Patent 6,773,705

The invention provides a method of detecting autoimmune disease in a mammal, comprising providing a biological sample from a mammal and detecting proteasome activity, wherein a reduction in proteasome activity from a basal state is indicative of autoimmune disease. In addition, the invention encompasses a method of treating an autoimmune disease in a mammal, comprising administering to a mammal suspected of suffering from an autoimmune disease an agent which restores NF.kappa.B activity in an amount and for a time sufficient to result in normal NF.kappa.B activity in the mammal.

DESCRIPTION OF THE INVENTION

The present invention is predicated on the discovery that NOD mice are deficient for NF.kappa.B activity. As described herein below, the methods and of the present invention comprise restoration of proteasome function, or simply that of NF.kappa.B, in the treatment of autoimmune disorders. The inventive methods are therefore contrary to prior art methods and, indeed, unexpected, based upon prior art references, which teach suppression of NF.kappa.B or of proteasome activity (and, consequently, that of NF.kappa.B) as a method of treating autoimmune disorders (see above). Restoration of proteasome function or of NF.kappa.B activity may be directed at the proteasome, the ubiquitinating machinery or protein kinases. Alternatively, therapy may involve providing functional (active forms of ) NF.kappa.B that is independent of the proteasome for activation or even the products of downstream genes normally under the transcriptional control of NF.kappa.B or providing the cell with cytoplasmic forms of NF.kappa.B for cell cycle control and cell differentiation/viability. The object of such treatment is to inhibit the progression of an autoimmune disease or to prevent its clinical initiation, where "clinical initiation" refers to the presentation of symptoms and to organ destruction.

Detection Defect in Proteolytic Processing

The invention contemplates detection of autoimmune disease by detecting a defect in proteasome activity. Such defects may be detected using the following assays.

i The Proteasome

Proteasome activity may be assayed as previously described (Gaczynska et al., 1994, Proc. Natl. Acad. Sci. U.S.A., 91: 9213-9217, incorporated herein by reference). Briefly, cells (whether cultured cells, or those of an model animal, such as a mouse) in which the efficacy of a stimulator of proteasome activity is to be assayed prior to administration to a human are homogenized in a Dounce homogenizer or other grinding device (e.g. a mortar and pestle or a blender) and then by vortex mixing with glass beads in a homogenization buffer (40 mM Tris.HCl, 5 mM MgCl₂, 2 mM ATP, 250 mM sucrose, pH 7.4). Fractions containing total 20S and 26 S proteasomes are isolated by differential centrifugation of homogenates: for 20 minutes at 10,000xg, then for 1 hour at 100,000xg or for hours at 100,000xg. Pellets are solubilized in 50 mM Tris.HCl, 5 mM MgCl₂, 2 mM ATP, 20% (volume/volume) glycerol, pH 7.4. Resulting "proteasome fractions" are used for peptidase assays and Western blot analysis. Degradation of the fluorogenic peptides, N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-MCA), N-tert-butoxycarbonyl-Leu Arg-Arg-7-amido-4-methylcoumarin (Boc-LRR-MCA) and N-carbobenzoyx-Leu-Leu-Glu-.beta.-naphthylamide (Cbz-LLE-.beta.NA) is assayed at 37^oC., for 40 minutes or 1 hour in the presence of apyrase (5 units/ml), as described previously described (Gaczynska et al., 1993, Nature, 365: 552-554, also incorporated herein by reference).

ii. Ubquitination

The invention contemplates detection of autoimmune disease by detecting a defect in the activity of ubiquitinating enzymes. Such defects may be detected using the following assays; Western analysis with antibodies directed against active forms of ubiquitinating enzymes, observation of eletrophoretic mobility of on a Western blot of the ubiquitinated form of a test protein or peptide relative to its non-ubiquitinated form or its proteolytically processed form relative to its unprocessed form in cytoplasmic extracts of unknown ubiquitinating capacity, Northern analysis to detect loss of mRNAs whose transcription is dependent upon a protein which required ubiquitination or enzymatic or other assay to determine the function of a protein or peptide incubated in a cytoplasmic extract of unknown ubiquitinating capacity, wherein the protein or peptide requires ubiquitination in order to undergo proteolytic activation. In vitro ubiquitination assays are known in the art (see Chen et al., 1995, Genes Dev., 9: 1586-1597; Corsi et al., 1995, J. Biol. Chem., 270: 8928-8935; Corsi et al., 1997, J. Biol. Chem., 272: 2977-2983; Mori et al., 1997, Eur. J. Biochem., 247: 1190-1196; Verma et al., 1997, Mol. Cell Biol., 8: 1427-1437; Kumar et al., 1997, J. Biol. Chem., 272: 13548-13554).

iii. NF.kappa.B

The invention contemplates detection of autoimmune disease by detecting a defect in the activation of NF.kappa.B. Such defects may be detected using the following assays.

The presence or absence of NF.kappa.B activity may be assayed by immunological analysis of protein from cells or individuals using anti-NF.kappa.B antibodies (in which one would expect to observe a band the size of I.kappa.B-free NF.kappa.B). Such protein may be derived from a biological sample, including, but not limited to, a tissue, cell, cell lysate or body fluid from an individual. Northern analysis using labeled nucleic acid probes specific for transcripts that may be produced by the downstream targets of NF.kappa.B (i.e., genes which are transcriptionally activated by that protein) may be performed. Alternatively, nuclear protein extracts may be prepared from such cells and tested for the ability to activate transcription in vitro of a marker gene which is operatively linked to an NF.kappa.B-inducible gene regulatory sequence. Assays may be directed at individual NF.kappa.B subunits, such as p50 and p65, as in Examples 1 and 2 below, wherein the cytoplasmic and nuclear functions of these subunits are tested in normal and autoimmune mice. In addition to activity, their processing from a larger protein or release from inhibitory substances may be assessed by molecular and biochemical methods known in the art (such as PAGE or Western analysis, as described below).

While these approaches are technically feasible, they may not be medically expedient or even safe, as they entail removal of treated cells from the patient. It is recommended that immunological analysis be performed on serum protein extracts, using antibodies which are directed against products of genes under the control of NF.kappa.B which are secreted proteins, by methods described below.

Restoration of Normal Proteolytic Processing

The invention contemplates methods of treating autoimmunity by restoring proteolytic processing, based upon the observation that NF.kappa.B activity is absent in the NOD mouse model of autoimmune disease. Restoration of proteolytic processing, such as would result in the restoration of NF.kappa.B activity, may be directed at the proteasome, the ubiquitinating machinery or protein kinases.

A Therapeutic Targets

Suppression of Proteasome Inhibitors

The invention contemplates methods of treating autoimmunity by restoring proteolytic processing by blocking the activity of inhibitors of proteasome function or changing the specificity of a proteasome subunit to favor activation of the substrate(s) deficient in an autoimmune disease, so that correct protein processing is restored.

Inhibition of proteasome activity blocks the production of activated NF.kappa.B and other essential proteins, as described above; therefore, in order to promote correct protein processing, it may be necessary to inactivate cellular inhibitors of the proteasome. Such endogenous inhibitors of proteasome activities have been isolated. These include the 240 kD and the 200 kD inhibitors isolated from human erythrocytes (Murakami et al., 1986, Proc. Natl. Acad. Sci. U.S.A., 83: 7589-7592; Li et al., 1991, Biochemistry, 30: 9709-715) and purified OF-2 (Goldberg, 1992, Eur. J. Biochem., 203: 9-23).

Proteasome processed proteins leading to activation include P100and P105. Proteasome processed proteins leading to degradation include TFIIH, Stat proteins, Jak proteins (Jak2, Jak1), Shc, Sp1, CDC25B, Kip1, p27, Serotonin N-acetyl transferase, IkB, P53, Cyclins, c-Fos, c-Jun, presenilin 1 FosL, tyrosine aminotransferase, and ornithine decarboxylase.

Endogenous proteasome inhibitors may be inactivated by methods known in the art, which methods include the administration of antibodies which bind them specifically, the use of antisense RNA or ribozymes directed against the mRNAs which encode them (see below). Antibodies against numerous proteins are now publicly available, both through commercial and non-profit suppliers (e.g. ATCC); however, antibodies of use in the invention may, if necessary, be prepared as described below.

Restoration of Wild-type Proteasome Function

The invention contemplates methods of treating autoimmune disease by direct stimulation of proteasome function, thereby restoring or preserving correct proteolytic processing.

Japanese Patent No. JP8322576, which is herein incorporated in full by reference, discloses proteasome activator PA28.beta.(see also Chu-ping et al., 1992, J. Biol. Chem., 267: 10515; Dubiel et al., 1992, J. Biol. Chem., 267: 22369); both cloning of a cDNA from bovine tissues (e.g. liver, heart and red blood cells) and a method for the production of the recombinant polypeptide encoded by the cloned nucleic acids are described by these references. PA28 (or PA28.beta.) has a subunit molecular weight of 28,000, as judged by denaturing gel electrophoresis and a native molecular weight of approximately 180,000 as determined by gel filtration and density gradient centrifugation; therefore, it is thought to exist as a hexameric protein complex. Dubiel et al. (1992, supra) further describe the isolation of a human protein of M_r approximately 200,000 that activates proteasomes; this complex is a hexamer comprising subunits that display M_r of approximately 29,000 and 31,000 on danaturing electrophoretic gels. This activator complex lacks intrinsic peptidase activity, but stimulates proteolysis of certain substrates about 60-fold, although activated proteasomes are unable to degrade ubiquitin-lysozyme conjugates, bovine serum albumin or lysozyme; activation involves reversible binding of the activator complex to proteasomes. WO 95/27058 discloses a human protein complex (M_r approximately 29,000) which is a .gamma.-interferon-inducible activator of proteasome function. The sequences encoding each of these polypeptides are of use in gene therapy according to the invention, as described below. Alternatively, the proteins themselves may be administered by methods known in the art (see also below).

In addition to proteasome-stimulating proteins, wild-type proteasome subunits or other associated proteins (e.g. Lmp2, Lmp7) may be administered if inactivating mutations are found within the sequences encoding them or in the regulatory elements controlling the transcription or these genes. While there exist many targets for such specifically-directed treatment, it should be noted that the discovery of one such mutant (that found in the shared Lmp2/Tap promoter) is described herein above (Yan et al., 1997, supra).

Restoration of Correct Ubiquitination/Phosphorylation

The invention contemplates methods of treating an autoimmune disease by restoring correct patterns of ubiquitination and/or phosphorylation.

If proteolytic failure has been traced to a deficiency in ubiquitination or phosphorylation, the missing activity may be supplemented either through the administration of a wild-type protein whose absence or inactivation is responsible for the deficiency or through gene therapy, in which a gene encoding such a protein is administered under the influence of transcriptional control elements (e.g., its own wild-type element or another strong promoter, e.g. thymidine kinase, heat-shock or others as are known in the art). Such proteins may include ubiquitinating proteins of the E1, E2 and E3 families as well as "glue" proteins (all as described above); alternatively, protein kinases (e.g., cyclin-dependent kinases; see also above) or cyclins may be administered.

Restoration of NF.kappa.B Function

The invention contemplates methods of treating autoimmune diseases by restoring NF.kappa.B function, which, in turn, restores the transcription of NF.kappa.B-dependent genes.

As is true of the proteasome and of the ubiquitination and protein phosphorylation machinery described above, it is possible to administer to cells of an organism in which NF.kappa.B carries an inactivating mutation, either in coding or regulatory sequences, a wild-type sample of the NF.kappa.B protein or one or more copies of the gene encoding it; however, a second scenario may instead be envisioned.

