JP2004331668A - Pharmaceuticals, compositions and systems for administering naked polynucleotides encoding bioactive peptides - Google Patents

Abstract

A method for introducing a biologically active peptide into a host by administering a polynucleotide operably encoding the biologically active peptide is provided.
Kind Code: A1 A naked polynucleotide is administered in the sense that it is not complexed with a colloid preparation such as a liposome and is not bound to a genomic integration vector. In one embodiment, the naked polynucleotide is administered to a host tissue that has a higher concentration of antigen presenting cells as compared to other tissues of the host. The polynucleotide is taken up and expressed locally, systemically, and / or at a location remote from the point of entry of the host. In another embodiment, the polynucleotide expresses a peptide that is effective in delaying, treating and preventing age-related diseases and disease resistance in mammals. In this embodiment, subtherapeutic levels of the peptide are expressed at an appropriate stage in host development. The equipment and compositions used are also mentioned.
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Description

  The present invention provides a method of administering a biologically active peptide to a mammalian host by introducing one or more polynucleotides operatively encoding the peptide into the mammalian host, preferably by non-invasive means. About. The present invention also relates to a method for preventing and treating diseases associated with aging of a mammal and loss of immune function by administering the polynucleotide.

Related U.S. Patent Application This application is a continuation-in-part of U.S. Patent Application Serial No. 08 / 112,440, filed August 26, 1993 with the United States Patent and Trademark Office.

STATEMENT OF US GOVERNMENT RIGHTS The present invention may also be made with government support under grant numbers AR07567 and AR25443 awarded by the United States National Institutes of Health. The United States government may have certain rights in the invention.

  The direct introduction of biologically active peptides or proteins into patient cells is of great therapeutic value. However, this approach has several disadvantages. An important issue is that there may be potential toxicity, especially at doses sufficient to produce a biological response to the peptide. In practice, there are cost issues associated with isolating / purifying or synthesizing these peptides. In addition, the clinical efficacy of these peptides is also limited by their relatively short half-life in vivo, which is usually due to degradation of these peptides by proteases present in the target tissue. .

  For these reasons, introducing a protein into a patient by delivering a gene that expresses the protein to the patient / host is an interesting alternative to administering the protein. However, to date, the main means of introducing heterologous genetic material into a host has been to integrate the gene into the host genome, for example, by transforming the host cell with a viral vector. There has also been reported a method of using a DNA encapsulated in a liposome containing a DNA encapsulated in a proteoliposome containing a virus envelope receptor protein to directly transfer a gene into a living body to a postnatal animal.

  In 1984, injection of naked cloned plasmid DNA of squirrel hepatitis into the liver of squirrels showed that the squirrels had undergone viral infection and production of antiviral antibodies at the National Institutes of Health (NIH). A study was reported (Seeeger et al., Proc. Nat'l. Acad. Sci. USA, 81, 5849-5852, 1984). Several years later, Felgner et al. Reported that proteins were expressed from naked polynucleotides injected into skeletal muscle tissue (ie, DNA or RNA not associated with liposome or viral expression vectors) (Felgner et al. Science, 247, 1465, 1990; see also PCT application WO 90/11092). Felgner et al. Describe that because muscle tissue comprises multinucleated cells, the sarcoplasmic reticulum, and the T-tube (transverse tubule) system that extends deep into the muscle cells and has a unique structure, the muscle cells can carry out polynucleotides. It was expected to be efficiently incorporated and expressed.

  Although it is believed that cells of other tissues can also take up the naked polynucleotide, expression in other tissues has heretofore been dependent on the delivery of the expressed gene to a delivery system, e. Only confirmed by conversion. Indeed, other researchers have suggested that naked polynucleotide uptake and expression does not occur at detectable or biologically active levels in tissues other than skeletal muscle (see, for example, Stribling et al., Proc. Natl.Acad.Sci.USA 89, 11277-11281, 1992 [When a gene was delivered by aerosol, expression occurred using a liposome delivery system, but expression did not occur when only DNA was introduced. And Tang et al., Nature, 356, 152-154, 1992 [hGH plasmid conjugated to colloidal gold beads did not elicit an immune response when injected into the skin of mice with the vaccine "cancer". ]).

  Injecting DNA or RNA into muscle tissue with long-term therapy is generally effective for expressing the gene in muscle cells, but requires repeated injections to compensate for expression lost due to gene degradation . This technique is not only practical because it is time-consuming and expensive, but also causes inflammation at and near the injection site. Such inflammation can cause somatic cells, such as muscle, into which the nucleotides are introduced, to target themselves for an immune response (see, eg, Example I), resulting in severe muscle necrosis. In addition, intramuscular injection of DNA not only risks damaging muscle tissue, but also obviously reduces the efficacy of the treatment. For example, researchers at the University of Ottawa recently stated, "Striated muscle is the only tissue found to be able to absorb and express reporter genes transmitted in the form of plasmid DNA ... According to the findings, the fibers damaged by the injection method do not take up and express plasmid DNA. "(Davis et al., Human Gene Therapy, 4: 151-159, 1993).

  In addition, the use of intramuscular injections, while effective for at least a short period of time when treating muscle tissue itself, is a tissue-specific immunization to peptides expressed elsewhere in the patient's body. Seems to be less effective at stimulating responses or other biological responses.

  As a result, intramuscular injection is not a particularly practical way to express the peptide at the main entry point of many infections, namely the skin and mucous membranes.

  Furthermore, injection of the polynucleotide intramuscularly appears to result in the production of both antibodies and cytotoxic T cells in the tissue due to the release of the encoded protein by target muscle cells. In contrast, injection of the protein (eg, in the case of a vaccination regime) does not usually trigger the generation of cytotoxic T cells, since exogenous proteins do not enter the class I processing pathway efficiently.

  In PCT Patent Application Publication No. WO 90/11092 (discussed above), we show that when naked DNA is injected into skeletal muscle and other body tissues, the gene is expressed in the cytoplasm of the injected cells. Proposed to be. In addition, the inventors believe that the encoded protein then enters the class I processing pathway and triggers the production of cytotoxic T cells (necessary to control established viral infections and cancer). . However, as discussed above, alternatively, any somatic cells expressing the antigen are first used for uptake by antigen presenting cells before a class I restricted cytotoxic T cell response is induced against the antigen. It appears that the antigen must be released into the extracellular space. This conclusion is supported by recent work on antigen presentation, which states that "priming an immune response to a class I-restricted antigen that is exclusively expressed in non-hematopoietic cells, To the host bone marrow-derived cells "(Huang et al., Science, 264: 961-965, 1994). Therefore, at least one assumption based on which the method of introducing genetic material into muscle cells to express the protein of PCT application WO 90/11092 is not accurate.

  However, with intramuscular injection, relatively high levels of protein are expressed systemically before the injected gene degrades. Such a response, while desirable for therapies aimed at protein replacement, may result in unintended toxicity in immunization protocols where relatively fast clearance or relatively low levels of expression are optimal. There is. As a result, gene transfer into tissues that regularly shed or regenerate (eg, skin) and / or cells that have relatively high abrasion rates in vivo (eg, antigen presenting cells) can be used for gene immunization Would be a more useful route.

  For gene delivery systems, means such as viral vectors that introduce the gene into the genome of the host have potential health risks with damage to the genetic material within the host cell. The use of cationic liposomes or biolistic devices (ie, vaccine "cancers" that "fire" polynucleotides attached to beads to tissues) to deliver genes in vivo is generous. Yes, and some experiments need to be performed to select the appropriate particle size to deliver to the target cells. In addition, invasive means of introducing nucleotides (eg, injection) have the problem of tissue trauma (especially in the case of long-term treatment), which limits access to certain target tissues, eg, organs.

  Means for non-invasively delivering pharmaceutical preparations of peptides, such as iontophoresis and other transdermal delivery methods, have the advantage of at least minimizing tissue trauma. However, it is believed that the bioavailability of peptides transdermally or transmucosally transmitted is limited due to the relatively high levels of proteases in these tissues. Unfortunately, a reliable means of delivering peptides by transdermal or mucosal delivery of the gene encoding the peptide is not yet available.

  The advantage of successful administration of the peptide by expressing the naked gene in vivo is that cytokine proteins (eg, interleukin-2, hereinafter referred to as “IL-2”) are administered to the patient and associated with aging. This can be explained by comparing with the current state of immunotherapy to treat or prevent the disease.

  Despite the differences in lifestyle and longevity of mammalian species, almost all mammalian species develop certain diseases as part of the aging process. These diseases include insulin resistance, which can lead to cancer, hypertension, vascular disorders and diabetes. Since the timing of the onset of these diseases cannot be attributed solely to environmental factors, their onset is believed to be genetically programmed. However, processes that actually control the aging process and the occurrence of age-related diseases are unknown.

  In general, aging is associated with a reduced ability to make an immune response to exogenous antigens, a reduced capacity for function and response to stress, and an increased tendency to fibrosis. All of these conditions involve or are partly regulated in vivo by circulating cytokines.

  For example, interleukin-1 (IL-1) protein affects glucose homeostasis and can act as a hypoglycemic agent in insulin-resistant C57BL / Ks db and C57BL / 6G ob / ob mice ( Del Rey et al., Proc. Natl. Acad. Sci. USA, 86, 5943, 1989). In these animal models of adult-onset diabetes, a single injection of human recombinant IL-1 results in normal glucose blood levels for several hours. IL-1 is also known to act on the hypothalamus-pituitary axis, which affects appetite, and on response to stress. Thus, abnormal IL-1 production or responsiveness is the basis for both aging-related non-insulin-dependent diabetes and obesity. Thus, IL-1 gene therapy (administered exactly the same as described for IL-2) prevents the development of diabetes in mice and humans.

  Hypertension is another complication of aging that often occurs with diabetes. Recent studies have shown that the primary regulator of blood pressure is the endothelial relaxing factor, nitric oxide. Nitric oxide production is regulated by IL-1. Thus, the rise in blood pressure that occurs with aging can also be prevented by IL-1 gene therapy. When IL-1 protein is rapidly administered to animals, it may cause fever and cause shock. However, the continued production of low concentrations of IL-1 by body gene therapy avoids these side effects.

  Studies on the biological effects of administering pharmaceutical doses of cytokine proteins have recently been performed by investigators, stimulating the immune system to increase its response to certain pathogens and, for example, the human immunodeficiency virus (HIV). The search for maintaining the immune function of immunocompromised patients such as those infected with) has been advanced.

  Specifically, several attempts to administer IL-2 as a therapeutic agent for immune system diseases have recently been reported as follows. That is, Teppler et al. Exp. Med. 177, 483-492, 1993 (Administration of a conjugate of recombinant IL-2 protein and polyethylene glycol to HIV-infected patients), Caligiri et al., J. Am. Clin. Invest. 91, 123-132, 1993 (long-term infusion of recombinant IL-2 protein into patients with advanced cancer), and Kaplan et al., Bio / Technology, 10, 157-162, 1992 (Malabar et al. M. leprae) or administration of IL-2 to patients infected with HIV). The focus of these studies is to develop means to administer the IL-2 protein in a manner that minimizes its toxicity.

  The toxicity of the IL-2 protein has been a major obstacle to its use as an effective therapeutic. IL-2 therapy is generally sufficient for the administered protein to saturate the high affinity IL-2 receptor and significantly swell circulating natural killer (NK) cells in order to be effective. Must be present in serum in amounts. However, high doses of IL-2 cause fatal toxicities such as severe hypotension, pulmonary edema, renal failure, cardiac arrhythmias and neurological dysfunction. In order to overcome this problem, the method adopted by the above-mentioned researchers was a method in which IL-2 protein was administered for a long period of time at a relatively small dose after an infection and / or disease had developed, and an experiment was conducted. However, this method must define effective parameters for consistent and predictable IL-2 therapy. In particular, the concentration of circulating proteins resulting from the introduction of cytokines varies considerably over time, partly due to degradation by proteases. Therefore, repeated injections are required, resulting in "peaks and valleys" in the amount of protein available to the patient. In addition, studies with IL-2 have shown that similar methods, even after considerable clinical trials, use other cytokines that play a role in various medical conditions, including age-related diseases. Does not indicate whether or not.

  Thus, there is a need for an effective means of introducing proteins into hosts in a manner that minimizes the toxicity of the proteins, including but not limited to cytokines and antigens. More specifically, there is also a need for a means of introducing a protein into a host in a manner that ensures but expresses the protein at sub-therapeutic concentrations for long periods of time. In the latter case, there is a need for means for administering cytokines, particularly for preventing and treating age-related diseases.

  Broadly speaking, the above discussion suggests that the introduction of naked nucleotides that induce local immunity in the skin and mucous membranes and express in vivo a peptide that can be vaccinated into the host against sexually transmitted diseases and respiratory diseases, for example. It also shows the demand for appropriate measures. There is also a need for means for introducing a gene encoding a bioactive peptide into a host in a tissue-specific manner without significantly trauma to the tissue.

  The present invention addresses all these needs.

BRIEF DESCRIPTION OF THE DRAWINGS The details of a preferred embodiment of the invention are set forth in the accompanying drawings and the description below. Once the details of the present invention are known, many additional innovations and modifications will be apparent to those skilled in the art.

