressing cell surface expression of MHC class I molecules 20 and preventing antigen presentation. More specifically, a ribozyme designed with flanking regions complimentary to a sequence of the PSF 1 message cleaves the transcript and inhibits transport of peptides to the ER, thereby preventing assembly of the MHC class I complex and antigen presentation.
As tiiccussed above, it will be evident to those skilled in the art that the 25 sequences of other proteins involved in the antigen presentation pathway may be identified and used to design a recombinant vector construct capable of transcribing a ribozyme that inhibits MHC antigen presentation. Down-regulation of ~C class I
e~ylt:ssion or antigen presentation may be assayed by FACS analysis or CTL assay as described in more detail in Examples 13 and 15 or other means as described above for 30 proteins and ~nti~ense messages capable of inhibiting ~vIHC class I presentation.
Within another aspect of the invention, multivalent recombinant vector constructs are provided. Briefly, the efficiency of suppressing an autoimmune response can be enhanced by transforming cells with a multivalent recombinant vector construct.
Upon eAyression~ the gene products increase the degree of interference with MHC
35 antigen presentation by attacking a single component via two different routes, or through two dirrele"~ components via the same or diIre~en~ route. The construction of ~158~ 16 multivalent recombinant vector constructs may require two promoters because one promoter may not ensure adequate levels of gene ~ ssion of the second gene. A~
noted above, a second promoter, such as an internal ribozyme binding site (IRBS)promoter, or herpes simplex virus thymidine kinase (HSVTK) promoter placed in conjunction with the second gene of interest boosts the levels of gene e~Jl es~,ion of the second gene.
Within pl e~. ed embodiments, the vector construct expresses or transcribes at least two of the following components in any combination: (a) a protein or active portion of the proteins E3/19K or H301; (b) an ~ntiCçnse message that binds the ll ans~-l ipt of a conserved region of the MHC class I heavy chain, .~2-microglobulin or PSFl l,~nspo-Ler protein; and (c) a ribozyme that cleaves the transcript of the proteins listed in (b) above. In addition, multivalent recolllbinant vector constructs are provided which express two proteins or active portions of proteins as described herein, two anticçnce messages, or two ribozymes.
Within related embodiments, a number of specific combinations may be utilized to form a multivalent recombinant vector construct. For example, a multivalent reco".binant vector construct may consist of a gene e~y~essing E3/19K or H301 incombination with the ~nticçnce message or ribozyme sequence for a conserved region of the MHC class I heavy chain, ~2-microglobulin, or PSFl transporter protein.
Within another aspect of the present invention, pharmaceutical compositions are provided comprising one of the above described recombinant vector constructs or a recombinant virus carrying the vector construct, such as a retrovirus, poliovirus, rhinovirus, vaccinia virus, influen7~ virus, adenovirus, adeno-associated virus, herpes simplex virus, measles virus, coronavirus or Sindbis virus, in combination with a pharm~ceutically acceptable carrier or diluent. The composition may be prepared either as a liquid solution, or as a solid form (e.g., Iyophilized) which is resuspended in a solution prior to ~lminictration. In addition, the composition may be prepared with suitable carriers or ~iluentc for either injection, oral, nasal or rectal administration or other means approp.iate to the carrier. Generally, the recombinant virus carrying the vector construct is purified to a concentration ranging from 0.25% to 25%, and preferably about 5% to 20% before forrnulation. Subsequently, after preparation of the composition, the reco-llbh1ant virus carrying the vector construct will constitute about 10 ng to 1 llg of material per dose, with about 10 times this amount of material present as copurified cont~min~nts. Preferably, the composition is prepared in 0.1-1.0 ml of aqueous solution formlll~ted as described below.

17 2I~ 2 Pharrn~ceutically acceptable carriers or diluents are those which are nontoxic to recipients at the dosages and concentrations employed. Repres~:,.lali~,re examples of carriers or ~ uçntc for injectable solutions include water, isotonic solutions which are preferably buffered at a physiological pH (such as phosphate-buffered saline 5 or Tris-buffered saline) and co..~ g one or more of m~nnitol, lactose, trehalose, dextrose, glycerol and ethanol, as well as polypeptides or proteins such as human serum albumin (HSA). One suitable composition comprises a reco,..b.nanl virus carrying a vector construct in 10 mg/ml mannitol, l mg/ml HSA, 20mM Tris pH=7.2 and 150mM
NaCI. In this case, since the recombinant virus carrying the vector construct represents 10 applo~,l.ately 10 ng to 1 ~g of material, it may be less than 1% of the total high molecular weight material, and less than 1/100,000 of the total material (inclu~in~
water). This composition is generally stable at -70C for at least six months. It will be evident that subst~nti~lly equivalent dosages of the recombinant vector construct may be prepared. In this regard, the vector construct will constitute 100ng to 100ug of15 material per dose, with about 10 times this amount of material present as copurified contaminants.
The composition may be a~minictered through a variety of routes (as diccllssed above), including intravenous (i.v.), subcutaneous (s.c.), or intramuscular (i.m.) injection. In this regard, it will be evident that the mode of adminictration will be 20 influenced by the specific therapeutic app!ication. For recombinant viruses carrying the vector construct, the individual doses norrnally used are 106 to 1010 c.f.u. (e.g., colony forming units of neomycin resistance titered on HT1080 cells). These compositions are administered at one- to four-week intervals for three or four doses (at least initially).
Subsequent booster shots may be given as one or two doses after 6-12 months, and25 thereafter annually.
The following examples are offered by way of illustration and not by way of limitation.

,S~32 18 EXAMPLES

Example I
PREPARATION OF MURINE RETROVIRAL PROVECTOR DNA
5 A. PREPARATION OF RETROVIRAL BACKBONE KT-3B

The Moloney murine le~lkPni~ virus (MoMLV) 5' long terminal repeat (LTR) EcoR I-EcoR I fragm~nt inel~ ing gag sequences, from N2 vector (Allllt;ll~al1o et al., J.
Vir. 61:1647, 1987, Eglitas et al., Science 230:1395, 1985) in pUC31 plasmid is ligated 10 into the plasmid SK+ (Stratagene, San Diego, CA). The resulting construct is called N2R5. The N2R5 construct is mllt7~ted by site-directed in vitro mutagenesis to change the ATG start codon to ATT preventing gag expression. This mutagenized fragment is 200 base pairs (bp) in length and flanked by Pst I restriction sites. The Pst I-Pst I
mllt~ted fragment is purified from the SK+ plasmid and inserted into the Pst I site of N2 15 MoMLV 5' LTR in plasmid pUC31 to replace the non-mllt~ted 200 bp fr~gment. The plasmid pUC31 is derived from pUC 19 (Stratagene, San Diego, CA) in which additional restriction sites Xho I, Bgl II, BssH II and Nco I are inserted between the EcoR I and Sac I sites of the polylinker. This construct is called pUC31/N2R5gM.
The 1.0 kilobase (Kb) MoMLV 3' LTR EcoR I-EcoR I fragment from N2 was 20 cloned into plasmid SK+ resulting in a construct called N2R3-. A 1.0 Kb Cla I-Hind III
fragment is purified from this construct.
The Cla I-Cla I dominant selectable marker gene fragment from pAFVXM
retroviral vector (Kriegler et al., Cell 38:483, 1984, St. .,ouis et al., PNAS 85:3150, 1988), comprising a SV40 early promoter driving exl)l es~ion of the neomycin 25 phosphotransferase gene, is cloned into plasmid SK+. A 1.3 Kb Cla I-BstB I gene fragment is purified from the SK+ plasmid.
An alternative selectable marker, phleomycin resistance (Mulsant et al., Som.
Cell and Mol. Gen. 14: 243, 1988, availab~e from Cayla, Cedex, FR) may be used to make the retroviral backbone KT-3C, for use in transforming genes to cells that are 30 already neomycin resistant. The plasmid pUT507 (Mulsant et al., Som.Cell and Mol Gen _4 243, 1988) is digested with Nde I and the ends blunted with Vlenow polymerase I.
~e sample is then further digested with Hpa I, Cla linhers ligated to the mix offragments and the sample further digested with Cla I. The excess Cla I linkers are removed by Cla I digestion and the 1.2 Kb Cla I fragment carrying the RSV LTR and 35 the phleomycin resistance gene isolated by agarose gel electrophoresis followed by WO 95tO6718 PCT/US94/09860 19 21S~9~'2 purification using Gene Clean (BiolOl, San Diego, CA). This fragment is used in place of the 1.3Kb Cla I - Bs+B I neomycin re~ict~nce fragrnent to give the backbone KT-3C.
A further modification ofthe select~ble marker c~sette is to simply use the ClaI-Cla I SV2 Neo fragment from PAFV~. ~nti~çnce or Ribozyme sequences can be 5 inserted into the HWC II S/TE in the 3-untr~ncl~ted region of the MEO gene. This vector is desi n~ted KT3D.
The eApress;on vector is constructed by a three part ligation in which the Xho I-Cla I fragment cont~ining the gene of interest and the 1.0 Kb MoMLV 3' LTR Cla I-Hind III fragment are inserted into the Xho I-Hind III site of pUC31/N2R5gM plasmid.
10 The 1.3 Kb Cla I-BstB I Neor gene, or 1.2 Kb ClaI PHLEOMYCIN, fragment is then inserted into the Cla I site of this plasmid in the sense orientation.

