WO1997046253A9 - Immunotherapie pour maladie auto-immune - Google Patents

Immunotherapie pour maladie auto-immune

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Publication number
WO1997046253A9
WO1997046253A9 PCT/US1997/009427 US9709427W WO9746253A9 WO 1997046253 A9 WO1997046253 A9 WO 1997046253A9 US 9709427 W US9709427 W US 9709427W WO 9746253 A9 WO9746253 A9 WO 9746253A9
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antigen
cell
ala
autoimmune disease
immune response
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PCT/US1997/009427
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English (en)
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  • the present invention relates to the use of immunotherapies for the treatment of autoimmune disorders in general and relates, in particular, to the use of techniques of in si tu antigen or gene delivery for the treatment of autoimmune diseases.
  • Autoimmune diseases are characterized by cytotoxic immune responses to epitopes on self antigens natively found in the diseased individual.
  • the immune system of the individual then activates an inflammatory cascade aimed at cells and tissues presenting those specific self antigens.
  • the destruction of the antigen, tissue, cell type, or organ attacked by the individual ' s own immune system gives rise to the symptoms of the disease.
  • Clinically significant autoimmune diseases include, for example, rheumatoid arthritis, multiple sclerosis, juvenile-onset diabetes, systemic lupus erythematosus , autoimmune uveoretinitis, autoimmune vasculitis, bullous pemphigus, myasthenia gravis, autoimmune thyroiditis or Hashimoto's disease, Sjogren's syndrome, granulomatous orchitis, autoimmune oophoritis, Crohn' s disease, sarcoidosis, rheumatic carditis, ankylosing spondylitis, Grave's disease, and autoimmune thrombocytopenic purpura . See e . g. , Paul, .E. (1993) Fundamental Immunology, Third Edition, Raven Press, New York, Chapter 30, pp. 1033-1097; and Cohen et al . (1994) Autoimmune Disease Models, A Guidebook, Academic Press, 1994.
  • autoimmune disease may be triggered by infection, either clinical or sub-clinical, by an infectious agent which presents the immune system with an epitope that is similar to an epitope natively present on some cell type in the individual.
  • This theory may explain the semi-inheritable nature of many autoimmune diseases since some familial alleles of particular cell surface markers may be more or less similar to the epitope on the actuating infectious agent, and therefore more or less likely to be recognized by antibodies or T-cells that are specific to the infectious agent.
  • Thl refers to a cytotoxic T lymphocytic response (a CTL response) , also called a delayed hypersensitivity type reaction (DTH) .
  • CTL response cytotoxic T lymphocytic response
  • DTH delayed hypersensitivity type reaction
  • This type of response generally results in destruction of the antigen, cell, tissue, or organ which expresses the antigen.
  • Th2 T lymphocytic response In contrast to the cytotoxic Thl type of response, the Th2 T lymphocytic response generally does not participate in delayed type hypersensitivity or cytotoxic responses. Thus, elimination of cytotoxic responses and development of desensitizing responses are considered to be indicia of development of tolerance to the antigen.
  • an immune response in either of these two categories tends to down-regulate the immune response in the other category.
  • interferon- gamma released by cytotoxic T cells inhibits proliferation of Th2 cells
  • interleukin-10 released by Th2 cells inhibits the stimulation of cytotoxic cells by monocytic antigen presenting cells.
  • This mutual inhibition tends to favor the development of only one type of response, cytotoxic or desensitizing, to a given antigen. Because of the mutual inhibition and because an animal's immune system is constantly exposed to self antigens, it has been difficult to intervene in the autoimmune process.
  • Rheumatoid arthritis is an inflammatory, autoimmune disease affecting multiple systems, but primarily affecting multiple joints. The disease is accompanied by inflammation of the synovial membranes and other joint structures, muscle atrophy, bone erosion and rarefaction, and formation of pannus (fibrovascular inflammatory membrane) on the joint surfaces. Rheumatoid arthritis generally is quite painful and often severely debilitating.
  • Rheumatoid arthritis is not associated with known infection.
  • the disease is commonly associated with formation of autoantibodies reactive with the subject's own IgG (rheumatoid factor), leading to immunoglobulin aggregates, the role of which in the disease is unclear.
  • IgG rheumatoid factor
  • rheumatoid arthritis is more common (-3 -fold) in human females than in males.
  • Family studies, including studies of identical and fraternal twins clearly show that the causation of rheumatoid arthritis is partly genetic and partly non-genetic.
  • the non-genetic component may be either environmental and/or caused by random chance (e.g. somatic mutations, chance biochemical events, stochastic rearrangement of T-cell receptor genes) .
  • HLA-DR4 (and DQ8) on one copy of chromosome 6 and HLA- DR1 on the other.
  • Recent transgenic mouse studies suggest that HLA-DR and HLA-DQ affect rheumatoid arthritis susceptibility via different mechanisms.
  • One animal model for the study of human rheumatoid arthritis is an autoimmune arthritis induced in rodents. In order to generate disease, Lewis rats or DBA/1 mice, are intradermally injected with type II collagen (CII) in adjuvant (see, e.g., Stuart et al . , Ann. .Rev. Immunol . 2 . :199-218 (1984)). This model is referred to as collagen-induced arthritis or CIA.
  • CII type II collagen
  • Trentham et al . Science 261 : 1727-1730 (1993) conducted a double-blind placebo-controlled clinical trial of 28 chicken Cll-fed and 31 placebo- fed patients with rheumatoid arthritis.
  • the authors reported that clinical and lab variables were similar in the collagen and placebo groups at entry. However, despite the stated random allocation to collagen or placebo groups, the placebo group started out significantly worse than the collagen group by many of the assessment criteria used.
  • desensitization by oral feeding type II collagen is safe and may be clinically effective, although its effectiveness in rheumatoid arthritis patients has not been clearly demonstrated. Its effectiveness is highly dose-dependent , which would clearly complicate its application in the heterogeneous human population.
  • CIA model involves the injection of collagen or collagen-related peptides into an individual.
  • the CII protein or Cll-derived peptides have also been used to treat or prevent CIA and in other autoimmune arthritis models (not collagen-induced) .
  • a synthetic modified collagen peptide (designed to competitively inhibit binding of the corresponding native collagen peptide to MHC) reduced the incidence and severity of CIA in mice.
  • this approach is less promising than induction of an anti-inflammatory "Th2-like" response since it likely would require the continued presence of large quantities of the synthetic competitor peptide and is not expected to have a bystander effect .
  • Mt Hsp65 65-kDa heat-shock protein from Mycobacterium tuberculosis, or Mt Hsp65.
  • Mt Hsp65 is known to cross-react with a component of articular cartilage, now known to be human Hsp60. This and other heat-shock proteins are up-regulated in the joints of arthritic patients.
  • Rheumatoid arthritis patients have T-cell reactivity to Hsp65, and arthritogenic T cell clones from CFA-treated rats recognize a specific epitope of Hsp65.
  • Hsp65 emulsified in oil did not induce adjuvant arthritis (AA) , but instead protected against a subsequent attempt to induce adjuvant arthritis with whole Mycobacterium tuberculosis.
  • the 9-amino acid immunodominant epitope was similarly protective.