1. Definitions The following definitions are provided to facilitate the discussion of the present invention. However, those skilled in the art will appreciate that these definitions can be extended to include equivalents without departing from the scope and spirit of the invention. For this reason, these definitions should not be construed as limiting the invention.

  a. "Naked polynucleotide" means DNA or RNA and includes sense and antisense strands that are appropriate for the purpose of the therapy to be practiced according to the present invention. The polynucleotide in this case may include an oligonucleotide. "Naked" in this context means a polynucleotide that is not complexed with a colloidal material (including liposome formulations) or contained in a vector that allows the polynucleotide to be integrated into the host genome.

  b. "Operatively encoding" refers to a polyprotein modified to contain sequences such as a promoter necessary for expression of a desired translation product, eg, a peptide or protein, and, if desired, secretion. Means nucleotide. All of the embodiments of the present invention can be carried out using known plasmid expression vectors. Preferably, these vectors contain cDNA encoding the desired translation product. Thus, unless otherwise specified, "polynucleotide" or "naked polynucleotide" refers to an operable coding sequence contained in a suitable plasmid expression vector.

  c. “Mixture of polynucleotides” shall mean two or more and up to 200 species of polynucleotides under the control of the same promoter.

  d. "Synthesis" means a known technique for synthesizing a polynucleotide sequence, including the isolation and purification of the native polynucleotide.

  e. "Peptide" means small peptides, polynucleotides, oligopeptides and proteins that have a desired biological effect in vivo.

  f. "Iontophoresis" refers to known transdermal penetration techniques currently used to continuously deliver peptides to a host. More specifically, the technique is a process that facilitates the transport of ionic species by applying a physiologically acceptable current. This process and other transdermal penetration techniques are described in Chien et al., Transdermal Drug Delivery, "Novel Drug Delivery Systems", Chapter 7, Section C (Marcel Dekker, 1992), the relevant disclosure of which is incorporated herein by reference. It is hereby incorporated by reference to indicate the state of the art in the art of drug delivery.

  g. "Surfactant / absorption enhancer" means a chemical agent currently known in the art that facilitates the absorption and transfer of certain small molecules and peptides.

  h. “Antigen-presenting cells” or “APCs” include known APCs such as Langerhans cells, veil cells of imported lymphatic vessels, dendritic cells, and finger-like process cells of lymphoid organs. This definition also includes (1) lymphocytes and macrophages that absorb and express the polynucleotides of the present invention into the skin, and (2) mononuclear cells such as those shown in the histological photographs contained herein. Mononuclear cells are also included. These cells are not tissue cells, but are considered antigen presenting cells. The most important of these cells for the present invention are the epidermis and the epithelial and lymphoid tissues, including the buccal mucosa, the vagina, the cervix and the squamous epithelium of the esophagus (areas where the concentration of APC is "relatively high") Is an APC that is known to be present in a large number in the thymus-dependent region. Thus, as used herein, the terms "skin" and "mucosa" refer specifically to the site at which APCs aggregate in addition to their definitions below.

  i. "Host" means a recipient of a therapy performed according to the present invention. The host can be any vertebrate, but is preferably a mammal. The host is preferably a human in the case of a mammal, but may be a domestic animal or a pet animal.

  j. "Target tissue" refers to the tissue of the host in which expression of the naked polynucleotide is sought.

  k. "Skin" as used herein means the epidermis, dermis and subcutaneous tissue of a host.

  l. "Mucosal" means the mucosal tissue of the host located within the body, including but not limited to respiratory channels (including bronchial channels, lung epithelium and nasal epithelium), genital channels (vaginal, penis and anal). , The urinary flow path (eg, urethra, bladder, etc.), mouth, eyes, and vocal cords.

  m. "Invasion point" refers to the site in a tissue immediately adjacent to which introduces a naked polynucleotide into a host.

  n. "Surrogate End Point" means the biological state of the host that occurs just before the onset of the disease. Examples include loss of glucose tolerance (diabetes), increased cholesterol levels (heart disease) and the presence of free radical amino acids in the blood and urine (which are enzymes that detoxify free radicals in the central nervous system). Loss).

  o. "Biological deficiency" refers to the loss of immune function or good health associated with aging of a mammal, including deficiencies in disease and host disease resistance.

  p. "Subtherapeutic concentration" and "subtherapeutic dose" refer to an acute detectable response of a host to a peptide expressed by a single non-cumulative administration of a naked polynucleotide to a host. Means expression of the peptide in the host in an amount that is insufficient to cause the naked polynucleotide.

  q. "Dermal" and "epidermal administration" refer to routes of administration in which a naked polynucleotide is provided to or through the skin. Dermal routes include intradermal and subcutaneous injection and transdermal penetration. The epidermal route involves stimulating the outermost layer of the skin sufficiently to elicit an immune response against the irritant. The stimulants include mechanical or chemical (preferably topical) agents.

  r. "Epithelial administration" is in much the same way as epidermal administration of chemical agents, except that the chemical stimulant is applied to the epithelium of the mucosa.

  s. "IL" means interleukin.

  t. "TH1 response" means a humoral immune response that is preferentially triggered by an antigen that binds and activates certain APCs, macrophages II and dendritic cells.

  u. "Bioactive peptide" means a peptide that, when administered to a host, exerts a therapeutic benefit or elicits an immune response in the host.

2. DISCUSSION In one aspect, the present invention provides for local immunization or therapy against an antigen by delivering a naked polynucleotide operably encoding the antigen or peptide (having the genetic code to make it) to the cells of the host. For eliciting a systemic response to the peptide or polynucleotide for use. More particularly, naked polynucleotides are preferably delivered to tissues that contain relatively high concentrations of antigen presenting cells as compared to other tissues in the body. The present invention is not completely limited by the specific principles of the expression mechanisms involved, but since polynucleotides are expressed by mononuclear cells, presumably host antigen presenting cells, in these tissues by administration of naked polynucleotides. It is believed that a biological response of The mononuclear cells are also thought to be involved in the inflammatory immune response to the introduction of the naked polynucleotide.

  Histological studies show that naked polynucleotides, in significant amounts, are not directly absorbed by tissue cells such as fibroblasts (see Example 9 and FIG. 15). This conclusion suggests that (1) even a small amount of naked polynucleotide administered to mice intradermally induced a significant TH1 response (indicating antigen presentation by macrophages and dendritic cells, Example 17 and FIGS. 24-25). (2) that intradermal administration of naked polynucleotide to mice induced the production of cytotoxic T cells without stimulating the production of detectable concentrations of antibodies (see Example 15 and (See FIG. 21), and (3) the results of studies showing that long-term immune memory is induced against the polynucleotide as an antigen (Example 16 and FIGS. 22-23).

  Assuming that inflammation plays a definite role in this method of the present invention, the inflammation increases the permeability of the cell membrane of the target tissue to allow for uptake of naked polynucleotides (especially across barriers such as skin and mucous membranes). It will be well appreciated by those skilled in the art.

  The ideal target tissue is the skin or mucous membrane. These tissues are particularly preferred when treating for an infection or disease where it is desired to elicit a localized therapeutic or immune response. For example, administration by the mucosal route is preferred for the treatment of sexually transmitted diseases where treatment is performed to boost the immune response to the antigen in the infected tissue. Administration by the intranasal route (by inhalation or insufflation) is also particularly useful for therapies for treating respiratory disorders and related disorders. In addition, mucosal or dermal routes are useful for immunizing against allergens. In addition, since these tissues have a regenerating performance, the period during which the introduced substance stays at the entry point is preferably limited.

  Because the antigen presenting cells present in the target tissue serve to mediate the expression of the naked polynucleotide, the method of the present invention provides a systemic approach to the expressed peptide that is useful to elicit a localized response. It is not useful for eliciting a response. However, if the dose is sufficient, a transient systemic effect can be induced. Thus, a useful application of this aspect of the invention for inducing a systemic response to an expressed peptide would be as an adjuvant for other systemic treatments.

  In another aspect of the invention, the APC acts as a vehicle and delivers naked polynucleotides to mucosal tissues other than lymphoid organs and entry points. This embodiment is described with reference to the following assumptions, but the mechanism described should not be construed as limiting the invention.

  In this embodiment, the APC absorbs the naked polynucleotide at or near the point of entry and then sends it to the lymphatic circulation. When the APC reaches the lymph nodes, it presents the expressed protein as an antigen and stimulates an immune response. From there, these APCs carrying "homing" receptors, for example, from the mucosa, re-enter the lymphatic circulation and circulate until they colonize target cells other than the tissue at the point of entry. If desired, the sequence of the homing receptor (a specific membrane protein that binds to the target cell ligand) is determined and incorporated into the naked polynucleotide.

  For expression in the lymphatic system, this embodiment also provides a means to increase the responsiveness of the host by delivering the cytokines and increase the concentration of specific cytokines present in the host. In particular, increased host concentrations of circulating cytokines (administered with or shortly after antigen administration) in lymphoid organs can further increase the host's immune response to pathogen antigens, and (1) vaccines And (2) reduce the immune response to autoantigens in autoimmune diseases, or (3) reduce the immune response to alloantigens (eg, produced after transplantation of a tissue or organ).

  If the APC carrying the gene of interest migrates from the lymph nodes and circulates to the tissue that has the homing receptor, the gene is administered to an accessible entry point, and at an inconvenient and difficult-to-access site. Can be expressed. For example, naked polynucleotides delivered intranasally, under appropriate conditions, are expressed in the genital mucosa.

  Another use of the present invention is in the relief of allergic reactions to antigens. In this regard, the nasal route of administration is particularly useful.

  For example, the gene for IL-2, gamma interferon and / or transforming growth factor (TGFβ) can be administered to suppress the production of IgE molecules. The above method is of particular interest as recent clinical trials have confirmed that IL-2 and gamma interferon are toxic at doses sufficient to prevent the production of IgE. Moreover, since IgE molecules are predominantly present in the skin and mucous membranes, using these pathways as entry points in the present invention is particularly effective in moderating allergic reactions in these tissues. Can be expected.

  Examples exist where it is useful to elicit a localized response in the skin or mucous membranes. In particular, administration by the mucosal route is preferred when treating sexually transmitted diseases. This therapy can be used to modulate the local immune response to infectious agents such as HIV, human papilloma virus (such as those involved in the production of genital warts) or skin viral infections. Also, where immunosuppression is of therapeutic value, a gene that effectively encodes an immunosuppressive factor (eg, TGFβ) can also be provided by the methods of the present invention. An example where this method is useful is in treating inflammatory bowel disease.

  A particularly useful aspect of the present invention is the use of the present invention to supply cytokines and biochemicals associated with the development of aging-related diseases to hosts at sub-therapeutic concentrations for long periods of time. In this aspect, the naked polynucleotide operably encodes proteins such as cytokines or related growth factors, which facilitate the development of aging-related diseases depending on whether or not these proteins are present in the mammalian circulatory system. Stimulates the immune system, such as slowing or delaying. Such diseases include peptides whose serum levels usually decrease with age, such as cytokines, growth hormones and enzymes (eg, human α-L fucosidase, which mediates the inflammatory response to antigens). Can be suppressed by increasing the concentration of

  Another particular advantage of the present invention is that relatively small doses of the antigen are administered. More specifically, the amount of foreign material introduced into the host is relatively small since the polynucleotide that will operatively encode the antigen is administered instead of the antigen itself. In addition, routes of administration of naked polynucleotides through the skin or mucous membranes require lower concentrations of DNA to produce an immune response of the same strength than do intramuscular routes of administration (eg, about 1/10 to 1/50). For example, see Example 16 and FIGS. As a result, the invention is useful for administering naked polynucleotides encoding hundreds of different antigens, for example, for use as a multivalent vaccine.

  Another particular advantage of the present invention is its use in antisense therapy. Briefly, if a particular disorder is associated with the expression of a particular mutated nucleic acid sequence, use a nucleotide sequence that prevents the mutated gene from undergoing specific expression at the transcription or translation stage. Can be. This technique uses antisense oligonucleotides and / or ribozymes to block transcription or translation of the mRNA by masking the specific mutant mRNA with an antisense nucleic acid or cleaving the mRNA with a ribozyme. .

  Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub, Scientific American, 262, 40, 1990). To date, several genes and oncogenes have been targeted for suppression or down-regulation, including: That is, although not limited, p53 (VS Prasolov et al., Mol. Biol. (Moscow), 22, 1105-1112, 1988), ras (SK Anderson et al., Mol. Immunol., 26) Pp. 985-991, 1989, D. Brown et al., Oncogene Res., 4, 243-249, 1989), fos (B. Levi et al., Cell. Differ. Devl, 25 (Suppl), 95-102, 1988, D. Mercola et al., Gene, 72, 253-265, 1988), and myc (SO Freytag, Mol. Cell. Biol., 8, 1614-1624). 1988, EV Prochonik, et al., Mol. Cell. Bio. ., Vol. 8, pp. 3683 to 3695, 1988, S.L.Loke other, Curr.Top.Microbiol.Immunol., 141, pp. 282-288, which is 1988).

  It is not enough to block production of the target mutant gene in all cases. As reported by Levine et al. (Biochimica et Biophysica Acta, 1032, 119-136, 1990), there are at least five mutations that contribute to the tumor phenotype. Therefore, the benefits of antisense therapy must be optimized by targeting each mutation in the multiple mutant gene disorder. The methods of the invention are particularly useful for multiple mutation antisense therapy, as they are particularly suitable for delivering mixtures of polynucleotides.

Antisense therapy is also a type V mutation similar to type III described in, for example, types III, IV and Levine et al., Which acts directly to increase the probability of producing neoplastic cells. It can also be used to block protein production. Antisense polynucleotides are also therapeutically effective if the mutation is not significant, for example, if the unmutated allele encoding the correct protein remains. However, when the mutation is prominent, as in the case of a type I mutation, and as in the case of certain type III mutations, either alleles are deleted or one is deleted and the other is deleted When a mutant, antisense therapy is preferably followed by replacement therapy.

  In replacement therapy, a target cell identified as having a mutated gene or proto-oncogene is required to prevent a disorder or overgrowth associated with the identified mutated gene from occurring. Is introduced.

  In the case of tumor suppressor genes, introduction of the suppressor gene into cultured cells results in cell death or no discernible changes, but these cells are no longer known to be tumorigenic in animals. I have. Thus, when ribozyme and / or antisense therapy is administered in conjunction with gene replacement therapy, the cell population containing the mutated gene for which the ribozyme or antisense oligonucleotide is specific may cause abnormal growth in the patient being treated. The chance of no longer contributing increases.

  The invention also provides gene therapy for treating a cancer condition, ie, a cell proliferation disorder mediated by a deletion of a particular gene or a polymorphism within a particular gene. This therapy achieves its effect by introducing a specific antisense polynucleotide and / or a substituted wild-type gene into cells identified by the method of the present invention as having a growth disorder caused by a mutant gene. Whether the cell requires replacement of the wild-type gene as well as antisense therapy to prevent duplication of the polymorphic gene must be determined on a case-by-case basis, and the mutations are prominent. Effect, ie, whether the allele of the wild-type gene is destroyed and the absence of the gene has a cell growth effect.

