WO2008144508A2 - Modulating mhc class ii antigen presentation in dendritic cells prevents diabetes - Google Patents

Abstract

The instant invention provides method and compositions for the treatment of autoimmune disorders. The instant invention provides modulators of the DO/DM ratio or the activity of DO and/or DM.

Description

TITLE OF THE INVENTION

MODULATING MHC CLASS II ANTIGEN PRESENTATION IN DENDRITIC CELLS

PREVENTS DIABETES

RELATED APPLICATIONS/PATENTS & INCORPORATION BY REFERENCE

This application claims priority to the following U.S. provisional patent applications Ser. Nos.: 60/930,756, filed May 17, 2007, and 60/940,145, filed May 25, 2007, the entire contents of which are incorporated herein by this reference.

Each of the applications and patents cited in this text, as well as each document or reference cited in each of the applications and patents (including during the prosecution of each issued patent; "application cited documents"), and each of the PCT and foreign applications or patents corresponding to and/or claiming priority from any of these applications and patents, and each of the documents cited or referenced in each of the application cited documents, are hereby expressly incorporated herein by reference, and may be employed in the practice of the invention. More generally, documents or references are cited in this text, either in a Reference List before the claims, or in the text itself; and, each of these documents or references ("herein cited references"), as well as each document or reference cited in each of the herein cited references (including any manufacturer's specifications, instructions, etc.), is hereby expressly incorporated herein by reference.

BACKGROUND OF THE INVENTION

Diabetes mellitus is a metabolic disorder characterized by hyperglycemia (high blood sugar) and other signs, as distinct from a single illness or condition. The three main forms of diabetes - type 1, type 2, and gestational diabetes — have similar signs, symptoms, and consequences, but different causes and population distributions. Ultimately, all forms are due to the beta cells of the pancreas being unable to produce sufficient insulin to prevent hyperglycemia. Type 1 is usually due to autoimmune destruction of the pancreatic beta cells, which produce insulin. Type 1 diabetes comprises up to 10% of total cases in North America and Europe, though this varies by geographical location. It can affect children or adults but was traditionally termed "juvenile diabetes", because it represents a majority of cases of diabetes affecting children. Though an incurable, chronic condition, it has been treatable since insulin became medically available in 1921, and, today, is usually managed with a combination of dietary treatment and, frequently, insulin supplementation.

The recognition of MHCII molecules loaded with self and pathogen-derived peptides by CD4 T cells is one mechanism by which immune responses are activated. Inappropriate recognition of MHCII-self peptide complexes can result in autoimmune disorders such as Type I diabetes (Mathis, D. and Benoist, C. 2004 Immunity 20:509-516).

SUMMARY OF THE INVENTION

MHCII molecules acquire peptides from self and non-self proteins in endosomes of antigen presenting cells. Peptide loading of MHCII molecules is shown to be directly catalyzed by the action of the MHCII-like molecule, HLA-DM (DM) (Denzin, L.K., and P. Cresswell. 1995 Cell 82:155-165; Sloan, V.S., et al. 1995 Nature 375:802-806; Weber, D.A., et al. 1996 Science 274:618-620). HLA-DOA and HLA-DOB are structurally similar to the α and β chains of conventional MHCII molecules (Servenius, B., et al. 1987 J Biol Chem 262:8759-8766; Tonnelle, C, et al. 1985 Embo J 4:2839-2847; Trowsdale, J., and Kelly, A. 1985 Embo J 4:2231-2237). HLA-DO (DO), the heterodimer formed by HLA-DOA and HLA-DOB was initially thought to be mainly expressed in B cells, but has recently been shown to also be highly expressed in dendritic cells (DCs) (Chen, X., et al. 2006 J Immunol 176:3548-3556; Fallas, JX. , et al. 2007 J Immunol In Press; Hornell, T.M., et al. 2006 J Immunol 176:3536-3547). DO strongly associates with DM (Liljedahl, M., et al. 1996 Embo J 15:4817-4824). It has been shown that DO modulates the peptide loading activity of DM (Denzin, L. K., et al. 2005 Immunol Rev 207:279-292; Kropshofer, H., et al. 1998 Embo J 17:2971-2981; van Ham, M., et al. 2000 J Exp Med 191 :1127-1136). Importantly, DO modifies MHCII peptide loading and, thus, DO expression alters the MHCII Ag presentation capabilities of DCs and B cells by reducing or modulating the complexity or level of the endogenous self-peptide repertoire (Karlsson, L. 2005 Curr Opin Immunol 17:65-70). Modulation of the MHCII pathway may play a role in autoimmune-mediated diseases, since dampening the antigen processing ability of B cells and DCs should reduce the possibility of activating self-reactive CD4 T cells.

Accordingly, in one aspect, the invention provides a method of treating an auto-immune disease in a subject, comprising administering DO or a functional mimetic thereof to the subject, thereby treating the auto-immune disease in the subject. In another embodiment of the invention, the auto-immune disease is type 1 diabetes. In still another embodiment of the invention, the type I diabetes has progressed to the insulitis stage. In still another embodiment of the invention, the administering of DO or a functional mimetic thereof normalizes the DO/DM ratio in the subject.

In another aspect, the invention provides a method of treating an auto-immune disease in a subject, comprising administering to the subject an agent that increases DO expression or activity in the subject, thereby treating the auto-immune disease in the subject. In another embodiment of the invention, DO expression is increased in immune cells or immune cell precursors. In still another embodiment of the invention, the auto-immune disease is type 1 diabetes. In still another embodiment of the invention, the type 1 diabetes has progressed to the insulitis stage. In still another embodiment of the invention, the administering of the agent normalizes the DO/DM ratio in the immune cells of the subject.

In another aspect, the invention provides a method of treating auto-immune disease in a subject, comprising decreasing the expression or activity of DM in a subject, thereby treating auto-immune disease in the subject. In another embodiment of the invention, the auto-immune disease is type 1 diabetes. In still another embodiment of the invention, the type 1 diabetes has progressed to the insulitis stage. In still another embodiment of the invention, the decreasing of the expression or activity of DM normalizes the DO/DM ratio in immune cells of the subject.

In another embodiment of the invention, DM is over-produced in immune cells of the subject.

In a further embodiment of the invention, the immune cells are selected from the group consisting of dendritic cells, T cells, and B cells.

In another embodiment of the invention, DO expression is increased via transfection, transduction, or infection of the cells with a vector comprising a nucleic acid molecule encoding DO-alpha and a nucleic acid molecule encoding DO-beta. In still another embodiment of the method of the invention, a) the nucleic acid molecule encoding DO-alpha (i) comprises the nucleic acid sequence of SEQ ID NO:1 or a complement thereof or (ii) encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or a fragment thereof; and b) the nucleic acid molecule encoding DO-beta (i) comprises the nucleic acid sequence of SEQ ID NO:3 or a complement thereof or (ii) encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:4 or a fragment thereof. In still another embodiment of the invention, the vector comprises an immune cell- specific promoter. In still another embodiment of the invention, the vector is a retroviral vector.