Example 2 15 A. CLONING OF E3/19K GENE INTO KT-3B

i. ISOLATION AND PURIFICATION OF ADENOVIRUS

The isolation and purification of adenovirus is described by Green et al., 20 Methods in Enzymolo~y 58: 425, 1979. Specifically, five liters of Hela cells (3-6 x 105 cells/ml) are infected with 100-500 plaque forming units (pfu) per ml of adenovirus type 2 (Ad2) virions (ATCC VR-846). After incubation at 37C for 30-40 hours, the cells are placed on ice, harvested by centrifugation at 230g for 20 minutes at 4C, and resuspended in Tris-HCI buffer (pH 8.1). The pellets are mechanically disrupted by 25 sonication and homogenized in trichlorotrifluoroethane prior to centrifugation at 1,000g for 10 rnin. The upper aqueous layer is removed and layered over 10 mls of CsCI (1.43 g/cm3 ) and centrifuged in a SW27 rotor for I hour at 20,000 rpm. The opalescent viral band is removed and adjusted to 1.34 g/cm3 with CsCI and further centrifuged in a Ti 50 rotor for 16-20 hours at 30,000 rpm. The visible viral band in the middle of the gradient 30 is removed and stored at 4C until purification of adenoviral DNA.

ii. ISOLATION AND PURIFICATION OF ADENOVIRUS DNA

The adenovirus band is incubated with protease for 1 hour at 37C to digest 35 proteins. Af'ter centrifugation at 7,800g for 10 minutes at 4C, the particles are solubilized in 5% sodium dodecyl sulfate (SDS) at room temperature for 30 minutes WO 9!i/06718 PCT/US94/09860 9~ 20 before being extracted with equal volume of phenol. The upper aqueous phase is removed"~-e,.l-~cted with phenol, extracted three times with ether, and dialyzed in Tris buffer for 24 hours. The viral Ad2 DNA is precipitated in ethanol, washed in ethanol, and resuspended in Tris-EDTA buffer, pH 8.1. Approximately 0.5 mg of viral Ad2 5 DNA is isolated from virus produced in 1.0 Iiter of cells.

iii. ISOLATION OF E3/19K GENE

The viral Ad2 DNA is digested with EcoR I (New F.llg]~nd Biolabs, Beverly, 10 MA) and separated by electrophoresis on a 1% agarose gel. The resulting 2.7 Kb Ad2 EcoR I D fra~rnents, located in the Ad2 coordinate region 75.9 to 83.4, CO,lt~ -g the E3/19K gene (Herisse et al., Nucleic Acids Research 8:2173, 1980, Cladaras et al., Virolog~ 140:28, 1985) are eluted by electrophoresis, phenol extracted, ethanol precipi~aled, and dissolved in Tris-EDTA (pH 8.1).
iv. CLONING OF E3/19K GENE INTO KT-3B

The E3/19K gene is cloned into the EcoR I site of PUC1813. PUC1813 is prepa,ed as essenti~lly described by Kay et al., Nucleic Acids Research 15:2778, 1987 20 and Gray et al., PNAS 80:5842, 1983). The E3119K is retrieved by EcoR I digestion and the isolated fragment is cloned into the EcoR I sit- ~f phosphatase-treated pSP73 plasmid, (Promega, Madison, WI). This construct is ~esi~:n~ted SP-E3/19K. The orientation of the SP-E3/19K cDNA is verified by using applop,iate restriction enzyme digestion and DNA sequencing. In the sense orientation, the 5' end of the cDNA is 25 adj~cent to the Xho I site of the pSP73 polvlinker and the 3' end ~djacent to the Cla I
site. The Xho I-Cla I fragment cort~ining -. E3/19K cDNA in either sense or antisense orientation is retrieved from the SP-E3/19K construct and cloned into the Xho I-Cla I
site of the KT-3B retroviral backbone. This construct is designated KT-3B/E3/19K.

30 B. CLONING OF PCR AMPLIFIED E3/19K GENE INTO KT-3B

i. PCR AMPLIFICATION OF E3/19K GENE

The Ad2 DNA E3/19K gene, including the amino terminal signal sequence, 35 followed by the intraluminal domain and carboxy terminal cytoplasmic tail which allow the E3/19K protein to embed itself in the endoplasmic reticulum (ER), is located 2i,rj8932 between viral nucleotides 28,812 and 29,288. Isolation of the Ad2 E3/19K gene from the viral genomic DNA is accomplished by PCR amplification, with the primer pairshown below:

S The forward primer cot~ ponds to the Ad2 nucleotide sequences 28,812 to 28,835.
(Sequence ID No.
5'-3': TATATCTCCAGATGAGGTACATGATTTTAGGCTTG

The reverse primer corresponds to the Ad2 nucleotide sequences 29,241 to 29,213.10 (Sequence ID No.
5'-3': TATATATCGATTCAAGGCATTTTCTTTTCATCAATAAAAC

In addition to the Ad2 complementary sequences, both primers contain a five nucleotide "buffer sequence" at their 5' ends for efficient enzyme digestion of the PCT
15 amplicon products. This sequence in the forward primer is followed by the Xho I
recognition site and by the Cla I recognition site in the reverse primer. Thus, in the 5' to 3' direction, the E3/19K gene is flanked by Xho I and Cla I recognition sites.
Amplification of the E3/19K gene from Ad2 DNA is accomplished with the following PCR cycle protocol:
Te."pe,~Lure C Time (min) No. Cycles 94 0.5 0.17 5 72 3.5 94 0.5 30 3.5 ii. LIGATION OF PCR A~IPLIFED E3/19K GENE INTO KT-3B

The E3/19K gene from the SK-E3/19K construct, approximately 780 bp in 25 length, is removed and isolated by 1% agarose/TBE gel electrophoresis as described in Example 2Bi. The Xho I-Cla I E3/19K fragment is then ligated into the KT-3B

?,~5~9~ 22 retroviral backbone. This construct is design~ted KT-3B/E3/19K . It is amplified by ro""ing DH5a bacterial strain with the KT-3B/E3/19K construct. Specifically, thebacteria is transformed with 1-1000 ng of ligation reaction mixture DNA. The îo"l-ed bacterial cells are plated on LB plates co.~ g ~rnpicillin The plates are 5 incub~ted overnight at 37C, bacterial colonies are selected and DNA is prepared from them. The DNA is digested with Xho I and Cla I. The expected endonllcle~ce restriction cleavage fragment sizes for plasmids co~ ning the E3/19K gene are 780 and 1300 bp.