  Preferred routes of administration that elicit local immunity at or near the skin are transdermal penetration, intradermal injection, or by scratching or irritating the surface of the outermost layer of epidermal cells (ie, epidermal administration), but subcutaneous injection Can also be used for certain applications. The preferred route of administration for inducing local immunity in the respiratory tract is by inhalation or insufflation, but the route of administration to other mucosal tissues will vary depending on its location.

  If naked polynucleotides are to be introduced into the skin or mucous membranes, the use of surfactants, absorption enhancers, chemical stimulants (eg, keratolytics) or mechanical stimulants allows the polynucleotide to be injected without the need for injection Preferably, the nucleotides can be easily delivered. Detergents and absorption enhancers that facilitate the uptake of small molecules other than genes are known in the art and should be adapted for use to facilitate gene uptake without undue testing. Can be. Another substantially non-invasive approach to introducing naked polynucleotides is the percutaneous permeation method (preferably iontophoresis) that has been used successfully for percutaneous penetration of peptides.

  For embodiments of the present invention that stimulate the production of circulating cytokines and related peptides, parenteral routes of administration may be used, but routes that cause little or no invasion of host tissues are very likely. preferable. However, intramuscular injection is not preferred because naked polynucleotides need to be administered repeatedly. Instead, introducing naked polynucleotides into regenerating body areas, such as skin and mucous membranes, since these areas have the ability to replace cells directly affected by the trauma associated with each administration, preferable. To ensure that the protein expressed in these embodiments of the invention is secreted, secretion control sequences known to those of skill in the art may be used if such a sequence is not already present in the full length gene. , Contained in the naked polynucleotide to be administered.

  Further, the polynucleotide may be conjugated to a liposome or delivered in a cell transfected with the polynucleotide in vitro, when used in embodiments of the invention that deliver the peptide at sub-therapeutic concentrations. It should be noted that it is good. However, for the reasons discussed above, it is still preferred in these embodiments to use a naked polynucleotide and a mucosal or dermal route of administration. In particular, the use of liposomes appears to reduce expression levels. This phenomenon seems to be due to the low performance of APCs that recognize liposomes as antigenic substances.

  The embodiments and examples shown throughout this description should be regarded as representative and not limiting of the present invention.

I. Introduction of Naked Polynucleotides into Target Tissues Having Significant Concentrations of Antigen Presenting Cells Production of Naked Polynucleotides The polynucleotide used in the present invention may be DNA or RNA, but preferably has a complementary DNA (cDNA) sequence. The polynucleotide sequences used in the present invention may be treated by means known in the art such that they are (a) expressible and (b) non-replicating or not replicated in the host genome. There must be. The preparation of polynucleotides suitable for use in the present invention is described below, followed by specific examples that show how to prepare compositions of particular polynucleotides. However, it will be apparent to those skilled in the art that other known methods for the production of non-replicating polynucleotides are also suitable.

  The polynucleotide used in the present invention can be obtained using a hybridization method known in the art. DNA and RNA can also be synthesized using an automatic nucleic acid synthesizer known in the art. It is particularly preferred to use the known polymerase chain reaction (PCR) to generate a mixture of polynucleotides. Genomic nucleic acids can be produced by methods known in the art, including protocols described in Ausubel et al., Current Protocols in Molecular Biology, Chapters 2 and 4 (Wiley Interscience, 1989). CDNA can be produced by methods known in the art (see, for example, Maniatis et al., Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Lab, New York, 1982)). Also, a cDNA expression library containing the polynucleotide of interest can be selected by methods known in the art. For reference, an embodiment of the above method is described by the following discussion.

  Preferred polynucleotides for use in particular applications are proposed in the Summary of the Invention section above. For example, a naked polynucleotide of the invention can operably encode a therapeutic peptide, but more preferably encodes an immunogenic peptide that can act as an antigen to stimulate a humoral and / or cellular response. Also, a naked polynucleotide can operably encode an antibody. In this regard, the term "antibody" includes whole immunoglobulins of all classes, chimeric antibodies, hybrid antibodies with dual or multiple antigen specificities, and fragments containing hybrid fragments. Also included within the meaning of "antibody" are conjugates of such fragments and so-called antigen binding proteins (single chain antibodies, e.g., as described in U.S. Patent No. 4,704,692). ) Is also included. Alternatively, the encoded antibody may be an anti-idiotype antibody (an antibody that captures another antibody), for example, as described in US Pat. No. 4,699,880.

  However, one of ordinary skill in the art will appreciate that the methods of the present invention are configured to administer a polynucleotide or mixture thereof operably encoding a therapeutic and / or immunogenic peptide in a subject. You will see that. Thus, the present invention is not limited to using a particular polynucleotide.

  As used herein, the term "polynucleotide" means deoxyribonucleotides or ribonucleotides in the form of discrete fragments or as a component of a larger structure. DNA encoding the therapeutic and / or immunogenic peptides of the present invention can be assembled from cDNA fragments or from oligonucleotides that provide synthetic genes that can be expressed in a recombinant transcription unit. The sequences of the polynucleotide of the present invention include DNA, RNA and cDNA sequences. Although the sequence of a polynucleotide can be deduced from the genetic code, the degeneracy of the genetic code must be considered. The polynucleotides of the present invention contain sequences that are degenerate as a result of their genetic code, which sequences can be readily determined by one of ordinary skill in the art.

  Polynucleotide sequences encoding the desired therapeutic and / or immunogenic peptides can be expressed in eukaryotes or prokaryotes. Hosts can include microorganisms, yeast, insects and mammalian organisms. Methods for expressing a DNA sequence having a eukaryotic or viral sequence in a prokaryote are known in the art. Biologically functional viral and plasmid DNA vectors capable of being expressed and replicated in a host are also known in the art. Such vectors are used to incorporate the DNA of the present invention.

  Polynucleotides of the invention include functional derivatives of known polynucleotides that operatively encode a therapeutic and / or immunogenic peptide for a subject. The term “functional derivative” means “fragments”, “variants”, “analogs” or “chemical derivatives” of a molecule. A "fragment" of one molecule, such as any of the DNA sequences of the present invention, includes any nucleotide subset of that molecule. By “variant” of such a molecule is meant a naturally occurring molecule that is substantially similar to the entire molecule or a fragment thereof. "Analog" of a molecule means a non-natural molecule that is substantially similar to the entire molecule or a fragment thereof.

  As used herein, a molecule is said to be a "chemical derivative" of another molecule if it contains additional chemical moieties that are not normally a part of the molecule. Such moieties can improve the solubility, absorption, biological half-life, etc. of the molecule. Alternatively, these moieties can reduce the toxicity of the molecule, eliminate or reduce unwanted side effects of the molecule, and the like. Portions known in the art that can mediate such effects are described, for example, in Remington's Pharmaceutical Sciences, 16th Edition, Mack Publishing Co. And Easton, Pennsylvania, USA (1980).

  Thus, the term "polynucleotide" as used herein includes functional derivatives, fragments, variants that are substantially similar and have similar activity to the polynucleotides described herein. , Analogs and chemical derivatives.

  Also, the DNA sequences used to produce the therapeutic and / or immunogenic peptides of the present invention can be obtained in a number of ways. For example, the DNA can be isolated using hybridization methods known in the art. These methods include, but are not limited to, 1) a method for detecting a shared nucleotide sequence by hybridizing a probe with a genomic or cDNA library, and 2) selecting and sharing an expression library with an antibody. 3) synthesis by polymerase chain reaction (PCR). Also, specific DNA sequences encoding the fragment include: 1) isolating a double-stranded DNA sequence from genomic DNA; and 2) chemically preparing the DNA sequence to provide the codons required for the polypeptide of interest. And 3) synthesizing a double-stranded DNA sequence in vitro by reverse transcribing mRNA isolated from eukaryotic cells of the donor. In the latter case, the complement of mRNA double-stranded DNA, commonly called cDNA, is ultimately formed.

  Hybridization methods involve the use of labeled mixed synthetic oligonucleotides where each probe is potentially a complete complement of specific DNA sequences in a hybridized sample containing a heterogeneous mixture of denatured double-stranded DNA. It is useful for selecting a recombinant clone using a probe. When performing such a selection, hybridization is preferably performed on single-stranded DNA or denatured double-stranded DNA. Hybridization methods are particularly useful for detecting cDNA clones of origin when the abundance of mRNA associated with the polypeptide of interest is extremely low. In other words, specific cDNA clones are autoradiographed by using stringent hybridization conditions aimed at avoiding non-specific binding, for example, by hybridizing the target DNA with a single probe in the mixture. Can be visualized.

  A cDNA library suspected of containing the polynucleotide of interest can be prepared by injecting various mRNAs from the cDNA into an oocyte, allowing sufficient time for the cDNA gene product to be expressed, and then, for example, Presence of the desired cDNA expression product by using an antibody specific for the nucleotide-encoded peptide or by utilizing a probe for the repetitive motif and the characteristic tissue expression pattern of the polynucleotide-encoded peptide of interest Can be tested and sorted. Alternatively, a cDNA library can be indirectly selected for expression of a therapeutic and / or immunogenic peptide having at least one epitope using an antibody specific for that peptide. Such antibodies can be derived as polyclonal or monoclonal antibodies and used to detect expression products that indicate the presence of the cDNA of interest.

  If a suitable probe is available, any gene sequence can be isolated from any organism by a selection method that relies on nucleic acid hybridization. Oligonucleotide probes corresponding to a part of the sequence encoding the protein in question can be synthesized chemically. For that, a short oligopeptide stretch of the amino acid sequence must be known. The DNA sequence encoding a protein can be deduced from its genetic code, but the degeneracy of the code must be considered. If the sequences are degenerate, mixed addition reactions can be performed. This includes heterogeneous mixtures of denatured double-stranded DNA. When performing such a selection, hybridization is preferably performed on single-stranded DNA or denatured double-stranded DNA.

  The naked polynucleotides of the present invention may be conjugated to or used in combination with other polynucleotides that operably encode regulatory proteins that control the expression of these polypeptides, Alternatively, it may contain recognition, promoter and secretion sequences. One of skill in the art can select, without undue testing, a regulatory polynucleotide and incorporate it into a naked polynucleotide of the invention (if the regulatory polynucleotide is not already present). it can. For example, suitable promoters for use in mouse or human systems and their use are described in Chapter 1 of Current Protocols in Molecular Biology, supra.

  A particularly preferred form of the naked polynucleotide used in the present invention is a form incorporated into a plasmid vector. The use of a plasmid vector, especially a plasmid vector containing a replicator, extends the expression of the gene in the target tissue. Also, certain plasmid vectors are excellent mediators of the immune response to immunogenic peptides because high levels of expression are achieved when the gene encoding the peptide is incorporated into this vector.

  Suitable plasmid vectors are known in the art and include the vectors described in Chapter 1 of Current Protocols in Molecular Biology, supra. Two particularly preferred plasmid vectors are pRSV (Rous sarcoma virus) and pCMV (cytomegalovirus) promoter vectors. Of these promoters, CMV is preferred for polynucleotides introduced into tissues other than muscle. This preference is based on the observation that utilizing the CMV promoter achieves high levels of expression in the context herein.

  For a suitable protocol for isolating the RSV promoter and its use in constructing plasmid vectors, see Gorman et al., Proc. Natl. Acad. Sci. USA, 79, 6777 (1982). Other preferred plasmid vectors are pREP7 and pREV, which are commercially available from Invitrogen, San Diego, CA, USA. For cloning polynucleotides, particularly suitable plasmids for producing mRNA are those described in Kreig et al., Nucleic Acids Res. 12, p. 7057-7070 (1984). Any cDNA containing an initiation codon can be introduced into this plasmid, and mRNA is produced using standard techniques from the expressed DNA template.

  Various viral vectors that can be used in the present invention include adenovirus, herpes virus, vaccinia, or, preferably, an RNA virus such as a retrovirus. As the retrovirus vector, a derivative of a mouse or avian retrovirus vector is preferable. Examples of retroviral vectors into which a single heterologous gene can be inserted include, but are not limited to, Moloney murine leukemia virus (MoMuLV), Harvey mouse sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV) and Rous sarcoma virus (RSV). . Many additional retroviral vectors can incorporate multiple genes. All of these vectors can carry or incorporate a gene for a selectable marker, so that transduced cells can be identified and generated.

  By inserting one or more sequences of interest into a viral vector, for example, together with other genes encoding ligands for receptors on specific target cells, the vector is made target-specific. Retroviral vectors can be made target-specific by inserting, for example, a polynucleotide encoding a sugar, glycolipid, or protein. Preferred targeting is achieved by targeting the retroviral vector with an antibody. One of ordinary skill in the art knows or has to experiment with specific polynucleotide sequences that can be inserted into the genome of a retrovirus to achieve target-specific delivery of a retroviral vector containing the polynucleotide of interest. Can be easily confirmed without performing the above. If necessary, the replacement gene can be targeted to the cell using another vector. In the case of antisense therapy, the antisense oligonucleotide and the replacement gene can be delivered in the same vector, since the antisense oligonucleotide is specific only to the target gene having the polymorphism.

  Recombinant retroviruses are defective and require assistance in producing infectious vector particles. This assistance is provided, for example, by using a helper cell line containing a plasmid encoding the entire structural gene of the retrovirus under the control of regulatory sequences in the LTR. These plasmids lack nucleotide sequences that allow the packaging machinery to recognize RNA transcripts for encapsidation. Helper cell lines in which the packaging signal has been deleted include, but are not limited to, for example, Ψ2, PA317 and PA12. These cell lines produce hollow virions because the genome is not packaged. If the retroviral vector is introduced into a helper cell where the packaging signal is intact, but the structural gene has been replaced by another gene of interest, the vector can be packaged to produce a vector virion. it can.