In another aspect, the invention provides cell therapy methods to treat or prevent autoimmune disease. For example, the invneiton provides methods of treating an auto-immune disease in a subject by administering to the subject a cell that expresses DO, thereby treating the auto-immune disease in the subject. In one embodimetn, the cell overexpresses DO. In another embodiment, the cell expresses wild-type levels of DO and DM. In one embodiment, the cell expresses a) the nucleic acid molecule encoding DO-alpha (i) comprises the nucleic acid sequence of SEQ ID NO:1 or a complement thereof or (ii) encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or a fragment thereof; and b) the nucleic acid molecule encoding DO-beta (i) comprises the nucleic acid sequence of SEQ ID NO:3 or a complement thereof or (ii) encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:4 or a fragment thereof.

In another aspect, the invention provides a method of identifying an inhibitor of DM expression or activity for use in the treatment of an auto-immune disease or disorder, comprising: (i) contacting a population of immune cells or immune cell precursors over- expressing DM with a test agent; and (ii) detecting a decrease in the level of expression or activity of DM in the cells, thereby identifying an inhibitor of DM expression or activity for use in the treatment of an auto-immune disease or disorder. In another embodiment of the invention, the auto-immune disease or disorder is type 1 diabetes. In still another embodiment of the invention, the immune cell precursors comprise stem cells.

In another aspect, the invention provides a method of decreasing DM gene expression in a cell, comprising contacting the cell with a vector comprising a nucleic acid sequence comprising an interfering RNA, thereby decreasing DM gene expression in the cell. In another embodiment of the invention, the cell over-expresses DM. In still another embodiment of the invention, the interfering RNA comprises a small interfering RNA.

In another aspect, the invention provides a packaged pharmaceutical comprising DO or a functional mimetic thereof and instructions for use.

In another aspect, the invention provides a kit for treating type 1 diabetes in a subject in need thereof, comprising DO or a functional mimetic thereof and instructions for use. In another aspect, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of a nucleic acid molecule encoding a polypeptide comprising DO or a functional mimetic thereof and a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 graphically depicts the incidence of diabetes in NOD vs. NOD.DO mice over time.

Figures 2A and 2B show by FACS analysis that transgenic DO in NOD.DO mice is expressed in all DCs (Figure 2A) and a subset of B cells (Figure 2B), compared to control NOD mice.

Figures 3A-3F show by immunofluorescence histology that DO is expressed in pancreatic DCs in NOD.DO mice, compared to pancreatic DCs in control NOD mice. Figures 3A-3C show co-immuno fluorescence staining in pancreatic tissue in the NOD mouse genetic background using antibodies against CDl Ic (Fig. 3A) and DO (Fig. 3B), and the merged image of CDl Ic and DO immunofluorescence (Fig. 3C). Figures 3D-3F show the co-localization of CDl Ic and DO pancreatic tissue in the NOD.DO mouse genetic background. Figures 3D-3F show the co-immuno fluorescence staining in pancreatic tissue in the NOD.DO mouse genetic background using antibodies against CDl Ic (Fig. 3D) and DO (Fig. 3E), and the merged image of CDl Ic and DO immunofluorescence (Fig. 3F).

Figure 4 graphically depicts the incidence of diabetes over time in irradiated mice receiving T-depleted bone marrow from NOD mice, T-depleted bone marrow from NOD.DO mice, or a combination of T-depleted bone marrows from NOD mice and NOD.DO mice (50:50).

Figure 5 shows a histogram depicting staining with specific monoclonal antibodies for H2-0 (mouse homologue of DO) (Figure 2A) and H2-M (mouse homologue of DM) (Figure 2B) in splenic dendritic cells of NOD vs. B6 mice.

Figure 6 sets forth the nucleic acid and polypeptide sequences of DO-α (HLA-DOA) and DO-β (HLA-DOB). The nucleic acid sequences of DOA and DOB are set forth as SEQ ID NOs:l and 3, respectively. The polypeptide sequences of DO-α and DO-β are set forth as SEQ ID NOs :2 and 4, respectively. Figure 7 sets forth the nucleic acid and polypeptide sequences of DM-α (HLA-DMA) and DM-β (HLA-DMB). The nucleic acid and polypeptide sequences of DMA and DMB are set forth as SEQ ID N0s:5 and 7, respectively. The polypeptide sequences of DM-α and DM-β are set forth as SEQ ID NOs: 6 and 8, respectively.

The following Detailed Description, given by way of example, but not intended to limit the invention to specific embodiments described, may be understood in conjunction with the accompanying drawings, incorporated herein by reference. Various preferred features and embodiments of the present invention will now be described by way of non- limiting example and with reference to the accompanying figures, in which:

DETAILED DESCRIPTION

Definitions

The term "treating" as used herein refers to administering an agent (for example, DO or a DM modulator) in amount, manner, and/or mode effective to improve a condition, symptom, or parameter associated with a disorder or to prevent progression of a disorder (herein, an autoimmune disease, for example, type I diabetes), to either a statistically significant degree or to a degree detectable to one skilled in the art. An effective amount, manner, or mode can vary depending on the subject and may be tailored to the subject. By preventing progression of a disorder, a treatment can prevent deterioration of a disorder in an affected or diagnosed subject or a subject suspected of having the disorder, but, also, a treatment may prevent the onset of the disorder or a symptom of the disorder in a subject at risk for the disorder or suspected of having the disorder.

Furthermore, the terms "treatment", "treating", and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse affect attributable to the disease. "Treatment", as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, e.g., causing regression of the disease, e.g., to completely or partially remove symptoms of the disease.

HLA-DO (DO) is an unconventional class II molecule of the major histocompatibility complex. It is a functional heterodimer formed by the alpha and beta gene products. HLA-DO (DO), as used herein, includes the heterodimer, the alpha or beta chain, and biologically active fragments thereof. The nucleic acid and amino acid sequences for HLA-DOA (DO-alpha) are set forth herein as SEQ ID Nos: 1 and 2, respectively. The nucleic acid and amino acid sequences for HLA-DOB (DO-beta) are set forth herein as SEQ ID Nos:3 and 4, respectively. HLA-DO is a negative modulator of HLA-DM-mediated MHC class II peptide loading (van Ham, S. M., et al. 1997 Curr Biol 7(12):950-7). The nucleic acid and amino acid sequences for HLA-DMA (DM-alpha) are set forth herein as SEQ ID Nos:5 and 6, respectively. The nucleic acid and amino acid sequences for HLA-DMB (DM-beta) are set forth herein as SEQ ID Nos: 7 and 8, respectively.