In the case in which NF.kappa.B activity is reduced or absent due to an `upstream` defect (that is, one involving activation by the proteasome, instead of- or in addition to a mutation in the NF.kappa.B gene itself), it is possible to circumvent the need for proteolytic activation of NF.kappa.B by introducing a constitutively-active version of the protein, such as one in which the I.kappa.B recognition site has been mutated such that I.kappa.B can no longer bind to- and inactivate NF.kappa.B. Binding of NF.kappa.B to I.kappa.B occurs through ankyrin repeats (as reviewed by Siebenlist et al., 1994, Ann. Rev. Cell. Biol., 10: 405-455); it is contemplated that sequences encoding these repeats be deleted or mutated in an NF.kappa.B subunit p100 or p105 gene expression construct such that binding to I.kappa.B is significantly impaired or is eliminated. As a transcription/signalling factor which remains active when it is no longer required may have undesirable consequences, particularly in the absence of proteolytic which would normally inactivate it under such circumstances, administration of such a protein in limited doses or of a gene encoding it under a tightly-regulated (i.e. inducible, rather than constitutive, promoter) may be necessary. Alternatively, such a protein may be expressed at all times, provided that an inhibitor thereof is co-administered; such an inhibitor may be an antibody directed against the protein, or an antisense RNA or ribozyme directed against the message encoding it, as described below.

Inactivation of I.kappa.B may also be performed by methods described below, such as by the use of antibodies directed against it or of antisense RNA or ribozymes directed against the mRNA transcript encoding it. Preferably, such inactivation is transient, as it would otherwise lead to constitutive activation of NF.kappa.B, which activation is not, itself, normal.

The invention contemplates treatment of autoimmune disease using methods directed at the potential therapeutic targets discussed above. In the section following, methods by which such treatment may be carried out are presented.

B. Therapeutic Methods

Autoimmune Disorders in Humans

In order to provide effective treatment according to methods contemplated by the invention, it is first necessary to identify those individuals in need of treatment.

Genetic linkage studies have confirmed the MHC to be an important contributor to human autoimmune diseases such as type I diabetes, rheumatoid arthritis, lupus erythematosus, Hashimoto's disease, and multiple sclerosis (Bach et al., 1994, Endocr Rev., 15: 516; Cudworth and Woodrow, 1976, Br. Med. J., 2: 846; Festenstein et al., 1986, Nature, 322: 64; Nerup et al., 1977, HLA and Disease, Munksgaard, Copenhagen; Todd et al., 1987, Nature, 329: 599; Van Endert et al., 1994, Diabetes, 43: 110). Other autoimmune disorders include Graves' disease, ulcerative colitis, Crohn's disease, polyendocrine failure, Sjogren's syndrome and others as listed above in the Summary.

The present invention is of use in the treatment of HLA class II-linked autoimmune diseases such as those listed above. Diagnostic symptoms or other indicators may be used either to assess a patient for the presence of- or susceptibility to such a disorder; in addition, improvement (i.e., a change toward the basal state, as defined above) in one or more of these indicators is indicative of the efficacy of a given method of treatment for such a disease.

Examples of autoimmune disease-related symptoms for several representative diseases are as follows:

Addisons's Disease

Addison's disease is a disorder characterised by failure of the adrenal gland and is often an autoimmune disorder involving destruction of the adrenal cortex and the presence of adrenal autoantibodies in the patient's serum. The adrenal cortex is responsible for producing several steroid hormones including cortisol, aldosterone and testosterone. In autoimmune Addison's disease and other forms of the disease, levels of these hormones are reduced. This reduction in hormone levels is responsible for the clinical symptoms of the disease which include low blood pressure, muscle weakness, increased skin pigmentation and electrolyte imbalance.

Autoantibodies to the adrenal cortex may be identified for diagnosis of Addison's disease using the technique of complement fixation or immunofluorescence (Anderson et al., 1957, Lancet, 1: 1123-1124; Blizzard and Kyle, 1963, J. Clin. Invest., 42: 1653-1660; Goudie et al., 1968, Clin. Exp. Immunol., 3: 119-131; Sotosiou et al., 1980, Clin. Exp. Immunol., 39: 97-111). Radioimmunoassay and ELISA techniques using crude adrenal membrane preparations are also of use in the invention (Stechemesser et al., 1985, J. Immunol. Methods, 80: 67-76; Kosowicz et al., 1986, Clin. Exp. Immunol., 671-679).

U.S. Pat. No. 5,705,400 discloses methods for the detection of adrenal autoantigen. Such assays are useful for the diagnosis of latent or actual autoimmune Addison's disease. These methods are briefly summarized as follows:

1. Assay Based on a Radioactive Label

Purified adrenal autoantigen is labeled with a radioactive label such as ¹²⁵ I using one of many well-known techniques. The labeled material is then incubated (1 hour at room temperature) with a suitably diluted (e.g. 1:20 in phosphate buffered saline) serum sample. Adrenal autoantibodies present in the test sample bind to the ¹²⁵ I-labeled adrenal autoantigen and the resulting complex is precipitated by addition of antibodies to human immunoglobulins or a similar reagent (e.g. solid phase Protein A). The amount of ¹²⁵ I-labelled antigen in the precipitate is then determined. The amount of adrenal autoantibody in the test serum sample is a function of the amount of radioactivity precipitated. The amount of adrenal autoantibody can be expressed as the amount of radioactivity in the pellet or more usually by including dilution of an adrenal autoantibody-positive reference serum in the assay. Note that such techniques using autoantigens such as have been identified in other diseases may be broadly applied to the detection of autoantibodies.

2. Assay Based on an Enzyme Label

Purified adrenal autoantigen is coated onto plastic wells of ELISA plates either directly onto plain wells or indirectly. The indirect method may involve coating the wells first with a monoclonal or polyclonal antibody to adrenal autoantigen (the antibody is selected so as not to bind to the same site as adrenal autoantibodies) followed by addition of adrenal autoantigen. Several other indirect coating methods are well known in the art. After coating with autoantigen, suitably diluted (e.g., 1:20 in phosphate buffered saline) test sera are added to the wells and incubated (1 hour at room temperature) to allow binding of adrenal autoantibody to the antigen coated onto the wells. The wells are then washed and a reagent such as antihuman IgG conjugated to horseradish peroxide is added. After further incubation (e.g., 1 hour at room temperature) and washing, an enzyme substrate such as orthophenylene diamine is added and the color generated measured by light absorbance. The amount of adrenal autoantibody in the test sample is a function of the final color intensity generated. Results are expressed as light absorbance or, more usually, by including dilution of an adrenal autoantibody positive reference serum in the assay.

Ulcerative Colitis and Crohn's Disease

A number of human diseases result in the subject having a diseased gut in which digestion or absorption is impaired. Examples of autoimmune diseases in humans include chronic ulcerative gut diseases (e.g., ulcerative colitis) and inflammatory gut diseases such as colitis and Crohn's disease.

In addition to impaired digestion and inflammation and/or ulteration of the intestinal tract, symptoms include pain, bleeding, abnormal stool production and weight loss. Such symptoms may be assessed either by patient interview or through techniques such as endoscopy and other imaging techniques such as heavy metal (e.g. barium enema followed by X-ray), and scanning using CAT, positron emission tomography (PET), (magnetic resonance imaging) MRI or histological analysis (biopsy).

Lupus Erythematosus

As described by U.S. Pat. Nos. 5,695,785 and 5,700,641, and briefly summarized here, lupus erythematosus is an autoimmune disease which is not specific to a particular organ. The common type of lupus erythematosus, Discoid Lupus Erythematosus (DLE), affects exposed areas of the skin. The more serious and fatal form of the disease, Systemic Lupus Erythematosus (SLE), affects a large number of organs and has a chronic course with acute episodes. The external manifestations of SLE are lesions on the facial skin. In most cases, other areas of skin and the mucosa are affected. Also observed are nephritis, endocarditis, hemolytic anemia, leukopenia and involvement of the central nervous system.

Many immunological phenomena have been observed with SLE. For example, the formation of antibodies against certain endogenous antigens has been seen. These antibodies are directed against, for example, the basement membrane of the skin, and against lymphocytes, erythrocytes and nuclear antigens. Antibodies which are directed against double-stranded DNA (ds-DNA) form with the latter complexes. These antibodies, together with complement, are deposited on small blood vessels and frequently result in vasculitis. These deposits are especially dangerous when they occur in the renal glomeruli because they result in glomerulonephritis and kidney failure. The incidence of clinically detectable kidney involvement is reported in the literature to be between 50 and 80%.

Of the multitude of autoreactive antibodies that spontaneously arise during the disease, high levels of circulating autoantibodies to DNA are the best evidence of the pathogenesis. In SLE, there is almost invariable presence in the blood of antibodies directed against one or more components of cell nuclei. Certain manifestations in SLE seem to be associated with the presence of different antinuclear antibodies and genetic markers, which have suggested that SLE may be a family of diseases (Mills, 1994, Medical Progress, 33: 1871-1879). Lupus nephritis, especially diffuse proliferative glomerulonephritis, has been known to be associated with circulating antibodies to double stranded (native) DNA (Casals et al., 1964, Arthritis Rheum., 7: 379-390; Tan et al., 1964, J. Clin. Invest., 82: 1288-1294). The detection of antinuclear antibodies is a sensitive screening test for SLE. Antinuclear antibodies occur in more than 95% of patients (Hochberg, 1990, Rheum. Dis. Clin. North Am., 16: 617-639). Such autoantibodies may be detected using DNA or other cellular components (such as small nuclear ribonucleoprotein complexes) by the methods described above.

Sjogren's Syndrome

Tear film dysfunctions are collectively diagnosed as keratoconjunctivitis sicca (KCS) or, simply, dry eye (Holly et al., 1987, Internat. Opthalmol. Clin., 27: 2-6; Whitcher, 1987, Internat. Opthalmol. Clin., 27: 7-24). Lacrimal gland abnormalties falling into the category of aqueous tear deficiencies, which are most frequently responsible for dry eye states, include autoimmune disease. By far, the greatest single cause of KCS worldwide, excluding those countries wherein trachoma remains epidemic, is Sjogren's syndrome (Whitcher, 1987, supra). This syndrome which is the second most common autoimmune disease (Tabbara, 1983, "Sjogren's Syndrome" in The conrnea. Scientific Foundations and Clinical Practice, Smolin and Thoft, eds., Little Brown and Co., Boston, Mass., pp. 309-314; Daniels, 1990, "Sjogren's Syndrome--in a nut shell" in Sjogren's Syndrome Foundation Inc. Report, Port Washington, N.Y.). This disease occurs almost exclusively in females and is characterized by an insidious and progressive lymphocytic infiltration into the main and accessory lacrimal glands, an immune mediated extensive destruction of lacrimal acinar and ductal tissues and the consequent development of persistent KCS (Tabbara, 1983, supra; Moutsopoulos and Talal, 1987, in Sjogren's Syndrome Clinical and Immunological Aspects, Talal et al., eds., Springer Verlag, Berlin, pp. 258-265; Talal and Moutsopoulos, 1987, in Sjogren's Syndrome. Clinical and Immunological Aspects, Talal et al., eds., Springer Verlag, Berlin, pp. 291-295; Kincaid, 1987, in Sjogren's Syndrome. Clinical and Immunological Aspects, Talal et al., eds., Springer Verlag, Berlin, pp. 25-33). In primary Sjogren's syndrome, which afflicts about 50% of the patient population, the disease is also associated with an immunological disruption of the salivary gland and pronounced xerostomia In secondary Sjogren's, the disorder is accompanied by another autoimmune disease, which is most often rheumatoid arthritis and, less frequently, systemic lupus.

Dryness of the eyes, infiltration of lymphocytes into the lacrymal glands and the presence of autoantibodies are diagnostic criteria for Sjogren's disease that are of use in the invention. The restoration one, more than one or even all of these indices to the basal state is indicative of effective treatment.

Type I Diabetes

Insulin dependent diabetes mellitus (IDDM) (also known as type I diabetes) primarily afflicts young people. Although insulin is available for treatment the several-fold increased morbidity and mortality associated with this disease require the development of early diagnostic and preventive methods, as well as methods for the restoration of normal insulin secretion (e.g., with islet therapy or regeneration os endogenous islets by methods described in detail below). As described in U.S. Pat. No. 5,691,448 and summarized briefly herein, the disappearance of pancreatic .beta.-cells (which are the insulin-secreting cells of the islets of Langerhans) precedes the clinical onset of IDDM. Among the most thoroughly studied autoimmune abnormalities associated with the disease is the high incidence of circulating .beta. cell-specific autoantibodies years prior to frank hyperglycemia, the typical clinical diagnosis. Family studies have shown that the autoantibodies appear prior to overt IDDM by years, suggesting a long prodromal period of humoral autoimmunity before clinical symptoms emerge, and have also documented a slow, progressive loss of insulin response to intravenous glucose in the years preceding diagnosis. The presence of .beta. cell-specific autoantibodies in the prediabetic period allows for diagnosis according to the invention prior to critical .beta.-cell depletion and insulin dependency. It has been estimated that only 10% of the total .beta.-cell mass remains at the time of clinical onset (i.e., presentation of elevated blood glucose levels relative to those observed in unaffected individuals, who represent the basal state, as defined above).