  To monitor expression, these vectors may be modified to include a known reporter gene. See, for example, Norton et al., Mol. Cell. Biol. 5, p. 281, 1985 (pRSV lac-Z DNA vector) is capable of producing β-galactosidase by protein expression. Luciferase and chloramphenicol acetyltransferase ("CAT", see Gorman et al., Supra for the construction of the pRSV-CAT plasmid) can also be used. Convenient plasmid propagation can be performed in E. coli (see, eg, Molecular Cloning, A Laboratory Manual, supra).

  When used as a tolerizing vaccine, a mixture of polynucleotides or separately co-administered groups of polynucleotides comprises genes encoding operably for immunosuppressive cytokines (eg, TGFβ) and It may contain another gene operably coding for the relevant tissue compatible protein. This approach is adapted for use in inducing resistance to heterologous antigens (including alloantigens) and self-antigens.

  When used in antisense therapy, synthetic antisense oligonucleotides are generally 15 to 25 bases in length. Assuming that the human genome is a random tissue, statistically, the 17-mer forms a unique sequence in the cellular mRNA of human DNA and the 15-mer forms a unique sequence in a component of the cellular mRNA. It is suggested that Thus, substantial specificity for a selected genetic target can be readily obtained using the synthetic oligomers of the present invention.

  In the cell, the antisense nucleic acid hybridizes with the corresponding mRNA to produce a double-stranded molecule. Since the cell does not translate double-stranded mRNA, the antisense nucleic acid interferes with translation of the mRNA. Antisense oligomers of about 15 nucleotides are preferred because they are easily synthesized and are less likely to cause problems than large molecules when introduced into target nucleotide variant-producing cells. The use of antisense methods to inhibit in vitro translation of genes is known in the art (Marcus-Sakura, Anal. Biochem., 172, 289, 1988). Less commonly, antisense molecules that bind directly to DNA can also be used.

  Ribozymes are RNA molecules that have the ability to specifically cleave other single-stranded RNAs in a manner similar to DNA restriction endonucleases. Modification of the nucleotide sequences encoding these RNAs creates molecules that recognize and cleave specific nucleotide sequences associated with the generation of mutant proto-oncogenes or tumor suppressor genes within the RNA molecule. (Cech, J. Amer. Med. Assn., 260, 3030, 1988). The main advantage of this approach is that since the ribozyme is sequence specific, only the target mRNA with the particular mutated sequence is inactivated.

  There are two basic types of ribozymes. That is, a tetrahymena-type (Hasselhoff, Nature, vol. 334, p. 585, 1988) and a "hammerhead" -type. Tetrahymena-type ribozymes recognize sequences that are 4 bases in length, while "hammerhead" -type ribozymes recognize sequences that are 11 to 18 bases in length. The longer the recognition sequence, the greater the likelihood that the sequence will exclusively appear in the target mRNA species. Thus, to inactivate specific mRNA species, hammerhead ribozymes are preferred over tetrahymena ribozymes, and 18 base recognition sequences are preferred over shorter recognition sequences.

  Unmodified oligodeoxyribonucleotides are readily degraded by serum and cellular nucleases. Therefore, certain modifications were made to the phosphate backbone to confer nuclease resistance to the antisense DNA, as is well known in the art. For example, backbone-modified oligomers such as phosphorothioates, methylphosphonates and α-anomeric sugar phosphates have increased resistance to serum and cellular nucleases. Moreover, methyl phosphonate is non-ionic, increases lipophilicity and improves uptake through cell membranes. When using modified oligonucleotides as antisense agents, slightly longer or shorter sequences may be required. This is because chemical changes in molecular structure affect hybridization (LA Crisey et al., BioPharm, 4, 36-42, 1991). These backbone modified oligos bind to the target sequence and either block the translational machinery of the cell from binding to specific RNA or form an RNA / DNA duplex to form ribonuclease H activity. Exerts its inhibitory action.

B. Pharmaceutical Formulations of Naked Polynucleotides The naked polynucleotide composition and the mixture of polynucleotides may be in a pharmaceutically acceptable suspension, solution or emulsion. Suitable vehicles include saline, but in those embodiments that do not rely on antigen presenting cells to deliver the polynucleotide to the target tissue, may be liposome formulations.

  More specifically, pharmaceutically acceptable carriers include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / water solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (eg, based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobial agents, antioxidants, chelating agents and inert gases. Further, the composition of the naked polynucleotide may be lyophilized using means known in the art in preparation for later reconstitution and use according to the present invention.

  For embodiments of the present invention that do not rely on APC recognition as an antigen of a naked polynucleotide, in addition to the targeted vector delivery systems discussed above, it would also be possible to use a colloidal dispersion system for targeted delivery. However, there are advantages to using the methods of the present invention to administer naked nucleotides and to administering these nucleotides to tissues having relatively high concentrations of antigen presenting cells, but not to deliver polynucleotides. It will be appreciated by those skilled in the art that using a colloidal dispersion may not be a very preferred method. Accordingly, the following discussion of such a system is provided primarily as a reference to determine that the preferred methods of the present invention are not available for use in a particular indication.

  Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloid system of the present invention is a liposome.

  Liposomes are artificial membrane vesicles useful as delivery vehicles in vitro and in vivo. It has been found that large single membrane vesicles (LUV) in the size range of 0.2-4.0 μm can encapsulate a significant percentage of aqueous buffers containing large macromolecules. RNA, DNA and intact virions can be encapsulated in aqueous contents and delivered to cells in a biologically active form (Fraley et al., Trends Biochem. Sci., 6, 77). 1981). Liposomes have been used to deliver polynucleotides to plant, yeast and bacterial cells in addition to mammalian cells. In order for liposomes to be an efficient gene delivery vehicle, they must have the following properties: That is, (1) encapsulating a gene encoding an antisense polynucleotide with high efficiency without reducing its biological activity, (2) preferentially and substantially in a target cell as compared to a non-target cell. (3) delivering the aqueous content of vesicles to the cytoplasm of target cells with high efficiency, and (4) accurately and efficiently expressing genetic information (Mannino et al., Biotechniques). 6, 682 (1988)).

  The composition of the liposome is usually a combination of phospholipids, especially phospholipids having a high phase transition temperature, and is usually a combination of steroids, especially cholesterol. Other phospholipids or other lipids can also be used. The physical properties of liposomes depend on pH, ionic strength and the presence of divalent cations.

  Examples of lipids useful for making liposomes include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides and gangliosides. Particularly useful are diacylphosphatidylglycerols, whose lipid moieties contain 14-18 carbon atoms, especially those containing 16-18 carbon atoms, and are saturated. Illustrative examples of phospholipids are egg phosphatidylcholine, dipalmitoyl phosphatidylcholine and distearoyl phosphatidylcholine.

  The targeting of liposomes can be categorized based on anatomical and mechanistic factors. Anatomical classification is based, for example, on the level of organ-specific, cell-specific and organelle-specific selectivity. Mechanistic targeting can be distinguished based on whether it is passive or active. Passive targeting takes advantage of the natural tendency of liposomes to be distributed in cells of the reticuloendothelial system (RES) in organs with sinusoidal capillaries. On the other hand, active targeting involves coupling liposomes to specific ligands, such as monoclonal antibodies, sugars, glycolipids or proteins, or achieving targeting to organs and cell types other than the naturally occurring localized sites. By changing the composition or size of the liposome, the liposome is modified.

  The surface of the targeted delivery system can be modified in various ways. In the case of targeted delivery systems for liposomes, the lipid group is incorporated into the lipid bilayer of the liposome, and the targeting ligand maintains stable association with the liposome bilayer. Various linking groups can be used to link the lipid chain to the targeting ligand.

  For these embodiments of the invention that rely on the expression of APC, liposome formulations should not be used because they significantly limit in vivo uptake of naked polynucleotides. In such embodiments, an isotonic buffer solution is instead the preferred medium for maximizing uptake of the naked polynucleotide. In addition, absorption enhancers, surfactants, chemical stimulants or mechanical stimulus means are also preferred to facilitate the penetration of the naked polynucleotide composition through the point of entry. For general principles on accelerators and surfactants that have been successfully used for mucosal delivery of organic or peptide-based drugs, see Chien, Novell Drug Delivery Systems, Chapter 4 (Marcel Dekker, 1992). I want to be. Specific information regarding known means and principles of intranasal delivery of medicaments is described in Chapter 5 of Chien, supra. Examples of suitable intranasal absorption enhancers are provided in Tables 2 and 3 of Chapter 5, although milder agents are preferred. In addition, known means and principles of transdermal delivery of medicaments are also discussed in Chapter 7 of Chien, supra. Also, suitable agents for use in the method of the present invention for mucosal / intranasal delivery are described in Chang et al., Nasal Drug Delivery, Chapter 9 of "Treaties on Controlled Drug Delivery" and Table 3-4B (Marcel Dekker, 1992). It is described in. Suitable agents known to promote the absorption of drugs through the skin are described in Sloan's Use of Solubility Parameters from the Regular Solution Theory to Discretion Particulation-Driving Procedures-Dragon Procedure-Dragon Procedure- Delivery "(Marcel Dekker, 1992) and other parts of that text.

These methods (and other methods that are conveniently used to facilitate drug delivery) produce naked polynucleotides for use in the methods of the invention without undue testing by those skilled in the art. It is believed that it can be adapted to: In particular, the methods discussed in the preceding paragraph, according to the findings of the inventor of the present invention, have not been used previously for the delivery of polynucleotides, but are considered to be suitable for this purpose. For that reason, the above cited references are not essential to the method of the present invention, but are hereby incorporated by reference. A specific example illustrating this suitability is described below.
C. Means and Routes of Naked Polynucleotide Administration In the case of the dermal administration route, the means of introduction is by epidermal, subcutaneous or intradermal injection. Of these means, epidermal administration is preferred when APCs are considered to be present at high concentrations in intradermal tissue.

However, the most preferred means of introduction for the route of dermal administration is the least invasive. Preferred among these means are:
Transdermal penetration method and epidermal administration method.

  In the case of transdermal transmission, iontophoresis is the appropriate method. Iontophoresis permeation can be performed using a commercially available "patch," which delivers the product continuously through intact skin for several days or more. Using this method, the permeation of the relatively concentrated pharmaceutical composition can be controlled, the infusion of the combination drug can be performed, and the absorption enhancer can be used simultaneously.

  A typical patch product used in this method is LECTRO PATCH, a registered product of General Medical Company, Los Angeles, California, USA. The product can be adapted to provide various concentrations and to administer continuously and / or periodically, maintaining the reservoir electrode at neutral pH by electronic control. The preparation and use of the patch must be performed according to the manufacturer's printed instructions accompanying the LECTRO PATCH product. The instructions are incorporated herein.

  Epidermal administration basically stimulates the outermost layer of the epidermis mechanically or chemically sufficiently to elicit an immune response to the irritant. Specifically, the stimulus must be sufficient to attract the APC to the stimulation site. As discussed earlier, the APC is then believed to take up and express the administered naked polynucleotide.

  A typical method of mechanical stimulation uses a large number of tines, which are very small and short in diameter, which are used to irritate the skin, attract the APC to the stimulation site, and transfer from the ends of the tines. The naked polynucleotide obtained is incorporated. For example, the MONO-VACC old tuberculin test manufactured by Pasteur Merieux, located in Lyon, France, includes a device suitable for introducing naked polynucleotides.

  The device (sold in the United States by Connaught Laboratories, Inc. of Swiftwater, PA) is a plastic with a syringe plunger at one end and a tine disk at the other end. It consists of a container made of. The tines desk has a number of small diameter tines that are long enough to scratch the outermost layer of epidermal cells. Each of the tines of the MONO-VACC kit is coated with old tuberculin. In the case of the present invention, each needle is coated with a naked polynucleotide or a pharmaceutical composition of a mixture thereof. The device is used in accordance with the manufacturer's instructions included in the product for the device. These instructions for use and administration are hereby incorporated by reference to describe the normal use of this device. A similar device that can also be used in this embodiment is the device currently used to perform allergy tests.

  Another method suitable for epidermal administration of naked polynucleotides is to use a chemical agent that stimulates the outermost cells of the epidermis to elicit a sufficient immune response to attract APCs to the area. One example is a keratolytic, such as salicylic acid used in a commercially available topical depilatory cream sold under the trademark NAIR by Noxema Corporation. This approach can also be used to effect administration to the mucosal epithelium. The chemical stimulant may be applied with a mechanical stimulant (eg, if the MONO-VACC type tines are also coated with a chemical stimulant). The naked polynucleotide may be suspended in a carrier that also contains the chemical stimulant, or may be co-administered with the stimulant.

  For mucosal administration, the method of introduction will depend on the location of the point of entry. In particular, when immunizing and treating respiratory infections, the intranasal administration method is most preferable. These methods include inhalation of an aerosol suspension of the naked polynucleotide or mixture thereof or insufflation of the naked polynucleotide or mixture thereof. Suppositories and topical formulations are also suitable for introduction into certain mucous membranes, such as the genital and ocular cell sites. Of particular interest for vaginal delivery of naked polynucleotides are vaginal sandwich rings and pessaries. Examples of these devices and their use are described in Chapter 9 of Chien, supra.

  The dosage of each naked polynucleotide or mixture thereof provided using the methods of the invention will depend on the response desired by the host and the polynucleotide used. Generally, up to 100-200 μg of DNA can be administered in a single administration, but it is believed that as little as about 0.3 μg of DNA can be administered through the skin or mucous membranes to elicit a long lasting immune response. .

  However, to achieve the objects of the present invention, it is sufficient that the naked polynucleotide be provided in a dosage sufficient to express the biologically active peptide encoded by the polynucleotide. . Suitable dosages for particular conditions (eg, when delivering sub-therapeutic dosages of cytokines) are described in the discussion and examples below.

  These dosages may be modified to achieve therapeutic, sub-therapeutic, or immunogenic levels. Methods for determining the presence and amount of expressed peptide are known to those skilled in the art and will not be described in detail. Certain of these methods are described in the Examples below, which generally include immunoassays (eg, enzyme-linked immunosorbent assays), PCR, and methods known in the art. There is an immunohistological analysis performed according to the following. Based on these assays and quantification methods and the information provided by in vivo clinical microbiology known to those skilled in the clinical arts, the dosage of the polynucleotide to be administered will achieve the desired expression level. Can be adjusted.