The term "functional mimetic", as used herein, refers to an agent that imitates at least one biological function exhibited by a biological agent. For example, a functional mimetic of DO is an agent other than DO that mimics the latter's normalization of the DO/DM ratio in a subject exhibiting over-production of DM in their immune cells. Exemplary functional mimetics of DO include fragments of DO-alpha or DO-beta.

A normalized DO/DM ratio, as referred to herein, is an increase in DO expression levels such that DO expression is equal to or more equal to DM expression levels such that the DO/DM ratio approaches one. Alternatively, normalizing the DO/DM ratio includes measurably modulating the DO/DM ratio.

The term "insulitis stage", as used herein, refers to the stage of type 1 diabetes marked by a histologic change in the islets of Langerhans characterized by edema and the infiltration of small numbers of white blood cells.

The term "stem cells", as used herein, refers to multipotent or pluripotent cells having the capacity to self-renew and to differentiate into multiple cell lineages.

The term "subject", as used herein, refers to any member of the class mammalia, including humans, domestic and farm animals, and zoo, sports or pet animals, such as mouse, rabbit, pig, sheep, goat, cattle and higher primates. The term "vector", as used herein, refers to a nucleic acid-based delivery vehicle comprising regulatory sequences and a gene of interest (for example, DO), which can be used to transfer its contents into a cell.

The term "interfering RNA", as used herein, refers to any double stranded or single stranded RNA sequence, capable — either directly or indirectly (i.e., upon conversion) — of inhibiting or down regulating gene expression by mediating RNA interference. Interfering RNA includes but is not limited to small interfering RNA ("siRNA") and small hairpin RNA ("shRNA"). "RNA interference" refers to the selective degradation of a sequence-compatible messenger RNA transcript.

The term "small interfering RNA" or "siRNA", as used herein, refers to any small RNA molecule capable of inhibiting or down regulating gene expression by mediating RNA interference in a sequence-specific manner. The small RNA molecule can be, for example, about 18 to about 21 (e.g., about 18, 19, 20, 21) nucleotides long.

The terms "comprises," "comprising," "containing" and "having" and the like, as used herein, can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

These and other objects of the invention will be described in further detail in connection with the detailed description of the invention. Autoimmune Diseases

Autoimmune diseases arise from an overactive immune response of the body against substances and tissues normally present in the body. Autoimmune diseases include, without limitation, acute disseminated encephalomyelitis (ADEM), Addison's disease, ankylosing spondylitisis, antiphospho lipid antibody syndrome (APS), aplastic anemia, autoimmune hepatitis, autoimmune oophoritis, coeliac disease, Crohn's disease, diabetes, e.g., diabetes mellitus (type 1), gestational pemphigoid, Goodpasture's syndrome, Graves' disease, Guillain- Barre syndrome (GBS), Hashimoto's disease, idiopathic thrombocytopenic purpura, Kawasaki's disease, lupus erythematosus, multiple sclerosis, myasthenia gravis, opsoclonus myoclonus syndrome (OMS), optic neuritis, Ord's thyroiditis, pemphigus, pernicious anaemia, polyarthritis, primary biliary cirrhosis, rheumatoid arthritis, Reiter's syndrome, Sjogren's syndrome,

Takayasu's arteritis, temporal arteritis, warm autoimmune hemolytic anemia, and Wegener's granulomatosis.

Compositions and Methods of the Invention

Compositions and methods of the present invention are directed towards the treatment of autoimmune diseases and disorders in a subject. Such treatment is sought by administering DO or a functional mimetic thereof to a subject, by effecting an increase in the expression of DO in a subject, or by inhibiting DM expression or activity in a subject. In certain embodiments, DM is over-produced in immune cells of the subject. Thus, the administration of DO, increase in DO expression, or inhibition of DM expression or activity seeks to normalize the physical or functional DO/DM ratio in the subject.

In certain embodiments, DO or the functional mimetic thereof has at least about 60%, 70% 80% 90% 95%, 99%, or 100% sequence identity with DOA or DOB, or a fragment thereof.

In additional embodiments of the present invention, methods are contemplated that identify inhibitors of DM expression or activity. Design of assays (e.g., cell-based assays) to identify such inhibitors is well within the skill in the art. The end point of the assays will typically measure protein expression or a physiologic effect.

The pharmaceutical compositions of the invention can be administered to any subject that may experience the beneficial effects of DO or a functional mimetic thereof, or of a DM inhibitor. The pharmaceutical compositions can be administered by any means that achieve their intended purpose. For example, administration can be by topical, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, administration can be by the oral route. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired.

In addition to DO, a functional mimetic thereof or a DM inhibitor, the pharmaceutical compositions can contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of DO, a functional mimetic thereof or a DM inhibitor into preparations that can be used pharmaceutically. The preparations contain from about 0.001 to about 99 percent, preferably from about 0.01 to about 95 percent, about 1.0 to about 90 percent, or about 10 to about 50 percent of DO or the functional mimetic thereof or the DM inhibitor, together with the excipient. Standard texts, such as "Remington's Pharmaceutical

Science", 17th edition, 1985, "Hand Book of Pharmaceutical Excipients, 4^tn edition 2003, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation. Suitable dosages can also be based upon the text herein and documents cited herein.

The dose ranges for the administration of the compositions of the present invention are those large enough to produce the desired effect, but not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Counter indications, if any, immune tolerance, and other variables will also affect the proper dosage. The pharmaceutical preparations are manufactured in a manner that is itself known, for example, by means of conventional mixing, granulating, dragee making, dissolving, or lyophilizing processes.

Suitable formulations for parenteral administration include aqueous solutions of DO, a functional mimetic thereof or a DM inhibitor in water-soluble form, for example, water-soluble salts. In addition, suspensions of DO, a functional mimetic thereof or a DM inhibitor as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers.

Additional pharmaceutical methods can be employed to control the duration of action. Delivery systems can include time-release, delayed release or sustained release delivery systems (collectively referred to herein as controlled release). Such systems can avoid repeated administrations of DO, a functional mimetic thereof or a DM inhibitor, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent 5,075,109. Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which the anti- inflammatory agent is contained in a form within a matrix such as those described in U.S. Patent Nos. 4,452,775, 4,667,014, 4,748,034 and 5,239,660 and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Patent Nos. 3,832,253, and 3,854,480.

Cell Therapy

In one embodiment, the methods of the invention comprises administering to a subject a cell expressing DO in order to treat or prevent an auto-immune disease. In one embodimetn, the cells over-express DO. In another embodiment, the cells express wild-type levels of DO and DM.