The target of autoantibodies in pancreatic .beta.-cells in IDDM were originally identified as both insulin and a 64 kD autoantigen by immunoprecipitation experiments using detergent lysates of human islets (Baekkeskov et al., 1982, Nature, 298: 167-169). Antibodies to the 64 kD autoantigen precede the clinical onset of IDDM and have been shown to have an incidence of about 80% at clinical onset and during the prediabetic period (Baekkeskov et al., 1987, J. Clin. Invest., 79: 926-934; Atkinson et al., 1990, Lancet 335: 1357-1360; and Christie et al., 1988, Diabetologia, 31: 597-602. Many other autoantibodies exist, most directed against intracellular proteins.

A therapeutic agent is administered to a patient suspected of suffering- or suffering from established diabetes in an amount suffcient to inhibit or prevent further .beta.-cell destruction/death. For individuals at risk of IDDM or stiff man syndrome, the pharmaceutical agent is administered prophylactically in an amount sufficient to either prevent or inhibit destruction and death of the 0-cell. According to the invention, a therapeutic agent is administered in an amount and for a time sufficient to prevent or inhibit .beta. cell destruction; .beta. cell survival, as judged by immunological detection of insulin, the level of serum glucose levels or restoration of vigorous insulin stimulation to glucose challenge (intravenous glucose tolerance test, or IVGTT; Joslin, 1985, Diabetes Mellitus, 20th Edition, eds. Marble et al., Lea & Febiger, Philadelphia, Pa.), is indicative of effective treatment.

Multiple Sclerosis

The symntoms of multiple sclerosis, such as those described in Treatment of Multiple Sclerosis: Trial Design, Results and Future Perspectives, eds. Rudick and Goodkin, Springer-Verlag, N.Y., 1992 (particularly those symptoms described on pages 48-52), incorporated by reference as if fully set forth herein.

These multiple sclerosis symptoms include perturbations of pyramidal functions, for example the developement of paraparesis, hemiparesis, monoparesis, quadriparesis and the developement of monoplegia, paraplegia, quadriplegia, and hemiplegia. The symptoms of multiple sclerosis also include perturbations in cerebellular functions. These perturbations include the developement of ataxia, including truncal and limb ataxia. When we refer to "paralytic symptoms of multiple sclerosis" we are refering to these perturbations in pyramidal and cerebellar funtions. The symptoms of multiple sclerosis also include changes in brain stem funtions, including development of nystamus and extraocular weakness along with dysarthria. Further symptoms include loss of sensory function including decrease in touch or position sense and loss of sensation in limbs. Perturbations in bowel and bladder function, including hesitancy, urgency, retention of bowel or bladder or incontinence, can also occur. Visual funtions, such as the development of scotoma, are also affected by multiple sclerosis. Cerebral function degeneration, including a decrease in mentation and the developemnt of dementia, is also a symptom.

Inflamed MS and EAE (see below) lesions, but not normal white matter, sometimes have infiltrating CD4 T cells that respond to self antigens presented by MHC class II-linked molecules like human HLA-DR2 (MS) or murine I-A^M (EAE). The infiltrating CD4 Tcells (Th1 cells) produce proinflammatory cytokines interleukin(IL)-2, interferon (IFN)-.gamma., and tumor necrosis factor (TNF)-.alpha. that activate antigen-presenting cells like macrophage to produce inflammatory cytokines (IL-1.beta., IL-6, and IL-8) and IL-12. The I-12 induces further IFN-.gamma. synthesis. The imbalance of one or more of these proteins relative to other cellular factors may be assayed by biochemical or immunological methods as are known in the art. Such methods are described below.

The disclosure of the present invention of poor NF.kappa.B function inside cells of autoimmune mammals implicates decreased resistance of target tissues to such inflammatory cytokine insults.

To evaluate whether a patient is benefitting from treatment, the patient's symptoms are examined in a quantitative way, such as by the EDSS (Rudick and Goodkin, supra), or decrease in the frequency of relapses, or increase in the time to sustained progression, or improvement in the magnetic resonance imaging (MRI) behavior in frequent, serial MRI studies and compare the patient's status measurement before and after treatment. In a successful treatment, the patient status will have improved, ie., the EDSS measurement number or frequency of relapses will have decreased, or the MRI scans will show less pathology.

Preferably, treatment should continue as long as multiple sclerosis symtoms are suspected or observed.

Rheumatoid Arthritis

In rheumatoid arthritis, the main presenting symptoms are pain, stiffness, swelling, and loss of function (Bennett, 1984, "The etiology of rheumatoid arthritis" in Textbook of Rheumatology, Kelley et al., eds., W. B. Saunders, Philadelphia, pp. 879-886). The multitude of drugs used in controlling such symptoms seems largely to reflect the fact that none is ideal. Although there have been many years of intense research into the biochemical, genetic, microbiological, and immunological aspects of rheumatoid arthritis, its pathogenesis is not completely understood, and none of the treatments clearly stop progression of joint destruction (Harris, 1985, "Rheumatoid Arthritis: The clinical spectrum" in Textbook of Rheumatology, Kelley. et al., eds., W. B. Saunders, Philadelphia, pp. 915-990).

TNF-.alpha. is present in rheumatoid joint tissues and synovial fluid at the protein and mRNA level (Buchan et al., 1988, Clin. Exp. Immunol., 73: 449-455), indicating local synthesis. Detection of this protein by methods described herein below (e.g. enzyme immunoassay, EIA, or enzyme-linked immunosorbent assay, ELISA) provides a diagnotic indicator of arthritis independent of clinical symptoms. In addition, autoantibodies may be quantified as described above.

Analysis of improvement in individual patients following treatment is made using two separate indices. Firstly, an index of disease activity (IDA) is calculated for each time point according to the method of Mallya and Mace (Mallya et al., 1981, Rheumatol. Rehab., 20: 14-17, the contents of which are fully incorporated herein by reference) with input variable of morning stiffniess, pain score, Richie Index grip strength, ESR and Hgb. The second index calculated was that of Paulus (Paulus et al., 1990, Arthritis Rheum., 33: 477-484, the contents of which are fully incorporated herein by reference) which uses input variables of morning stiffness, ESR, joint pain/tenderness, joint swelling, patient's and Physician's global assessment of disease severity.

Rheumatoid factors may be measured using the rheumatoid arthritis particle agglutination assay (FAPA, FujiBerio Inc., Tokyo, Japan), in which titers of 1/160 or greater are considered significant. Rheumatoid factors are measured by ELISA (e.g. using a kit supplied by Cambridge Life Sciences, Ely, UK).

Hashimoto's Disease (Hypothyroidism)

Symptoms include low levels of circulating thryoid hormone, tiredness, yellow skin discoloration, delayed reflexes, slowed heartrate, with eventual edema leading to coma and death.

Graves's Disease (Hyperthyroidism)

Symptoms include high levels of circulating thyroid hormone, hyperactivity, inability to sleep, thinning hair, irritable bowel and orbital abnormality (protruding eyes).

Vitiligo

This disorder is characterized by melanocyte loss in a characteristic pattern on the body. It is initially diagnosed; as is true of other autoimmune diseases affecting the skin (see "psoriasis" and "pemphigus vulgaris", below), tissue biopsy is performed to confirm diagnosis.

Psoriasis

The symptom of psoriasis, also present for visual diagnosis, is scaly skin.

Pemphigus Vulgaris

Symptoms of pemphigus vulgaris include skin peeling and scaling. It, too, is diagnosed visually and by skin biopsy.

In addition, genetic diagnosis of autoimmune disease, which is an effective means of early diagnosis, is possible for diseases for which genetic linkage (pedigree) studies have been performed for large (or, alternatively, small but numerous) families of affected individuals. Early diagnosis may, additionally, be facilitated by the simple assay of NF.kappa.B activity in individuals deemed to be at risk of disease; methods by which NF.kappa.B are described herein, and include in, vitro DNA/protein binding and/or transcriptional activation assays.

In order to ensure the safety of treatments according to the invention, following treatment of arthritis or another autoimmune disease, vital signs are recorded at intervals for up to 24 hours following administration of the therapeutic agent. Patients are later questioned concerning possible adverse events before each treatment. Preferably, a complete physical examination is performed at the time of initial diagnosis. In addition, patients may be monitored by standard laboratory tests including complete blood count, C3 and C4 components of complement, IgG, IgM and IgA, serum electrolytes, creatinine, urea, alkaline phosphatase, aspartate transaminase and total bilirubin. Urine analysis may, additionally, be performed.

Prior to testing potential therapeutic compositions and methods on human subjects, testing is performed in an animal model. It is generally accepted by those of skill in the art that results obtained through the use of animal models are predictive of the efficacy of a given treatment in a human clinical patient. The following section describes a selection of animal models which are of use in assessing the efficacy of proposed treatments of autoimmune disease according to the invention.

Animal Models of Autoimmune Disease

i. Mouse Models

Animal models such as the NOD³ (or, simply, NOD) mouse, which is prone to diabetes, Sjogren's syndrome and hemolytic anemia have also demonstrated the importance of the H2 (again, the mouse MHC) genomic region, in combination with non-H2 genes in autoimmunity. The inheritance of MHC and MHC-linked genes with minimal recombinations (linkage disequilibrium), together with the fact that most of these genes contribute to immune responses, has hampered the identification of the genes that underlie autoimmunity. Polymorphisms are abundant in the MHC and are readily detected but the challenge remains to identify those polymorphisms that contribute to disease susceptibility and have functional consequences, and to define the disease-causing mechanisms.

NOD mice, like humans with type I diabetes, exhibit a phenotype in which conformationally abnormal forms of class I molecules (which can be detected with conformationally specific antibodies) are present on the surface of APCs (Faustrnan et al., 1992, supra). The exit of class I molecules from the endoplasmic reticulum (ER) of NOD mouse APCs is delayed, and the presentation of test antigens by these cells is markedly impaired in in vitro assays of cytotoxic T cell lysis (Li et al., 1994, supra). Surface class I molecules of NOD mouse APCs can be stabilized by culture at low temperature or by the addition of allele-specific peptides that presumably occupy the empty peptide-binding pockets of the class I protein.

Impaired antigen presentation and class I assembly may be essential for disease expression in diabetes-prone NOD mice and humans. Only NOD females who progress to hyperglycemia or salivary gland destruction possess the defect; normoglycemic NOD males, 15% of which develop diabetes, lack the APC defect.

The NOD mouse exhibits a rare MHC haplotype known as H-2⁸^.sup.7 , in which many polymorphisms are apparent (Hattori et al., 1986, supra; Lund et al., 1990, J. Autoimmun., 3: 289; Prochazka et al., 1987, Science, 237: 286; Acha-Orbea and McDevitt, 1987, Proc. Natl. Acad. Sci. U.S.A., 84: 2435). For instance, the NOD mouse has a rare Tap1 allele with a transcription defect (Faustman et al., 1991, supra), an uncommon Lmp2 allele with a transcription defect, and a unique MHC class II gene at the I-A locus. The quantitative defect in Tap1 transcription, like the class I cell surface assembly abnormality, correlates with disease expression in NOD mice, again demonstrating a pattern of gene expression that can be influenced by the environment (Huang et al., 1995, Diabetes, 44: 1114), gender or noninherited gene phenomena (e.g. somatic gene rearrangements or changes in gene methlyation pattern). Many of these genes have similar promoters and respond in unison to external stimuli. In the case of Tap1 and Lmp2, the genes even share the same promoter in opposing orientations. Therapies based on nonspecific immunostimulation, such as injection with CFA or infection with mouse hepatitis virus, ameliorate diabetes in NOD mice. These treatments also increase the rate of Tap1 transcription, and re-educated or reselected the 7 cell repertoire so that T cell autoreactivity to class I and syngeneic peptides is eliminated (Huang et al., 1995, supra). These data suggest transcription or quantitative issues of gene expression could be dominant in patterns of disease expression.