  • Multiple sclerosis is a chronic central nervous system disease of considerable medical importance. See Martin et al . , Immunological aspects of demyelinating diseases. Ann. .Rev. Immunol . 10 : 153- 187 (1992) . It is the most common demyelinating disorder of the brain and spinal cord. It affects males and females equally, usually beginning between the ages of 20 and 40 years. It can have a subtle onset, with spontaneous remissions and relapses, but its course is often progressive and sometimes relentless .
  • multiple sclerosis is characterized by localized demyelination of white matter with formation of characteristic plaques.
  • T cells reactive to myelin basic protein (MBP) and proteolipid protein (PLP) have been found in the cerebrospinal fluid and blood of patients with multiple sclerosis.
  • the MBP molecule contains multiple epitopes, and those recognized by multiple sclerosis and normal T cells may differ.
  • T cell lines derived from patients with multiple sclerosis recognize an HLA-DR2- restricted epitope in residues 84-106 (Ota et al . , Nature 346:183-187 (1989)). Additionally, Martin et al . , J. Exp. Med. 173:19 (1991), identified an epitope in residues 87-106 that was recognized by multiple sclerosis T cells.
  • EAE Experimental autoimmune encephalomyelitis
  • MS multiple sclerosis
  • the pathology of EAE is characterized by lymphocytic and mononuclear cell inflammation, an increase in blood-brain barrier permeability, and demyelination (Raine et al . , Lab Invest . 43_:150-157 (1980); Paterson et al . , Immunol . Rev. 55: 89-120 (1981)) resulting in partial or complete paralysis and in severe cases death of the affected patient.
  • neural antigen-specific CD4+ T lymphocytes appear to be the initiators of the response since in vivo depletion of CD4+ T cells inhibits induction of EAE (Waldor et al .
  • the autoreactive T cells that induce organ specific autoimmune diseases, including EAE generally display a helper T cell type 1 (Thl) phenotype (Miller et al., Immunol . Today 15:356 (1994)).
  • Thl helper T cell type 1
  • the adoptive transfer of myelin-reactive Thl cells but not Th2 cells can induce EAE and cytokines associated with a Thl type response are present in the inflammatory lesions of the central nervous system of animals affected by EAE, whereas cytokines associated with a
  • Th2 type are absent from such lesions. Based on these findings, it has been suggested that Thl cytokines may play a role in the pathology of EAE (Kuchroo et al . , J “ . Immunol . 151:3776 (1992); Zamvil et al . , Ann . Rev. Immunol . 8:579 (1990)).
  • regulatory cells involved in suppressing the development of EAE are those associated with a cytokine profile of the Th2 phenotype, and recovery from EAE is associated with the presence of Th2 cells and cytokines in the CNS
  • Diabetes Mellitus Diabetes mellitus is a disorder of glucose metabolism due to a relative or absolute lack of insulin (reviewed in Atkinson et al . , Sci . Am. 263 : 62- 71 (1990) and further described in Paul, W.E., Fundamental Immunology, 3rd edition, Raven Press, New York, 1993) .
  • Type I diabetes is characterized by the patient's dependency upon exogenous insulin to maintain normal glucose metabolism.
  • Type I diabetes is an autoimmune disease that destroys the insulin-producing beta cells of the islets of Langerhans in the pancreas.
  • insulitis The specific lesion, called “insulitis” is characterized by an infiltration of the islets of Langerhans by mononuclear cells, mainly CD8+ T cells, and to a lesser extent by CD4+ T cells (Rossini et al . , Annu . Rev. Immunol . 3:289-320 (1985)).
  • Autoantibodies against islet cells are characteristic in type I diabetes, some bind to cytoplasmic constituents, and others bind to proteins on the beta-cell membrane.
  • a notable feature of these anti-islet cell antibodies is that they precede the onset of the disease, and some subsets (such as complement-fixing IgG anti-islet cell antibodies) can predict the disease in susceptible individuals.
  • Antibodies against a cytoplasmic 64 kD beta-cell protein occur in about 80% of newly diagnosed patients, and over 90% of patients have antibodies against particular tryptic fragments of the protein (Christie et al . , J. Exp . Med . 172 :789-794 (1990)). These antibodies may be present for years before the onset of disease, and thus may be predictive markers.
  • Another potential immunogen is a 38-kD beta-cell granule protein. It stimulates in vi tro proliferation of T cells from diabetics (Roep et al . , Nature 345:632-634 (1990)). This immune response may provide the link between cytotoxic T-cell responses and type I diabetes.
  • current therapy for type I diabetes primarily consists of injection of insulin.
  • the ability to predict the disease by means of assays for anti-islet cell antibodies, along with the availability of tests for residual beta-cell function raises the possibility of preventive therapy. This possibility is of substantial interest because in many cases, almost all beta-cells have been destroyed by the time of diagnosis.
  • the present invention provides an effective method for treating an autoimmune disease.
  • the method is based on the induction of self antigen desensitization in an individual by introduction of the self antigen, or a gene coding therefor, into a cell of the individual.
  • the antigen is selected on the basis of its involvement in the autoimmune process.
  • the subject invention is directed to a method for treating or preventing an autoimmune disease in a mammal comprising the steps of : (a) providing a particle coated with an antigen against which an immune response is mounted in the autoimmune disease, wherein the particle is suitable for delivery into a recipient cell, the particle having an average diameter of about 0.5 to about 5 ⁇ m and a density sufficient to allow delivery into the recipient cell; (b) delivering the particle into the recipient cell of the mammal; and
  • step (c) repeating step (b) a sufficient number of times until either a reduction in a cytotoxic immune response or a desensitizing immune response is induced in the mammal .
  • the subject invention is directed to a method for treating or preventing an autoimmune disease in a mammal comprising the steps of:
  • nucleic acid construct which comprises a coding sequence for an antigen against which an immune response is mounted in the autoimmune disease, operably linked to control elements such that the coding sequence can be transcribed and translated in a recipient cell;
  • step (c) repeating step (b) a sufficient number of times until either a reduction in a cytotoxic immune response or a desensitizing immune response is induced in the mammal .
  • the invention is directed to a method for treating or preventing an autoimmune disease in a mammal, wherein the method comprises :
  • step (c) repeating step (b) a sufficient number of times until either a reduction in a cytotoxic immune response or a desensitizing immune response is induced in the mammal .
  • the cytotoxic immune response is characterized by the secretion from a T- lymphocyte of one or more mediators selected from the group consisting of interleukin-2 , interferon-gamma and tumor necrosis factor.
  • the desensitizing immune response is characterized by the secretion from a T- lymphocyte of one or more mediators selected from the group consisting of interleukin-4 , interleukin-5 , interleukin-6 , and interleukin-10.
  • the desensitizing immune response is characterized by augmented production of one or more mediators selected from the group consisting of interleukin-4, interleukin-10, and, surprisingly, interferon-gamma.
  • the autoimmune disease is rheumatoid arthritis and the antigen is selected from the group consisting of collagen, the Mycobacterium tuberculosis heat shock protein Mt Hsp65, and epitopes thereof.
  • the autoimmune disease is multiple sclerosis and the antigen is selected from the group consisting of myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein, and epitopes thereof.