Cells for cell therapy may be obtained from a number of sources, including healthy individuals. In alternative embodiments, the cells can be genetically engineered as described below to express, for example, DO and/or DM.

Methods of Gene Transfer

In one embodiment of the invention, cells are trans fected with DO-alpha and DO-beta chains. In one embodiment, the cells can be stem cells, e.g., mesenchymal stem cells. The cells are then transplanted into a subject having or suspected of having diabetes. The cells would, subsequently, differentiate into dendritic cells, T cells, and B cells having a normal ratio of DO/DM (or, at least, not exhibiting an over-production of DM) and prevent the onset or ongoing destruction caused by the immune system in patients having diabetes. In another embodiment of the invention, the stem cell transfection/transplantation approach is conducted in the setting of a bone marrow transplant - bone marrow stem cells are transfected, the host undergoes irradiation, and a bone marrow transplant is performed using a population comprising the transfected cells.

Various techniques may be employed for introducing vectors comprising nucleic acid molecules of interest (for example, DO-alpha and DO-beta) into cells. Such techniques include transfection of nucleic acid-CaPO4 precipitates, transfection of nucleic acids associated with DEAE, transfection with a retrovirus including the nucleic acid of interest, liposome mediated transfection, and the like. For certain uses, it is preferred to target the nucleic acids of the invention to particular cells. In such instances, a vehicle used for delivering a nucleic acid according to the invention into a cell (e.g., a retrovirus, or other virus; a liposome) can have a targeting molecule attached thereto. For example, where liposomes are employed to deliver the nucleic acid molecules of interest, proteins which bind to a surface membrane protein associated with endocytosis may be incorporated into the liposome formulation for targeting and/or to facilitate uptake. Such proteins include proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half life, and the like. Polymeric delivery systems also have been used successfully to deliver nucleic acids into cells, as is known by those skilled in the art. Such systems even permit oral delivery of nucleic acid molecules.

Methods for the delivery of nucleic acid molecules are described in Akhtar, et al, Trends Cell Bio., 2, 139, (1992); Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, (1995), Maurer, et al, MoI. Membr. Biol, 16, 129-140, (1999); Hofland and Huang, Handb. Exp. Pharmacol, 137, 165-192, (1999); and Lee, et al, ACS Symp. Ser., 752, 184-192, (2000), all of which are incorporated herein by reference. Beigelman, et al, U.S. Pat. No. 6,395,713 and Sullivan, et al, PCT WO 94/02595 further describe the general methods for delivery of nucleic acid molecules. These protocols can be utilized for the delivery of virtually any nucleic acid molecule.

Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins (see for example Gonzalez et al., Bioconjugate Chem., 10, 1068-1074, (1999); Wang et al., International PCT publication Nos. WO 03/47518 and WO 03/46185), poly(lactic-co-glycolic)ac- id (PLGA) and PLCA microspheres (see for example U.S. Pat. No. 6,447,796 and U.S. Patent Application Publication No. U.S. 2002130430), biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (O'Hare and Normand, International PCT Publication No. WO 00/53722). In another embodiment, the nucleic acids of the invention can also be formulated or complexed with polyethyleneimine and derivatives thereof, such as polyethyleneimine-polyethyleneglycol-N-a- cetylgalactosamine (PEI-PEG- GAL) or polyethyleneimine-polyethyleneglycol-t- ri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives.

Delivery of vectors of the invention can be systemic, such as by intravenous or intramuscular administration, or by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell.

In other specific embodiments, the invention features the use of methods to deliver nucleic acid molecules of interest to stem cells. These methods are described, for example, by Hartmann, et al, J. Phamacol. Exp. Ther., 285(2), 920-928, (1998); Kronenwett, et al., Blood, 91(3), 852-862, (1998); Filion and Phillips, Biochim. Biophys. Acta., 1329(2), 345-356, (1997); Ma and Wei, Leuk. Res., 20(11/12), 925-930, (1996); and Bongartz, et al., Nucleic Acids Research, 22(22), 4681-8, (1994). Vector Design

Nucleic acid molecules of interest (for example, encoding DO), as well as, in another embodiment, interfering RNA molecules that interact with target RNA molecules, thereby down- regulating genes encoding target RNA molecules, are inserted into delivery vectors and expressed from transcription units within the vectors. The recombinant vectors can be DNA plasmids or viral vectors. Generation of the vector construct can be accomplished using any suitable genetic engineering techniques well known in the art, including, without limitation, the standard techniques of PCR, oligonucleotide synthesis, restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, for example as described in Sambrook, et al. Molecular Cloning: A Laboratory Manual. (1989)), Coffin, et al. (Retroviruses. (1997)) and "RNA Viruses: A Practical Approach" (Alan J. Cann, Ed., Oxford University Press, (2000)).

As will be apparent to one of ordinary skill in the art, a variety of suitable vectors are available for transferring nucleic acids of the invention into cells. The selection of an appropriate vector to deliver nucleic acid molecules and the optimization of the conditions for insertion of the selected expression vector into the cell are within the scope of one of ordinary skill in the art without the need for undue experimentation. Viral vectors comprise a nucleotide sequence having sequences for the production of recombinant virus in a packaging cell. Viral vectors expressing nucleic acids of the invention can be constructed based on viral backbones including, but not limited to, a retrovirus, lentivirus, adenovirus, adeno-associated virus, pox virus or alphavirus. The recombinant vectors capable of expressing the nucleic acids of the invention can be delivered as described herein, and persist in target cells (e.g., stable transformants). Alternatively, viral vectors can be used that provide for transient expression of the nucleic acid molecules of interest.

In one embodiment of the present invention, nucleic acid molecules of interest are introduced into cells by transducing the cells, in vitro or in vivo, using replication-deficient retroviruses. Replication-deficient retroviruses are capable of directing synthesis of all virion proteins, but are incapable of making infectious particles. Accordingly, these genetically altered retroviral vectors have general utility for high-efficiency transduction of genes in cultured cells, and specific utility for use in the methods of the present invention.

Retroviruses have been used extensively for transferring genetic material into cells. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell line with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with the viral particles) are provided in the art. Retroviruses insert efficiently a single copy of the nucleic acid molecules of interest into the host cell genome, thereby permitting the integrated genetic material to be passed on to the progeny of the cell when it divides. In addition, gene promoter sequences in the LTR region have been reported to enhance expression of an inserted coding sequence in a variety of cell types. Delivery of a therapeutically effective amount of nucleic acids of the invention via a retrovirus can be efficacious if the efficiency of transduction is high and/or the number of target cells available for transduction is high.