10 C. CLONING OF SYNTHESI~ED E3/19K GENE INTO KT-3B

i. SYNlHESIS OF E3/19K GENE DNA

Chemical synthesis of synthetic DNA has been previously described (Caruthers et 15 al., Methods in Enzymolo~y 211:3, 1992). Sequences which encode the E3/19K gene are synthPsi7ed by the phosphotriester method on an Applied Biosystems Inc. DNA
synthP~i7er, model 392 (Foster City, CA) using the PCR primer as the 5' and 3' limits and Ic eping the same Xho I and Cla I linkers on the ends. Short oligonucleotides of approximately 14-40 nucleotides in length are purified by polyacrylamide gel 20 electrophoresis and ligated together to form the single-stranded DNA molecule (Ferretti et al., PNAS 83 :599, 1986) .

ii. SEQUENCING OF E3/19K GENE DNA

Fragments are cloned into the bacteriophage vectors M13mpl8, and M13mpl9 (GIBCO, Gaithersburg, MD), for amplification of the DNA. The nucleotide sequence of each fragment is determined by the dideoxy method using the single-stranded M13mpl8 and M13mpl9 recombinant phage r~ - ~ as templates and selected synthetic oligonucleotides as primers. This confirn.. .he identity and structural integrity of the 30 gene.

iii LIGATION OF E3/19K GENE INTO KT-3B

The E3/19K gene is ligated into the KT-3B or KT-3C vector as previously 35 described in Example 2Bii.

WO 95/06718 PCT/US91~ 5~
21~8~2 Example 3 CLONING OF AN ANTISENSE SEQUENCE OF A CONSERVED REGION OF

A. CONSTRUCTION OF KT-3CneoaMHC

The cDNA clone of the MHC class I allele CW3 (7Pmmour et al., Tissue Antigens 39:249, 1992) is used as a template in a PCR reaction for the amplification of 10 specific sequences to be inserted into the untran~l~ted region of the neomycin resistance gene of the KT-3C backbone vector, .
The MHC class I allele CWl cDNA is amplified between nucleotide sequence 147 to 1,075 using the following primer pairs:

15 The forward primer corresponds to MHC CW3 cDNA nucleotide sequence 147 to 166:
(Sequence ID No.
5'-3': TATATGTCGACGGGCTACGTGGACGACACGC

The reverse primer corresponds to MHC CW3 cDNA nucleotide sequence 1,075 to 20 1,056:
(Sequence ID No.
5'-3': TATATGTCGACCATCAGAGCCCTGGGCACTG

In addition to the MHC class I allele CW3 complementary sequences, both 25 primers contain a five nucleotide "buffer sequence" at their 5' ends for efficient enzyme digestion of the PCR amplicon products. The buffer sequence is followed by the Hinc II
recognition sequence in both primers. Generation of the ~IC amplicon with the primers shown above is accomplished using the PCR protocol described in section 2Bi..
This protocol is modified by using Vent polymerase (New F.ngl~ Biolabs, Beverly,30 MA) and further modified to include I minute extension times instead of 3.5 min~ltes.
-The Vent polyrnerase generates amplicons with blunt ends. Alternatively, the forward and reverse primers may contain only the MHC CW3 complementary sequences.
-The MHC CW3 cDNA 950 bp amplicon product is purified with Gene Clean (BiolOl, San Diego, CA) and digested with Hinc II. The digested fr~gment, 938 bp, is 35 isolated by 1% agarose/TBE gel electrophoresis and purified with Gene Clean.

2~ 932 24 The MHC CW3 cDNA 938 bp fragment is inserted in the 3' untrancl~ted region of the neomycin recict~nce gene in the ~nticçnce orientation. Sperific~lly, the Hinc II
recognition sequence at nucleotide sequence number 676 of the pBluescript II SK+(pSK+) (Stratagene, San Diego, CA) plasmid is removed by digestion with Hinc II and 5 Kpn I. The Kpn I 3' end is blunted with T4 DNA polymerase and the blunt ends are ligated. This plasmid is desi~n~ted as pSKdlHII. As described in Example lA, the 1.3 Kb Cla I- Cla I dominant selectable marker gene fragment from pAFVXM retroviral vector is cloned into the Cla I site of pSKdlHII. This plasmid is design~ted as pSKdlHII/SVneo. The MHC CW3 cDNA 938 bp fragment is inserted in an ~ntic~nce 10 orientation into the Hinc II site of pSKdlHII/SVneo, located in the 3' untr~nc~ted region of the neomycin resistance gene. Conr~ alion that the MHC CWl cDNA 938 bp fragment is present in the neomycin gene in an ~nSicen.ce orientation is determined by restriction endonuclease digestion and sequence analysis. This clone is design~ted as pSKdlHII/SVneo/aMHC .
Construction of KT-3D/SVneo/aMHC is accomplished by a three way ligation, in which the Cla I 2.2 Kb SVneoaMHC fragment, and the 1.0 Kb MoMLV 3' LTR Cla I-Hind III fragment from N2R3-, are inserted between the Cla I and Hind III sites of pUC3 1/N2RSgM pl2sr.lid as described in Example 1.

20 B. CONSTRUCTION OF KT-3C/S~ eo/VARNA/aMHC

High level MHC CW3 ~ntisence RNA eA~., ession is accomplished by insertion of this sequence downstream of the Ad2 VARNA1 promoter. The Ad2 VARNA
promoter-MHC ~ntisen.se cDNA is assembled as a RNA polymerase III (pol III) 25 e~pression ç~csette then inserted into the KT-3C backbone. In this pol III expression ç~csette, the Ad2 VARNAl promoter is followed by the antisense aMHC cDNA, which in turn is followed by the pol III concen.C~s te"".nalion signal.
The double stranded -30/+70 Ad2 VARNA1 promoter is chemically synthesized (Railey et al., Me Cell. Biol. 8:1147, 1988) and incl~ldes Xho I and Bgl II sites at the 5' 30 and 3', te~Je~ e.y.

The VARNAl promoter forward strand:
(Sequence ID No.

W O 9~/06718 2 1 S 8 9 3 2 PCT~US94/09860 5'-3': CGAGTCTAGACCGTGCAAAAG&AGAGCCTGTAAGCGGGCACTCTTCC
GTGGTCTGGTGGATAAATI'CGCAAGGGTATCATGGCGGACGACCGGGGl~
CGAACCCCGGA

The VARNAl promoter reverse strand:
(Sequence ID No. ~

5'-3': GATCTCCGGGGl-rCGAACCCCGGTCGTCCGCCATGATACCCl~GCGAA
l-rTATCCACCAGACCACG&AAGAGTGCCCGCTTACAGGCTCTCC m TGCA
CGGTCTAGAC

In order to form the double stranded VARNA1 promoter with Xho I and Bgl II
cohesive ends, equal amounts of the single strands are mixed together in 10 mM MgC12, heated at 95C for 5 min then cooled slowly to room temperature to allow the strands to anneal.
The MHC class I allele CW3 fragment, nucleotide sequence 653 to 854, from the plasmid pSKdlHII/SVneo/aMHC is amplified using the following primer pair:

The forward primer corresponds to nucleotide sequence 653 to 680:

5'-3': TATATCCTAGGTCTCTGACCATGAGGCCACCCTGAGGTG

The reverse primer corresponds to nucleotide sequence 854 to 827:

5'-3': TATATAGATCTACATGGCACGTGTATCTCTGCTCTTCTC
In addition to the MHC class I allele CW3 complementary sequences, both primers contain a five nucleotide "buffer sequence" at their 5' ends for efficient enzyme digestion of the PCR amplicon products. The buffer sequence is followed by the Avr II
recognition sequence in the forward primer and by the Bgl II recognition sequence in the 25 reverse primer, which allows insertion in an ~nticen.ce orientation, relative to the Ad2 VARNAl promoter in the pol III expression cassette. Generation of the MHC amplicon with the primers r~i.cc~lssed above is accomplished with the PCR protocol described in Example 2Bi modified to include 0.5 minute extension times instead of 3.5 minlltes~
The ~vIHC CW3 cDNA 223 bp amplicon product is purified with Gene Clean 30 (BiolOl, San Diego, CA), then digested with AvrII and BglII, and isolated by 2%

2~ 8932 26 NuSeive-1% agarose/TBE gel electrophoresis. The 211 bp band is then excised fromthe gel and the DNA purified with Gene Clean.
The double stranded pol III con.ce~ )s tel,l.inalion sequence is chemically synth~ci7ed (Geidnsch~l~ et al., Annu. Rev. Biochem. 57:873, 1988) and includes Avr II
5 and Cla I sites at the 5' and 3' ends, respectively.