As in humans, lymphocytic developmental errors are characteristic of mouse (NOD) and rat (BB; see below) models of Type I diabetes (Shimada et al., 1996, Diabetes, 45: 71-78; Serreze et al., 1993, Proc. Natl. Acad. Sci. U.S.A., 90: 9625-9629; Li et al., 1994, Proc. Natl. Acad. Sci. U.S.A., 91: 11128-11132). For instance, mature T lymphocytes in peripheral blood, spleen and lymph nodes are markedly absent in autoimmune disease-prone BB animals (Crisa et al., 1992, Diabetes Metaholism Rev., 8: 9-37). As might be expected of an immature lymphoid cell, diabetic lymphocytes in animal and human models demonstrate defective intracellular activation of signal transduction pathways, including responses to TNF, lipopolysaccharides (LPS, which are non-specific immunostimulants) and signal transduction along the microtubule-associated protein kinase (MAP kinase) pathway of T cell activation (Serreze et al., 1993, supra; Rapoport et al., 1993, J. Exp. Med., 177: 1221-1226).

Given the established role of antigen presentation in T cell education and its impairment in numerous autoimmune diseases in both humans and mice, mutations which contribute to the abnormal antigen presentation and processing in the NOD mouse (made apparent, in part, by altered class I assembly and altered presentation of syngeneic peptides) are of significant interest; therefore, the NOD mouse provides a good model system in which genetic and environmental factors influencing autoimmune diseases can be studied. Recently, a mutation in the shared, bidirectional Lmp2/Tap1 promoter has been found to reduce expression of these genes in the NOD mouse (Yan et al., 1997, J. Immunol., 159: 3068-3080)

ii. The BB Rat

Diabetes-prone BB rats have profound peripheral T lymphocyte immunodeficiencies and lack a surface maturation molecule or lymphocytes RT6, a member of the src tyrosine kinase family (Elder and Maclaren, 1983, J. Immunol., 130: 1723-1731; Rigby et al., 1996, Diabetes, 45: 1419-1426; Jackson et al., 1983, Metabolism, 32: 83-86; Woda et al., 1986, J. Immunol., 136: 856-859; Greiner et al., 1986, J. Immunol., 136: 148-151).

iii Other Models

Other animal models of autoimmune disease as are known in the art are as follows:

Experimental autoimmune encephalomyelitis (EAE) in mice and rats serves as a model for multiple sclerosis (M.S.) in humans. It is a CD4⁺ T-cell mediated autoimmune disease that is directed against protein components of CNS myelin (Miller and Karpus, supra, 1994). In this model, the demyelinating disease is induced by administration, typically by injection, of myelin basic protein (MBP), as described by Paterson, P. Y. (1986, Textbook of Immunopathology, eds. Mischer et al., Grune and Stratton, New York, pp. 179-213), McFarlin et al. (1973, Science, 179: 487480) and Satoh et al. (1987, J. Immunol., 138: 179-184). B10.PL mice are known to have histopathological and clinical similarities to the relapsing-remitting form of human M.S. (Miller and Karpus, 1994, Immun. Today, 15: 356); these mice develop EAE in response to injection with MBP. EAE is characterized by transient asscending paralysis of the affected mouse's limbs.

Systemic lupus erythematosis (SLE) is tested in susceptible mice as disclosed by Knight et al. (1978, J. Exp. Med., 147: 1653). Myasthenia gravis (MG) is tested in SJL/J female mice by inducing the disease with soluble acetyl-cholinesterase receptor (AChR) protein from another species, as described by Lindstrom et al., (1988, Adv. Immunol., 42: 233-284). Arthritis is induced in a susceptible strain of mice by injection of type II collagen, as described by Stuart et al., (1984, Ann. Rev. Immunol., 42: 233-284). Thyroiditis is induced in mice by administration of thyroglobulin as described by Maron et al., (1980, J. Exp. Med., 152: 1115-1120). Insulin-dependent diabetes mellitus (IDDM) occurs naturally or can be induced in certain strains of mice.

The contents of the above references relating to animal models of autoimmune disease are all herein fully incorporated by reference.

NF.kappa.B

i. Activation

Rather than treating defects in proteolytic processing at the stage of the proteolytic processing, it is possible to target treatment according to the invention at the restoration of an important downstream target of proteasome activation, the transcription factor, NF.kappa.B and/or its downstream targets.

NF.kappa.B is a heterodimeric transcription factor composed of 50 and 65 kD subunits that belong to the rel family; it is present with inhibitory factor I.kappa.B in the cytoplasm of most cells (Baeuerle and Henkel, 1994, Ann. Rev. Immunol., 12: 141-179; Verma et al., 1995, Genes Dev., 9; 2723-2735). This transcription factor is responsive to cell surface cytokines, such as tumor necrosis factor .alpha., interleukin-1 and cytoplasmic activation of this factor is required prior to nuclear localization. NF.kappa.B plays an active role in lymphocytic development and in cell survival (Wang et al., 1996Science, 274: 784-787; Beg and Baltimore, 1996, Science, 274: 782-784; Van Antwerp et al., 1996, Science, 274: 787-789; Arsura et al., 1997, Cell Growth Differ., 8: 1049-1059; Liu et al., 1996, Cell, 87: 565-576). In B cells, NF.kappa.B is constitutively expressed (Wu et al., 1996, EMBO J., 15: 4682-4690). Knock-out mice missing Re1A (p65) die before birth, in part, due to a described developmental defect of the immune system (macrophages, B and T cells) and massive death of liver cells (Arsura et al., 1997, supra; Beg et al., 1995, Nature, 376: 167-170; Bargou et al., 1997, J. Clin. Invest., 100: 2961-2969). In vitro inhibition of NF.kappa.B induces similar developmental arrest and death of B cells (Liu et al., 1996, supra).

In the NF.kappa.B pathway, it has been observed that phosphorylation and ubiquitination work in concert to transmit a message to the nucleus and to activate the cell-cycle genes and proteins in the cytoplasm, thus activating cell signalling, division, development (e.g., differentiation) and proliferation; stimulating the the human epithelial HeLa cell line with TNF-.alpha. switches on a stress-activated MAP (mitogen-activated protein) cascade that promotes the phosphorylation of I.kappa.B.alpha. kinase (Lee et al., 1997, Cell, 88: 213-222). The kinase, in turn, phosphorylates the NF.kappa.B inhibitor protein I.alpha.B.kappa.marking it for ubiquitination. In unstimulated cells, I.kappa.B binds to- and inhibits the activity of NF.kappa.B. When ubiquitinated I.kappa.B is degraded by the proteasome, NF.kappa.B translocates to the nucleus where it activates transcription. As is stated in Hopkin (1997, supra), the combination of two highly specific processes, phosphorylation and ubiquitination, has been utilized by cells to control complex signal-transduction pathways precisely. Such a mechanism which allows for a rapid return to normal is critical in the activation and de-activation of molecules such as cytokines, which are said to act transiently, as constitutive activation would be cytotoxic.

Cell surface signals on lymphocytes activate NF.kappa.B through cascades of kinases (Verma et al., 1995, supra; Baeuerle and Baltimore, 1996, Cell, 87: 13-20). A previous report shows a possible association of NF-.kappa.B with a cellular serine kinase, resulting phosphorylation and activation of NF-.kappa.B (Ostrowski et al., 1991, J. Biol. Chem., 266: 12722-12733; Hayashi et al., 1993, J. Biol. Chem., 268: 26790-26795). NF.kappa.B also can interact with cyclin dependent kinases (Cdk), phosphorylation steps regulating cell cycle progression and conveyance of signals for differentiation and apoptosis. Specifically, Cdk8 or Cdk7 (in combination with cyclins) coordinate the metabolism of differentiated cells with extracellular stimuli and regulate transcriptional activation.

ii Activity in the Nucleus

NF.kappa.B and other members of the rel family of protein complexes play a central role in the transcriptional regulation of a remarkably diverse set of genes involved in the immune and inflammatory responses (Grilli et al., 1993, Int. Cytology, 143: 1-62). For example, NF.kappa.B is required for the expression of a number of immune response genes, the Ig-.kappa. light chain immunoglobulin gene, the IL-2 receptor a chain gene, the T cell receptor .beta. chain gene, and class I and II major histocompatibility genes. In addition, NF.kappa.B has been shown to be required for a number of genes involved in the inflammatory response, such as the TNF-.alpha. gene and the cell adhesion genes, E-selectin, I-cam, and V-cam. NF.kappa.B is also required for the expression of a large number of cytokine genes such as IL-2, IL-6, G-CSF, and IFN-.beta.. Finally, NF.kappa.B is essential for the expression of the human immunodeficiency virus (HIV).

iii. Role in the Cytoplasm

In addition to its role as a transcription factor, NF.kappa.B is believed mediate events occurring in the cytoplasm. Subunit p65 binds cyclin-dependent kinases (cdk's), cdc's and other cell cycle activators, which are part of a multiprotein complex; the data presented in Example 1, below, demonstrates such binding. These proteins control the cell cycle, differentiation, DNA replication and cell proliferation. It is thought that p50 may have similar binding affinities.

iv Role in Autoimmune Disease

Developmental arrest of lymphocytes has been observed in humans with type I diabetes; such an arrest often manifests itself as an increase in the number of CD45RA-naive cells (Faustman et al., 1989, Diabetes 38: 1462-1468; Faustman, 1993, Diabete Metab. 19: 446-457; Faustman et al., 1990, J. Autoimmunity, 3: 111-116; Faustman et al., 1991, Diabetes, 40: 590-597). Functional assays of antigen presentation and analysis of surface antigens on lymphocytes have confirmed the existence of diverse and immature lineages of lymphocytes in type I diabetics (Faustman et al., 1991, Science, 254: 1756-1761; Peakman et al., 1993, Lancet, 342: 1296; Peakman et al., 1994, Lancet, 343: 424; Peakman et al., 1994, Diabetes, 43: 712-717).

Regardless of the level at which an autoimmune disease is treated according to the methods of the invention, it is necessary to deliver therapeutic agents in a safe and medically expedient manner. Gene therapy provides one set of methods by which bioactive substances, such as proteins and nucleic acids, may be delivered in active form to- or synthesized at their intended sites of action. Gene therapy methods are discussed in the following section.

Gene Therapy According to the Invention

i. Therapeutic Nucleic Acids

Sequences

A therapeutic gene may be transfected for use in the invention using a viral or non-viral DNA or RNA vector, where non-viral vectors include, but are not limited to, plasmids, linear nucleic acid molecules, artificial chromomosomes and episomal vectors. Expression of heterologous genes has been observed after injection of plasmid DNA into muscle (Wolff J. A. et al., 1990, Science. 247: 1465-1468; Carson D.A. et al., U.S. Pat. No. 5,580,859), thyroid (Sykes et al., 1994, Human Gene Therapy, 5: 837-844), melanoma (Vile et al., 1993, Cancer Res., 53: 962-967), skin (Hengge et al., 1995, Nature Genet., 10: 161-166), liver (Hickman et al., 1994, Human Gene Therapy, 5: 1477-1483) and after exposure of airway epithelium (Meyer et al., 1995, Gene Therapy, 2: 450-460).