It is possible to propagate and isolate quantities of retroviral vectors (e.g. to prepare suitable titres of the retroviral vector) for subsequent transduction of, for example, a site of interest by using a combination of a packaging or helper cell line and a recombinant vector. In some instances, propagation and isolation may entail isolation of the retroviral gag, pol and env genes and their separate introduction into a host cell to produce a "packaging cell line". The packaging cell line produces the proteins required for packaging retroviral DNA, but it cannot bring about encapsidation due to the lack of a psi region. However, when a recombinant vector carrying a nucleotide sequence of the invention and a psi region is introduced into the packaging cell line, the helper proteins can package the psi-positive recombinant vector to produce the recombinant virus stock. This can be used to infect cells to introduce the nucleotide sequence of the invention into the genome of the cells. A summary of the available packaging lines is presented in "Retroviruses" (1997 Cold Spring Harbour Laboratory Press Eds: J M Coffin, S M Hughes, H. E. Varmus pp. 449).

Transient transfection can also be used to measure vector production when vectors are being developed. Cell lines have been developed using transient infection that produce vector titre levels that are comparable to the levels obtained from stable vector-producing cell lines (Pear, et al. 1993, PNAS 90:8392-8396).

Both viral and non- viral vectors employed in the methods of the invention will contain regulatory sequences, for example, transcription units (e.g., promoters, enhancers), internal ribosome binding site (IRES) elements, signal sequences, terminators and origins of replication in operable association with the nucleic acids of the invention. An IRES element can be used, for example, to separate multiple genes of interest driven by a common promoter. The vectors may also contain, for example, a selectable marker to facilitate manipulation in vitro.

Transcription units drive expression of the nucleic acid molecules of interest. Such transcription units include, for example, promoters and enhancers. A promoter characteristically has a specific nucleotide sequence to initiate transcription. In some cases, additional sequences known as enhancers are desired to obtain the desired gene transcription activity. An "enhancer" is simply any non-translated DNA sequence that works contiguous with the coding sequence (in cis) to change the basal transcription level dictated by the promoter. Preferably, the nucleic acid molecules of interest are introduced into the cell genome immediately downstream from the promoter, so that the promoter and coding sequence are operatively linked so as to permit transcription of the coding sequence.

Promoters include both constitutive and inducible promoters. Naturally-occurring constitutive promoters control the expression of essential cell functions. As a result, a gene under the control of a constitutive promoter is expressed under all conditions of cell growth. Exemplary constitutive promoters include the promoters for the following genes which encode certain constitutive or "housekeeping" functions: hypoxanthine phosphoribosyl transferase (HPRT), dihydrofolate reductase (DHFR) (Scharfmann et al, Proc. Natl. Acad. ScL USA, 88:4626-4630, (1991)), ubiquitin, CMV (U.S. Pat. No. 5,168,062; Karasuyama et al., 1989, J. Exp. Med. 169:13), phosphoglycerol kinase (PGK) (U.S. Pat. Nos. 4,615,974 and 5,104,795; Adra et al., Gene 60:65-74 (1987), Singer-Sam et al. Gene 32:409-417, (1984) and Dobson et al. Nucleic Acids Res. 10:2635-2637, (1982)), adenosine deaminase, pyruvate kinase, phosphoglycerol mutase, the actin promoter (Lai et al., Proc. Natl. Acad. Sci. USA, 86:10006- 10010, (1989)), and other constitutive promoters known to those of skill in the art.

Genes that are under the control of inducible promoters are expressed only or to a greater degree, in the presence of an inducing agent, (e.g., transcription under control of the metallothionein promoter is greatly increased in presence of certain metal ions). Inducible promoters include responsive elements (REs) which stimulate transcription when their inducing factors are bound. For example, there are REs for serum factors, steroid hormones, retinoic acid and cyclic AMP. Promoters containing a particular RE can be chosen in order to obtain an inducible response and in some cases, the RE itself may be attached to a different promoter, thereby conferring inducibility to the recombinant gene. Thus, by selecting the appropriate promoter (constitutive versus inducible; strong versus weak), it is possible to control both the existence and level of expression of nucleic acids of the invention in a cell. A number of systems for inducible expression using such a promoter are known in the art, including the tetracycline responsive system and the lac operator-repressor system. Selection and optimization of these factors for delivery of a therapeutically effective dose of a nucleic acid molecule of interest (for example, encoding DO) or of a particular interfering RNA is deemed to be within the scope of one of ordinary skill in the art without undue experimentation, taking into account the above-disclosed factors and the clinical profile of the patient.

The promoter may also be an immune cell-specific promoter. The skilled artisan will be readily able to select a promoter based on the desired expression pattern. Promoters having cell specificity are advantageous, in that they can specifically direct expression of, for example, DO, thereby controlling the biological effect as desired.

The expression vector may include a selectable marker, for example, a neomycin resistance gene, for facilitating selection of cells that have been transfected or transduced with the expression vector. Alternatively, the cells are transfected with two or more expression vectors, at least one vector containing the nucleic acid molecules of interest and the other vector containing a selection gene. The selection of suitable regulatory sequences is deemed to be within the scope of one of ordinary skill in the art without undue experimentation. Interfering RNA

Interfering RNA is capable of reducing or inhibiting target gene (for example, DM) expression by mediating RNA interference. A "target gene" is a gene whose expression is to be selectively reduced or inhibited. By "reduce or inhibit" is meant the ability to cause an overall decrease preferably of about 20% or greater, more preferably of about 50% or greater, and even more preferably of about 75% or greater, in the level of target protein or nucleic acid. RNA interference involves the selective degradation of a sequence-compatible messenger RNA transcript. See for example Zamore et al, Cell, 101, 25-33 (2000); Bass, Nature, 411, 428-429 (2001); Elbashir et al., Nature, 411, 494-498, (2001); Kreutzer et al., International PCT Publication No. WO 00/44895; Zemicka-Goetz et al., International PCT Publication No. WO 01/36646; Fire, International PCT Publication No. WO 99/32619; Plaetinck et al., International PCT Publication No. WO 00/01846; Mello and Fire, International PCT Publication No. WO 01/29058; Deschamps-Depaillette, International PCT Publication No. WO 99/07409; and Li et al., International PCT Publication No. WO 00/44914; Allshire, Science, 297, 1818-1819, (2002); Volpe et al., Science, 297, 1833-1837, (2002); Jenuwein, Science, 297, 2215-2218, (2002); and Hall et al., Science, 297, 2232-2237, (2002); Hutvagner and Zamore, Science, 297, 2056-60, (2002); McManus et al., RNA, 8, 842-850, (2002); Reinhart et al., Gene & Dev., 16, 1616-1626, (2002); and Reinhart & Bartel, Science, 297, 1831, (2002)).