  • Fig. 1 depicts the results of an experiment where type II collagen (CII) was delivered to mice.
  • Fig. 2 shows IgG subclass responses in CII- challenged mice.
  • Fig. 3 shows IgG subclass responses in Hsp65-DNA-pretreated, CII -challenged mice.
  • Fig. 4 shows the protocol for immunizing rats for treatment of experimental autoimmune encephalomyelitis (EAE) .
  • autoimmune disease means a set of sustained organ-specific or systemic clinical symptoms and signs associated with altered immune homeostasis that is manifested by qualitative and/or quantitative defects of expressed autoimmune repertoires (Autoimmuni ty Physiology and Disease, Coutinho and Kazatchkine, eds, Wiley-Liss, 1993, Chapter 27, page 433).
  • an “antigen” refers to any agent, generally a macromolecule, which can elicit an immunological response in an individual .
  • the immunological response may be of B- and/or T-lymphocytic cells.
  • the term may be used to refer to an individual macromolecule or to a homogeneous or heterogeneous population of antigenic macromolecules .
  • "antigen” is used to refer to a protein molecule or portion thereof which contains one or more epitopes.
  • epitopes generally refers to the site on an antigen to which a specific antibody molecule binds. The identification of epitopes which are able to elicit an antibody response is readily accomplished using techniques well known in the art.
  • T-cell epitopes are generally those features of a peptide structure capable of inducing a T-cell response. In this regard, it is accepted in the art that T-cell epitopes comprise linear peptide determinants that assume extended conformations within the peptide-binding cleft of MHC molecules, (Unanue et al . , Science (1987) 236 : 551- 557) .
  • an epitope is generally a peptide having about 3-5, preferably 5-10 or more amino acid residues.
  • Particular epitopes useful in the practice of the invention include, but are not limited to, the 9 amino acid immunodominant epitope of Hsp65 and the epitope present in residues 84-106 of the MBP molecule.
  • self antigen which is used interchangeably herein with the term “autoantigen,” means an antigen, or a molecule capable of being recognized during an immune response, that is normally part of the individual. This is in contrast with antigens which are foreign, or exogenous, which are not normally part of the individual's milieu.
  • Each autoimmune disease is characterized by an immune response directed at a self antigen. Normally, there are no active immune responses to self antigens, and no symptoms appear. With the development of an immune response to a self antigen, autoimmune diseases may appear. Autoimmune diseases present clinically with different symptoms depending upon the specific self antigen against which an immune response is raised.
  • This immune response results in the destruction of the structure containing the self antigen, and it is the loss of that structure with concurrent loss of that structure's normal function which results in symptoms of autoimmune disease.
  • the term "anergy” means a reversible antiproliferative state which results in decreased responsiveness of an immune cell or cells to an antigen.
  • antigen desensitization refers to the process of decreasing an immune response by delivering to the cell or animal, over a period of time, the antigen against which an immune response is mounted. With repeated exposure of the immune cells to the antigen, a decrease in the cytotoxic response is seen. Such desensitization includes, but is not limited to, a switch from a "Thl” to a "Th2" response. Antigen desensitization may result in the cessation of the autoimmune process, and may ultimately result in the replenishment of the individual's self antigen, and the associated structures.
  • Thl means that response of T- lymphocytes to antigen characterized by, but not limited to, T-lymphocyte secretion of one or more of the following mediators: interleukin-2 (IL-2); interferon-gamma (IFN-gamma) ; and tumor necrosis factor-beta (TNF-beta) .
  • IL-2 interleukin-2
  • IFN-gamma interferon-gamma
  • TNF-beta tumor necrosis factor-beta
  • Th2 means that response of T- lymphocytes to antigen characterized by, but not limited to, T-lymphocyte secretion of one or more of the following mediators: interleukin-4 (IL-4); interleukin-5 (IL-5) ; interleukin-6 (IL-6) ; and interleukin-10 (IL-10) .
  • the lymphokines produced by Th2 cells are primarily mediators of helper T-cell function for B-cell antibody production, and are not generally part of the delayed hypersensitivity type response (DTH) or of the cytotoxic response. Th2 responses are generally desensitizing to the antigen. It is understood that other mediators may be released without changing the meaning of "Th2.
  • vector any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is generally capable of replication when associated with the proper control elements and present in an appropriate host cell.
  • Gene delivery refers to methods or systems for reliably inserting foreign DNA into host cells. Such methods can result in expression of non- integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episomes) , integration of transferred genetic material into the genomic DNA of host cells, or eventual loss of the DNA through degradative processes .
  • nucleotide sequence or a “nucleic acid molecule” refers to DNA and RNA sequences.
  • the term captures molecules that include any of the known base analogues of DNA and RNA.
  • a "coding sequence” or a sequence which "encodes” a particular polypeptide is a nucleic acid sequence which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vi tro or in vivo when placed under the control of appropriate regulatory sequences.
  • the boundaries of the coding sequence are conventionally determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
  • a coding sequence can include, but is not limited to, cDNA from procaryotic or eukaryotic mRNA, genomic DNA sequences from procaryotic or eukaryotic DNA, and even synthetic DNA sequences. In eukaryotic DNA, a transcription termination sequence will usually be located 3' to the coding sequence.
  • control sequences refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected gene is capable of being transcribed and translated in an appropriate recipient cell .
  • “Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
  • control sequences operably linked to a coding sequence are capable of effecting the expression of the coding sequence.
  • the control sequences need not be contiguous with the coding sequence, so long as they function to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.
  • the present invention allows for the treatment and/or prevention of a wide variety of autoimmune diseases including, but not limited to, rheumatoid arthritis, multiple sclerosis, juvenile-onset diabetes, systemic lupus erythematosus, autoimmune uveoretinitis, autoimmune vasculitis, bullous pemphigus, myasthenia gravis, autoimmune thyroiditis or Hashimoto's disease, Sjogren's syndrome, granulomatous orchitis, autoimmune oophoritis, Crohn' s disease, sarcoidosis, rheumatic carditis, ankylosing spondylitis, Grave's disease, and autoimmune thrombocytopenic purpura.
  • autoimmune diseases including, but not limited to, rheumatoid arthritis, multiple sclerosis, juvenile-onset diabetes, systemic lupus erythematosus, autoimmune uveoretinitis, autoimmune vasculitis, bullous pemph
  • the method utilizes autoantigen desensitization carried out using, for example, particle-mediated delivery techniques (e.g., gene gun delivery) .
  • particle-mediated delivery techniques e.g., gene gun delivery
  • a wide variety of nucleic acid molecules or peptide antigens can be delivered using the methods of the invention. The particular molecule selected depends on the autoimmune disease being treated, for example, if the autoimmune disease to be treated is rheumatoid arthritis, the autoantigen can be selected from the group of collagen, the Mycobacterium tuberculosis heat shock protein Mt Hsp65, epitopes thereof capable of eliciting the appropriate immune response, and nucleic acid molecules encoding the same.
  • the autoantigen can be selected from the group of myelin basic protein, myelin oligodendrocyte protein, proteolipid protein, epitopes thereof, and nucleic acid molecules encoding the same, as well as coding sequences encoding the entire cDNA library of human oligodendrocytes or an oligodendrocyte gene library.