The pol III tell, inalion sequence forward primer:
(Sequence ID No.
5'-3': CTAGGGCG~ l l l l l GCGCAT
The pol m telll~nalion sequence reverse primer:
(Sequence ID No.
5'-3': CGATGCGCAAAAAGCGCC

In order to forrn the double stranded pol nI llanscliplion termination sequence with Avr II and Cla I cohesive ends, equal amounts of the single strands are mixed together in 10 mM MgCI2, heated at 95C for 5 min then cooled slowly to room te~llpelalure to allow the strands to anneal.
The pol nI e"~lression c~csette for ~l~t;cellce aMHC class I allele CW3 is 20 assembled in a four way ligation in which the Xho I-Bgl II Ad2 VARNA1 promoter fr~nent, the Bg! II-Avr II aMHC CW3 fragment, and the Avr II-Cla I transcriptiontelll inalion fragment, are cloned into pSKII+ between the Xho I and Cla I sites. This constluct is design~ted pSK/VARNA/aMHC.
Construction of KT3B/SVneo/VARNA/aMHC is accomplished in a two step 25 ligation. The first step is a three way ligation in which the Xho I-Cla I VARNA/aMHC
fragrnent and the 1.0 Kb MoMLV 3' LTR Cla I-Hind III frag-rnent from N2R3-, are inserted between the Xho I and Hind III sites of pUC3 1/N2R5gM plasmid as described in Example 1. This construct is design~ted KT3B/VARNA/aMHC. In the second ligation step the 1.3 Kb Cla I-BstB I SVneo fragment into the Cla I site of 30 KT3B/VARNA/aMHC. This construct is designated KT3B/SVneo/VARNA/aMHC.

2~ 3 2 Example 4 , CLONING A RIBOZYME THAT WILL CLEAVE A CONSERVED REGION OF

s A. CONSTRUCTION OF pSK/VARNA/MHCHRBZ

In order to efficiently inhibit ~ ression of MHC class I in tr~n.c-luced cells, a hairpin ribozyme with target specificity for the MHC class I allele is inserted into the 10 KT3B/SVneo vector. The ribozyme is eAp.essed at high levels from the Ad2 VARNA1 promoter. The ~C hairpin ribozyme (HRBZ) is inserted into the pol III
pSK/VARNA/aMHC tAp~t:ssion c~ccette described in Example 3.
The HRBZ and the MHC class I allele CW3 have the homologous sequence shown below:
15 (Sequence ID No. ~
5'-3': GATGAGTCTCTCATCG

The HRBZ is designed to cleave after the A residue in the AGTC hairpin substrate motif contained in the target sequence. Following cleavage, the HRBZ is 20 recycled and able to hybridize to, and cleave, other ~IC class I RNA molecule.
Double stranded HRBZ as defined previously (Hampel et al., Nucleic Acids Research 18:299, 1990), cont~ining a four base "tetraloop" 3 and an extended helix 4, with specificity for the MHC class I homologous sequence shown above, is chemically synth~ci7ed and includes Bgl II and Avr II sites at the 5' and 3' ends, respectively.
The ~IC HRBZ sense strand:
(Sequence ID No.

5'-3': GATCTCGATGAGAAGAACATCACCAGAGAAACACACGGACTTCGGT
CCGTGGTATATTACCTGGTAC

30 The MHC HRBZ antisense strand:
(SequenceIDNo.

5'-3': CTAGGTACCAGGTAATATACCACGGACCGAAGTCCGTGTG l l-l CTCT
GGTGATGTTCTTCTCATCGA

~,t5~93~ 28 In order to form the double stranded MHC class I specific HRBZ with Bgl II and Avr II cohesive ends, equal amounts of the single strands are mixed together in 10 mM
MgCl2, heated at 95C for 5 rnin then cooled slowly to room temperature to allow the 5 strands to anneal.
The pol III eA~Jleasion c~sette for the MHC ~BZ is assembled by ligation of the chemically synthesized double stranded MHC class I specific HRBZ with Bgl II and Avr II cohesive ends into Bgl II and Avr II di~este~l and CIAP treated pSK/VARNA/c~IC, in which the aMHC sequence has been gel purified away from 10 the eAp-es~ion vector. This plasmid is de~i~n~ted pSK/VARNAlMHCHRBZ and cont~inc the Ad2 VARNAl promoter followed by the MHC HRBZ, which in turn is followed by the pol III con~en.cl1s termination sequence. The pol III eAp. cssion components is flanked by Xho I and Cla I recognition sites.

15 B. CONSTRUCTION OF KT3B/SVneo/VARNA/~ICHRBZ

Construction of KT3B/SVneo/VARNA/MHCHRBZ is accomplished in a two step ligation. The first step is a three way ligation in which the Xho I-Cla I VARNAIMHCHRBZ
fragment and the 1.0 Kb MoMLV 3' LTR Cla I-Hind III fragment from N2R3-, are inserted 20 between the Xho I and Hind III sites of pUC31/N2R5gM plasmid described in Example 1.
This construct is dçci~:n~ted KT3B/VARNA/MHC~BZ. In the second step, the 1.3 Kb Cla I-BstB I SVneo fragment is ligated into the Cla I site of KT3B/VARNA/MHCHRBZ. I his construct is designated KT3B/SVneo/VARNA/~ICHRBZ.

Example 5 CLONING OF PSFl ANTISENSE cDNA

30 A. CONSTRUCTION OF KT-3C/SVneo/aPSFl The cDNA clone of PSFl (Spies et al., Nature 351: 323, 1991; Spies et al., Nature 348: 744, 1990) is used as a template in a PCR reaction for the amplification of specific sequences to be inserted into the KT-3C backbone vector, into the untr~n~l~ted 35 region of the neomycin resistant gene. The PSFl cDNA is amplified between nucleotide sequence 91 to 1,124 using the following primer pairs:

WO 95/06718 21~ 8 ~ 3 ~ PCT/11S94/09860 The forward primer col I es~ onds to nucleotide sequence 91 to 111:
(Sequence ID No.
5'-3': TATATGTCGACGAGCCATGCGGCTCCCTGAC

5 The reverse primer corresponds to nucleotide sequence 1,124 to 1,105:
(Sequence ID No.
5'-3': TATATGTCGACCGAACGGTCTGCAGCCCTCC

In addition to the PSF1 complementary sequences, both primers contain a five 10 nucleotide "buffer sequence" at their 5' ends for efficient enzyme digestion of the PCR
amplicon products. The buffer sequence is followed by the Hinc II recognition sequence in both primers. Generation of the PSFl amplicon with the primers disc~lssed above is accomplished with the PCR protocol described in Example 2Bi. This protocol is modified by using Vent polymerase (New Fng]~nd Biolabs, Beverly, MA) and further15 modified to include 1 minute extension times instead of 3.5 min~ltes The Vent polyrnerase generates amplicons with blunt ends.

B. CONSTRUCTION OF KT3B/SVneo/VARNA/aPSFl High level PSFl antisense expression is accomplished by insertion of this sequence downstream of the Ad2 VARNAl promoter. The Ad2 VARNA promoter-PSFl anticeTlce cDNA is first assembled as a pol III expression c~csette then inserted into the KT-3B backbone. In this pol III expression c~sette, the Ad2 VARNAl promoter is followed by the ~nticence PSFI cDNA, which in turn is followed by the pol m concenc~ls te,l"inalion signal.
The nucleotide sequence 91 to 309 of the PSFl cDNA are amplified in a PCR
reaction using the following primer pair:

The forward primer corresponds to nucleotide sequence 91 to 1 1 1:
(Sequence ID No.
5'-3': TATATCCTAGGGAGCCATGCGGCTCCCTGAC

- The reverse primer corresponds to nucleotide sequence 309 to 288:
(Sequence ID No.
3 5 5'-3': TATATAGATCTCAGACAGAGCGGGAGCAGCAG

~,~5a93Z 30 In addition to the PSFl complementary sequences, both primers contain a five nucleotide "buffer seq~lencell at their 5' ends for efficient enzyme digestion of the PCR
amplicon products. The buffer sequence is followed by the Avr II recognition sequence in the f~.w~d primer and by the Bgl II recognition sequence in the reverse primer, 5 which allows insertion in an ~nticçnce orientation, relative to the Ad2 VARNAlpromoter in the RNA polymerase III eAp.es~ion c~csette. Generation of the PSFl amplicon with the primers described above is accomplished with the PCR protocol described in Example 2Bi modified to include 0.5 minutes extension times instead of 3.5 minlltes.
The MHC CW3 cDNA 240 bp amplicon product is purified with Gene Clean (BiolOl, San Diego, CA), then digested with Avr II and Bgl II, and isolated by 2%
NuSeive-1% agarose/TBE gel electrophoresis. The 211 bp band is then excised fromthe gel and purified with Gene Clean.
Construction of KT3B/SVneo/VARNA/aPSFl is accomplished in two step ligation. The first step is a three-way ligation in which the Xho I-Cla I VARNA/aPSFl fragment and the 1.0 Kb MoMLV 3' LTR Cla I-Hind III fragment from N2R3-, are inserted between the Xho I and Hind III sites of pUC3 1/N2R5gM plasmid as described in Example 1. This construct is designated as KT3B/VARNA/aPSFl. In the second ligation step, the 1.3 kb Cla I-BstB I SVneo fragment is ligated into the Cla I site of KT3B/VARNA/aPSFl. This construct is design~ted KT3B/SVneo/VARNA/aPSFl.