Interfering RNA includes, but is not limited to small interfering RNA ("siRNA") and small hairpin RNA ("shRNA"). Methods for constructing interfering RNAs are well known in the art. For example, the interfering RNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e., each strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure); the antisense strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof (i.e., an undesired gene) and the sense strand comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. Alternatively, interfering RNA is assembled from a single oligonucleotide, where the self- complementary sense and antisense regions are linked by means of nucleic acid based or non- nucleic acid-based linker(s).

The interfering RNA can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The interfering can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNA interference.

An interfering RNA coding region may encode a self-complementary RNA molecule having a sense region, an antisense region and a loop region. Such an RNA molecule when expressed desirably forms a "hairpin" structure, and is referred to herein as an "shRNA." The loop region is generally between about 2 and about 10 nucleotides in length. In a preferred embodiment, the loop region is from about 6 to about 9 nucleotides in length. In one such embodiment of the invention, the sense region and the antisense region are between about 15 and about 30 nucleotides in length. Following post-transcriptional processing, the small hairpin RNA is converted into a siRNA by a cleavage event mediated by the enzyme Dicer, which is a member of the RNase III family. The siRNA is then capable of inhibiting the expression of a gene with which it shares homology. For details, see Brummelkamp, et al, Science 296:550- 553, (2002); Lee, et al, Nature Biotechnol., 20, 500-505, (2002); Miyagishi and Taira, Nature Biotechnol. 20:497-500, (2002); Paddison et al. Genes & Dev. 16:948-958, (2002); Paul, Nature Biotechnol, 20, 505-508, (2002); Sui, Proc. Natl Acad. Sci. USA, 99(6), 5515-5520, (2002); Yu, et al Proc Natl Acad Sci USA 99:6047-6052, (2002).

The target RNA cleavage reaction guided by siRNAs is highly sequence-specific. In general, siRNA containing a nucleotide sequence identical to a portion of the target gene are preferred for inhibition. However, 100% sequence identity between the siRNA and the target gene is not required to practice the present invention. Thus the invention has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence. For example, siRNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition. Alternatively, siRNA sequences with nucleotide analog substitutions or insertions can be effective for inhibition. Screening Assays

A cDNA encoding DO or DM may be used to screen a library or a plurality of molecules or compounds for active fragments of DO or DM based on specific binding affinity. The libraries may be aptamers, DNA molecules, RNA molecules, PNAs, peptides, proteins such as transcription factors, enhancers, or repressors, and other ligands which regulate the replication, transcription, or translation of the endogenous gene. The assay involves combining a polynucleotide with a library or plurality of molecules or compounds under conditions allowing specific binding, and detecting specific binding to identify at least one molecule which specifically binds the single-stranded or double-stranded molecule.

In one embodiment, the cDNA encoding DO or DM may be incubated with a plurality of purified molecules or compounds and binding activity determined by methods well known in the art, e.g., a gel-retardation assay (U.S. Pat. No. 6,010,849) or a commercially available reticulocyte lysate transcriptional assay. In another embodiment, the cDNA may be incubated with nuclear extracts from biopsied and/or cultured cells and tissues. Specific binding between the cDNA and a molecule or compound in the nuclear extract is initially determined by gel shift assay and may be later confirmed by recovering and raising antibodies against that molecule or compound. When these antibodies are added into the assay, they cause a supershift in the gel- retardation assay.

In another embodiment, the cDNA may be used to purify a molecule or compound using affinity chromatography methods well known in the art. In one embodiment, the cDNA is chemically reacted with cyanogen bromide groups on a polymeric resin or gel. Then a sample is passed over and reacts with or binds to the cDNA. The molecule or compound which is bound to the cDNA may be released from the cDNA by increasing the salt concentration of the flow- through medium and collected. In a further embodiment, the protein or a portion thereof may be used to purify a ligand from a sample. A method for using a protein or a portion thereof to purify a ligand would involve combining the protein or a portion thereof with a sample under conditions to allow specific binding, detecting specific binding between the protein and ligand, recovering the bound protein, and using a chaotropic agent to separate the protein from the purified ligand.

In a further embodiment, a DO- or DM-containing protein may be used to screen a plurality of molecules or compounds in any of a variety of screening assays. The portion of the protein employed in such screening may be free in solution, affixed to an abiotic or biotic substrate (e.g. borne on a cell surface), or located intracellularly. For example, in one method, viable or fixed prokaryotic host cells that are stably transformed with recombinant nucleic acids that have expressed and positioned a peptide on their cell surface can be used in screening assays. The cells are screened against a plurality or libraries of ligands, and the specificity of binding or formation of complexes between the expressed protein and the ligand can be measured. Depending on the particular kind of molecules or compounds being screened, the assay may be used to identify DNA molecules, RNA molecules, peptide nucleic acids, peptides, proteins, mimetics, agonists, antagonists, antibodies, immunoglobulins, inhibitors, and drugs or any other ligand, which specifically binds the protein.

In one aspect, this invention contemplates a method for high throughput screening using very small assay volumes and very small amounts of test compound as described in U.S. Pat. No. 5,876,946, incorporated herein by reference. This method is used to screen large numbers of molecules and compounds via specific binding. In another aspect, this invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding the protein specifically compete with a test compound capable of binding to the protein. Molecules or compounds identified by screening may be used in a model system to evaluate their toxicity, diagnostic, or therapeutic potential.

The following examples are provided as a further description of the invention, and to illustrate but not limit the invention.

EXAMPLES The presentation of disease-relevant self-peptides is clearly essential for type 1 diabetes (TlD). General inhibition of antigen (Ag) presentation, however, would severely compromise a patient's immune system. Overexpression or the complete loss of the MHC-like DO protein has not been shown to cause any significant deviation in immune function (1-8). Variations in the presentation of both antigenic and self-peptides have, however, been documented (1-8). It was, therefore, sought to subtly alter MHC class II (MHCII) Ag presentation in NOD mice by overexpressing the HLA-DM (DM; H2-M in mice) modulator, HLA-DO (DO; H2-0 in mice), in DCs. Studies described herein show that NOD mice expressing human DO specifically in DCs rarely develop TlD.

Example 1. Protection of NOD.DO mice from diabetes

NOD mice expressing DO under the control of a heterologous promoter were examined for the incidence and time of onset of diabetes (Fig. 1). Transgenic (Tg) mice expressing the human HLA-DOA and HLA-DOB genes under the control of the DC-specific CDl Ic promoter (6) were crossed into the NOD background for 10 generations (NOD.DO). Age-matched female NOD.DO (n=36) and transgenic-negative littermate control NOD mice (n=32) were tested for the onset of diabetes by monitoring urine glucose levels. Mice with 2 consecutive measurements exceeding 250 mg/dl were considered diabetic, with the first positive reading scored as diabetic (P<0.05 (X² test)). As shown in Fig. 1, diabetes onset in NOD mice was at 16 weeks of age and about 90% were diabetic by 41 weeks. No diabetes was observed in NOD.DO mice over a 50 week period.