  • nucleic acid molecules used in the subject methods contain coding regions with suitable control sequences and, optionally, ancillary therapeutic nucleotide sequences.
  • the nucleic acid molecules are prepared in the form of vectors which include the necessary elements to direct transcription and translation in a target cell. If expression is desired using the host's enzymes (such as by the use of endogenous RNA polymerase) , the gene or genes will be present in the vectors operatively linked to control sequences recognized by the particular host, or even particular cells within the host. Thus, eukaryotic control elements will be present for expression in mammalian hosts. Such sequences are known in the art and are discussed more fully below.
  • the above-described autoantigens can be administered in conjunction with ancillary substances, such as pharmacological agents, adjuvants, cytokines, or in conjunction with delivery of vectors encoding cytokines.
  • ancillary substances such as pharmacological agents, adjuvants, cytokines, or in conjunction with delivery of vectors encoding cytokines.
  • chemical immunomodulatory agents such as the active form of vitamin D3 can also be used.
  • 1 , 25-dihydroxy vitamin D3 has been shown to exert an adjuvant effect via intramuscular DNA immunization.
  • genes encoding for peptides known to stimulate, modify, or modulate a host's immune response can be coadministered with the above-described antigens.
  • genes encoding one or more of the various cytokines (or functional fragments thereof), such as the interleukins, interferons, and colony stimulating factors will find use with the instant invention.
  • the gene sequences for a number of these substances are known.
  • genes encoding IL-4 and IL-10 can be codelivered with autoantigen preparations.
  • autoantigen delivery to the epidermis elicits an autoantigen-specific anti-inflammatory immune response that is characterized by suppression of IgG2 antibody response, T cell proliferation and IL-2 production, coupled with augmentation of IL-4, IL-10 and interferon gamma (IFN- ⁇ ) production.
  • IFN- ⁇ interferon gamma
  • the suppression of IgG2 and IL-2 production, coupled with augmented production of IL-4 and IL-10 is reminiscent of a Thl to Th2 shift.
  • the corresponding augmentation of IFN- ⁇ production is a surprising and unexpected event in an anti -inflammatory response. Elevated IFN-7 responses may thus play a role in the protective mechanism, rather than contributing to autoimmune disease as previously believed.
  • IFN- ⁇ plays an important down-regulatory role in both the induction and effector phases of myelin oligodendrocyte glycoprotein- induced EAE in mice. Willenborg et al . , (1996) J “ . Immunol . 157 -.3223-3227.
  • delivery of an autoantigen is coupled with codelivery of one or more of the following immunological response modifiers: IL-4; IL-10; and IFN- ⁇ .
  • ancillary agents can be used to enhance desensitization, such as where the cytotoxic response is pharmacologically suppressed.
  • CIA and AA in rodents, and RA in humans each appear to be mediated by cytotoxic immune responses .
  • This observation is supported by data showing that both anti-IL-2 receptor monoclonal antibodies and IL-4 can suppress CIA in mice, while IL-12 and interferon- gamma exacerbate disease.
  • autoantigen-specific TH1 immunity can be transiently suppressed using suitable pharmacological agents such as IL-4, anti-IL-2 receptor antibody molecules, anti-TNF-alpha antibodies, and anti-CD4 antibody molecules.
  • vectors encoding other articular cartilage antigens can be codelivered with RA-specific autoantigens to help decrease cytotoxic responses.
  • RA-specific autoantigens can be codelivered with RA-specific autoantigens to help decrease cytotoxic responses.
  • expression vectors encoding such articular cartilage antigens can be constructed using, for example, cDNA sequences of human and other vertebrate proteoglycan core protein genes, which are readily available.
  • Nucleotide sequences selected for use in the present invention can be derived from known sources, for example, by isolating the same from cells containing a desired gene or nucleotide sequence using standard techniques. Similarly, the nucleotide sequences can be generated synthetically using standard modes of polynucleotide synthesis that are well known in the art. See, e.g., Edge et al . , Nature 292:756 (1981); ⁇ ambair et al . , Science 223.: 1299 (1984); Jay et al . , J " . Biol . Chem . 259:6311 (1984).
  • synthetic oligonucleotides can be prepared by either the phosphotriester method as described by Edge et al . ⁇ supra) and Duckworth et al . , Nucleic Acids Res . . 9:1691 (1981), or the phosphoramidite method as described by Beaucage et al . , Tet. Letts. 22 . :1859 (1981), and Matteucci et al . , J " . Am . Chem . Soc . .103 . : 3185 (1981) .
  • Synthetic oligonucleotides can also be prepared using commercially available automated oligonucleotide synthesizers.
  • the nucleotide sequences can thus be designed with appropriate codons for a particular amino acid sequence. In general, one will select preferred codons for expression in the intended host.
  • the complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e . g. , Edge et al . ⁇ supra) ; ⁇ ambair et al . (supra) and Jay et al . ( supra) .
  • nucleic acid sequences for use herein is by recombinant means.
  • a desired nucleotide sequence can be excised from a plasmid carrying the same using standard restriction enzymes and procedures.
  • Site specific D ⁇ A cleavage is performed by treating with the suitable restriction enzyme (or enzymes) under conditions which are generally understood in the art, and the particulars of which are specified by manufacturers of commercially available restriction enzymes.
  • size separation of the cleaved fragments may be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques.
  • Restriction cleaved fragments may be blunt ended by treating with the large fragment of E . coli DNA polymerase I (Klenow) in the presence of the four deoxynucleotide triphosphates (dNTPs) using standard techniques.
  • the Klenow fragment fills in at 5' single-stranded overhangs but digests protruding 3' single strands, even though the four dNTPs are present.
  • selective repair can be performed by supplying only one, or several, selected dNTPs within the limitations dictated by the nature of the overhang.
  • the mixture can be extracted with e.g. phenol/chloroform, and ethanol precipitated. Treatment under appropriate conditions with SI nuclease or BAL-31 results in hydrolysis of any single-stranded portion.
  • PCR polymerase chain reaction
  • Mullis et al . Methods Enzymol . 155.: 335-350 (1987) .
  • This technique uses DNA polymerase, usually a thermostable DNA polymerase, to replicate a desired region of DNA.
  • the region of DNA to be replicated is identified by oligonucleotides of specified sequence complementary to opposite ends and opposite strands of the desired DNA to prime the replication reaction.
  • the product of the first round of replication is itself a template for subsequent replication, thus repeated successive cycles of replication result in geometric amplification of the DNA fragment delimited by the primer pair used.
  • This method also allows for the facile addition of nucleotide sequences onto the ends of the DNA product by incorporating these added sequences onto the oligonucleotide primers (see, e.g., PCR Protocols , A Guide to Methods and Applications, Innis et al (eds) Harcourt Brace Jovanovich Publishers, NY (1994)).
  • sequences for desired proteins can be cloned into any suitable vector or replicon.
  • Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. Ligations to other sequences are performed using standard procedures, known in the art.
  • Selected nucleotide sequences can be placed under the control of regulatory sequences such as a promoter or ribosome binding site (collectively referred to herein as "control" elements) , so that the sequence encoding the desired protein is transcribed into RNA in the host tissue transformed by a vector containing this expression construct .