Therapeutic nucleic acid sequences useful according to the methods of the invention include those encoding receptors, enzymes, ligands, regulatory factors, and structural proteins. Therapeutic nucleic acid sequences also include sequences encoding nuclear proteins, cytoplasmic proteins, mitochondrial proteins, secreted proteins, plasmalemma-associated proteins, serum proteins, viral antigens, bacterial antigens, protozoal antigens and parasitic antigens. Therapeutic nucleic acid sequences useful according to the invention also include sequences encoding proteins, lipoproteins, glycoproteins, phosphoproteins and nucleic acids (e.g., RNAs such as ribozymes or antisense nucleic acids). Proteins or polypeptides which can be expressed using the methods of the present invention include hormones, growth factors, neurotransmitters, enzymes, clotting factors, apolipoproteins, receptors, drugs, oncogenes, tumor antigens, tumor suppressors, structural proteins, viral antigens, parasitic antigens and bacterial antigens. The compounds which can be incorporated are only limited by the availability of the nucleic acid sequence encoding a given protein or polypeptide. One skilled in the art will readily recognize that as more proteins and polypeptides become identified, their corresponding genes can be cloned into the gene expression vector(s) of choice, administered to a tissue of a recipient organism such as a mammalian tissue (including human tissue), and expressed in that tissue.

Therapeutic sequences according to the invention may encode products which restore proteasome activity; such genes are referred to as being `upstream` of NF.kappa.B. For example, gene expression constructs encoding proteasome components or associated proteins (e.g. the Lmp2/Tap1 gene pair, or Lmp2, Lmp7, Tap1 or Tap2) comprising cDNA sequences functionally linked to the corresponding wild-type transcriptional regulatory sequences are of use. Genes which restore proper ubiquitination include those encoding members of the superfamily of ubiquitination-mediating enzymes of the classes E1, E2 and E3; as stated above, human homologues of the yeast ubiquitination enzymes have been discovered, among them the UbcH5 (which functions as an E2) and the MDM2 oncoprotein, which acts as a ubiquitin ligase, or E3 (see Honda et al., 1997, supra).

Sequences encoding wild-type NF.kappa.B subunits for use in the reconstitution of missing activity resulting from inactivating mutations in either or both of p65 and p50; genes encoding these proteins may be administered according to the invention. Genes which might compensate for a loss of proteasome function to activate NF.kappa.B by removing the need for proteasome-mediated cleavage of I.kappa.B are also of use, for example, a recombinant NF.kappa.B cDNA engineered such that its product can no longer be bound by I.kappa.B, as discussed above.

Other genes requiring activation by the proteasome encode apolipoprotein B100 (apoB), transcription factors, e.g. STAT transcription factor or DNA repair factor TFIIH, are also of use.

Genes downstream of NF.kappa.B (i.e. those which are under NF.kappa.B transcriptional control) may, themselves be expressed as cDNA constructs in a recipient host; however, this requires a knowledge of all downstream activation targets of NF.kappa.B in cells which are to receive treatment, as well as designing individual expression constructs for each such gene and ensuring that they are expressed in the proper ratios relative to one another an to other cellular proteins. As stated above, such genes include, but are not limited to, those which encode the Ig-.kappa., light chain immunoglobulin, the IL2 receptor a chain, the T cell receptor .beta. chain, class I and II major histocompatibility proteins, TNF-.alpha., E-selectin, I-cam, and V-cam, IL-2, IL-6, G-CSF, and IFN-.beta..

Nucleic acids of use in the invention include those that encode proteins for which a patient might be deficient or that might be clinically effective in higher-than-normal concentration as well as those that are designed to eliminate the translation of unwanted proteins. As discussed above, nucleic acids of use according to the invention for the elimination of deleterious proteins arc antisense RNA and ribozymes, as well as DNA expression constructs that encode them. Note that antisense RNA molecules, ribozymes or genes encoding them may be administered to a patient by a method of nucleic acid delivery that is known in the art, such as an in vivo or an ex vivo method, as described below.

Therapeutic genes of use in the invention include those whose products may suppress the function of inhibitors or other negative regulators of proteasome function. One such regulator is the 40 kD-, ATP-dependent protein mentioned above whose release from the proteasome complex permits proteolytic cleavage of target proteins to occur. Inactivating nucleic acid sequences such may encode a ribozyme or antisense RNA specific for the mRNA which encodes the 40 kD protein or, alternatively, may encode an antibody directed against the 40 kD protein or a polypeptide of like sequence with the site on the proteasome complex to which the 40 kD protein binds in vivo; such a polypeptide could, if present at several-fold molar excess (e.g. 10-fold or more) over the endogenous proteasome component bound by the 40 kD species, serve as to compete the inhibitory protein off of it. Note that the 40 kD proteasome regulator is said to exist as a 250 kD multimer when released (see again WO 95/25533). Japanese patent JP 95121484 discloses a non-functional mutant of this protein which may be of use to titrate functional 40 kD molecules away from the proteasome complex.

In addition to the need to suppress the activity of inhibitors of proteasome function, it may be equally necessary to suppress that of proteins normally targeted for inactivation by the proteasome. These include oncogene c-Fos, ornithine decarboxylase, tyrosine aminotransferase, c-myb, HMG-R (a key enzyme of sterol synthesis) and apoB (also activated by proteasomes).

Successful methods for the therapeutic administration of antibodies for the treatment of autoimmune disease (in this case, rheumatoid arthritis) have been disclosed in U.S. Pat. No. 5,698,195, the contents of which are herein incorporated by reference.

Ribozymes of the hammerhead class are the smallest known, and lend themselves both to in vitro synthesis and delivery to cells (summarized by Sullivan, 1994, J. Invest. Dermatol., 103: 85S-98S; Usman et al., 1996, Curr. Opin. Struct. Biol., 6: 527-533).

Physical Properties and Delivery Vehicles

A nucleic acid of use according to the methods of the invention may be either double- or single stranded and either naked or associated with protein, carbohydrate, proteoglycan and/or lipid or other molecules. Such vectors may contain modified and/or unmodified nucleotides or ribonucleotides. Examples of some therapeutic nucleic acid sequences are enumerated above. In the event that the gene to be transfected is without its native transcriptional regulatory sequences, the vector must provide such sequences to the gene, so that it can be expressed once inside the target cell. Such sequences may direct transcription in a tissue-specific manner, thereby limiting expression of the gene to its target cell population, even if it is taken up by other surrounding cells. Alternatively, such sequences may be general regulators of transcription, such as those that regulate housekeeping genes, which will allow for expression of the transfected gene in more than one cell type; this assumes that the majority of vector molecules will associate preferentially with the cells of the tissue into which they were injected, and that leakage of the vector into other cell types will not be significantly deleterious to the recipient mammal. It is also possible to design a vector that will express the gene of choice in the target cells at a specific time, by using an inducible promoter, which will not direct transcription unless a specific stimulus, such as heat shock, is applied.

Delivery of a nucleic acid may be performed using a delivery technique selected from the group that includes, but is not limited to, the use of viral vectors and non-viral vectors, such as episomal vectors, artificial chromosomes, liposomes, cationic peptides, tissue-specific cell transfection and transplantation, administration of genes in general vectors with tissue-specific promoters, etc.

ii. Dosage

Generally, nucleic acid molecules are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective. When the end product (e.g. an antisense RNA molecule or ribozyme) is administered directly, the dosage to be administered is directly proportional to the the amount needed per cell and the number of cells to be transfected, with a correction factor for the efficiency of uptake of the molecules. In cases in which a gene must be expressed from the nucleic acid molecules, the strength of the associated transcriptional regulatory seuqences also must be considered in calculating the number of nucleic acid molecules per target cell that will result in adequate levels of the encoded product. Suitable dosage ranges are on the order of, where a gene expression construct is administered, 0.5- to 1 .mu.g, or 1-10 .mu.g, or optionally 10-100 .mu.g of nucleic acid in a single dose. It is conceivable that dosages of up to 1 mg may be advantageously used. Note that the number of molar equivalents per cell vary with the size of the construct, and that absolute amounts of DNA used should be adjusted accordingly to ensure adequate gene copy number when large constructs are injected.

iii. Administration

Nucleic acid molecules to be administered according to the invention also may be formulated in a physiologically acceptable diluent such as water, phosphate buffered saline, or saline, and further may include an adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are materials well known in the art. Administration of a nucleic acid molecule as described herein may be either localized or systemic.

Localized Adminstration

It is contemplated that global administration of a therapeutic composition to an animal is not needed in order to achieve a highly localized effect. Localized administration of a nucleic acid is preferably by via injection or by means of a drip device, drug pump or drug-saturated solid matrix from which the nucleic acid can diffuse implanted at the target site. When a tissue that is the target of treatment according to the invention is on a surface of an organism, topical administration of a pharmaceutical composition is possible. For example, antibiotics are commonly applied directly to surface wounds as an alternative to oral or intravenous administration, which methods necessitate a much higher absolute dosage in order to counter the effect of systemic dilution, resulting both in possible side-effects in otherwise unaffected tissues and in increased cost.

Compositions comprising a therapeutic composition which are suitable for topical administration can take one of several physical forms, as summarized below:

(i) A liquid, such as a tincture or lotion, which may be applied by pouring, dropping or "painting" (i.e. spreading manually or with a brush or other applicator such as a spatula) or injection.

(ii) An ointment or cream, which may be spread either manually or with a brush or other applicator (e.g. a spatula), or may be extruded through a nozzle or other small opening from a container such as a collapsible tube.

(iii) A dry powder, which may be shaken or sifted onto the target tissue or, alternatively, applied as a nebulized spray.

(iv) An liquid-based aerosol, which may be dispensed from a container selected from the group that comprises pressure-driven spray bottles (such as are activated by squeezing), natural atomizers (or "pump-spray" bottles that work without a compressed propellant) or pressurized canisters.

(v) A carbowax or glycerin preparation, such as a suppository, which may be used for rectal or vaginal administration of a therapeutic composition.

In a specialized instance, the tissue to which a therapeutic composition is the lung. In such a case the route of administration is via inhalation, either of a liquid aerosol or of a nebulized powder of Drug delivery by inhalation, whether for topical or systemic distribution, is well known in the art for the treatment of asthma, bronchitis and anaphylaxis. In particular, it has been demonstrated that it is possible to deliver a protein via aerosol inhalation such that it retains its native activity in vivo (see Hubbard et al., 1989, Clin. Invest., 84: 1349-1354).

Note that in some cases, the surface in question is internal, for example, the gastric lining; in such a case, topical application would comprise taking the therapeutic composition via an oral route, whether in liquid, gel or solid form.

Systemic Administration

Systemic administration of a nucleic acid or other therapeutic composition according to the invention may be performed by methods of whole-body drug delivery are well known in the art. These include, but are not limited to, intravenous drip or injection, subcutaneous, intramuscular, intraperitoneal, intracranial and spinal injection, ingestion via the oral route, inhalation, trans-epithelial diffusion (such as via a drug-impregnated, adhesive patch) or by the use of an implantable, time-release drug delivery device, which may comprise a reservoir of exogenously-produced nucleic acid or other material or may, instead, comprise cells that produce and secrete a therapeutic protein or other agent (see "Ex vivo therapy", below). Note that injection may be performed either by conventional means (i.e. using a hypodermic needle) or by hypospray (see Clarke and Woodland, 1975, Rheumatol. Rehabil., 14: 47-49).

Systemic administration is advantageous when a pharmaceutical composition must be delivered to a target tissue that is widely-dispersed, inaccessible to direct contact or, while accessible to topical or other localized application, is resident in an environment (such as the digestive tract) wherein the native activity of the nucleic acid or other agent might be compromised, e.g. by digestive enzymes or extremes of pH.

Nucleic acid constructs of use in the invention can be given in a single- or multiple dose. A multiple dose schedule is one in which a primary course of administration can include 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the cellular level of the transfected nucleic acid. Such intervals are dependent on the continued need of the recipient for the therapeutic nucleic acid, the ability of a given nucleic acid to self-replicate in a mammalian cell if it does not become integrated into the recipient's genome and the half-life of a non-renewable nucleic acid (e.g. a molecule that will not self-replicate). Preferably, when the medical needs of the recipient mammal dictate that a nucleic acid or a product thereof will be required throughout its lifetime, or at least over an extended period of time, such as a year or more, a nucleic acid may be encoded by sequences of a vector that will self-replicate in the target cells. The efficacy of transfection and subsequent maintenance of the nucleic acid molecules may be assayed either by monitoring the activity of a marker gene, which may additionally be comprised by the transfected construct, or by the direct measurement of either the protein product encoded by the gene of interest or the reduction in the levels of a protein the production of which it is designed to inhibit. The assays can be performed using conventional molecular and biochemical techniques, such as are known to one skilled in the art.