To examine the expression of DO in NOD.DO transgenic mice, splenic cells were stained with antibodies to define DCs (CDl Ic⁺) and B cells (CD 19 ), co-stained with an antibody specific for human DO, and subjected to FACS analysis. The results of the FACS analysis showed that most if not all DCs in the NOD.DO Tg mice expressed DO, compared to DCs isolated from the NOD mice, which did not detectably show expression of DO (Fig. 2A). A substantial percentage of B cells (-25%) in the NOD.DO Tg mice also expressed DO, compared to B cells isolated from the NOD mice, which did not express DO (Fig. 2B). Most of the DO expressing B cells were marginal zone B cells (data not shown). These results show that DO is expressed in NOD.DO transgenic mice in splenic DCs and B cells. Pancreatic sections from NOD and NOD. DO mice were analyzed by immuno fluorescent histology to examine DO expression in DCs (Fig. 3 A-3F). Pancreatic sections were co- immunostained using anti-CD 1 Ic to define DCs and anti-DO (red) to identify DO expressing cells. DO expression was observed in pancreas tissue from NOD.DO (Fig. 3B), but not NOD mice (Fig. 3A). DO expression was present in CDl Ic⁺ cells (Fig. 3F; yellow arrow in bottom panel denotes example of colocalization). A subset of pancreatic cells expressed DO but were not CDl Ic⁺ (Fig. 3F; red arrow denotes one example). These cells were CD19⁺ B cells (data not shown). These results show that DO is expressed in NOD.DO transgenic mice in pancreatic DCs and B cells.

Based on these results, we believe that DO modulates the loading of disease-relevant peptides by H2-M, thereby preventing the activation of self-reactive CD4 T cells.

Example 2. Determination of the mechanism by which DO expression in dendritic cells (DCs) blocks diabetes in NOD mice

Previous studies have shown that ectopic DO expression in mouse DCs inhibited and/or modulated MHCII peptide loading in C57BI/6 (H2-b) and Bl O.BR mice (H2-k) (6). DCs from the DO Tg mice expressed lower levels of MHCII-endogenous peptide complexes on their surface (6). Having shown that ectopic expression of DO in DCs of NOD mice prevents the onset of TID (Fig. 1), efforts are made to define the molecular mechanism by which NOD.DO mice are protected from TlD.

About 30 Female NOD and NOD.DO mice were analyzed for diabetes onset by measuring urine glucose levels from 10 weeks of age. The level of insulitis in NOD and NOD.DO mice (n>5/group) was determined at weeks 10-11, 14-19, and 23-38 by histology on fixed, paraffin embedded pancreas tissue. The severity of insulitis was scored on individual islets. It was then confirmed that NOD.DO mice have the linkage markers associated with NOD-derived recessive Idd loci by microsatellite analysis (Charles River). Microsatellite analysis has demonstrated that the NOD.DO mice have all Idd loci. Example 3. DO over-expression in bone-marrow derived cells protects NOD mice from diabetes.

Six week old NOD (CD45.1) recipient mice were irradiated and reconstituted with 100% NOD (CD45.2) T-depleted bone marrow, 100% NOD.DO (CD45.1) T-depleted bone marrow, or a 50:50 mix of NOD and NOD.DO T-depleted bone marrows. Diabetes frequency was determined by measurement of urine glucose levels in female mice. Mice with 2 consecutive measurements exceeding 250mg/dl were considered diabetic with the first positive reading scored as diabetic. Chimerism of the T-depleted bone marrow was confirmed by FACs analysis. The data are representative results obtained from 1 of 2 similar experiments.

As shown in Fig. 4, about 70% of the mice receiving 100% NOD (CD45.2) T-depleted bone marrow were diabetic by 26 weeks, with the earliest onset of diabetes at 12 weeks after bone marrow transfer. Mice receiving 100% NOD.DO (CD45.1) T-depleted bone marrow did not develop diabetes during the course of the study, up to 30 weeks after bone marrow transfer. Diabetes development in mice receiving the chimeric T-depleted bone marrow was also drastically decreased and the incidence of diabetes was observed to be about 8%. These results show that DO over-expression in 50% of bone marrow derived cells is sufficient to protect NOD mice from diabetes.

Example 4. Determination of Mechanism for Inhibition of Diabetes in NOD.DO mice

In order to determine how DO expression in DCs might function to prevent TlD onset in NOD mice, a number of studies are carried out, as follows:

A. To test whether DO modulates presentation of islet-specific Ags to CD4 T cells, CSFE-labeled BDC2.5 NOD T cells are transferred into NOD and NOD.DO mice as previously described (44, 45). BDC2.5 TCR Tg T cells express CD4 T cells that express a TCR specific for an unknown islet Ag presented by I-A^g7 (46). The proliferation (CSFE dilution) of the transferred BDC2.5 T cells 3 days post transfer is measured by FACs. If presentation of islet Ags is inhibited in NOD.DO mice, in vitro Ag presentation assays are carried out with NOD and NOD.DO derived pancreatic DCs or bmDCs.

B. I-A^g7 MHC tetramers loaded with the peptide mimitope 2.5 (A^g7/2.5mi) can be used to specifically stain BDC2.5 Tg T cells (48). Importantly, A^g7/2.5mi tetramers also stain a distinct population of CD4 T cells that are positively selected in the thymus of NOD mice (48). To test whether DO expression in DC alters the negative selection of diabetic promoting T cells in the thymus, thymocytes and peripheral T cell populations from NOD and NOD. DO mice are stained with A^g7/2.5mi tetramers and analyzed by FACs (48).

C. To determine if NOD. DO mice have altered regulator T cell populations, the percentages and absolute numbers of CD4, CD8 and regulatory T cells in the thymus, spleen and pancreatic lymph nodes are determined by FACs analysis. Regulatory T cells are identified as cells expressing both CD4 and FOXP3 (51). If the regulatory T cell populations are normal in NOD. DO mice, the functionality of the cells is confirmed using the in vitro assay for regulatory T cell function (52). Regulatory T cell populations will be purified using the CD4⁺CD25⁺ Regulatory T Cell Isolation Kit (Miltenyi).

Example 5. H2-M and H2-O levels in NOD and NOD-related (B6) strains of mice.