Example 6 CLONING A RIBOZYME THAT WILL CLEAVE A CONSERVED REGION OF
PSFl INTO KT-3B

A. CONSTRUCTION OF pSK/VARNA/PSFlHRBZ

In order to efficiently inhibit expression of PSFl in tr~n~duced cells, a hairpin ribozyme with target specificity for the PSFl RNA is inserted into the KT3B/SVneo vector. The riboz,vme is expressed at high levels from the Ad2 VARNAl promoter.
The PSFl hairpin ribozyme (HRBZ) is inserted into the pol III pSK/VARNA/aMHC
eApres~;on c~csette described in Example 3 . The PSF I HRBZ-pol III eAI~I essionr~ette is then inserted into the KT3B/SVneo backbone vector.
The HRBZ and the PSFl RNA have the homologous sequence shown below:
(Sequence ID No. ~

W O 95/06718 PCTrUS94/09860 3j~1S89~2 5'-3': GCTCTGTCTGGCCAC

The HRBZ is designed to cleave after the T residue in the TGTC hairpin substrate motif con~ained in the target sequence. Following c1eavage, the HRBZ is 5 recycled and able to hybridize to, and cleave, other PSFl RNA molecule.
Double stranded HRBZ as defined previously (Hampel et al., Nucleic Acids Research 18:299, 1990), co..lA;,.;..g a four base "tetraloop" 3 and an extended helix 4, with specificity for the PSFl homologous sequence shown above, is chemically synth.oci~ed and includes Bgl II and Avr II sites at the 5' and 3' ends, respectively.
The PSFl HRBZ, sense strand:
(Sequence ID No.
5'-3':
GATCTGTGGCCAGACAGAGCACCAGAGAAACACACGGACTTCGGTCC

The PSFl HRBZ, antisense strand:
(Sequence ID No.
5'-3':
CTAGGTACCAGGTAATATACCACGGACCGAAGTCCGTGTGTTTCTCT
GGTGCTCTGTCTGGCCACA

In order to form the double stranded PSFl specific HRBZ with Bgl II and Avr n cohesive ends, equal amounts of the single strands are mixed together in 10 mM MgCl2 heated at 95C for 5 min then cooled slowly to room temperature to allow the strands to anneal.
The pol m expression c~csette for the PSFl HRBZ is assembled by ligation of the chemically synth~si7ed double stranded PSFl specific HRBZ with Bgl II and Avr II
cohesive ends into Bgl II and Avr II digested and CIAP treated pSK/VARNA/a~IHC, in which the ctMHC sequence has been gel purified away from the pol III eApression vector. This plasmid is designated pSK/VARNA/PSFl~BZ and contains the Ad2 VARNAI promoter followed by the PSFI HRBZ, which in turn is followed by the pol - III consensus termination sequence. The pol III expression component is flanked by Xho I and Cla I recognition sites.
B. CONSTRUCTION OF KT3B/SVneo/VARNA/PSFlHRBZ

WO 95/06718 PCT/US9 11~5~5~

2~58932 Construction of KT3B/SVneo/VARNA/MHCHRBZ is accomplished in a two step ligation. The first step is a three way ligation in which the Xho I-Cla I VARNA/PSFlHRBZ
fragment and the 1.0 Kb MoMLV 3' LTR Cla I-Hind III fragrnent from N2R3-, are inserted S between the Xho I and Hind m sites of pUC31/N2R5gM plasmid as described in Exarnple 1.
This construct is de~i~n~ted KT3B/VARNA/PSFlHRBZ. In the second ligation step, the 1.3 Kb Cla I-BstB I SVneo fragment is ligated into the Cla I site of KT3B/VARNA/PSFlHRBZ. This construct is desi~n~ted KT3B/SVneo/VARNA/PSFlHRBZ.

Example 7 CONSTRUCTION OF THE MULTIVALENT RECOMBINANT RETROVIRAL
VECTOR KT3B-E3/19K/aMHC

A variation of the retroviral vector KT3B-E3/19K can also be constructed cont~ining both the E3/19K sequences and anti-sense sequences specific for a conserved region between the three class I MHC alleles A2, CW3 and B27, Example 2 and 3. This vector, known as KT3B-E3/19K/aMHC, is designed to incorporate the MHC class I
anti-sense sequences at the 3' end of the E3/19K sequence which would be expressed as 20 a chimeric molecule. The retroviral vector, KT3B-E3/19K/ccMHC, can be constructed by ligating a Cla I di~çsted PCR amplified product cons~ining the MHC anti-sensesequences into the Cla I site of the KT3B-E3/19K vector. More specifically, the cDNA
clone of the MHC class I allele CW3 (Zemmour et al., Tissue Anti~ens 39:249, 1992) is amplified by PCR between nucleotides 653 and 854 using the following primer pair:
The forward primer of aMHC is:
(Sequence ID No.
S'-3': ATTATCGATTCTCTGACCATGAGGCCACCCTGAGGTG

30 The reverse primer of aMHC is:
(Sequence ID No.
5'-3': ATTAATCGATACATGGCACGTGTATCTCTGCTCTTCTC

The primer pairs are flanked by Cla I restriction enzyme sites in order to insert an 35 amplified Cla I digested product into the partially pre-digested KT-3B-E3/19K vector in 33 ~IS~g~

the anti-sense orientation. By placing the Cla I fragment in reverse G,ienLa~ion the vector will express the negative anti-sense strand upon transcription.

Example 8 TRANSDUCTION OF PACKAGING CELL LIMES DA WITH THE

A. PLASMID DNA TRANSFECTION
293 2-3 cells (a cell line derived from 293 cells ATCC No. CRL 1573, WO
92/05266) 5 x 105 cells are seeded at approximately 50% confl~lence on a 6 cm tissue culture dish. The following day, the media is replaced with 4 ml fresh media 4 hours prior to transfection. A standard calcium phosphate-DNA coprecipitation is performed 15 by mixing 10.0 '~Ig of KT-3B-E3/19K plasmid and 10 0 llg MLP G plasmid with a 2M
CaC12 solution, adding a lx Hepes buffered saline solution, pH 6.9, and incubating for 15 min~ltes at room temperature. The calcium phosphate-DNA copreeipitale is transferred to the 293 2-3 cells, which are then incubated overnight at 37C, 5% CO2.
The following morning, the cells are rinsed three times in lx PBS, pH 7Ø Fresh media 20 is added to the cells, followed by overnight incubation at 37C, 10% CO2 The following day, the media is collected offthe cells and passed through a 0.45 ~ filter. This supe--.dLa--l is used to transduce pack~ging and tumor cell lines. Transient vector supernatant for other vectors are generated in a similar fashion.