Ex Vivo Therapy

As alluded to earlier, it is possible to administer a therapeutic nucleic acid for use not only in in vivo therapy (i.e., that in which a nucleic acid is administered directly to a patient for uptake by- and subsequent expression in cells in situ) but also in ex vivo therapy (i.e., that in which a nucleic acid is administered to cultured or explanted cells in vitro, which transfected cells are subsequently transplanted into the clinical patient in order to supply a therapeutic product). Methods of ex vivo gene therapy are described in detail herein By these methods, a plasmid which continues to be maintained in a transformed or transfected cell after such a cell has been administered (e.g. via transplantation) to a multicellular host, such as a mammal, delivers a gene product to that individual. It is contemplated that a gene of interest, particularly a therapeutic gene, will be expressed by the transplanted cell, thereby providing the recipient organism particularly a human, with a needed RNA (e.g., an antisense RNA or ribozyme) or protein.

As discussed above, a cell type may be used according to the invention which is amenable to methods of nucleic acid transfection such as are known in the art. Such cells may include cells of an organism of the same species as the recipient organism, or even cells harvested from the recipient organism itself for ex vivo nucleic acid transfection prior to re-introduction Such autologous cell transplants are known in the art. One common example is that of bone marrow transplantation, in which bone marrow is drawn either from a donor or from a clinical patient (for example, one who is about to receive a cytotoxic treatment, such as high doses of ionizing radiation), and then transplanted into the patient via injection, whereupon the cells re-colonize bones and other organs of the hematopoietic system.

a. Cell Dosage

The number of transfected cells which are administered to a recipient organism is determined by dividing the absolute amount of therapeutic or other gene product required by the organism by the average amount of such an agent which is produced by a transfected cell. Note that steady-state plasmid copy number varies depending on the strength of its origin of replication as well as factors determined by the host cell environment, the availability of nucleotides and replicative enzyme complexes, as does the level of expression of the gene of interest encompassed by the plasmid, which level likewise is determined by the strength of its associated promoter and the availability of nucleotides and transcription factors in a given host cell background. As a result, the level of expression per cell of a given gene of interest must be determined empirically prior to administration of cells to a recipient.

While efficient methods of cell transfection and transplantation are known in the art, they do not ensure that the transfected cell is immortal. In addition, the requirements of the recipient organism for the product encoded by the transgene may change over time. In light of these considerations, it is contemplated that cells may be administered in a single dose or in multiple doses, as needed. A multiple dose schedule is one in which a primary course of administration can include 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the cellular level of the transfected nucleic acid. Such intervals are dependent on the continued need of the recipient for the therapeutic gene product. Preferably, when the medical needs of the recipient mammal dictate that a gene product will be required throughout its lifetime, or at least over an extended period of time, such as a year or more, the transfected cells will be replenished on a regular schedule, such as monthly or semi-monthly, unless such cells are able to colonize the recipient patient in permanent fashion, such as is true in the case of a successful bone-marrow cell transplant.

b. Nucleic Acid Dosage

Provided a nucleic acid vector capable of replication in the transfected cell is used, the absolute amount of nucleic acid which is transfected into cells prior to transplantation is not critical, since in cells receiving at least one copy of such a vector, the vector will replicate until an equilibrium copy-number is achieved. As a first approximation, an amount of vector equivalent to between 1 and 10 copies thereof per cell to be transfected may be used; one of skill in the art may adjust the ratio of plasmid molecules to cells as is necessary to optimize vector uptake. Of particular used in the invention are vectors or transfection techniques which result in the stable integration of the gene of interest into the chromosome of the transfected cell, so as to aviod the need to maintain selection for cells bearing the vector following transplantation into a recipient multicellular organism, such as a human.

c. Administration of Autologous or Syngeneic Cells

A cell type which is commonly transplanted between individuals of a single species (or, even, from an individual to a cell culture system and back to the same individual) is that of hematopoietic stem cells (HSCs), which are found in bone marrow; such cells have the advantage that they are amenable to nucleic acid transfection while in culture, and are, therefore, well suited for use in the invention. Cultures of HSCs are transfected with a minimal plasmid comprising an operator sequence and a gene of interest and the transfected cells administered to a recipient mammal in need of the product of this gene. Transfection of hematopoietic stem cells is described in Mannion-Henderson et al., 1995, Exp. Hematol., 23: 1628; Schiffmann et al., 1995, Blood, 86: 1218; Williams, 1990, Bone Marrow Transplant, 5: 141; Boggs, 1990, Int. J. Cell Cloning, 8: 80; Martensson et al., 1987, Eur. J. Immunol., 17: 1499; Okabe et al., 1992, Eur. J. Immunol., 22: 37-43; and Banerji et al., 1983, Cell, 33: 729. Such methods may advantageously be used according to the present invention. Administration of transfected cells proceeds according to methods established for that of non-transfected cells, as described below.

The transplantation of hematopoietic cells, such as in a bone marrow transplant, is commonly performed in the art by procedures such as those described by Thomas et al. (1975, New England J. Med., 292: 832-843) and modifications thereof Such a procedure is briefly summarized: In the case of a syngeneic gaft or of a patient suffering from an immunological deficiency, no immunosuppressive pre-treatment regiment is required; however, in cases in which a cells of a non-self donor are to be administered to a patient with a responsive immune system, an immunosuppressive drug must be administered, e.g. cyclophosphamide (50 mg/kg body weight on each of four days, with the last does followed 36 hours later by the transplant). Leukemic patients routinely receive a 1000-rad midline dose of total-body irradiation in order to ablate cancerous blood cells; this irradiation also has an immune-suppressive effect. Following pre-treatment, bone marrow cells (which population comprises a small number of pluripotent hematopoietic stem cells, or HSCs), are administered via injection, after which point they colonize the hematopoietic system of the recipient host Success of the graft is measured by monitoring the re-appearance of the numerous adult blood cell types by the immunological and molecular methods which are well known in the art. While as few as 1-10 HSCs are, in theory, able to colonize and repopulate a lethally-irradiated recipient mammal over time, it is advantageous to optimize the rate at which repopulation occurs in a human bone marrow transplant patient; therefore, a transplanted bone marrow sample comprising 10 to 100, or even 100 to 1000 HSCs should be administered in order to be therapeutically effective.

It is contemplated that both lymphoid and parenchymal cells, particularly those which are targeted for destruction in autoimmune disease, are of use in the invention. Such parenchymal include those of the islets of Langerhans, the thyroid, the adrenal cortex, muscles, cartilagenous- or other synovial tissue, the kidneys, epithelial tissues (both external and internal, particularly that of the intestinal lumen, lung, heart, liver, kidney, neurons and synovial cells) and the nervous system.

In that such cells are meant to either to replace those lost to autoimmune destruction or to provide a pool of autoimmune-resistant cells prior to massive cell death, it is necessary to ensure that such cells indeed are not susceptible to autoimmune disease. Provided that early treatment is undertaken, it is possible to harvest small (or, in some cases, large) numbers of cells of the target tissue directly from the patient for transfection and reintroduction; alternatively, cells of a donor of matching tissue type may be used.

To render the transplanted cells resistant, at least collectively, to immune rejection by the recipient organism, it is contemplated that transplanted cells expressing a high level of activated NF.kappa.B (a high NF.kappa.B "set point"), while still subject to destruction by autoimmune host lymphocytes, would enjoy the advantage of robust proliferative capacity in order to multiply at a rate surpassing that of cell killing, thereby providing a long-lived population of therapeutic cells to the recipient organism. Such cells may be transfected with gene expression constructs which result in the production of high levels of activated NF.kappa.B, or may be cells obtained from a donor selected for high endogenous NF.kappa.B activity, as may be determined in an in vitro transcription assay or DNA/protein binding assay (as described in Example 2, below) using protein extracts drawn from such a donor, which may, itself, be a transgenic mammal.

As an alternative, a procedure has been developed which allows for the shielding of transplanted cells, even those transplanted from a members of one species to another (see also below, for other such methods). As a protective measure against viral infection, a mechanism has evolved in the immune system of vertebrates in which viral proteins being produced within the infected cells are broken down into peptides by intracellular proteolytic enzymes. Some of the peptides are enfolded by a particular class (Class I) of proteins of the major histocompatibility complex (MHC) of genes and are transported to the cell surface, where the viral peptide/MHC protein complex is displayed as a surface antigen. Circulating cytotoxic T lymphocytes (CTLs) having the appropriate specificity recognize the displayed MHC Class I antigen as foreign and proceed, through activation and a complex lytic cascade, to kill the infected cell. The MHC Class I proteins are expressed in essentially all nucleated cells of the body and are a key element in the immune system's ability to distinguish between "self" molecules and "foreign" (non-self) molecules. They can be distinguished from the other class of proteins of the major histocompatibility complex of genes, known as MHC Class II proteins.

Although MHC Class I antigens are a magnificent mechanism for combating infection, they also are primarily responsible for the failure of tissues, e.g., cells, organs, or parts of organs, that are transplanted from one mammal (donor) to another (host). This rejection of tissue by the host organism was first observed in mouse skin graft experiments in the 1950s and was named the transplant reaction. The search for the factor on donor cells that was evidently recognized and attacked by the host's immune system led finally to the characterization of the two classes of MHC proteins (see, Snell, 1957, Ann. Rev. Micrbiol., 2: 439-57).

Recognition of donor MHC Class I antigens as foreign by host CTLs occurs not only where the donor tissue is different from a different species (a xenogeneic transplant) but also where the tissues are from a donor of the same species as the host (an allogeneic transplant). The specificity of the T cell receptors on CTLs and other T cells that bind to Class I and Class II antigens is such that a single amino acid difference in the structure of a MHC antigen can be detected as foreign, leading to an immune response. The MHC proteins are expressed from DNA formed by rearrangement of several gene segments in the MHC loci, leading to a high degree of polymorphism in MHC proteins.

A method applicable to inhibiting the rejection of transplanted tissues mediated by recognition of MHC class I antigens is as follows: Transplanted allogeneic or xenogeneic tissue comprising treating the transplant tissue with an enzyme capable of cleaving MHC Class I antigens. Removal of Class I antigens from the donor tissue attenuates the extent of the immune response mounted by the host mammal receiving the transplant. Furthermore, the enzyme treatment is an effective preparatory treatment for all tissues intended for transplant, without regard to the specific MHC antigens displayed on the donor tissue or the specificities of the immune system cells of the host.

The method of treating tissues to render them suitable for transplant comprises incubating the donor tissue with an enzyme capable of cleaving MHC Class I antigens, e.g., in an amount and for a sufficient period to remove sufficient MHC Class I antigens to significantly attenuate the host's immune response to the donor tissue. Such incubation is performed in a medium which allows both enzymatic cleavage of the surface antigens to proceed, but is still amenable to tissue survival (e.g. a physiological salt buffer, such as PBS, or a cell-, tissue- or organ culture medium, such as are known in the art. Typically the mean cell density of Class I antigens will be reduced below about 10% of untreated levels, preferably below 1%. One such useful enzyme is papain.

The enzyme selected for use in this method must be capable of cleaving MHC Class I antigens, that is, removing a MHC Class I protein/peptide complex from the surface of a cell on which it was displayed. Useful cleavage is that which alters the MHC Class I antigen as displayed sufficiently to avoid interaction with the immune system cells of the recipient mammal; the object of this cleavage step is to remove substantially all of the extracellular portion of the MHC Class I antigen from the cell. Any amount of MHC Class I antigen that can be removed from the donor tissue is helpful in avoiding rejection of the transplant; however, as a practical matter, removal of as much of the MHC Class I antigens as possible without killing the tissue is desired, e.g. a reduction in MHC Class I density of at least 90% or even as much as 99% is desirable.