II. Introduction of Naked Polynucleotides to Express Sub-Therapeutic Levels of Cytokines and Other Proteins Important in the Development of Age-Related Plague The above method of producing and introducing naked polynucleotides is described in Suitable for use in this embodiment of the invention. In this embodiment, the methods described herein are employed to produce subtherapeutic levels of circulating cytokines and related proteins. As can be seen, the amount of naked polynucleotide required to produce sub-therapeutic levels of the expressed protein can be readily determined and adjusted as needed by a skilled clinician.

  This aspect of the invention involves administering to a mammal a polynucleotide operably encoding a desired protein, or a recombinant mixture thereof, preferably prior to the onset of a biological defect associated with aging. Will be implemented. For example, in patients at high risk of developing a plague associated with a weakened immune system, for example, to perform protein expression at a continuous level prior to the onset of the disease as determined by observation of surrogate endpoints for the disease, Sub-therapeutic levels of immunostimulatory interleukins (eg, IL-2) are administered. For patients at high risk of disease, this treatment is continued throughout the patient's life. Individual dosages of the polynucleotide (and the resulting level of protein expression) must be low (ie, sub-therapeutic) in order for the patient to accept this long-term course of treatment. However, polynucleotides are considered to degrade much more slowly than proteins (ie, months apart) and need not be administered as frequently, thus minimizing toxicity and trauma risk to the patient. Become.

  More specifically, a disease state associated with aging, or other biological events caused by weakening of the immune system, such as loss of CD4 T cells during HIV-1 infection, occur or occur. At the same time, administration of the desired polynucleotide is started. A disease state or loss of immune function occurs. It is preferred that long-term, continuous treatment be initiated before the disease state or loss of immune function occurs. Treatment has been achieved by sub-therapeutic expression of genes of interest, particularly genes having codes for cytokines (eg, interleukins and lymphotoxins).

  Most preferably, at least one of the genes of interest has a code for IL-1, IL-2, growth hormone (GH), somatomedins (eg, insulin-like growth factor [IGF-1]) and / or TGFβ. It may be a gene having These proteins include the magnitude of the immune response (eg, IL-2, IL-1, GH and IGF-1), the systemic response to stress or loss (eg, IL-1), the cellularity of tissues (GH and IGF-1) and may control or affect the deposition of extracellular matrix proteins and connective tissue (TGFβ). And the latter exert a greater biological effect as the activity of IL-1 and IL-2 decreases.

  This particular method of the invention is, in part, a subset of the method described in Section I above. However, those skilled in the art will appreciate that this embodiment of the invention can be practiced with non- "naked" polynucleotides, ie, polynucleotides that are bound to a colloidal dispersion. However, while the method disclosed in Section I above is superior, its practice (i.e., using "naked" polynucleotides introduced into tissues containing a relatively high proportion of APCs) is not It is much better.

III. Administration of Naked Polynucleotide Cocktail Another aspect of the invention is a peptide cocktail, under the control of a single promoter, eg, by expressing a genetic construct containing up to 200 polynucleotide sequences. (Ie, a mixture of polynucleotides). This embodiment is particularly useful for treating infections by different species of material that cause similar symptoms. For example, there are more than 100 known species of Rhinowilles that cause respiratory illness with similar clinical symptoms. Rather than identifying a particular infecting species (an laborious and often inaccurate process), a cocktail vaccine can be administered according to the methods of the present invention, thereby stimulating an immune response to a number of different rhinoviruses. can do. This approach also makes it possible to construct vaccines against various strains of HIV using pooled isolates of envelope genes from different patients, which genes may be amplified if necessary. it can.

  Administering a mixture of polynucleotides also serves to deliver a peptide having more than one biological activity. For example, a naked polynucleotide operably encoding for an immunogenic peptide is linked or administered together with a naked polynucleotide operably encoding an antibody such that both the peptide and the antibody are expressed. Can be

  Illustratively, administration of genes jointly expressing IL-2 and anti-gp71 (based on results obtained with the IL-2 protein) in response to murine leukemia virus (MuLV) in mice. Results were obtained in which the antibody was localized in the developed tumor tissue (for results obtained by co-administration of IL-2 / anti-gp71 mAb, see Schulz et al., Cancer Res., 50, 5421-5425, 1990). See).

  Examples illustrating aspects of each embodiment of the present invention are provided below. These examples should be considered as illustrative and not limiting on the invention.

Example I
Delayed type hypersensitivity occurs in mice after intramuscular injection of naked polynucleotide. When naked plasmid cDNA is injected intramuscularly, the peptide encoded by the polynucleotide is expressed (reported earlier). (Consistent with the results reported above), but elicit an immune response against the gene in muscle tissue (as opposed to the results reported previously). When the two plasmids are injected simultaneously, the inflammatory response becomes chronic and muscle necrosis appears. Both responses are consistent with a diagnosis of a localized delayed type hypersensitivity response to the gene at the point of entry of the gene or muscle tissue. Contrary to previous expectations, this inflammatory response, rather than uptake by muscle cells, also appears to be the cause (if not the only cause) of naked polynucleotide expression following the intramuscular injection. .

To account for the immune response caused by intramuscular injection of naked cDNA, pREVk3 and pRSVIL2 were adjusted as follows.
Production of Plasmids A rearranged kappa light gene from a human patient with chronic lymphocytic leukemia was isolated. This gene contains Humkv325, which encodes a 17.109 cross-reactive idiotype commonly expressed by IgM autoantibodies and chronic lymphocytic leukemia cells. This gene is known in the art and is described, for example, in Martin et al. Exp. Med. 175, 983 (1992). This document is incorporated herein by reference.

  A 1040 bp HindIII-XhoI fragment containing the VJ region of this gene was excised and inserted into the polycloning site of the mammalian expression vector pREP7 (Invitrogen, San Diego, Calif., USA) with the Rous sarcoma virus (RSV) long terminal repeat (RSV). (LTR), and a vector was prepared and named pREVk3. Downstream of the rearranged JK1 segment is a natural stop codon that terminates translation.

To produce an IL-2 expression vector named pRSVIL-2, the luciferase cDNA in the vector pRSVL (Wolff et al., Science, 247, 1465, 1990) was transformed with Cullen, Cell, 46, 937 (1986). 680 bp of the HindIII-BamHI fragment of pBC12 / HIV / IL-2 (American Type Culture Collection, No. 67618). This Wolff et al. And Cullen document are hereby incorporated by reference to demonstrate their knowledge in the art of constructing these expression vectors.
Intramuscular injection of plasmid cDNA into mice Eight week old BALB / c mice were anesthetized with methoxyflurane. Plasmid cDNA (100 μg / injection) was depleted in 100 μl saline and then injected four times at weekly intervals into the quadriceps using a 28 gauge needle. One group of six mice was injected with 100 μg of pREVk3. Another group of six mice were injected with 100 μg each of pREVk3 and pRSVIL-2, and a third group of mice were injected with only 100 μg saline. Immediately prior to making these injections, blood samples were collected from the medial frontal basilar artery.
ELISA method for confirming gene expression in vivo by plasmids Antibodies to Humkv325 products were measured by ELISA method (enzyme-linked immunosorbent assay). IgM rheumatoid factor Glo is encoded by the Humkv325 gene and has a positive kappa light chain of 17.109 idiotype. The purified protein was dissolved at a concentration of 10 μg / ml in buffered saline borate at pH 8.2 containing 0.1 M borate, 0.2 M NaCl, ie, BBS, and then 100 μl aliquots were made of plastic. Added to wells of microtiter plate. After overnight incubation at 4 ° C., the plates were washed twice with BBS containing 0.5% Tween-20 (BBS / Tween), then BBS supplemented with 1% bovine serum albumin (BBS / BSA). ) Was quenched at room temperature for 4 hours. After washing twice with BBS / Tween, samples serially diluted with BBS / BSA were distributed to each well. After incubation at room temperature for 3 hours, the plates were washed four times with BBS / Tween, then biotinylated gout anti-mouse IgG (Kirkegaard & Gaithersburg, Ga., USA) diluted 1: 2000 with BBS / BSA. (Perry). After 1 hour, the plates were washed four times with BBS / Tween and then incubated with 25 μl of TMB peroxidase substrate (Kirkegaard & Perry). After 30 minutes, the absorbance at 450 nm wavelength light was measured with a microplate reader (Molecular Devices, Melon Park, CA, USA). To estimate the antibody content in the immune serum, the test results were compared to a standard curve generated with monoclonal antibody 17.109 (eg, Carson et al., (1983) Mol. Immunol., Vol. 20, 1081- 1081). (See description of this mAb on page 1087).

The results of these assays indicate that the production of the antibody of interest was increased, thereby confirming the expression of the gene by the plasmid.
Histological evaluation Mice injected with intramuscular injection were sacrificed on day 49. The injected muscle was fixed in 10% formalin and processed for histological evaluation.

  The portion of the muscle that had been co-injected with pREVk3 and pRSVIL2 demonstrated chronic inflammation and muscle necrosis, consistent with the response of delayed localization-type hypersensitivity (FIGS. 1A and 1B). In contrast, muscle injected with pREVk3 or pRSVIL2 alone had localized lymphoid infiltrates at the site of subcutaneous injection (FIG. 1C).

Example II
Gene Expression After Intradermal Injection of Naked Polynucleotide To explore an alternative to intramuscular injection of naked polynucleotide, mice were injected intradermally with naked cDNA plasmid. Since gene expression was observed, measurements were taken.

  The gene for influenza ribonucleoprotein (RNP) was subcloned into the pCMV plasmid described above. RNP genes from many strains of influenza are known in the art and are highly conserved in sequences within various strains (eg, Gorman et al., J. Virol, 65, 3704; 1991).

  Four 8-week-old Balb / c mice were injected three times with 15 μg of pCMV-PNP depleted in 100 μl of HBSS. Intradermal injections were given at two week intervals at the base of the tail. Cytotoxic T lymphocytes (CTLs) recognize the antigen presented by class I MHC molecules and play an important role in eliminating cells infected with the virus. Intramuscular (im) immunization with a cDNA expression vector has been regarded as an effective method for introducing antigens into class I MHC molecules to stimulate a CTL response. In this study, intradermal (id) injection of a plasmid containing the influenza nucleoprotein (NP) antigen gene resulted in the release of both NP-specific CTLs and high titers of anti-NP antibodies. Was. These antibodies reached the highest dose 6 weeks after injection without local inflammation and remained unchanged for at least 28 weeks.

  Plasmid DNA was purified by CsCl staining in the presence of ethidium bromide, frozen and stored in 10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0. Prior to injection, the plasmid was precipitated with ethanol and dissolved in normal saline containing 0.1 mM EDTA.

  Viara et al., Int. Immunl. 2, p. 487, (1990), the amount of anti-NP IgG present in the serum was measured by ELISA. The test results of this assay are shown in FIG. 2A. All of the animals developed high titers of anti-NP antibodies and were retained for more than 20 weeks. As shown in FIG. 2B, the intradermal injection appeared to be about 4-fold higher in antibody titer than the intramuscular injection of an equivalent amount of plasmid (performed as described in Example I).

  Each of the coordinate axes in FIG. 2 shows the ELSA titer over time (average, 1 ounce). The serum dilution for all graph points is 2560.

Example III
Gene Expression Following Intranasal Induction of Naked Polynucleotides Using the same plasmid (pCMV-RNP) in the same HBBS buffer as described in Example II, a naked polynucleotide encoding influenza ribonucleoprotein was purified. , Were introduced intranasally into three groups of six Balb / c mice. The concentration of anti-NP IgG in peripheral blood before and after the introduction of plasmids at various dilutions in serum was measured by ELISA as described in Example II. Blood was collected from each mouse 6 weeks after being introduced intranasally.

  FIG. 3 is a graph showing the results of ELISA assays performed before and after introduction of the plasmid into the nasal cavity. The graph plots the ELISA titer against the serum dilution. In FIG. 3, the numerical values indicate the value for each mouse in each group (# 1-3) and the average value for all mice in each group (# G1-G3).

  Without anesthesia, a second group of mice injected with 3 × 7.5 μg of plasmid showed a higher titer of antibody compared to background (FIG. 3). These data are shown in FIG.

  A third group of mice was injected with the same weight of plasmid under anesthesia. The anti-NPG IgG titers in these mice indicated that RNP expression was substantially similar to that achieved in non-anesthetized mice. The data for the anesthetized mouse is shown in FIG.

  The onset can be increased by the additional use of absorption enhancers and the duration can be extended with aging enhancers. The properties and use of aging enhancers are well known in the art, as suggested in Chapter 5 of Chien, supra.

Example IV
Subtherapeutic levels of expression of cytokines and systemic response to their cytokines after administration of the naked genes for IL-2, TGF-β1 and IL-4. Construction of Expression Vectors for Human (h) IL-2 (ATCC 67618), TGF-β1 (ATCC 59954) and Mouse IL-4 (ATCC 37561) were converted into cDNA using the vector pBSII SK (San Diego, CA, USA). (Obtained from Invitrogen) to generate 5'-HindIII and 3'-Smal sites. These sites were used to replace the HindIII-BamH1 luciferase cDNA fragment in the expression vector pRSVL described in Example I. The resulting expression vectors (pRSVIL2, pRSVTGFβl and pRSVIL4) contain the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter and the SV40 polyadenylation site. Plasmid DNA was purified from transformed DH5α E. coli using the QIAGEN kit obtained from Qiagen, Chatsworth, CA, USA.