25 B. PACKAGING CELL LlNE TRANSDUCTION

DA cells (an amphotropic cell line derived from D-17 cells ATCC No. 183, WO
92/05266) are seeded at 5 x 10~ cells/10 cm dish. Approximately 0.5 ml of the freshly collected 293 2-3 supernatant (or supernatant that has been stored at -70 C) is added to 30 the DA cells. The following day, phleomycin is added to these cells and a drug resistant pool is generated over a period of a week. This pool of cells is dilution cloned to yield a single cell per well of 96 well plates. Twenty-four clones are expanded to 24 well plates, then to 6 well plates, at which time cell supernatants are collected for titering. DA
clones are selected for vector production and called DA-E3/19K. Vector supernatants 35 are collected from 10cm confluent plates of DA-E3/19K clones cultured in normal media ~-15893% 34 co"~Ai~ g polybrene or protal.unc sulfate. Alternatively, vector supe-.la~a-l~ can be harvested from bioreactors or roller bottles, processed and purified further before use.
For those vectors without a drug rçcist~nce marker or with a marker already in the p~c~ in~ cell line, selection of stably tr~n~duced clones must be pclrollllcd by 5 dilution cloning the DA tr~ncduced cells one to two days after tr~nsduçing the cells with 293 2-3 generated supernatant. The dilution clones are then screened for the prt;sence of E3/19K e,~press;on by using reverse llanscliplion of mes.c~n~er RNA, followed by amplification of the cDNA mesS~ge by the polymerase chain reaction, a procedure known as the RT-PCR. A co..un~ ;al kit is available through Invitrogen Corp. (San 10 Diego, CA). RT-PCR should be pe~ro~ ed on clones which have been propagated for at least 10 days and app-o~i-..ately 50 to 100 clones will need to be screened in order to find a reasonable number of stably ll~llsrol~..cd clones. In order to p~,lro.,n RT-PCR, specific primers will be required for each message to be amplified. Primers designed to amplify a 401 bp product for E3/19K message screening are as follows:
Screening primers for E3/19K are:
(Sequence ID No.
5'-3': ATGAGGTACATGATTTTAGGCTTG

20 (SequenceID No.
5'-3': TCAAGGCATTTTCTTTTCATCAATAAAAC

Example 9 DETECTION OF REPLICATION COMPETENT RETROVIRUSES

The extended S+L- assay determines whether replication competent, infectious virus is present in the supernatant of the cell line of interest. The assay is based on the en.p;lical observation that infectious retroviruses generate foci on the inrlic~tQr cell line 30 MiCIl (ATCC CCL 64.1). The MiCII cell line is derived from the MvlLu mink cell line (ATCC CCL 64) by transduction with Murine Sarcoma Virus (MSV). It is a non-producer, non-transformed, revertant clone containing a murine sarcoma provirus that forms sarcoma (S+) indicating the presence of the MSV genome but does not cause lellkPmi~ (L-) indicating the absence of replication competent virus. Infection of MiCIl 35 cells with replication competent retrovirus "activates" the MSV genome to trigger "l.~ls~..,.&tion" which results in foci formation.

WO 95/06718 PCT/US91~58~11 ~ 1 ~ 8 ~ ~ 2 Supe.l.atal-t is removed from the cell line to be tested for pres~,nce of replication competent retrovirus and passed through a 0.45 11 filter to remove any cells. On day 1, Mv1Lu cells are seeded at 1 x 105 cells per well (one well per sample to be tested) of a 6 well plate in 2 ml DMEM, 10% FBS and 8 llg/ml polybrene. MvlLu cells are plated in 5 the same manner for positive and negative controls on separate 6 well plates. The cells are incuk~ted overnight at 37C, 10% C02. On day 2, 1.0 ml of test supellld~ isadded to the MvlLu cells. The negative control plates are incub~ted with 1.0 ml of media. The positive control consists of three dilutions (200 focus rO. ..f.,.g units (ffu), 20 ffu and 2 ffu each in 1.0 ml media) of MA virus (Miller et al., Molec. and Cell. Biol.
10 5:431, 1985) which is added to the cells in the positive control wells. The cells are inc~lb~ted overnight. On day 3, the media is aspirated and 3.0 ml of fresh DMEM and 10% FBS is added to the cells. The cells are allowed to grow to confluency and are split 1:10 on day 6 and day 10, amplifying any replication competent retrovirus. On day 13, the media on the MvlLu cells is aspirated and 2.0 ml DMEM and 10% FBS is added to 15 the cells. In addition, the MiCIl cells are seeded at 1 x 105 cells per well in 2.0 ml DMEM, 10% FBS and 8 llg/ml polybrene. On day 14, the supe...aLan~ from the MvlLucells is transferred to the corresponding well of the MiCIl cells and incubated overnight at 37C, 10% CO2. On day 15, the media is aspirated and 3.0 ml of fresh DMEM and10% FBS is added to the cells. On day 21, the cells are PY~mined for focus formation 20 (appearing as clustered, refractile cells that overgrow the monolayer and remain ~tt~çhed) on the monolayer of cells. The test article is determined to be cont~min~ted with replication competent retrovirus if foci appear on the MiCIl cells.

Example 10 The following adherent human and murine cell lines are seeded at 5 x 105 30 cells/10 cm dish with 4 ug/ml polybrene: HT 1080 (ATCC No. CCL 121), Hela (ATCC
No. CCL 2), BC10ME (Patek et al., Cell. Immuno. 72:113, 1982, ATCC No. TIB 85), BCenv, BC10~IE expressing HIV-l IIIBenv (Warner et al., AIDS Res. and Human Retroviruses 7:645, 1991, L33 obtained from Gunther Oennert, University of Southern California), and L33env. The following day, 1.0 ml of filtered supernatant from the DA
35 E3/19K pool is added to each of the cell culture plates. The following day, phleomycin is added to ehe media of all cell cultures. For cell lines that are already neomycin WO 95/06718 PCT/US9~ 60 ~, 1.s8932 36 r~cict~nt the E3/19K in the KT-3C backbone (phleomycin resistant) is used. Transient sup~.llalanls for 293 2-3 or from DA derived lines can be used." The cultures are ed until selection is complete and sufficient cell numbers are ~,enel~ed to testfor gene c~y.es~ion. The tr~ncduced cell lines are deci~n~ted HT 1080-E3/19K, Hela-5 E3/19K, BClOME-E3/19K, L33-E3/19K and L33env-E3/19K ~c~pecli~rely.
EBV transformed cell lines (BLCL), and other suspension cell lines, are tr~n~duced by co-cultivation with the irradiated producer cell line, DA-E3/19K.
Specifically, irradiated (10,000 rads) producer line cells are plated at 5 x 105 cells /6 cm dish in growth media co..~ ing 4 ~lg/ml polybrene. After the cells have been allowed 10 to attach for 2-24 hours, 106 suspension cells are added. After 2-3 days, the suspension cells are removed, pelleted by centrifugation, resuspended in growth media co.-t~ining lmg/ml phleomycin, and seeded in 10 wells of a round bottom 96 well plate. The cultures were exp~nded to 24 well plates, then to T-25 flasks.

Example 11 EXPRESSION OF E3/19K IN THE MULTIVALENT RECOMBrNANT

20 A. WESTERN BLOT ANALYSIS FOR E3/19K

Radio-immuno precipitation assay (RIPA) Iysates are made from selected cultures for analysis of E3/19K e~,res~ion. RIPA Iysates are prepared from confluent plates of cells. Specifically, the media is first aspirated off the cells. Depending upon the 25 size of the culture plate co..~inil~ the cells, a volume of 100 to 500 ~l ice cold RIPA
Iysis buffer (10 mM Tris, pH 7.4; 1% Nonidet P40 (Calbiochem, San Diego, CA); 0.1%
SDS: 150 mM NaCl) is added to the cells. Cells are removed from plates using a micropipet and the mixture is transferred to a microfuge tube. The tube is centrifuged for 5 mimltes to plecip;lale cellular debris and the supel-~a~al~ is ll~ns~"ed to another 30 tube. The s-~,e".alanls are electrophoresed on a 10% SDS-PAGE gel and the protein bands are lran~îe"ed to an Immobilon melllbl~ne in CAPS buffer (Aldrich, Milwaukee, WI) (10 mM CAPS, pH 11.0; 10% methanol) at 10 to 60 volts for 2 to 18 hours. Themembrane is t~nsre"ed from the CAPS buffer to 5% Blotto (5% non& dry milk; 50 mM Tris, pH 7.4; 150 mM NaCl; 0.02% sodium azide, and 0.05% Tween 20) and 35 probed with a mouse monoclonal antibody to E3/19K (Severinsson et al., J. Cell Biol.

wo 9~/06718 ~ 1 5 ~ ~ 3 2 PCT/US9 1~ 60 101:540, 1985). Antibody binding to the me,l,b,~ne is detected by the use of l25I-Protein A.