  • regulatory sequences such as a promoter or ribosome binding site (collectively referred to herein as "control" elements)
  • control elements will depend on the host being treated and the type of preparation used. Thus, if the host's endogenous transcription and translation machinery will be used to express the proteins, control elements compatible with the particular host will be utilized.
  • promoters for use in mammalian systems include, but are not limited to, promoters derived from SV40, CMV, HSV, RSV, MMTV, among others .
  • regulatory sequences which allow for regulation of the expression of protein sequences encoded by the delivered nucleotide sequences.
  • Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a coding sequence to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.
  • An expression vector is constructed so that the particular coding sequence is located in the vector with the appropriate control and, optionally, regulatory sequences such that the positioning and orientation of the coding sequence with respect to the control sequences allows the coding sequence to be transcribed under the "control" of the control sequences (i.e., RNA polymerase, which binds to the
  • DNA molecule at the control sequences transcribes the coding sequence
  • Modification of the sequences encoding the particular protein of interest may be desirable to achieve this end. For example, in some cases it may be necessary to modify the sequence so that it is attached to the control sequences with the appropriate orientation; i.e., to maintain the reading frame.
  • the control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector.
  • the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site.
  • Peptide antigens can be produced using a variety of methods known to those skilled in the art.
  • the antigens can be isolated directly from native sources, using standard purification techniques.
  • the antigens can be recombinantly produced using expression systems as described above and purified using known techniques.
  • the peptide antigens can also be synthesized, based on described amino acid sequences or amino acid sequences derived from the DNA sequence of a nucleic acid molecule of interest, via chemical polymer syntheses such as solid phase peptide synthesis. Such methods are known to those skilled in the art. See, e.g., J. M. Stewart and J. D.
  • Adeno-associated virus (AAV) vector systems have also been developed for nucleic acid delivery.
  • AAV vectors can be readily constructed using techniques well known in the art. See, e.g., U.S. Patent Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 (published 23 January 1992) and WO 93/03769 (published 4 March 1993); Lebkowski et al . , Molec. Cell . Biol .
  • the nucleic acid molecule of interest can also be delivered without a viral vector.
  • the molecule can be packaged in liposomes prior to delivery to the subject.
  • Lipid encapsulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid.
  • liposomes as carriers for delivery of nucleic acids, see, Hug et al . , Biochim. Biophys . Acta . 1097:1-17 (1991); Straubinger et al . , in Methods of Enzymology, Vol. 101, pp. 512-527 (1983) .
  • nucleic acid preparations of the present invention may also be encapsulated, adsorbed to, or associated with, particulate carriers for delivery to suitable recipient cells.
  • exemplary particulate systems and polymers include, for example, polymers such as polylysine, polyarginine, polyornithine, spermine, spermidine, as well as conjugates of these molecules. See, e.g., Feigner, P.L., Advanced Drug Delivery Reviews 5_:163-187 (1990), for a review of delivery systems useful for transfer of nucleic acid molecules. Preparation of Coated Carrier Particles
  • the autoantigen preparations are administered using particle mediated delivery techniques.
  • the above-described nucleic acid molecules and peptide antigens can be coated onto carrier particles using a variety of techniques known in the art.
  • Carrier particles are selected from materials which have a suitable density in the range of particle sizes typically used for intracellular delivery from a gene gun device. The optimum carrier particle size will, of course, depend on the diameter of the target cells.
  • tungsten, gold, platinum and iridium carrier particles can be used.
  • Tungsten and gold particles are preferred.
  • Tungsten particles are readily available in average sizes of 0.5 to 2.0 ⁇ m in diameter. Although such particles have optimal density for use in particle acceleration delivery methods, and allow highly efficient coating with DNA, tungsten may potentially be toxic to certain cell types.
  • Gold particles or microcrystalline gold e.g., gold powder A1570, available from Engelhard Corp., East Newark, NJ
  • Gold particles provide uniformity in size (available from Alpha Chemicals in particle sizes of 1-3 ⁇ m, or available from Degussa, South Plainfield, NJ in a particle size of 0.95 ⁇ m) and reduced toxicity.
  • Microcrystalline gold provides a diverse particle size distribution, typically in the range of 0.5-5 ⁇ m. However, the irregular surface area of microcrystalline gold provides for highly efficient coating with DNAs .
  • a number of methods are known and have been described for coating or precipitating DNA or RNA onto gold or tungsten particles. Most such methods generally combine a predetermined amount of gold or tungsten with plasmid DNA, CaCl 2 and spermidine . The resulting solution is vortexed continually during the coating procedure to ensure uniformity of the reaction mixture. After precipitation of the DNA, the coated particles can be transferred to suitable membranes and allowed to dry prior to use, coated onto surfaces of a sample module or cassette, or loaded into a delivery cassette for use in particular gene gun instruments.
  • Peptide or protein antigens can also be coated onto suitable carrier particles, e.g., gold or tungsten.
  • suitable carrier particles e.g., gold or tungsten.
  • peptides can be attached to the carrier particle by simply mixing the two components in an empirically determined ratio, by ammonium sulfate precipitation or other solvent precipitation methods familiar to those skilled in the art, or by chemical coupling of the peptide to the carrier particle.
  • the coupling of L-cysteine residues to gold has been previously described (Brown et al . , Chemical Society Reviews 9:271-311 (1980)).
  • peptide antigens can be dried onto carrier particles by centrifugation under vacuum. Once dried, the coated particles can be resuspended in a suitable solvent (e.g., ethyl acetate or acetone), and triturated (e.g., by sonication) to provide a substantially uniform suspension.
  • a suitable solvent e.g., ethyl acetate or acetone
  • carrier particles coated with either nucleic acid preparations, or peptide or protein antigen preparations are delivered to mammalian tissue using particle-mediated delivery techniques .
  • Various particle acceleration devices suitable for particle-mediated delivery are known in the art, and are all suited for use in the practice of the invention.
  • Current device designs employ an explosive, electric or gaseous discharge to propel the coated carrier particles toward target cells.
  • the coated carrier particles can themselves be releasably attached to a movable carrier sheet, or removably attached to a surface along which a gas stream passes, lifting the particles from the surface and accelerating them toward the target .
  • An example of a gaseous discharge device is described in U.S. Patent No. 5,204,253.
  • An explosive-type device is described in U.S. Patent No. 4,945,050.
  • an electric discharge-type particle acceleration apparatus is the ACCELL ® instrument manufactured by Auragen, Inc., Madison, WI , which instrument is described in U.S. Patent No. 5,120,657.
  • Another electric discharge apparatus suitable for use herein is described in U.S. Patent No. 5,149,655.
  • the coated particles are administered to the subject to be treated in a manner compatible with the dosage formulation, and in an amount that will be prophylactically and/or therapeutically effective.
  • the amount of the composition to be delivered which, in the case of nucleic acid molecules is generally in the range of from 0.001 to 10.0 ⁇ g, more preferably
  • nucleic acid molecule per dose 0.01 to 10.0 ⁇ g of nucleic acid molecule per dose, and in the case of peptide or protein molecules is 1 ⁇ g to 1 mg, more preferably 1 to 50 ⁇ g of peptide, depends on the subject to be treated. The exact amount necessary will vary depending on the age and general condition of the individual to be treated, the severity of the autoimmune condition being treated and the particular nucleotide sequence or peptide antigens selected, the site of administration, as well as other factors. An appropriate effective amount can be readily determined by one of skill in the art upon reading the instant specification.