The behavior of these three vectors is described by Lotz et al. Exp. Med. Transient lipofection of mouse C2C12 myoblasts (ATCC [American Type Culture Collection, Rockford, Md., CRL 1772]) as described in Chem., 167, 1253 (1988). transient lipofection). The above document by Lotz et al. Is incorporated herein by reference. In each case, the supernatant obtained 48 hours after lipofection was used in mice in the presence or absence of the appropriate specific neutralizing antibody (R & D Systems, Minneapolis, MN, USA) in the C3H / HeF thymus of mice. Cells contained bioactive cytokines as determined by lymphocyte activating factor (LAF) assay using cells.
Experimental Summary Five week old Balb / c mice were purchased from Jackson Laboratory (Bar Harbor, Maine, USA). At six weeks of age (day zero), the animals were divided into four groups of four mice each. On days Sero, 7 and 14, groups 2 to 4 had a total of 100 μg of plasmid DNA (pRSVIL2, PRSVTGFβ1 or PRSVIL2) dissolved in 100 μl of normal saline at five different locations on the right thigh. pRSVIL4) was injected intramuscularly (im) with a 28 gauge needle. On days 3, 10 and 17 (3 days after cytokine gene injection), 100 μg of key dispersed in 100 μl of IMJECT ALUM (aluminum hydroxide, Pierce, Rockford, Ill., USA) All animals were immunized by intramuscular injection of whole limpet hemocyanin (KLH) (Sigma, St. Louis, Mo., USA) into the same thigh.

  A second set of experiments was performed with a similar protocol. 100 μg of plasmid DNA (pRSVIL2, pRSVTGFβ1 or pRSVIL4 for the second to fourth groups, respectively) was injected into the right thigh of each group of mice consisting of eight mice. The first group was injected with normal saline, while the fifth group was injected with 100 μg of pRSVIL2 and 100 μg of pRSVTGFβ.

  Unlike the first experiment, the antigen (human transferrin (Sigma)) was injected intramuscularly in the left shoulder under the same conditions. On days 56 or 63, 50 μg of antigen (KLH or transferrin, respectively) depleted in 50 μl of normal saline was injected subcutaneously with a boost. All animals collected blood from the retro-orbital plexus weekly to determine antibody levels.

Six week old MRL / lpr / lpr mice (Jackson Laboratory, 10 mice / group) were injected in the same manner with 100 μg of pRSVIL2, pRSVTGFβ1 or pRSVnull (ie, no transgene) three times at 4 week intervals. Mice were bled at 16 weeks, two weeks after the last injection, to measure anti-chromatin antibody levels. In the transferrin experiment, three mice in the pRSVTGFβ1 group, one mouse in the control group and one mouse in the pRSVIL2 group died during blood collection or anesthesia.
Antibody assay to confirm effect on antigen response caused by expression of cytokines by cDNA plasmids. Microtiter plate wells (Costar # 3590, Cambridge, Mass., USA) were coated with human transferrin (100 μl / well, borate buffered saline). Washed with water (BBS) pH 8.0 at 10 μg / ml overnight) and the same buffer, then quenched with 1% bovine serum albumin (BSA) in BBS. After washing twice with 0.5% Tween 20 in BBS, serum samples diluted 1: 1000 in phosphate buffered saline pH 7.4 (PBS) were added to double the wells. After overnight incubation at 4 ° C., the plates were washed with BBS / Tween, then with biotinylated anti-mouse IgG and anti-mouse IgM (Jackson Laboratories, West Grove, Pa., USA) diluted 1: 8000 in BBS. Incubated. After 1 hour incubation with peroxidase-labeled streptavidin (Kirkegaard & Perry, Gaithersburg, MD) diluted 1: 2000 in BBS containing 1% BSA, the plates were washed 4 times with BBS / Tween, It was then incubated with TMB peroxidase substrate (Kirkegaard & Perry). After 30 minutes, the absorbance at 450 nm wavelength light was measured with a TITERTEK MULTISCAN METER (Flow Laboratories, Rockville, MD, USA). Each assay included serially diluted standard mouse anti-KLH or anti-transferrin antisera starting at 1: 5000. In transferrin experiments, absorbance values were converted to relative antibody concentrations. In the following test results, 1 is defined as the level of the antibody in a 1: 5000 dilution of the standard antiserum.

  In the transferrin experiments, total IgG and total IgG1 concentrations were measured using a radial immunodiffusion kit (The Binding Site, San Diego, CA, USA) according to the manufacturer's instructions.

Antibodies to chromatin were measured by ELISA as described in each of the above examples. The OD values in the ELISA table were obtained by reference to a standard curve generated for a strongly positive reference serum. The test result obtained is the dilution of the standard curve, given the same OD x 106 as the test serum, and shown to represent a measure of the amount of antibody present. The absolute unit is arbitrary, and is represented in FIGS. 6 and 7 by an equivalent dilution factor.
Measurement of circulating levels of TGF-β1 protein To determine whether the administration of the gene would result in a sustained increase in circulating cytokine levels, mice were injected with the 6th week, ie, the first gene injection. Blood was drawn 4 weeks after, and the plasma samples were assayed for TGF-β1 activity using the CCL64 mink lung cell proliferation assay. This assay is described in Latz et al. Immunol. 144, p. 4189. This document is incorporated herein by reference. Rabbit antibodies that specifically neutralize TGF-β1 but not TGF-β2 or TGF-β3 were purchased from R & D Systems (Minneapolis, MN, USA).
Measurement of Delayed Type Hypersensitivity Response To determine whether injection of cytokine genes modulates cellular immunity, delayed type hypersensitivity (DTH) response was measured by footpad swelling 48 hours after challenge. Tested. On day 70, 100 μg of transferrin in 100 μl of normal saline was injected into the right hind footpad. Footpad thickness was measured with calipers before and 48 hours after injection, and the difference between these measurements was calculated.
Test Results Effect of Cytokine Gene Injection on Antibody Response to Heterogeneous Antigen In a first set of experiments, a heterologous antigen, key hold limpet hemocyanin (KLH), was injected at the same site as the plasmid, but exerted a cytokine on antibody response to The effect of intramuscular injection of the gene was measured. Antibodies against KLH reached higher levels in the pRSVIL2 and pRSVIL4 groups than in the control group (FIG. 6, panel A). In contrast, anti-KLH antibodies were lower in the pRSVTGFβ1 group than in the control group (FIG. 6, panel B). To determine whether the cytokine gene exerted its effects not only on the regional lymph nodes but also throughout the body, a second set of experiments was performed in which the antigen (transferrin) and the cytokine vector were injected at two different sites. Antibodies to transferrin were highest in the pRSVIL2 group (FIG. 7, panel A) and lowest in the pRSVTGFβ1 group (FIG. 7, panel B). These test results indicated that injection of the two plasmids could stimulate or inhibit the antibody response.

In some in vitro systems, TGFβ counteracts the effects of IL-2. It is not known whether TGFβ has the same activity in vivo. Therefore, experiments were performed in which a plasmid encoding IL-2 or TGF-β1 was co-injected. The level of anti-transferrin antibody in the group injected with both pRSVIL2 and pRSVTGFβ1 was indistinguishable from the pRSVTGFβ group (FIG. 10, panel C). That is, it has been demonstrated that the expression of TGF-β1 completely neutralized the action of IL-2. The mean level of anti-transferrin antibodies in the pRSVIL4 group was higher than the control group, but not significantly different (2.3 ± 0.4 vs. 1.7 ± 0.36 mg / L at 11 weeks).
Total IgG and IgG1 levels Total IgG levels were measured in sera from mice injected with transferrin. The highest IgG levels were observed in the pRSVIL2 and pRSVIL4 groups after 10 and 11 weeks. On the other hand, the lowest level was found in the pRSVTGFβ1 group (Table 1). Injection of the TGF-β1 plasmid completely inhibited the increase by IL-2. The levels of total IgG in the pRSVIL2 / pRSVTGFβ1 group were similar to the pRSVTGFβ1 group. Mice injected with pRSVIL4 had significantly higher IgG1 concentrations than the other groups.

IgG1 levels were measured by radial immunodiffusion. Numerical values are mean ± SEM. The numbers in parentheses indicate the IgG1 / IgG ratio at the same time point. (* Indicates p <0.05 for IL-4).
Delayed Type Hypersensitivity (DTH) To determine whether injection of the cytokine gene could modulate cellular immunity, a footpad swelling assay was performed following antigen challenge. As shown in FIG. 8, the pRSVIL2 injected mice had a highly significant increase in footpad swelling as compared to the control group. Co-injection of pRSVTGFβ1 completely inhibited the effect of IL-2.
Levels of circulating TGF-β1 The average plasma concentration of TGFβ1 at week 6 or 4 weeks after the last injection of pRSVTGFβ1 is only 0.32 ng / ml for animals injected with pRSVIL2 or untreated. 2.6 ng / ml, which is an eight-fold difference. Its TGF-β activity was neutralized by specific antibodies to TGF-β1 (Table 2).

Four weeks after the last plasmid injection, plasma samples were collected from mice injected with either pRSVTGF-β1 or pRSVIL-2. These samples were diluted 1:10, acidified to pH 4, neutralized, and then tested in triplicate for TGF-β activity in the CCL64 assay. Some of these samples were also incubated with a rabbit neutralizing antibody specific for TGF-β1 (10 ng / ml) before being subjected to the CCL64 assay. Pre-immune rabbit IgG did not alter the level of TGFβ activity. TGFβ levels were determined by comparison to a standard curve containing recombinant TGF-β1 (R & D Systems), expressed in units of ng / ml, and expressed as mean ± SE of three samples per group.
The test results obtained from the test of anti-chromatin antibody transferrin show that injection of plasmid DNA elicited the characteristic biological effects of the three cytokines during a cellular or humoral immune response to the heterologous antigen. Indicated. To determine whether this method was also effective in modulating the ongoing pathological immune response, IL-2 and TGF-β1 plasmids were introduced into MRL / lpr / lpr mice by intramuscular injection. . In addition, this mouse produces a high-titer autoantibody and has been used as a model of systemic lupus erythematosus. Autoantibody titers to chromatin in MRL / lpr / lpr mice were significantly increased in the pRSVIL2 group. The lowest titer was found in the pRSVTGFβ1 group. The difference between the mean values of these two groups was almost 7 times (FIG. 9).

  Gene expression in muscle tissue following injection of plasmid cDNA has been previously demonstrated in the literature by Wolff et al., Nature, supra, in experiments using reporter gene constructs. However, the test results provided herein demonstrate, for the first time, that direct injection of a cDNA expression vector intramuscularly can induce the production of bioactive proteins with systemic effects. Cytokine genes and antigens were injected into experimental animals at different times and at different sites. Furthermore, the immunity of these cytokine genes persisted for weeks after administration. These test results indicate that the effects of these cytokine genes were exerted systemically.

Example V
Prophylactic IL-2 gene immunotherapy with subtherapeutic expression of naked polynucleotides in a mouse lymphoma model (testing whether expressed proteins can prevent or delay the onset of disease)
Experimental Design and Injection into Mice Six-month-old AKR / J retired breeder female mice, sixteen-month-old retired female ICR crossbred mice, sixteen-month-old BALB / c retired breeding female mice and six-week-old BALB / c female mice were purchased from Jackson Laboratory (Bar Harbor, Maine, USA). 90% of AKR female mice develop lymphoma by 9 months of age. Prior to conducting the experiment, AKR / J mice were pre-screened for the presence of circulating lymphoblasts by analysis of blood smear slides. Lymphoma-free animals were divided into two groups of 23 mice for DNA injection. AKR and ICR mice were injected with DNA three times weekly, followed by injections every two weeks during the experiment.

  The gene for IL-2 was subcloned into an appropriate expression vector as described in the above example. As described in the experimental examples above, the vector called pRSVIL-2 contains an IL-IL sandwiched between the Rous sarcoma virus long terminal repeat promoter sequence and the SV40 polyadenylation sequence. 2 (ATCC CRL # 67618, Rockville, MD, USA).

The control vector, pRSV, has no sequence encoding IL-2. Plasmid DNA was purified in large quantities using the Promega MEGAPREP kit (Madison, Wis., USA). Purified plasmid DNA (25 μg / injection) was suspended in 0.9% NaCl solution (100 μl / injection) and then injected directly into the right quadriceps of each mouse using a 28 gauge needle. .
Protein Expression and Detection Serum samples were purchased from Advanced Magnetics, Inc. Human IL-2 was assayed using an IL-2 ELISA kit obtained from Co. (Cambridge, Mass., USA). Each sample was assayed twice and compared to a standard curve using serum concentrations of recombinant human IL-2. The lowest limit of detection for this assay is 75 pg / ml. Cross-reactivity with mouse IL-2 was minimally observed.

The results of these tests are reported in FIG. 10 (time course of expression in individual mice).
Effect on animal longevity The survival status of the animals was measured from birth to the time of death. Animals were killed a) when they lost significant weight (ie, 30% or more in 2 weeks), b) when hunting or respiratory disorders appeared due to thymic hypertrophy, or c) in the peripheral blood. When a large number of lymphoblasts were found. In general, peripheral lymphoblasts emerged suddenly and became detectable 7-10 days before the death of physical signs of aging.

  Statistical analysis of survival curve data was performed using Kaplan-Meier survival curve estimators known in the art. Significance between groups was determined using the Mantel-Haentzel test known in the art.

Data on the longevity of the animals used in this experiment are summarized in FIG. One-half of the animals in the control group died of lymphoma at 7 months and 20 days, and overall died at 9 months. In contrast, of the six animals receiving naked pRSVIL-2, four survived nine months later.
Analysis of Natural Killer (NK) Cell Population to Determine the Effect of IL-2 Gene Expression on Lymphoblast Cytolytic Activity in Young AKR / J Mice. Measured using a standard 51Cr release assay known in the art. YAC-1 (NK-sensitive Maloney murine leukemia virus-induced mouse lymphoma cell line) and P815 (NK-resistant mouse mast cell tumor cell line) were obtained from ATCC (Rockville, MD, USA) and used as target cells. 51Cr-labeled target cells were added to the wells of a round-bottom 96-well microtiter plate, and then the effector cells were plated three times to obtain various effector-to-target ratios (E / T ratios). Non-adherent mouse PBLs (effector cells) were isolated by gradient centrifugation of lymphocytes M (Cedarlane Laboratories Ltd., Hornby, Ontario, Canada), followed by overnight incubation in culture dishes. Cells were depleted. Effector cells and target cells were incubated simultaneously for 4 hours. The supernatant was harvested and counted in a gamma counter to determine the amount of isotope release.