Example 12 DEMONSTRATE DECREASED LEVELS OF CLASS I EXPRESSION COMPARED
TO NON-TRANSDUCED CELLS.

Cell lines tr~n.cduced with the E3/19K-vector are eAa".ined for MHC class I
molecule eA~,ression by FACS analysis. Non-tr~n~duced cells are also analyzed for MHC class I molecule expression and col"pa,ed with E3/19K transduced cells to determine the effect of tr~n.~duction on MHC class I molecule eApl ession.
Murine cell lines, L33-E3/19K, L33env-E3/19K, L33, L33env, BC10ME, 15 BCenv, and BCenv-E3/19K, are tested for eAp,es~ion ofthe H-2Dd molecule on the cell surface. Cells grown to subconfluent density are removed from culture dishes by tre~tmçnt with Versene and washed two times with cold (4C) PBS plus 1% BSA and 0.02% Na-azide (wash buffer) by centrifugation at 200g. Two million cells are placed in microfuge tubes and pelleted by centrifugation, 200g, and the supe",atan~ is removed.
20 Cell pellets are resuspended with the H-2Dd-specific Mab 34-2-12s (50~1 of a 1:100 dilution of purified antibody, ATCC No. HB 87) and incubated for 30 min at 4C with occasional mixing. Antibody labeled cells are washed two times with 1 ml of washbuffer (4C) centrifuged and the supelllatanl is removed. Cells are resuspended with a biotinylated goat anti-mouse kappa light chain Mab (Amersham, Arlington Heights, IL) 25 (50~1, of a 1:100 dilution of purified antibody) and incubated for 30 min at 4C. Cells are washed, resuspended with 50~1 of avidin conjugated FITC (Pierce, Rockford, IL), and incubated for 30 min at 4C. The cells are washed once more, resuspended in 1 ml of wash buffer, and held on ice prior to analysis on a FACStar Analyzer (Becton Dickinson, Los Angeles, CA). The mean fluo-t;scence intensity of transduced cells is 30 compared with that of non-tr~n.cduced cells to determine the effect E3/19K protein has on surface MHC class I molecule c~)res~ion.

g93~ 38 Example 13 MURINE CTL ASSAY

Balb/c mice are injected with 107 irradiated (10,000 rads) BCenv cells. After 7 days the spleens are harvested, dispersed into single cell suspension and 3 x 106 s~,!e s ytes/ml are cultured in vi~ro with 6 x 104 cells/ml irradiated BCenv or BCenv-E3/19K cells for 7 days at 37C in T-25 flasks. Culture medillm consists of RPMI1640; 5% fetal bovine serum, heat-inactivated (FBS); 1 mM pyruvate; 50 ,ug/ml 10 ~nl~;cin and 10-5 M 2-me~captoethanol. Effector cells are harvested 7 days later and tested using various effect~ r~,el cell ratios in 96 well microtiter plates in a standard 4-6 hour assay. The assay employs Na25lCrO4-labeled, 100 IlCi, 1 hour at 37C, (Amersham, Arlington Heights, Illinois) target cells (BC, BCenv, Warner, et al., Aids Res. and Human Retroviruses 7: 645, 1991 or BCenv E3/19K) at 1.0 x 104 cells/well with the final total volume per well of 200 ~1. Following inc~lbation, 100 ~1 of culture medium is removed and analyzed in a WALLAC gamma spectrometer (Gaithersburg, MD.). Spontaneous release (SR) is determined as CPM from targets plus medi~lrn and maximum release (MR) is determined as counts per minute (CPM) from targets plus lM
H .. Percent target cell Iysis is calculated as: [(effector cell + target CPM) -(SR)]/[(MR) - (SR)] x 100. Spontaneous release values of targets are typically 10%-30% of the MR. Tumor cells that have been tr~ncduced with the gene of interest (ribozyme, E3/19K, ~nfisen~e, etc.) are used as stimulator and/or target cells in this assay to demonstrate the reduction of HIV-specific CTL induction and detection as co~pared to the non-tr~nsd~lced line which is the positive control.
Example 14 WHEN CLASS I MOLECllLE SURFACE EXPRESSION IS DECREASED BY THE
E3-VECTOR TRANSDUCTION.

The L33env cell is being employed as a model for gene therapy treated transformed cells. Gene therapy treated cells produce a foreign protein making them possible targets for clearance by CTL. It has been demonstrated that Balb/c mice35 injected with live L33 tumor cells will develop a solid tumor identifi~hle by caliper measurement within three weeks post-exposure. However, Balb/c mice injected with wo 95/06718 2 15 ~ ~ 3 2 PCTIUS94/09860 live L33env ~ roll-lcd tumor cells (L33 cells tr~ncduced and selected for cAplession of the H~V-lmg envelope protein) recognize H~V env in the context of H-2Dd and reject the tumor cells with no appale-ll tumor up to 15 weeks later (Warner et al., AIDS Res.
and Human Retroviruses 7:645, 1991). Tl~lsrolll'~Lion of L33env cells with the 5 E3/19K vector dec~eases cell surface ~Aplession of MHC class I molecules allowing these cells to evade immllne surveillance and thereby establish a tumor. Development of an L33env tumor indicates that ceU surface ,A~ression of MHC class I molecules has been decreased by co~ c~ çin~ cells with the El9 gene. This impedes optimal immune system clearance ...eçh~niem.c Three tumor cell lines L33, L33env, and E3/19K-L33env are grown in DMEM
cG..~ ing 10% FBS. The tumor cells are gently rinsed with cold (4C) PBS and treated with versene to remove them from the plate. After a.yil~ g cells from plates, single cell suspensions are added to sterile plastic tubes. Cell suspensions are washed two times in sterile PBS (4C), counted and resuspended in PBS to 107 cells/ml. Balb/c mice 15 (4-6 weeks old) are injected subcutaneous with 106 live tumor cells (0.1 ml) and ~csessed for tumor formation and tumor clearance. Different mice are injected with di~elt;lll tumor cell lines. Mice injected with L33 cells are positive control animals for tumor formation while those injected with L33env are negative controls and should reject the tumor cells because of the env specific CTL response. The group of mice 20 injected with E3/19K-transformed, L33env cells are monitored to show the effect that E3/19K eAp-ession in L33env cells has on the murine immune response to these tumor cells.

Example 15 DEMONSTRATE DECREASED LEVELS OF MHC CLASS I EXPRESSION
COMPARED TO NON-TRANSDUCED CELLS.

Cell lines tr~ncd~1ced with the E3/19K vector are examined for class I molecule eA~,~ession by FACS analysis. Non-tr~.cd~lced cells are analyzed for class I molecule eA~les~ion to compare with E3/19K tr~ncduced cells and determine the effect thattrancduction has on class I molecule eA~lession.
Two human cell lines, JY-E3/19K and JY (ATCC No. ) are tested for eA~ression of the HLA-A2 molecule on the cell surface. Suspension cells grown to 106 cells/ml are removed from culhlre flasks by pipet and washed two times with cold (4C) WO 9~i/06718 ~g9~ 40 PBS plus 1% BSA and 0.02% Na-azide (wash buffer) by centrifugation at 200g. Two million (2 x 106) cells are placed in microfuge tubes, pelleted in at 200g, and the supel,.dl~nl is removed. Cell pellets are resuspended with the HLA-A2-specific Mab BB7.2 (50~1 of a 1:100 dilutiQn of purified antibody, ATCC No. HB 82) and incub~ted 5 with antibody for 30 min at 4C with occasional mixing. Antibody labeled cells are washed two times with I ml of wash buffer (4C). Prior to removing the supe.,.alalll, the cells are resuspended with a biotinylated rat anti-mouse kappa light chain Mab (50~1, of a 1:100 dilution of purified antibody) and incl1b~ted for 30 min at 4C. Cells are washed, resuspended with 50111 of avidin conjug~ted FITC, and incub~ted for 30 rnin at 10 4C. The cells are washed once more, and resuspended in 1 ml of wash buffer, and held on ice prior to analysis on a FACStar Analyzer. The mean fluolçsce.-ce intensity of tr~nsduced cells is co--.~,aled with that of non-tr~ncduced cells to determine the effect E3/19K protein has on surface MHC class I molecule e,.~,ress;on.