  • a "therapeutically effective amount" of the antigens, or nucleic acids coding therefor will be sufficient to bring about treatment or prevention of autoimmune disease or condition symptoms, and will fall in a relatively broad range that can be determined through routine trials.
  • the coated particles are delivered to suitable target cells, most preferably to cells of the skin, such as the epidermis.
  • suitable target cells most preferably to cells of the skin, such as the epidermis.
  • the introduction of autoantigen into the epidermis of a mammal by gene gun can be performed in such a way so as to induce desensitization and decrease in cytotoxic T-cell responses.
  • Delivery of the particles is timed and repeated in such a manner so as to facilitate the suppression of cytotoxic immune responses and facilitate the creation of a desensitizing-type response which will, in turn, inhibit the arousal of a cytotoxic inflammatory immune response.
  • One way to elicit such a desensitizing response in the practice of the invention is to deliver antigen to a subject over a relatively prolonged period of time, for example over a period of several weeks. Such delivery may result in T-cell anergy, a state of reversible non-proliferation. In this condition, cytotoxic T cells do not proliferate and the cytotoxic effects of autoimmune diseases are minimal .
  • multiple and frequent deliveries of self antigen-encoding expression vectors via gene gun delivery to the epidermis generally provide transient bursts of localized antigen production that result in the induction of Th2-like responses characterized by a preponderance of IgGl antibodies, diminished interferon-gamma production, suppressed CTL activity, and elevated IL-4 production.
  • desensitization to antigens can be brought about using repeated gene gun delivery of antigens directly into the epidermis.
  • a method for the treatment of rheumatoid arthritis, multiple sclerosis, and other autoimmune diseases involves an active, rather than passive immunotherapeutic strategy.
  • This approach is based on the ability of epidermal antigen delivery to desensitize the immune system to the self antigen, and thereby eliminate the pro-inflammatory cytokine milieu at its source.
  • an example of this strategy involves the use of oral tolerance to anergize or actively suppress pro-inflammatory cytotoxic responses.
  • immunization by the nucleic acid-based methods described herein protects such animals from autoimmune disease of the central nervous system by inducing a switch in the type of immune response which they exhibit after antigen challenge with myelin basic protein (Paterson, P.Y., Textbook of Immunopathology (eds Miescher, P.A. and
  • Example 1 Prevention of Collagen Induced Arthritis with Type II Collagen
  • CIA collagen- induced arthritis
  • Bovine CII (Sigma, St. Louis) was coated onto 0.95 ⁇ m gold particles via ammonium sulfate precipitation. CII -coated gold particles were washed with ethanol and loaded into individual gene gun shots as previously described (Fuller et al . , supra) . Each shot contained approximately 5 ⁇ g CII protein and 0.5 mg of gold. Epidermal delivery of protein-coated gold particles was performed using the hand-held, helium driven Accell gene delivery system (PCT pat. appl . WO 95/19799) . Twenty 6 week old female DBA/1 mice each received single CII gene gun shots to the abdominal epidermis on days 0, 14 and 35 after fur removal by clipping.
  • mice Half of these animals, and 9 additional naive mice, received subcutaneous injections of 200 ⁇ g CII protein in Freund' s complete adjuvant, containing 100 ⁇ g M. tuberculosis, on day 28. Incidence of arthritis in each group was plotted. Disease was scored as described by Khare et al . , J. Immunol .
  • mice were monitored for redness or swelling in paws or toes, severe swelling and joint deformity, and joint ankylosis.
  • Figure 1 shows the incidence of arthritic disease in these three groups of animals as a function of time. While subcutaneous CII injections in complete Freund' s adjuvant induced arthritic disease in 8 of 9 animals that received the CII/Freund's injections only ( ⁇ ) , no animals in the groups given CII by gene gun (•), or CII/Freund's by gene gun ( ⁇ ) , exhibited any symptoms of arthritis. These data demonstrate that gene gun-mediated administration of CII protein to the epidermis not only failed to elicit CIA on its own, but completely protected against the development of CIA following CII/Freund's challenge.
  • mice immunized as described above were collected on day 70 (42 days after subcutaneous CII/Freund's challenge).
  • Cll-specific IgG subclass responses were measured by standard ELISA techniques using bovine CII adsorbed to ELISA plates as the immobilized phase.
  • the animals in the gene gun-only group exhibited no detectable Cll-specific IgG activity, but low IgM titers were seen. This was surprising in view of the relatively strong responses that gene gun immunizations normally elicit (using plasmid DNA vectors) , and could be related to the fact that gene gun delivery is largely an intracellular process.
  • the two groups of animals challenged with CII/Freund's exhibited significant Cll-specific IgG activity after challenge, but differed in the relative proportions of IgG subclasses, depending on whether or not they received prior gene gun CII treatments.
  • Hsp65 The nucleic acid sequence and corresponding amino acid sequence for the 65 kDa heat shock protein of M. tuberculosis (Hsp65) are known, and are disclosed herein as SEQ ID NOs 1 and 2.
  • Freund's adjuvant were collected 42 days after challenge. Half of the animals received two Hsp65 gene gun deliveries to the abdominal epidermis, at 28 and 14 days prior to challenge, respectively. Cll-specific IgG subclass responses were measured by standard ELISA techniques using adsorbed bovine CII as the immobilized phase. Although the Hsp65 DNA pretreatment, in theory, would not directly affect the induction of humoral and cellular immune responses to CII during challenge, the cytokine balance in these animals could have been altered, thus affecting the animals' recognition of the CII challenge in an indirect manner.
  • Examples 1 and 2 suggest that peptide antigens, or nucleic acids encoding the same, delivered to the epidermis via intracellular particle delivery, provide an effective means of stimulating anti-inflammatory immune responses. Since rheumatoid arthritis is a disease involving inflammatory autoimmune recognition, active immunotherapeutic approaches based on gene gun- mediated vector delivery to the epidermis are indicated in the prevention of rheumatoid arthritis. Protection against CIA development may also be achieved via delivery of a CII expression vector. Comparisons between gene gun delivery of protein- and DNA-based immunogens in murine influenza and hepatitis B immunization models have demonstrated that DNA immunization leads to significantly stronger humoral and cytotoxic T lymphocyte responses .
  • a human type II procollagen cDNA clone is inserted into a clinical grade CMV intron A-based expression construct.
  • a systematic study examining the efficacy of various viral and cellular promoters in a variety of tissues in vivo following gene gun- mediated DNA delivery has demonstrated the enhanced effectiveness of the human CMV immediate early promoter in skin. Cheng et al . , Proc . Natl . Acad . Sci . USA 90:4455-4459 (1993).
  • the hCMV promoter with and without its associated intron A sequence, has been the promoter of choice for most DNA immunization experiments.