Spleen cells from NOD and B6 mice were surface- stained with a monoclonal antibody specific for CDl Ic to identify splenic dendritic cells (DCs), permeabilized, stained intracellularly with monoclonal antibodies specific for H2-0 (Mags.Ob3) and H2-M (2C3A), and analyzed by FACS. DCs were defined as CDl Ic⁺, and histogram plots show staining for H2-0 (Figure 5A) and H2-M (Figure 5B) for spleen cells from two NOD and two B6 mice. NOD mice express low levels of H2-0 in splenic DCs (relative to B6 mice) - in fact, about 2- fold less (Fig. 5). H2-M levels are unaltered. This indicates that H2-M function is "over"-active in NOD mice (since H2-0 is not expressed at high levels to block H2-M function), further supporting the potential of H2-M as a therapeutic target.

Example 6. Further evaluation of H2-M function in NOD mice

H2-M and H2-0 levels are evaluated in the various DC and B cell populations by FACs and Western Blotting. An analysis of H2-M and H2-0 expression levels in splenic DC subsets using a mAb generated to H2-0 (24) is extended to NOD mice. H2-M and H2-0 levels in splenic and pancreatic lymphnode DC populations are determined by FACs analysis using the following markers to define the DC subsets: lymphoid, CDl Ic⁺ CDl Ib' B220' DEC205⁺ CD8a⁺; myeloid, CDl Ic⁺ CDl Ib⁺ B220' DE205' CD8a-; plasmacytoid CDl Ic⁺ CDl Ib' B220⁺ DEC205' CD8a' (53, 54). Levels of H2-M and H2-0 in NOD DCs are directly compared to the levels of these proteins in B6 mice, as well as B6.NOD, NOD.B1OSN-H2^b and NON.NOD mice. The herein-described finding that DO-mediated alteration of the MHCII pathway in DCs prevents TlD in NOD mice indicates that subtle changes in MHCII presentation are sufficient for disease prevention. Small molecules that can mimic or enhance DM-mediated peptide loading have been identified (56-59). Thus, H2-M/DM can be a target for potential interference with the disease process.

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Claims

WHAT IS CLAIMED IS:

1. A method of treating an auto-immune disease in a subject, comprising administering DO or a functional mimetic thereof to the subject, thereby treating the auto-immune disease in the subject.

2. The method of claim 1, wherein the auto-immune disease is type 1 diabetes.

3. The method of claim 3, wherein the type I diabetes has progressed to the insulitis stage.

4. The method of any one of claims 1-3, wherein the administering of DO or a functional mimetic thereof normalizes the DO/DM ratio in the subject.

5. A method of treating an auto-immune disease in a subject, comprising administering to the subject an agent that increases DO expression or activity in the subject, thereby treating the auto-immune disease in the subject.

6. The method of claim 5, wherein DO expression is increased in immune cells or immune cell precursors.

7. The method of claim 5, wherein the auto-immune disease is type 1 diabetes.

8. The method of claim 7, wherein the type I diabetes has progressed to the insulitis stage.

9. The method of any one of claims 5-8, wherein the administering of the agent normalizes the DO/DM ratio in the immune cells of the subject.

10. A method of treating an auto-immune disease in a subject, comprising decreasing the expression or activity of DM in a subject, thereby the auto-immune disease in the subject.

11. The method of claim 10, wherein the auto-immune disease is type 1 diabetes.

12. The method of claim 11 , wherein the type 1 diabetes has progressed to the insulitis stage.

13. The method of any one of claims 10-12, comprising administering to said subject an inhibitor of DM activity.

14. The method of any one of claims 10-12, wherein the decreasing of the expression or activity of DM normalizes the DO/DM ratio in immune cells of the subject.

15. The method of any one of claims 1 to 14, wherein DM is over-produced in immune cells of the subject.

16. The method of any one of claims 5-14, wherein the immune cells are selected from the group consisting of dendritic cells, T cells, and B cells.

17. The method of claim 5, wherein DO expression is increased via trans fection, transduction, or infection of the cells with a vector comprising a nucleic acid molecule encoding DO-alpha and a nucleic acid molecule encoding DO-beta.

18. The method of claim 17, wherein: a) the nucleic acid molecule encoding DO-alpha (i) comprises the nucleic acid sequence of SEQ ID NO:1 or a complement thereof or (ii) encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or a fragment thereof; and b) the nucleic acid molecule encoding DO-beta (i) comprises the nucleic acid sequence of SEQ ID NO:3 or a complement thereof or (ii) encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:4 or a fragment thereof.

19. The method of claim 17, wherein the vector comprises an immune cell-specific promoter.

20. The method of claim 17, wherein the vector is a retroviral vector.

21. A method of treating an auto-immune disease in a subject comprising: administering to the subject a cell that expresses DO, thereby treating the auto-immune disease in the subject.

22. The emthod of claims 21, wherein the cell overexpresses DO.

23. The method of claim 21, wherein the cell expresses wild-type levels of DO and DM.

24. The method of any one of claims 21-23, wherein the cell expresses: a) the nucleic acid molecule encoding DO-alpha (i) comprises the nucleic acid sequence of SEQ ID NO:1 or a complement thereof or (ii) encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or a fragment thereof; and b) the nucleic acid molecule encoding DO-beta (i) comprises the nucleic acid sequence of SEQ ID NO:3 or a complement thereof or (ii) encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:4 or a fragment thereof.

25. A method of identifying an inhibitor of DM expression or activity for use in the treatment of an auto-immune disease or disorder, comprising: (i) contacting a population of immune cells or immune cell precursors over-expressing DM with a test agent; and (ii) detecting a decrease in the level of expression or activity of DM in the cells, thereby identifying an inhibitor of DM expression or activity for use in the treatment of an auto-immune disease or disorder.

26. The method of claim 25, wherein the auto-immune disease or disorder is type I diabetes.

27. The method of claim 25, wherein the immune cell precursors comprise stem cells.

28. The inhibitor identified by the method of claim 25.

29. A method of decreasing DM gene expression in a cell, comprising contacting the cell with a vector comprising a nucleic acid sequence comprising an interfering RNA, thereby decreasing DM gene expression in the cell.

30. The method of claim 29, wherein the cell over-expresses DM .

31. The method of any one of claims 29 or 30, wherein the interfering RNA comprises a small interfering RNA.

32. A packaged pharmaceutical comprising DO or a functional mimetic thereof and instructions for use.

33. A kit for treating type 1 diabetes in a subject in need thereof, comprising DO or a functional mimetic thereof and instructions for use.

34. A kit for treatign an auto-immune disease comprising a cell that expresses DO and instructions for use.

35. A pharmaceutical composition comprising a therapeutically effective amount of a nucleic acid molecule encoding a polypeptide comprising DO or a functional mimetic thereof and a pharmaceutically acceptable carrier.