Example 16 AND NONTRANSDUCED EBV-TRANSFORMED HUMAN JY CELLS BY E~A-A2 RESTRICTED, EBV-SPECIFIC HUMAN CTL LrNES.
Human CTL lines propagated from donor blood samples using autologous EBV
l-~n~,ro~--ed cells as stim~ tors have been shown to be HLA-A2 restricted and specific for EBV proteins. These CTL lines, are propagated with autologous EBV transformed cells and can Iyse JY target cells (HLA-A2+ and EBV tl~nsru....ed). A chromium 25 release assay can be performed with these CTL lines and ~Y target cells that have been t~ ,folllled with the E3/19K gene or nontr~n.~duced The E3/19K transformed ~Y
target cell are used to demonstrate decreased recognition and Iysis of this cell when colllpal ed to no..~l ~nsrormed JY target cells. These results indic~te that cell tran~,folma~ion with agents that decrease MHC class I surface e,.~)ression also decreases 30 MHC class I restricted cell me~ ted immune responses in an in v ~ro human cell model system.
Approximately, 1 x 106 irradiated (10,000 rad) JY cells are cultured with 1 x 107 PBMC from a person that is HLA-A2 and verified to have an EBV response, in 10 mls of culture rne~lium at 37C 5% CO2 for 7-10 days. The culture rne~ium consists of 35 RPMI 1640 supplemented with 5% heat inactivated fetal bovine serum preselected for CTL growth, 1 mM sodium pyruvate and nonçssçnti~l amino acids. After the 7-10 day WO95tO6718 215 8 9 3 2 PCT/US9~ 50 incub~tion the effector cells are harvested and tested in a standard 4-6 hour chromium release assay using 5lCr labeled JY cells as the positive control and 51Cr labeled JY-E3/19K. lY and ~Y-E3/19K cells are labeled with 300 ~Ci of Na251CrO4 for 1 hour at 37C, then washed, counted, and used in the assay at 4 x 103 cells/well with the final total volume per well of 200 ul. Following incub~tion, 100 ~1 of culture medium is removed and analyzed in a WALLAC gamma ~,e~;L~u"leter. Spont~neous release (SR) is dete",~ned as counts per minute (CPM) CPM from targets plus medium and m~im~m release (MR) is determined as from targets plus lM HCI. Percent target cell Iysis is c~lc~ ted as: [(effector cell + target CPM) - (SR)]/[(MR) - (SR)] x 100.
Spont~neol.s release values of targets are typically 10%-30% of the MR. Tumor cells that have been tr~ncduced with the gene of interest (ribozyme, E3/19K, ~nticçnce, etc.) are used as stimul~tor and/or target cells in this assay to demonstrate the reduction of EBV-specific CTL induction and detection as compared to the non-transduced line which is the positive control.
The number of transformed cells used to infuse back into the patient per infusion is projected to be at a minimum of 107 lo8 cells per patient per injection. The site of the infusion may be directly into the patient's synovial membrane or i.v., into the peripheral hood stream.

From the foregoing, it will be appreciated that, although specific embo-iim~nt.c Of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the scope of the invention.
Accordingly, the invention is not limited except as by the appended claims.

Claims (25)

Claims

1. Tissue cells of an animal transformed with a recombinant vector construct that expresses a protein or active portion of a protein capable of inhibiting MHC
antigen presentation, for use in a method for suppressing an autoimmune response within an animal.

2. The cells of claim 1 wherein the recombinant vector construct directs the expression of a protein capable of binding .beta.2-microglobulin.

3. The cells of claim 1 wherein the recombinant vector construct directs the expression of a protein capable of binding the MHC class I heavy chain molecule intracellularly.

4. The cells of claim 1 wherein the recombinant vector construct directs the expression of a protein or active portion of a protein selected from the group consisting of E3/19K and H301.

5. Tissue cells of an animal transformed with a recombinant vector construct that transcribes an antisense message capable of inhibiting MHC antigen presentation, such that an autoimmune response against the cells is suppressed, for use in a method of suppressing an autoimmune response within an animal.

6. The cells of claim 5 wherein the recombinant vector construct transcribes an antisense message which binds a conserved region of MHC class I heavy chain transcripts.

7. The cells of claim 5 wherein the recombinant vector construct transcribes an antisense message which binds the .beta.2-microglobulin transcript.

8. The cells of claim 5 wherein the recombinant vector construct transcribes an antisense message which binds the PSF1 transporter protein transcript.

9. Tissue cells of an animal transformed with a recombinant vector construct that transcribes a ribozyme capable of inhibiting MHC antigen presentation, such that an autoimmune response against the cells is suppressed, for use in a method of suppressing an autoimmune response within an animal.

10. The cells of claim 9 wherein the recombinant vector construct transcribes a ribozyme that cleaves a conserved region of MHC class I heavy chain transcripts.

11. The cells of claim 9 wherein the recombinant vector construct transcribes a ribozyme that cleaves the .beta.2-microglobulin transcript.

12. The cells of claim 9 wherein the recombinant vector construct transcribes a ribozyme that cleaves the PSF1 transporter protein transcript.

13. Tissue cells of an animal transformed with a multivalent recombinant vector construct which directs the expression of a protein or active portion of a protein capable of inhibiting MHC antigen presentation, and an antisense message or ribozyme capable of inhibiting MHC antigen presentation, such that an autoimmune response against the cells is suppressed, for use in a method of suppressing an autoimmune response within an animal.

14. Tissue cells of an animal transformed with a multivalent recombinant vector construct which directs the expression of an antisense message and a ribozyme capable of inhibiting MHC antigen presentation, such that an autoimmune response against the cells is suppressed, for use in a method of suppressing an autoimmune response within an animal.

15. Tissue cells of an animal transformed with a multivalent recombinant vector construct which directs the expression of (a) two or more proteins or active portions of proteins, capable of inhibiting MHC antigen presentation; (b) two or more antisense messages capable of inhibiting MHC antigen presentation; or (c) two or more ribozymes capable of inhibiting MHC antigen presentation, such that an autoimmune response against the cells is suppressed, for use in a method of suppressing an autoimmune response within an animal.

16. The cells of claim 13 or 15 wherein the protein is E3/19K or H301 or an active portion thereof.

17. The cells of any one of claims 13, 14 or 15 wherein the antisense message binds to the transcript of a protein selected from the group consisting of a conserved region of the MHC class I heavy chains, .beta.2-microglobulin and PSF1 transporter protein.

18. The cells of any one of claims 13, 14 or 15 wherein the ribozyme cleaves the transcript of a protein selected from the group consisting of a conserved region of the MHC class I heavy chains, .beta.2-microglobulin and PSF1 transporter protein.

19. The cells of any one of claims 1, 5, 9, 13, 14 or 15 wherein said recombinant vector construct is a recombinant viral vector construct.

20. The cells of any one of claims 1, 5, 9, 13, 14 or 15 wherein the recombinant vector construct is a recombinant retroviral vector construct.

21. The cells of any one of claims 1, 5, 9, 13, 14 or 15 wherein the recombinant vector construct is carried by a recombinant virus selected from the group consisting of poliovirus, rhinovirus, vaccinia virus, influenza virus, adenovirus, adeno-associated virus, herpes simplex virus, and measles virus.

22. The cells of any one of claims 1, 5, 9, 13, 14 or 15 wherein the recombinant vector construct is carried by a recombinant virus selected from the group consisting of togaviridae, picornaviridae, poxviridae, adenoviridae, parvoviridae, herpesviridae, paramyxoviridae and coronaviridae viruses.

23. The cells of any one of claims 1, 5, 9, 13, 14 or 15 wherein the recombinant vector construct is carried by a recombinant corona virus.

24. The cells of any one of claims 1, 5, 9, 13, 14 or 15 wherein the recombinant vector construct is carried by a recombinant Sindbis virus.

25. The cells of any one of claims 1, 5, 9, 13, 14 or 15 wherein the tissue cells are selected from the group consisting of synovial membrane cells, pancreatic islet cells, hepatocytes and keratocytes.