  The test results are expressed as a percentage ratio 51Cr release amount calculated from the following formula: 100 × [(average cpm experimental amount−average cpm spontaneous release amount) / (average cpm total release amount−average cpm spontaneous release amount)]. Shown in The numerical values shown are the average + SEM for all samples at the indicated E / T ratio and at the time of display. Spontaneous release was measured after adding 2% SDS. The test results of this assay confirm the natural killer activity against lymphoblasts of AKR / J mice injected with the plasmid. This activity would normally be substantially absent in these mice.

Example VI
Response to Changes in Dosing Levels When Transfecting Naked IL-2 Gene into Young and Old Mice Using Balb / c mice and the plasmids described in Example V, pRS in 0.9% saline
VIL2 was injected into separate mice once a week at doses of 0 (ie, pRSV only), 5, 12.5, 25, 50, and 100 μg / injection, respectively. For each dose, three young mice (about 16 weeks of age) and three elderly mice (16 months of age and older) were injected intramuscularly in the right hind paw.

IL-2 expression was assayed for each group of mice as described in Example V. These data are shown in (a)-(b) of FIG. Animal weight and physical appearance were also monitored for signs of toxicity. At the test dose levels of 50 μg and 100 μg, some toxicity appeared.

  The best results for these mice were obtained at 12.5-25 μg in 100 ml of 0.9% saline. Expression was slightly larger in old mice at one month, but slightly larger in young mice at two weeks.

Example VII
Whole body expression after introduction of naked polynucleotides through various points of entry. A group of young Balb / c mice were exposed at weekly intervals to naked pRSVIL2 for (1) subcutaneous (back), (2) intramuscular (right Hind paw), (3) intradermal (base of tail), or (4) intranasal injection. The plasmid used was the same as described in Example V.

  Data on the level of systemic expression obtained by administering naked pRSVIL2 via each of these routes is shown in FIG.

Example VIII
Correlation of human IL-2 or expression in ICR mice with specific stimulation and expansion of mouse natural killer cell population Specific expansion of natural killer cell population in mice injected with IL-2 in AKR cancer model And stimulation are factors that have a beneficial effect on this group for tumor cell immune surveillance. Thus, in Example V, IL-2 levels (measured by ELISA) correlated with natural killer activity. Both values were significantly increased compared to the values of the group injected with the control. And the increase was experienced during the same time interval (0-3 months of injection). At the same time point, no changes in peripheral lymphocyte or granulocyte levels were detected by analysis of blood smear slides from the treated animals. This finding is important because it indicates that no inflammation occurred after injection and that a subset of the immune system other than the NK cell population was not affected by the level of IL-2 expression after injection. .

  The same information was obtained for aged animals as follows.

  Each group of animals consisted of 10 aged ICR mice (outbred mouse line with no inherent pathology). Animals were injected with pRSV or pRSV-IL-2 using the same protocol as the AKR animals in the previous example. The relative leukocyte counts of these animals (0-4 months after injection) were measured. The results are shown in Table 2 below.

  NK cell activity was measured in a 51Cr release assay as described in Example V. Target cells were p815 (NK-resistant mouse mastocytoma) or YAC-1 (NK-sensitive mouse lymphoma). Effector cells were isolated by lymphocyte M centrifugation. The formula used to measure NK cell activity is as follows:

Experimental release amount-spontaneous release release amount% = (...) x 100
Maximum release-spontaneous release These data are shown in Table 4 below.

Example IX
Histological study showing cellular uptake of naked polynucleotides by mononuclear cells at the point of entry of the skin. Mice were killed 3 days after the injection of naked pCMVlacz into the tail dermis of Balb / c mice. A tissue culture at the point of entry of the plasmid was obtained and stained for E. coli β-galactosidase activity. A photograph (× 40) of a slide obtained by histological examination of these cultures is shown in FIG.

  As shown in FIG. 15, it can be seen that the plasmid has been taken up by mononuclear cells (blue). The fibroblasts in the tissue samples were not stained, indicating that the plasmid was not taken up by these cells. Rounded mononuclear cells that have just taken up the plasmid appear to be macrophages and / or other antigen presenting cells. This indicates that the uptake of the plasmid is due to predation.

Example X
Epidermal Administration of Naked Polynucleotide Inducing Immune Response Using Mechanical Stimulator FIG. 16 shows the serum levels of anti-NP IgG after epidermal administration of pCMVRNP by mechanical means as described in Example I. Similarly, the results of analysis by the ELISA method are shown.

  Plasmid was coated on the tines of the uncoated MONO-VACC device as described above (or a naked polynucleotide was placed on the tines of the device for long-term storage stability). It should be noted that it can also be lyophilized). The total plasmid concentration over the entire tines of the device was about 50 μg in an isotonic normal saline carrier (about 150 μg plasmid / ml). The hair on the back of the Balb / c mouse was shaved and then the gently scratched skin with the above tincture device. As shown in FIG. 16, anti-NPGIgG was subsequently detected in the serum (eg, at day 42, the serum of this mouse contained a titer of 1: 10240 antibody).

Example XI
Epidermal Administration of Naked Polynucleotides to Induce an Immune Response Using Chemical Agents The serum concentration of anti-NP IgG after application of chemical agents and epidermal administration of pCMVRNP was determined by the ELISA method as described in Example I. FIG. 17 shows the results of the analysis.

  Plasmids were suspended in 40 μg of isotonic saline and contained about 150 μg of plasmid per ml. The solution was absorbed onto a non-adhesive pad (Johnson & Johnson) of a BAND-AID brand dressing.

  Balb / c mouse hair is sled off as described in Example X, and a commercially available keratolytic agent (ie, the previously described depilatory cream sold under the trade name NAIR) is shaved off. Was applied to the skin. After a few minutes, the keratolytic agent was washed off the skin and a bandage containing the plasmid was applied to the skin. As shown in FIG. 17, treated animals developed serum anti-NP IgG at a titer of 1: 640.

Example XII
Stability of IL-2 expression after administration of naked pRSV-IL-2 The stability of IL-2 expression was measured in the mice described in Example VII. After two injections of pRSV-IL-2 at one week intervals, the serum concentration of IL-2 was measured as described in Example V. In aged mice, serum levels of IL-2 decreased more slowly than in young mice. These data are shown in FIG. 18 and show that relatively stable gene expression can be achieved by introducing naked polynucleotides into tissues where the concentration of APC is relatively high, especially compared to muscle tissue. I have.

Example XIII
Immune Response to Viral Challenge by Mice Injected Intradermally with Naked pCMVRNP To test whether the immunity generated by vaccination with appropriate naked polynucleotides could protect animals from lethal viral challenge, Ten 15 Balb / c strains of 15 μg of the pCMVRNP plasmid containing the NP gene from the H1N1 strain of influenza virus (A / PR / 8/34, provided by Dr. Inocent N. Mawvike of Baylor College of Medicine, USA) Groups of mice received intradermal injections. Control groups include animals injected with a plasmid (pnBL3) that is not related to animals that were not injected.

  Six weeks after the initial plasmid injection, the animals were dosed with an LD90 dose of the H3N2 influenza strain (A / HK / 68, also provided by Dr. Mbawike). Mice vaccinated intradermally were significantly protected against the challenge (p <0.01) compared to unvaccinated control mice. See FIG. 19 (Kaplan-Meier survival curve).

Example XIV
Relative levels of gene expression after intradermal injection of a naked plasmid containing a naked cytomegalovirus or rous sarcoma virus promoter. The possible effect of the promoter region used in the expression vector was determined by using the RNP gene described in Example II. The evaluation was made by testing the two plasmids contained. One plasmid, pCMVRNP, had the cytomegalovirus immediate early promoter, enhancer and intron regions. The other plasmid had a promoter from the Rous sarcoma virus LTR region (pRSVRNP). As shown in FIG. 20, the antibody response to the NP protein expressed by these plasmids was consistently higher for the CMV promoter after dermal injection. Although this is different from the response seen after intramuscular injection of the NP gene, the levels of antibodies produced by the two plasmids are almost equivalent (data not shown).

Example XV
Selective induction of cytotoxic T lymphocyte response after intradermal administration of naked polynucleotide. CDM8 ova plasmid (see Shastri et al., J. Immunol., 150, 2724-) in the tail of C57 / B6 mice. 2736, 1993) was injected intradermally at two week intervals. This CDM8 ova plasmid contains the full-length (1.8 kb) cDNA for ovalbumin.

  Two weeks after the second gene administration, the spleen of the mouse was removed, and then a common gene pre-pulsed with a synthetic ovalbumin peptide and irradiated with lethal radiation (3000 rad) Was cultured in vitro with spleen cells. This peptide is a class I restricted target for cytotoxic T cells in mice and has the histocompatible haplotype Kb described by Shastri et al.

  After 5 days of culture, the cells were incubated with the two types of targets and tested for the generation of cytotoxic T cells by mice transfected with the gene encoding ovalbumin. These targets were mouse EL-4 lymphocytes pulsed with a synthetic ovalbumin peptide or EL-4 cells stably transfected with ovalbumin cDNA (see FIG. 21. The ovalbumin cDNA is shown in FIG. In the figure, “EG7” is indicated.) The percent lysis of these two targets was determined for different effector-to-target ratios (shown as E: T ratios in FIG. 21). As shown in FIG. 21, animals transfected with the naked CDM8 ova plasmid are specific for the ovalbumin target (ie, EL-4 and EG7 with the ovalbumin peptide), while the control EL-4 cells ( That is, non-specific cytotoxic T cells were produced for the ovalbumin peptide-free EL-4 cells).

  C57 / B6 mice vaccinated intradermally with the CDM8 ova plasmid were also screened for antibodies to ovalbumin. Serum collected 6 weeks after administration of the CDM8 ova plasmid contained no detectable levels of antibody (measured using an enzyme-linked immunosorbent assay on ovalbumin-coated microtiter plates). . Collectively, these data indicate that the method of administration of the naked polynucleotides of the present invention elicits MHC class I restricted cytotoxic T cells (in this case, T cells against ovalbumin) without inducing antibody production. It indicates that you want to.

Example XVI
Prolonged immune memory after intradermal administration of naked polynucleotides, induced by stimulation with antigen on T cells Plasmid-type naked polynucleotides encoding the E. coli enzyme β-galactosidase under the control of the CMV promoter (“pCMV Lac- Z ") (0.5-5 ng / 1 mg DNA endotoxin content) of 0.1, 1, 10 and 100 [mu] g in groups of four mice intramuscularly (" IM ") or intradermally (" IM "). ("ID"). For comparison, another group of four mice received 100 μg of β-galactosidase protein (“PR”) intradermally. All injections were made using 50 μl of normal saline as carrier. IM and ID injections were performed with a 0.5 ml syringe and a 28.5 gauge needle. Thereafter, at two-week intervals, antibodies were measured by an enzyme-linked immunosorbent assay.

  Briefly, total antibodies were measured using β-galactosidase (Calbiochem, CA, USA) as the solid phase antigen. Microtiter plates (Costar, Cambridge, Mass., USA) were coated overnight at room temperature with 5 μg of antigen dissolved in 90 mM borate (pH 8.3) +89 mM NaCl (ie borate buffered saline: BBS), then Blocked overnight with BBS containing 10 mg / ml bovine serum albumin.

  Serum samples were serially diluted with BBS at a 1:40 dilution for the first 8 weeks and thereafter at a 1: 320 dilution. These samples were added to the plates and stored overnight at room temperature. The plates are washed with BBS + 0.05% polysorbate 20 and then reacted with a 1: 2000 dilution of an alkaline phosphatase-labeled goat anti-mouse IgG antibody (Jackson Immunoresearch Labs., West Grove, Pa., USA) for 1 hour at room temperature. Or, under the same conditions, react with a 1: 2000 dilution of an alkaline phosphatase-labeled goat anti-mouse IgG antibody (Southern Bio-tech, Alabama, USA), or react with an alkaline phosphatase-labeled rat anti-mouse IgG2A antibody (California, USA) (Pharmingen, Calif.) At a 1: 500 dilution. The plate was washed again and then 1 mg / ml p-nitrophenol phosphate (Boehringer-Mannheim, Indianapolis, Ind., USA) in 0.05 M carbonate buffer (pH 9.8) containing 1 mM MgCl2. Was added. One hour after the substrate was added to the plate, the absorbance at 405 nm was read.

  As shown in FIG. 22, animals receiving ID injection of the pCMV Lac-Z plasmid and animals receiving PR elicited an equivalently large antibody response, whereas the pCMV Lac-Z plasmid received the pCMV Lac-Z plasmid. A smaller antibody response was measured in animals administered by IM injection.

  To assess T cell memory, animals were boosted with 0.5 μg of PR by ID injection at another site. If these animals had developed memory T cells to control the production of antibodies to β-galactosidase, they would respond to the first challenge after boosting the soluble protein antigen. Is likely to be a much more severe immune response than indicated in

  As shown in FIG. 23, it was clear that animals receiving the pCMV Lac-Z plasmid by ID injection developed significantly better immune memory than animals receiving the plasmid or PR by IM injection. is there. In addition, the memory generated by animals receiving ID injections persisted for a minimum of about 12 weeks.

Example XVII
Selective induction of a TH1 response after intradermal administration of a naked polynucleotide In mice, the IgG2A antibody is a serum marker for a TH1-type immune response, while an IgG1 antibody suggests a TH2-type immune response. TH2 responses include the allergic-associated IgE antibody class, and soluble protein antigens tend to stimulate relatively strong TH2 responses. In contrast, a TH1 response is triggered by antigens that bind to macrophages and dendritic cells. TH1 responses are particularly important for treating allergies and AIDS.

  Mice are treated with pCMV Lac-Z or the protein described in the preceding example to determine which response, if any, would be caused by the mice administered the naked polynucleotide of the invention. Inoculated. At two-week intervals, if any IgG2a and IgG1 to β-galactosidase are present, an enzyme-linked immunosorbent assay (using antibodies specific for the IgG1 and IgG2A subclasses) is performed on microtiter plates coated with the enzyme. ) To determine its presence.