Typically, this is accomplished by bathing the donor tissue in a solution of the enzyme for a period to allow the enzyme to react with the MHC proteins, e.g., from 20 minutes to 24 hours or more. At high enzyme concentration, incubation of tissues may be for even shorter periods, so long as the cells of the tissues are not damaged. In general, a minimum of 75% viability of the tissue cells is required, although 90% viability or more is sought. In order to retard resynthesis of the MHC class I molecules, the enzyme treatment is carried out at the optimal temperature for enzyme activity, but the treated tissue is thereafter maintained at a low temperature, for example at 4^oC., until ready for use.

There are several advantages to the use of enzymes as a treatment for avoiding transplant rejection: (a) the enzymes are comparatively inexpensive, and many are commercially available in high purity with well-characterized activity and specificity; (b) enzymes can be used locally or in vitro to avoid systemic treatments; (c) enzyme shaving of the transplant tissue can be used in combination with (i.e., without foreclosing) other complementary therapies; and (d) the use of enzymes is not species-restricted or allelically restricted, and thus the method is adaptable to veterinary, human and xenogenic tissue treatment without radical modification of the procedures or reagents. Since the tissues will remain viable after treatment, expression of MHC molecules will continue, and eventually reappearance of MHC antigens on the donor tissue will occur, e.g., after transplantation; consequently, it is this method may be used as part of an overall therapy that may include additional measures to avoid rejection of the transplanted cells, such as immunosuppression, plasmaphoresis, antigen blocking, transfection, and the like. Although pre-transplantation treatment of the tissues will be the most common practice, it is also contemplated that this method of the present invention may be employed in situ to effect local immune response inhibition to preserve previously translated tissue. In such cases, cleavage of the surface antigen produces a local, soluble, competitive receptor for the cells of the host's immune system, which may serve to effectively blunt immune attack on the transplanted tissue.

Useful enzynes include proteolytic enzymes, gycosidases, proteinases and combinations of such enzymes that may sufficiently alter the surface antigens to inhibit subsequent transplant rejection. Examples include, but are not limited to, endoproteinase, pepsin, papain, chymotrypsin, trypsin, collagenase, cyanogen bromide, enterokinase (Asp or Glu-specific), iodosobenzoate, lysobacter endoproteinase, N-bromosuccinimide, N-chlorosuccinimide, hydroxylamine, 2-nitro5-thiocyanobenzoate and endopeptidase. Papain particularly of use, as it is known to cut all MHC Class I molecules of different alleles and different species in the .alpha.3 domain. Papain does not cut the .alpha.1 or .alpha.2 domain.

Papain cutting characteristics are well described. Papain is the major ingredient of meat tenderizers and is sulfhydryl protease isolated from the latex green fruit of papaya It was first isolated in 1955 and its enzymatic capabilities have been extensively documentated. In its native state, the enzyme is inactive, and therefore donor tissue treatments may be advantageously carried out with a high degree of control, using native papain in the presence of activators such as cysteine (0.005 M) and/or EDTA (0.002 M). See generally, Stockell et al., 1957, J. Biol. Chem., 227: 1-26.

Additional such enzymatic reagents include, but are not limited to, oxidoreductases acting on: (1) OH-OH groups: (2) aldehyde or keto groups; (3) CH-CH groups; (4) CH-NH, groups; (5) reduced NAD or NADP; (6) nitrogenous compounds; (7) diphenols; (8) acting on H₂ O₂ ; (9) hydrogen; (10) acting on single donors with incorporation of oxygen: and (11) acting on paired donors with incorporation of oxygen into one donor, tranferases: (1) transferring onecarbon groups (methyltranferases, hydroxymethyl-, formyl-and related transferases, carboxyl- and carbamoyltransferases, amidinotransferases); (2) transferring aldehydic or ketonic residues; (3) acting on acyltranferases, aminoacyltransferases); (4) acting on glycosyltranferases (hexosyltranferases, pentosyltranferases); (5) transferring alkyl or related groups; (6) transferring nitrogenous groups; (7) transferring phosphorus-containing groups (phosphotranferases with an alcohol group as acceptor, phosphotransferases with a carboxyl group as acceptor, phosphotranferases with a nitrogenous group as acceptor, phosphotransferases with a phosphate group as acceptor, phosphotransferases, pyrophosphotransferases, nucleotidyltransferases, transferases for other substituted, phospho-groups); and, (8) transferring sulphur-containing groups (sulphurtransferases, sulphotransferases, CoA-transferases); hydrolases: (1) acting on ester bonds (carboxylic ester hydrolases, thiolester hydrolases, phosphoric monoester hydrolases, phosphoric diester hydrolases, triphosphoric monoester hydrolases, sulphuric ester hydrolases); (2) acting on glyeosyl compounds (glycoside hydrolases, hydrolysing N-glycosyl compounds, hydrolysing S-glycosal compounds); (3) acting on ether bonds (thioether hydrolases); (4) acting on peptide bonds (peptide hydrolases) (.alpha.-amino-acyl-peptide hydrolases, peptidyl-amino-acid hydrolases, dipetide hydrolases, peptidyl-peptide hydrolases); (5) acting on C--N bonds other tan peptide bonds (in linear amidines, in cylic amides, in linear amidines, in cylic amidines, in cyanides); (6) acting on acid-anhydride bonds (in phosphoryl-containing anhydrides); (7) acting on C=C bonds; (8) acting on carbon-halogen bonds; (9) acting on P--N; lyases (1) acting on carbon-carbon bonds (carboxyl-lyases, aldehyde-lyases, keto acid-lyases); (2) acting on carbon-oxygen bonds (hydrolyases and other carbonxygen lyases); (3) acting on carbon-nitrogen bonds (amonia-lyases and amidine-lyases); (4) carbon-sulphur lyases; (5) carbon-halogen lyases; (6) other lyases; isomerases: (1) racemases and epimerases (acting on amino acids and derivatives; acting on hydroxyacids and derivatives, acting on carbohydrates and derivatives, acting on other compounds; (2) acting on cis-trans isomerases; (3) acting on intramolecular oxidoreductases (interoconverting aldoses and ketoses, interoconverting keto- and enol-groups, transposing C=C bonds); (4) acting on intramolecular transferases (transferring acyl groups, transferring phosphoryl groups, transferring other groups); (5) acting on intramolecular lyases; (6) other isomerases; ligases: (1) acting on forming C--O bonds (amino-acid-RNA ligases); (2) acting on forming C-N bonds (acid-ammonia ligases (amide synthetases), acid-amino-acid ligases (peptide synthetases), cyclo-ligases, other C--N ligases, C--N ligases with glutamine as N-donor); (3) forming C--C bonds; and glycosidases, such as .alpha.-amylase, .beta.-amylase, glucoamylase, celulase, laminarinase, inulase, dextranase, chitinase, polygalacturonase, lysozymne, neuraminidase, .alpha.-glucosidase, .beta.-glucosidase, .alpha.-galactosidase, , .beta.-galactosidase, .alpha.-mannosidase, .beta.-fructofuranosidase, trehalase, chitobiase, .beta.-acetylglucosaminidase, .beta.-glucuronidase, dextrin-1,6-glucosidase, hyaluronidase, .beta.-D-fucosidase, metalopeptidases, phospholiphase C and nucleosidase.

d. Administration of Xenogeneic and Allogeneic Cells

While transfection and subsequent tranplantation of cells which are obtained from an individual or cell culture system of like species with the recipient organism may be performed, it is equally true that the invention may be practised using cells of another organism (such as a well-characterized eukaryotic microorganism, e.g. yeast, in which appropriate processing of proteins encoded by therapeutic genes is likely and in which useful origins of replication,are known). In such a case, certain concerns must be addressed.

First, when a protein is encoded by the gene of interest, the transplanted cells must produce the protein in a form that may is of use to the recipient organism. Post-translational processing (including, but not limited to, cleavage and patterns of glycosylation) must be consistent with proper function in the recipient. In addition, either a protein or an RNA molecule of interest must be made available to the recipient after synthesis, such as by secretion, excretion or exocytosis from the transplanted cell. To address the former, the protein produced by the transfected cells may be qualitatively compared to the native protein produced by an individual of the same species as the recipient organism by biochemical methods well known in the art of protein chemistry. The latter, release of the protein of interest by the cells to be transplanted, may be assayed by isolating protein from culture medium which has been decanted from the transfected cells or from which such cells have been separated (i.e. by centrifugation or filtration), and performing Western analysis using an antibody directed at the protein of interest. Antibodies against many proteins are commercially available; techniques for the production of antibody molecules are well known in the art.

Second, the cells must be shielded from immune rejection by the recipient organism. It is contemplated that such cells may be transfected with constructs expressing cell-surface markers (e.g. MHC antigens) characteristic of the recipient patient so as to provide them with biochemical camoflage.

In addition, methods for the encapsulation of living cultures of cells for growth either in an artificial growth environment, such as in a fermentor, or in a recipient organism have been developed, and are also of use in the administration of cells transfected according to the invention. Such an encapsulation system renders the cell invisible to immune detection and, in addition, allows for the free exchange of materials (e.g. the gene product of interest, oxygen, nutrients and waste materials) between the transplanted cells and the environment of the host organism.

Methods and devices for cell encapsulation are disclosed in numerous U.S. Patents; among these are U.S. Pat. Nos. 4,353,888; 4,409,311; 4,673,566; 4,744,933; 4,798,786; 4,803,168; 4,892,538; 5,011,472; 5,158,881; 5,182,111; 5,283,187; 5,474,547; 5,498,401 (which is particularly directed to the encapsulation of bacterial and yeast cells in chitosan); 5,550,050; 5,573,934; 5,578,314; 5,620,883; 5,626,561; 5,653,687; 5,686,115; 5,693,513; and 5,698,413, the contents of which are fully incorporated by reference herein. Typically required for the successful culture of encapsulated cells is a selectively-permeable outer covering or `skin` which is biocompatible (i.e., tolerated by both the encapsulated cells and the recipient host), and, optionally, a matrix in- or upon which cells are distributed such that the matrix provides structural support and a substrate to which anchorage-dependent cells may attach themselves. As relates to encapsulation devices applicable to use in the invention, the term "selectively-permeable" refers to materials comprising openings through which small molecules (including molecules of up to about 50,000 M.W.-100,000 M.W.) may pass, but from which larger molecules, such as antibodies (approximately 150,000 M.W.), are excluded. Suitable covering materials include, but are not limited to, porous and/or polymeric materials such as polyaspartate, polyglutamate, polyacrylates (e.g., acrylic copolymers or RL.RTM., Monsanto Corporation), polyvinylidene fluoride, polyvinylidienes, polyvinyl chloride, polyurethanes, polyurethane isocyanates, polystyrenes, polyamides, cellulose-based polymers (e.g. cellulose acetates and cellulose nitrates), polymethyl-acrylate, polyalginate, polysulfones, polyvinyl alcohols, polyethylene oxide, polyacrylonitriles and derivatives, copolymers and/or mixtures thereof, stretched polytetrafluoroethylene (U.S. Pat. Nos. 3,953,566 and 4,187,390, both incorporated herein by reference), stretched polypropylene, stretched polyethylene, porous polyvinylidene fluoride, woven or non-woven collections of fibers or yarns, such as "Angel Hair" (Anderson, Science, 246: 747-749; Thompson et al., 1989, Proc. Natl. Acad. Sci. U.S.A., 86: 7928-7932), fibrous matrices (see U.S. Pat. No. 5,387,237, incorporated herein by reference), either alone or in combination, or silicon-oxygen-silicon matrices (U.S. Pat. No. 5,693,513). Polylysine having a molecular weight of 10,000 to 30,000, preferably 15,000 to 25,000 and most preferably 17,000 is also of use in the invention (see U.S. Pat. No. 4,673,566). Alternatively, the matrix material, comprising the transfected cells of the invention, is exposed to conditions that induce it to form its own outer covering, as discussed below.