  • Gene gun DNA dosages in excess of 0.025 to 0.05 ⁇ g per shot generally yield the maximum level of expression obtainable in a given skin delivery site. Therefore, to increase the dosage beyond this point, the number of shots per immunization are increased.
  • the treatment protocol includes a group of animals that receive three shots per immunization. The number of treatments and the length of resting periods between treatments are based on results of the above study investigating these variables. The regimen chosen is the one resulting in Cll-specific immune responses exhibiting the greatest anti -inflammatory character and protection. The short resting period groups will likely exhibit the most favorable responses.
  • an appropriate CMV intron A vector can be used.
  • a heterologous signal peptide coding sequence can be inserted into the CMV Hsp65 vector to enhance immunogenicity via secretion.
  • Endpoints in this study include the incidence of arthritic disease and disease score and CII- and Hsp65-specific IgG isotype profiles, cytokine profiles, and proliferative T cell responses. Immunization protocols that favor the induction of maximum IgGl, TGF-beta, IL-4, and IL-10 production levels may correlate with enhanced protective effects.
  • This example illustrates the effectiveness of gene gun vaccination with a plasmid encoding the gene for myelin basic protein (MBP) , an encephalitogen, in preventing the induction of EAE in rats.
  • MBP myelin basic protein
  • a protein of 18.5 kD was detected by Western blot analysis, using a rabbit polyclonal antibody specific for rat MBP, in 143B cells transfected with pJWMBP. Closed circular plasmid D ⁇ A was purified from transformed DH5a-competent cells by CsCl-ethidium bromide gradient ultracentrifugation using standard protocols as described by Sambrook et al . , Molecular Cloning-A Laboratory Manual , 2d Ed. Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York, 1989.
  • DNA concentration was determined by optical density at 260nm and confirmed by agarose gel analysis comparing standards of known concentration with varying amounts of restriction endonuclease fragments of the plasmid. There was no detectible chromosomal DNA in the plasmid preparations as assessed by gel analysis. After ethanol precipitation, the DNA plasmid was aliquoted and stored at -70°C and when required, was re-precipitated and dissolved in normal saline at 4°C.
  • the plasmid DNA was attached to gold particles by adding 100 ⁇ l of 0.1 M spermidine (Sigma Chemical Co.) to a 1.5 ml centrifuge tube containing 50 mg of gold powder (0.95 ⁇ m diameter gold powder) .
  • the plasmid and gold were precipitated by adding 200 ⁇ l of 2.5 M CaCl 2 solution during vortex mixing and after settling for 10 minutes, the precipitate was washed extensively with absolute ethanol and resuspended in EtOH at 7.0 mg gold/ml .
  • the DNA/gold preparation was then loaded into a gene gun sample cartridge using known techniques, and then delivered to experimental animals using an electric discharge particle acceleration device as follows.
  • Optical density was measured using a microplate reader at 405 nm with reference to 490 nm. Levels of antibody were determined using an endpoint calculated by measuring three times the mean value of the background optical density and the standard deviation. A positive response was considered to be detection of antibody at dilutions greater than those of corresponding control animals.
  • MBP-specific antibodies were detected after they had received only three immunizations with pJWMBP, and by the 5th immunization, 8 of 9 rats had seroconverted (see Table 1 below) .
  • circulating anti- MBP antibody titres ranged from 1/160-1/2560.
  • analysis of the antibody isotype showed that animals immunized with pJWMBP produced primarily IgGl responses. Only one animal in this group produced any IgG2a, and this was at a low level (titer: 1/80) .
  • pJWMBP protected 8 of 9 animals from the development of autoimmune encephalomyelitis after challenge with MBP in complete Freund's adjuvant.
  • One animal developed symptoms of EAE.
  • this animal was also the one which failed to seroconvert to MBP after gene gun immunization, suggesting that priming had not been successful.
  • Seven out of 9 animals in the pJW control plasmid immunized group developed clinical symptoms of EAE within 14 days of challenging with MBP in complete Freund' s adjuvant.
  • Histological sections from the pJW control and nonimmunized control groups showed extensive inflammatory lesions in the brain and spinal cord, as noted in the histological scores shown in Table 2.
  • Two animals from the pJW control plasmid- immunized group did not develop clinical signs of disease, despite the fact that they were found to have severe CNS inflammation.
  • Histological sections of the pJWMBP-immunized group revealed that 7 of the 8 protected animals showed either no inflammatory response, or mild lesions in the meninges .
  • One animal from this group despite not developing clinical disease, exhibited extensive lesions both in the parenchyma and meninges.
  • IgGl/IgG2a antibody in protected animals given pJWMBP was consistently higher than in the control groups of animals exhibiting clinical EAE. This suggests that gene gun vaccination with pJWMBP desensitized the animals to MBP and decreased the cytotoxic immune response .
  • Example 5 Vaccination Against Myelin Oligodendrocyte Glycoprotein
  • This example demonstrates gene gun vaccination with a plasmid encoding the gene for the encephalitogen, myelin oligodendrocyte glycoprotein in preventing the induction of EAE in rats.
  • the human myelin oligodendrocyte glycoprotein (MOG) gene (Hilton et al . , J “ . Neurochem. 6_5:309-318 (1995)) is cloned into the expression vector pJW4303.
  • the plasmid DNA is attached to gold particles as described in Example 4.
  • Example 4 are given gene gun inoculations with the plasmid containing cDNA encoding the human MOG antigen. This procedure is repeated 3 to 5 times at weekly intervals in a similar protocol to that depicted in Figure 4. Rats vaccinated in this manner are not expected to develop autoimmune encephalomyelitis .
  • This example demonstrates gene gun vaccination with a plasmid encoding the gene for the encephalitogen, proteolipid protein (PLP) in preventing the induction of EAE in rats.
  • PLP proteolipid protein
  • the human PLP gene (Nadon et al . , Development 110 :529-537 (1990)) is cloned into the expression vector pJW4303.
  • the plasmid DNA is attached to gold particles as described in Example 4.
  • Specific pathogen-free female Lewis rats, as described in Example 4 are given gene gun inoculations with the plasmid containing DNA encoding the human MOG antigen. This procedure is repeated 3 to 5 times at weekly intervals for a total of 3-5 deliveries in a similar protocol to that shown in Figure 4. Rats vaccinated in this manner will not develop autoimmune encephalomyelitis.
  • Human Oligodendrocytes This example demonstrates gene gun vaccination with a plasmid encoding the entire cDNA library of human oligodendrocytes in preventing the induction of EAE in rats.
  • oligodendrocytes Human oligodendrocytes are isolated and grown in culture according to the method of Zhou et al . , J. Neurosci . Res . 42:504-515 (1995) and an expression cDNA library is prepared from these cells.
  • Total RNA is prepared from oligodendrocytes by extraction with RNAzol, according to the manufacturer's (Tel-Test, Inc.) instructions.
  • Messenger RNA is isolated using the "mRNA purification kit" (Pharmacia, 27-9258-01) .
  • First strand cDNA is synthesized from 2 ⁇ g mRNA, with 100 ng of random hexamer primer, using the "Superscript Preamplification System for First Strand cDNA Synthesis" (GIBCO BRL, 18089-011) .
  • Second strand cDNA is synthesized by a modification of the method of Gubler and Hoffman, Gene 25:263-269 (1983) . All reagents used in this preparation are from GIBCO BRL.