  As shown in FIG. 24, only mice that received the plasmid by ID injection produced high titers of IgG2A antibody. As shown in Figure 25, immunization of mice with the enzyme itself ("PR") induced the production of relatively high titers of IgGl antibodies. In the case of mice injected with IM, both IgG2A and IgG1 antibodies were produced at low titers with no apparent selectivity. The data shown in these figures is composed of the average value of numerical values obtained from each group of four mice.

  To determine the stability of the antibody response over time, animals in the same group received an additional injection of 0.5 μg of the enzyme intradermally. As shown in FIGS. 26 and 27, additional administration of enzyme to pre-ID injected animals elicited a nearly 10-fold increase in IgG2A antibody response (ie, increased antibody titers from 1: 640 to 1: 5120). Did not stimulate the IgG1 response. These data indicate that the selective TH1 response elicited by ID administration of a naked polynucleotide is maintained in the host despite subsequent exposure to the antigen.

Example XVIII
TH1 Response in Mice Following Administration of Naked Polynucleotide by Mechanical Stimulator The experiment described in Example XVII was repeated in another group of mice, with the following differences. That is, the difference is that (1) only the first challenge was tested, and (2) the pCMV Lac-Z plasmid was administered to a group of four mice using the tine device described in Example X. On the other hand, β-galactosidase protein (10 μg) was administered by intradermal (ID) injection to another group of four mice.

  As shown in FIG. 28, mice that received the plasmid produced relatively lower titers of IgG1 antibody compared to mice that received the protein. In contrast, as shown in FIG. 29, the mice to which the plasmid was administered produced a sufficiently high titer of IgG2A antibody as compared to the mice to which the protein was administered.

  These test results are similar to those obtained in Example XVII, but interestingly, there are the following differences. That is, mice that received the plasmid by scratching the skin with a tine device produced higher titers of IgG2A antibody than mice that received the same plasmid by ID injection (both groups had IM injection). Produced higher titers of IgG2A antibody than the mice that received the plasmid). These test results show that scratching the skin with a tincture device attracts a large number of APCs to the "damaged" entry point for the naked polynucleotide and that the APCs Consistent with the theory that it is a more effective target for gene administration and expression than somatic cells.

  The data shown in these figures consist of the average of the numerical values obtained from each group of four mice.

FIG. 1A shows a cross section of muscle tissue showing chronic inflammation after intramuscular injection of pREVk3 and pRSVIL-2. FIG. 1B shows a section of muscle tissue showing muscle necrosis (panel B) after intramuscular injection of pREVk3 and pRSVIL-2. FIG. 1C shows a cross section of a similar muscle tissue after subcutaneous injection of pREVK3 or pRSVIL-2. FIG. 2A shows the results of ELISA analysis of serum anti-NP IgG after intradermal injection of naked pCMVRNP. FIG. 2B shows the results of an ELISA analysis of anti-NP IgG in serum after intramuscular injection of naked pCMVRNP. FIG. 3 shows the results of ELISA analysis of anti-NP IgG before introducing naked pCMVRNP into the nasal cavity of Balb / c mice. FIG. 4 shows the results of ELISA analysis of anti-NP IgG from Balb / c mice in the non-paralyzed group. FIG. 5 shows the results of ELISA analysis of anti-NP IgG from Balb / c mice in the anesthetized group. FIG. 6A shows the results of ELISA analysis of the concentration of anti-KLH in the serum of mice injected intramuscularly with pRSVIL-2 and pRSVIL-4. FIG. 6B shows the results of ELISA analysis of the concentration of anti-KLH in the serum of mice injected with pRSVTGFβ1 intramuscularly. FIG. 7A shows the results of ELISA analysis of the concentration of anti-transferrin in the serum of mice after intramuscular injection of pRSVIL-2. FIG. 7B shows the results of ELISA analysis of the concentration of anti-transferrin in the serum of mice after intramuscular injection of pRSVTGFβ1. FIG. 7C shows the results of ELISA analysis of the concentration of anti-transferrin in the serum of mice after intramuscular injection of pRSVTGFβ. FIG. 8 shows the results of a footpad tumor assay after antigen challenge (in mice injected with pRSVIL2 and pRSVTGFβ). FIG. 9 shows the results of ELISA analysis of anti-chromatin serum concentrations of MRL / lpr / lpr mice injected with pRSVIL-2 and pRSVTGFβ. FIG. 10 shows the results of ELISA analysis of IL-2 expression levels of individual AKR / J mice over time. FIG. 11 shows the long-lived data of the mouse described in FIG. FIG. 12 shows cells of NK cells of AKR / J mice (model of mouse lymphoma) after introduction of naked polynucleotide at a dose sufficient to induce IL-2 gene expression at sub-therapeutic concentrations. The results of a 51Cr release assay test for lytic activity are shown. FIG. 13A shows the level of IL-2 expression following administration of naked pRSVIL-2 at different doses to young Balb / c mice. FIG. 13B shows the level of IL-2 expression after administration of naked pRSVIL-2 at different doses to aged Balb / c mice. FIG. 14 shows the levels of IL-2 expression detected in serum following administration of pRSVIL-2 at different entry points. FIG. 15 is a photograph showing the results of histological examination of the skin at the entry point of pCMVRNP in Balb / c, showing the uptake of the plasmid by mononuclear cells (APC). APCs are indicated by arrows, and tissue cells (containing no plasmid) are indicated by a slushed line. FIG. 16 shows the results of ELISA analysis of anti-NP IgG after naked pCMVRNP was mechanically administered to the epidermis to Balb / c mice. FIG. 17 shows the results of ELISA analysis of anti-NP IgG after naked administration of naked pCMVRNP to Balb / c mice. FIG. 18 shows the stability of IL-2 expression detected in serum over time after administration of a naked polynucleotide encoding IL-2. FIG. 19 shows a Kaplan-Meier survival curve showing the duration of survival after administration of virus to Balb / c mice injected intradermally with naked pCMVRNP. FIG. 20 graphically compares the expression of the NP gene after separate intradermal injection of a naked plasmid containing the CMV or RSV promoter sequence. FIG. 21 shows the levels of cytotoxic T cells detected in mice after injection of various naked plasmids into the dermis. FIG. 22 shows that (1) anti-β-galactosidase was injected and administered intramuscularly or intradermally, and (2) anti-β-galactosidase was injected and injected intradermally. -Shows the results of ELISA analysis of galactosidase antibodies. FIG. 23 shows the results of ELISA analysis of anti-β-galactosidase antibody in serum from the mice shown in FIG. 22 after booster injection of the antigen. FIG. 24 shows the results of ELISA analysis of IgG2A type antibody in the serum of (1) a mouse injected with a polynucleotide encoding β-galactosidase intradermally or intramuscularly or (2) a mouse injected with the enzyme intradermally. . FIG. 25 shows the results of ELISA analysis of the IgG1 type antibody in the serum of (1) a mouse injected with a polynucleotide encoding β-galactosidase intradermally or intramuscularly or (2) a mouse injected with the enzyme intradermally. . FIG. 26 shows the results of ELISA analysis of the IgG2A type antibody in the serum of the mouse shown in FIG. 25 after the booster injection of the antigen was performed. FIG. 27 shows the results of ELISA analysis of the IgG1 type antibody in the serum of the mice shown in FIG. 24 after the booster injection of the antigen was performed. FIG. 28 shows IgG2A in serum of (1) mice introduced by scratching the skin with a pointed tip coated with a polynucleotide encoding β-galactosidase or (2) mice injected intradermally with the enzyme. 5 shows the results of ELISA analysis of type antibodies. FIG. 29 shows IgG1 in serum of (1) mice introduced by scratching the skin with a pointed tip coated with a polynucleotide encoding β-galactosidase or (2) mice injected intradermally with the enzyme. 5 shows the results of ELISA analysis of type antibodies.

Claims (29)

A medicament for administering a polynucleotide not deeper than the subcutaneous layer of the skin of a host to introduce the polynucleotide into antigen-presenting cells present in the skin, for introducing the antigen into a vertebrate host. ,
The medicament comprises a polynucleotide as an active ingredient,
The polynucleotide is naked in that it is not complexed with (a) any colloidal substance that interferes with the uptake of the polynucleotide by antigen presenting cells, (b) operatively encodes the antigen, and A medicament characterized by expressing said antigen in antigen presenting cells to stimulate a TH1-type immune response of a host. The medicament according to claim 1, wherein
A medicament characterized in that the polynucleotide is administered by an administration route including transdermal penetration. The medicament according to claim 1, wherein
The polynucleotide is applied to the epidermis of a host in a pharmaceutically acceptable topical composition, and a chemical stimulant capable of eliciting an immune response against itself is administered in combination with the polynucleotide. A medicament characterized in that it is administered by an administration route that involves attracting additional antigen presenting cells to the stimulation site for uptake. The medicament according to claim 1, wherein
A medicament characterized in that the polynucleotide is administered using a means that mechanically stimulates the epidermis of the host to attract additional antigen presenting cells for uptake and expression of the polynucleotide. The medicament according to claim 1, wherein
A medicament characterized by being administered by an administration route including intradermal or subcutaneous injection.
The polynucleotide is administered by an administration route including transdermal penetration The medicament according to claim 3, wherein
A medicament characterized in that said permeation is achieved using an iontophoretic patch. The medicament according to claim 1, wherein
A medicament comprising as a mixture up to 200 operably encoding polynucleotides. The medicament according to claim 7, wherein
A medicament characterized in that at least some of the polynucleotides in said mixture separately and operatively encode different antigens. The medicament according to claim 1, wherein
A medicament characterized in that the polynucleotide is selected from the group of molecules consisting of DNA, RNA or cDNA. The medicament according to claim 7, wherein
Furthermore, a medicament characterized in that the immunostimulatory peptide is used by being administered to a host. (A) naked in that it can be taken up by antigen presenting cells and is not complexed with any colloidal substance that interferes with the uptake of polynucleotides by antigen presenting cells, and operably encodes an antigen or immunostimulatory peptide At least one nucleotide that is
(B) a pharmaceutically acceptable carrier containing the polynucleotide; and (c) a keratolytic agent capable of stimulating the epidermis or mucosal epithelium of a mammal and eliciting an immune response against itself. And
A composition comprising attracting additional antigen presenting cells for uptake and expression of said polynucleotide to stimulate a TH1-type immune response in a host. An epidermal administration system for administering a polynucleotide into antigen-presenting cells in the skin or mucous membrane of a host by scratching the skin or mucous membrane,
(A) mechanical stimulation means for introducing a polynucleotide into a host comprising handle means attached with a number of needles of a length equal to the expected thickness of the outermost layer of the epidermis or mucosal epithelium of the host;
(B) a polynucleotide coated on the needle in a pharmaceutically acceptable carrier,
The polynucleotide may be (a) without any colloidal material that interferes with the uptake of the polynucleotide by the antigen-presenting cell, and (b) expressed in the antigen-presenting cell to stimulate a host TH1-type immune response to the antigen. A system operably encoding an antigen to The system according to claim 12, wherein
The system wherein the carrier further comprises an absorption enhancer. The system according to claim 12, wherein
The system wherein the carrier contains a chemical stimulant that attracts additional antigen presenting cells for uptake of the polynucleotide and expression of the antigen. The medicament according to claim 3, wherein
A medicament characterized in that the chemical stimulant is a keratolytic agent. The system according to claim 14, wherein
The system wherein the chemical stimulant is a keratolytic agent. The system according to claim 14, wherein
The system wherein the needle is also coated with an immunostimulatory peptide. The system according to claim 14, wherein
The system wherein the polynucleotide further encodes an immunostimulatory peptide operatively. The medicament according to claim 1, wherein
A medicament characterized in that said TH1-type immune response is stimulated for a response to an antigen already present in the host or separately administered to the host. The medicament according to claim 10, wherein
A medicament characterized in that the immunostimulatory peptide is expressed from a polynucleotide. The medicament according to claim 10, wherein
A medicament characterized in that the immunostimulatory peptide is selected from the group of peptides consisting of antigens, hormones, adjuvants, cytokines and growth factors. In order to introduce the antigen into the vertebrate host, the polynucleotide contained in a pharmaceutically acceptable carrier is administered into mucosal tissue no deeper than the epithelial layer of the mucosa of the host, and present in the mucosa. A drug for introducing a polynucleotide into an antigen-presenting cell,
The medicament comprises a polynucleotide included in a pharmaceutically acceptable carrier,
The polynucleotide is naked in that it is not complexed with (a) any colloidal substance that interferes with the uptake of the polynucleotide by antigen presenting cells, (b) operatively encodes the antigen, and A medicament characterized by expressing said antigen in antigen presenting cells to stimulate a TH1-type immune response of a host. The medicament according to claim 22, wherein
The polynucleotide is applied to a mucosal epithelium of a host in a pharmaceutically acceptable topical composition, and a chemical stimulant is administered in conjunction with the polynucleotide for additional uptake and expression of the polynucleotide. A medicament characterized by being administered by an administration route including attracting antigen-presenting cells. The medicament according to claim 22, wherein
A medicament characterized in that said polynucleotide is administered using means for mechanically stimulating the mucosal epithelium of a host to attract additional antigen presenting cells for uptake and expression of said polynucleotide. The medicament according to claim 22, wherein
A medicament comprising as a mixture up to 200 operably encoding polynucleotides. The medicament according to claim 25,
A medicament characterized in that at least some of the polynucleotides in said mixture separately and operatively encode different antigens. The medicament according to claim 22, wherein
A medicament characterized in that the polynucleotide is selected from the group of polynucleotides consisting of DNA, RNA or cDNA. The medicament according to claim 22, wherein
A medicament characterized in that the immunostimulatory peptide is selected from the group of peptides consisting of antigens, cytokines, adjuvants, hormones and growth factors. The medicament according to claim 22, wherein
A medicament characterized in that said TH1-type immune response is stimulated for a response to an antigen already present in the host or separately administered to the host.

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