As described in U.S. Pat. No. 5,626,561, the selective permeability of such a covering may be varied by impregnating the void spaces of a porous polymeric material (e.g., stretched polytetrafluoroethylene) with a hydrogel material. Hydrogel material can be impregnated in substantially all of the void spaces of a porous polymeric material or in only a portion of the void spaces. For example, by impregnating a porous polymeric material with a hydrogel material in a continuous band within the material adjacent to and/or along the interior surface of a porous polymeric material, the selective permeability of the material is varied sharply from an outer cross-sectional area of the material to an inner cross-sectional area of the material. The amount and composition of hydrogel material impregnated in a porous polyhrneric material depends in large part on the particular porous polymeric material used to encapsulate cells for transplant. Examples of suitable hydrogel materials include, but are not limited to, HYPAN.RTM. Structural Hydrogel (Hymedix International, Inc.; Dayton, NJ), non-fibrogenic alginate, as taught by Dorian in PCT/US93/05461, which is incorporated herein by reference, agarose, alginic acid, carrageenan, collagen, gelatin, polyvinyl alcohol, poly(2-hydroxyethyl methacrylate), poly(N-vinyl-2-pyrrolidone) or gellan gum, either alone or in combination.

The matrix typically has a high surface-area:volume ratio, comprising pores or other spaces in- or on which cells may grow and through which fluids may pass; in addition, suitable matrix materials are stable following transplantation into a recipient organism. Preferably, the matrix comprises an aggregation of multiple particles, fibers or laminae. Alternatively, a matrix may comprise an aqueous solution, such as a physiological buffer or body fluid from the recipient organism (see U.S. Pat. No. 5,011,472). Suitable matrix materials include liquid, gelled, polymeric, copolymeric or particulate formulations of aminated glucopolysachharides (e.g., deacetylated chitin, or "chitosan", which is prepared from the pulverized shells of crabs or other crustaceans and is commercially available as a dry powder; Cat # C 3646, Sigma, St. Louis, Mo.), alginate (U.S. Pat. No. 4,409,331), poly-.beta.-1.fwdarw.5-N-acetylglucosamine (pGlcNAc) polysaccharide species (either alone of formulated as co-polymer with collagen; see U.S. Pat. No. 5,686,115), reconstituted extracellular matrix preparations (e.g. Matrigel.RTM.; Collaborative Research, Inc, Lexington, Mass.; Babensee et al., 1992, J. Biomed. Matr. Res., 26: 1401), proteins, polyacrylamide, agarose and others.

Methods by which cells become encapsulated using such materials are both numerous and varied. Encapsulation devices comprising a semi-permeable membrane material, as described above, may be pre-formed, filled with cells (e.g. by injection or other manual means) and then sealed (U.S. Pat. Nos. 4,892,538; 5,011,472; 5,626,56; and U.S. Pat. No. 5,653,687); such sealing may be effectively permanent (e.g. by the use of heat-sealing), semi-permanent (e.g. by the use of a biocompatible adhesive, such as an epoxy, which will not dissolve or degrade in an aqueous environment) or temporary (e.g. by the use of a removable cap or plug, or by shutting of a valve or stopcock). Methods of permanent and semi-permanent sealing are disclosed in U.S. Pat. No. 5,653,687. As an alternative to the use of a pre-formed, semi-permeable cell reservoir, methods by which cells suspended in matrix material and the substance which is to form the outer covering of the encapsulation device are co-extruded under conditions which cause the cell/matrix mixture, which may be in liquid or semi-liquid (i.e., gelled) form to be encased in a continuous tube of the semi-permeable polymer, which either forms, or becomes crosslinked, under the extrusion conditions; such an extrusion procedure may lead to the formation of capsules which have only one cell reservoir (U.S. Pat. No. 5,283,187) or which are divided into multiple, discrete compartments (U.S. Pat. No. 5,158,881). As an alternative to both types of procedure, a liquid or semi-liquid (i.e., gelled) cell/matrix mixture droplet is suspended either in an agent which induces `curing` or crosslinking of the outer layer of matrix material to form a semi-permeable barrier (U.S. Pat. Nos. 4,798,786 and 5,489,401) or in a solution of polymeric material (or monomers thereof), which will polymerize and/or crosslink upon contact with the cell/matrix droplet such that a semi-permeable membrane is deposited thereon (U.S. Pat. Nos. 4,353,888; 4,673,566; 4,744,933; 5,620,883; and 5,693,513).

One of such of skill in the art is well able to select the appropriate matrix and semi-permeable membrane materials and to construct a cell-encapsulation device as described above.

Implantation of such a device is achieved surgically, via standard techniques, to a site at or near the anatomical location to which the product encoded by the gene on the gene of interest is to be delivered, as is deemed safest and most expedient. Such a device may take a convenient shape, including, but not limited to, that of a sphere, pellet or other capsule shape, disk, rod or tube; often, the shape of the device is determined by its method of synthesis. For example, one which is formed by co-extrusion of a cell suspension and a polymeric covering material is typically tubular, while one formed by the deposition of a covering on droplets comprising cells in matrix material might be spherical. As discussed above, the number of cells which must be implanted (and, therefore, encapsulated) is dependent upon the requirements of the recipient organism for the product of the transfected gene. The encapsulation devices described above are typically small.(most usefully, 10 .mu.m to 1 mm in diameter, so as to permit efficient diffusion of substances back and forth between the outer covering and the cells most deeply embedded in the matrix), and it is contemplated that such devices may carry between and 10 and 10¹⁰ cells each. Should the need for larger numbers of cells be anticipated, a plurality (2, 10 or even 100 or more) of such in vivo culturing devices may be made and implanted in a given recipient organism.

An encapsulated cell device may be intended for permanent installation; alternatively, retrieval of the device may be desirable, whether to terminate delivery of the product of the gene of interest to the recipient organism at the discretion of one of skill in the art, such as a physician (who must determine on a case-by-case basis the length of time for which a given cell implant is beneficial to the recipient organism) or to replenish the device with fresh cells after long-term use (i.e. months to years). To the latter end, an implantation device may usefully comprise a retrieval aid, such as a guidewire, and a cap or other port, such as may be opened and re-sealed in order to gain access to the cell reservoir, both as described in U.S. Pat. No. 4,892,538.

Live cultures of encapsulated cells have been used successfully to deliver gene products to tissues of a recipient animal. U.S. Pat. No. 4,673,566 discloses successful maintenance of normal blood sugar levels in a diabetic rat into which encapsulated rat islet of Langerhans cells were implanted; two administrations of 3,000 cells each together were effective for six months, while a single dose of 1,000 cells was effective for two months.

Encapsulated GABA-secreting pancreatic cells implanted into subthalamic nucleus of monkeys in whom Parkinsonism has been clinically-induced have been observed relieve the symptoms of that syndrome (U.S. Pat. No. 5,474,547), demonstrating invisibility of encapsulated cells to the immune system, as well as efficacy in delivering a product of encapsulated, transplanted cells to a recipient organism.

More encouraging, as it demonstrates immunological shielding by cell encapsulation systems sufficient for cross-species cell transplants, as is advantageous for their use in practicing the present invention, is the finding that encapsulated embryonic mouse mesencephalon cells, when transplanted into recipient rats, alleviate symptoms of clinically-induced Parkinsonism (U.S. Pat. No. 4,892,538).

Similarly, heterospecific transplantation of encapsulated islet cells has been demonstrated to treat diabetes successfully (dog islet cells to a mouse recipient, U.S. Pat. No. 5.578,314; porcine islet cells to a mouse recipient, Sun et al., 1992, ASAIO J., 38: 124). It is believed that such an approach is promising for the clinical treatment of diabetes mellitus in humans (Calafiore, 1992, ASAIO J., 38: 34).

It is contemplated that these techniques, which have been applied successfully to untransfected cells, may be utilized advantageously with cells that are transfected with therapeutic nucleic acid molecules of use in the invention.

e. Assay of efficacy of transplanted cells in a recipient organism

The efficacy of the transfected cells so administered and their subsequent maintenance in the recipient host may be assayed either by monitoring the activity of a marker gene, which may additionally be comprised by the transfected construct, or by the direct measurement of either the product (e.g. a protein) encoded by the gene of interest or the reduction in the levels of a protein the production of which it (an antisense message or ribozyme) is designed to inhibit. The assays can be performed using conventional molecular and biochemical techniques, such as are known to one skilled in the art, or may comprise histological sampling (ie., biopsy) and examination of tranplanted cells or organs.

In addition to direct measurements of protein or nucleic acid levels in blood or target tissues encoded by the gene of interest borne by the vector in transfected/transplanted cells, it is possible to monitor changes in the disease state in patients receiving gene transfer via transplantation of cells in which the gene of interest is maintained and compare them to the progression or persistence of disease in patients receiving comparable cells transfected with vector constructs lacking the gene of interest.

Proteins and other Therapeutic Agents

In addition to nucleic acids, proteins and perhaps other bioactive substances may be used to stimulate proteosome activity in a recipient mammal. When the amount of a protein or other therapeutic agent to be used is considered, the lowest dose that provides the desired degree of enhancement of NF.kappa.B activity by the target cells should be used; lower doses may be advantageous in order to minimize the likelihood of possible adverse effects. Note that "NF.kappa.B activity" includes not only the presence of functional NF.kappa.B, but may also include the presence of the products of genes regulated by NF.kappa.B, regardless of the means by which they have arisen in the cell, as well as normal differentiations proliferation and survival of the cell. It will be apparent to those of skill in the art that the therapeutically-effective amount of a composition administered in the invention will depend, inter alia, upon the efficiency of cellular uptake of a composition, the administration schedule, the unit dose administered, whether the compositions are administered in combination with other therapeutic agents, the health of the recipient, and the therapeutic activity of the particular protein or other pharmaceutical substance.

As is also true of nucleic acids administered according to the invention, the precise amount of a protein or other pharmaceutical agent required to be administered depends on thejudgmnent of the practitioner and may be peculiar to each subject, within a limited range of values. An appropriate dose of a protein or other substance may be calculated as follows:

The NOD mouse model may be used to assay the effectiveness of varying doses of a protein or other agent in treating an autoimmune disease according to the invention. For a given therapeutic composition, it is necessary to establish an approximate range of dosages that are useful, yet relatively safe, in a clinical situation. The NOD mouse model may be employed to establish a dosage curve prior to use of the invention in human subjects. Alternatively, if a pharmaceutical agent useful according to the invention already has been granted regulatory approval, it stands that acceptable upper limits of dosage tolerance for humans and other mammals already will have been established for these drugs prior to testing, as have systemic concentrations useful for other clinical applications. These known dosages may serve as the basis upon which calculations may be made prior to use of the mouse model.

A therpeutic composition may be administered either systemically or locally. In the general case, a starting dosage to be administered locally to cells in the mice equals the optimal systemic concentration described for a known use of the therapeutic agent. Ideally, such a dosage has been established for mice; otherwise, the relevant human dosage is used for the purposes of calculation. As it is not known whether the concentration of a particular protein or other agent that is useful for enhancing NF.kappa.B activity is higher or lower than that used for other clinical purposes, a range of values above and below the recommended dosage may be assayed. In a first attempt, values spanning four orders of magnitude below this dosage are examined; if no effect is seen, or if enhancement of NF.kappa.B activity in the target cells is observed to increase at or near the starting dosage, values that exceed that dosage by up to four orders of magnitude are assayed. If no effect is seen within four orders of magnitude in either direction of the starting dosage, it is likely that the agent is not of use according to the invention. It is critical to note that when elevated dosages are used, the concentration must be kept below harmful levels, which are also known for all drugs that are approved for clinical use. Such a dosage should be one (or, preferably, two or more) orders of magnitude below the LD₅₀ value that is known for a laboratory mammal, whether or not that mammal is a mouse, and preferably below concentrations that are documented as producing serious, if non-lethal, side effects. If it determined that a therapeutic agent is optimally useful at levels that are harmful if achieved systemically, that agent should be used for local administration only, and then only at such doses where diffusion of the drug from the target site reduces its concentration to safe levels.

Claim 1 of 4 Claims

What is claimed is:

1. A method of restoring NF.kappa.B activity in a mammal afflicted with an automimmune disease resulting from a reduction in NF.kappa.B activity, comprising administering to a mammal suspected of suffering from said autoimmune disease a therapeutically effective amount of a protein which restores NF.kappa.B activity so as to treat said disease in said mammal.

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