  • the first strand cDNA reaction mixture (20 ul) is added to a premix of 16 ⁇ l of 10X E. coli ligase buffer, 2.4 ⁇ l 10 mN cNTP mix and H 2 0, on ice.
  • RNAse H 1.6 U
  • DNA polymerase I 40 U
  • E. coli DNA ligase 40 U
  • Glycogen carrier (10 mg; Boehringer- Mannheim) is added and the cDNA purified by phenol extraction and 2 ether extractions, followed by ethanol precipitation with 2 M ammonium acetate, washed in 70% ethanol and resuspended in 20 ml 10 mM Tris-HCl, 1 mM EDTA, pH 8.0 (TE) .
  • Double stranded cDNA (1 ⁇ l) is digested with 12 U of Sau3A (New England Biolabs) at 37°C overnight. The restriction enzyme is inactivated at 70°C for 30 min, and the DNA is ethanol precipitated, washed with 70% ethanol and resuspended in 20 ⁇ l TE buffer.
  • CMV-GH-F1, -F2, or -F3 vectors (20 ⁇ g) are digested with BamHI or Bglll according to the method of Durry et al . , Nature 377 :632-635 (1995) and treated with 2U of calf alkaline phosphatase (30 min at 37°C, 60 min at 50°C, 10 min at 75 °C; Boehringer Mannheim) .
  • the vectors were extracted with phenol/chloroform, ethanol precipitated and electrophoresed on 1% agarose (low melting temperature, D ⁇ A grade agarose, Progen industries) with 0.1 ⁇ g/ml ethidium bromide, excised in the minimum possible volume of agarose and melted at 65 °C before adding to the ligation mix.
  • 1% agarose low melting temperature, D ⁇ A grade agarose, Progen industries
  • the Sau3A digested cD ⁇ A (2 ⁇ l) is ligated to 100 ng of vector (in agarose) with 5U of T4 D ⁇ A ligase (Boehringer Mannheim) in 50 mM Tris-HCl, pH 7.5 , 10 mM MgCl 2 , 10 mM DTT, 0.5 mM ATP in a final volume of 20 ⁇ l , overnight at 16°C.
  • Electrocompetent E. coli are transformed with the ligated cD ⁇ A, using a Biorad Gene Pulser (25 uF, 2.5 kV) with Pulse Controller at 200 Ohms and 0.2 cm cuvettes (according to a protocol from Biorad) .
  • the transformation efficiency is approximately 5 X 10 8 colonies per ⁇ g D ⁇ A.
  • 2 ⁇ l Sau3A digested cD ⁇ A (equivalent to 10 ng mRNA) gives from 10 to 25,000 colonies .
  • the cDNA library is amplified.
  • the library is plated out at high density on L-broth containing 100 ⁇ g/ml ampicillin (approximately 30,000 per 9.5 cm square plate) .
  • the colonies are grown overnight to a diameter of 0.2-0.3 mm, and rinsed from the plate in a total of 10 ml L-broth per plate.
  • L-broth from several plates is combined, glycerol added to 15% v/v, and the library stored in 1 ml aliquots at -80°C. Separate libraries are generated for each of the 3 CMV-GH vectors.
  • DNA is prepared in the following manner. L- broth, containing 100 ⁇ g/ml ampicillin (70 ml) is inoculated with a 1 ml library aliquot and grown overnight at 37 °C with vigorous shaking. The next day, 1 liter baffled flasks containing 500 ml L-broth, containing 100 ⁇ g/ml ampicillin are inoculated with 10 ml of overnight culture and grown overnight at 37°C. Closed circular plasmid DNA is purified by CsCl- ethidium bromide gradient ultracentrifugation and attached to gold beads as described in Example 4. Immunization of rats with the Acell particle bombardment device (Auragen, Inc., Middleton, Wis) is used according to the protocol in Example 4.
  • Acell particle bombardment device Auragen, Inc., Middleton, Wis
  • MOLECULE TYPE DNA (genomic)
  • ATCACTTCGC A ATG GCC AAG ACA ATT GCG TAC GAC GAA GAG GCC CGT CGC 290 Met Ala Lys Thr lie Ala Tyr Asp Glu Glu Ala Arg Arg 1 5 10
  • GGT ATG CGG TTC GAC AAG GGC TAC ATC TCG GGG TAC TTC GTG ACC GAC 866 Gly Met Arg Phe Asp Lys Gly Tyr lie Ser Gly Tyr Phe Val Thr Asp 190 195 200 205
  • GTC AAG GTG ACC CGT TCG GCG CTG CAG AAT GCG GCG TCC ATC GCG GGG 1778 Val Lys Val Thr Arg Ser Ala Leu Gin Asn Ala Ala Ser He Ala Gly 495 500 505 CTG TTC CTG ACC ACC GAG GCC GTC GTT GCC GAC AAG CCG GAA AAG GAG 1826 Leu Phe Leu Thr Thr Glu Ala Val Val Ala Asp Lys Pro Glu Lys Glu 510 515 520 525
  • GTGTCGTCAT CGGGGCCTGC GTCCGACGCC TGGGCACGGC CGTCGACGAT CAGCGAGTAG 2114 CCGCTAGGAT CGGATGGCGG CCACAACAGG GTGACTTCGC TGCGGTGGGC CAGGTTTTGC 2174

Abstract

On décrit un traitement de maladies auto-immunes fondé sur l'apport dans la peau d'antigène à l'aide d'un pistolet à gènes, ou l'apport dans la peau d'ADN codant l'antigène. L'apport de collagène ou d'ADN codant le collagène est utile pour traiter l'arthrite rhumatoïde, l'apport de protéine de base de myéline (PBM) ou d'ADN codand le PBM étant quant à lui utile pour traiter la sclérose en plaques. Ces antigènes atténuent les réponses cytotoxiques et favorisent la désensibilisation aux antigènes. A l'aide d'autres antigènes on peut traiter d'autres maladies auto-immunes.
PCT/US1997/009427 1996-06-03 1997-06-03 Immunotherapie pour maladie auto-immune WO1997046253A2 (fr)

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US7030098B2 (en) 1999-03-12 2006-04-18 The Board Of Trustees Of The Leland Stanford Junior University DNA vaccination for treatment of autoimmune disease
US6884785B2 (en) * 1999-06-17 2005-04-26 The Scripps Research Institute Compositions and methods for the treatment or prevention of autoimmune diabetes
US7544669B2 (en) 2001-11-21 2009-06-09 The Board Of Trustees Of The Leland Stanford Junior University Polynucleotide therapy
EP2322186A3 (fr) 2002-11-21 2011-07-13 Bayhill Therapeutics, Inc. Procédés et compositions d'acide nucléique modulatoire immunitaire pour empêcher et traiter une maladie
SE535625C2 (sv) 2010-10-28 2012-10-16 Toleranzia Ab Nya kompositioner och förfaranden för behandling av autoimmuna och allergiska sjukdomar
WO2013160865A1 (fr) 2012-04-26 2013-10-31 Toleranzia Ab Protéines hybrides d'immuno-tolérisation pour le traitement de la sclérose en plaques

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